U.S. patent application number 14/897612 was filed with the patent office on 2016-05-12 for methods for detecting prostate cancer.
The applicant listed for this patent is UNIVERSITY OF SOUTH AUSTRALIA. Invention is credited to Doug Brooks, Lisa Butler, Ian Johnson, Emma Parkinson-Lawrence, Roberto WEIGERT.
Application Number | 20160130661 14/897612 |
Document ID | / |
Family ID | 52021494 |
Filed Date | 2016-05-12 |
United States Patent
Application |
20160130661 |
Kind Code |
A1 |
Brooks; Doug ; et
al. |
May 12, 2016 |
METHODS FOR DETECTING PROSTATE CANCER
Abstract
The present disclosure relates to methods for detecting a
prostate cancer. Certain embodiments of the present disclosure
provide a method of detecting a prostate cancer in a subject, the
method comprising detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject.
Inventors: |
Brooks; Doug; (North
Cheltenham, AU) ; Parkinson-Lawrence; Emma;
(Morphettville, AU) ; Johnson; Ian; (Woodside,
AU) ; Butler; Lisa; (Paradise, AU) ; WEIGERT;
Roberto; (Washington, DC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF SOUTH AUSTRALIA |
South Australia |
|
AU |
|
|
Family ID: |
52021494 |
Appl. No.: |
14/897612 |
Filed: |
June 13, 2014 |
PCT Filed: |
June 13, 2014 |
PCT NO: |
PCT/AU2014/000612 |
371 Date: |
December 10, 2015 |
Current U.S.
Class: |
435/6.12 ;
435/7.1; 435/7.92; 436/501; 506/16; 506/18; 506/9; 514/169;
530/387.9; 600/1 |
Current CPC
Class: |
C12Q 2600/118 20130101;
C12Q 2600/158 20130101; C12Q 2600/106 20130101; G01N 2333/96455
20130101; G01N 33/57434 20130101; A61N 5/10 20130101; C12Q 1/6886
20130101; G01N 2800/54 20130101; C07K 2317/34 20130101; C07K
16/3069 20130101; C12Q 2600/112 20130101; G01N 2333/4703 20130101;
G01N 2333/82 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07K 16/30 20060101 C07K016/30; A61N 5/10 20060101
A61N005/10; G01N 33/574 20060101 G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 2013 |
AU |
2013902141 |
Claims
1. A method of detecting a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject,
thereby detecting a prostate cancer m the subject.
2. The method according to claim 1, wherein the selected marker
comprises one or more of an early endosomal marker, a late
endosomal marker, a marker associated with endosomal biogenesis, a
marker associated with endosomal trafficking and a marker
associated with endosomal recycling.
3. The method according to claims 1 or 2, wherein the selected
marker comprises one or more of CATHEPSIN B, CAPTHESEN D,
.alpha.-GALACTOSIDASE, RAB7, LIMP-1, LIMP-2, TFR1, TFR2, STAMP2,
SORT1 (SORTILIN), APPL1, EEA-1, LAMP-1, RAB4, APPL2, RAB5, RAB11,
MPR, PAP, ACTIN, M6PR, IGFR2, MYO1B, PDCD6IP, SDCBP, SDC1, STX7,
STX12, FGF1, FGF2, FGF3, FGFR1, FGFR2, FGFR3, NOX2, NOX4, and/or a
mRNA encoding one of the aforementioned, a fragment of one of the
aforementioned, a derivative of one of the aforementioned, and a
processed form of one of the aforementioned.
4. The method according to any one of claims 1 to 3, wherein one or
more of an altered presence, altered level, altered secretion and
altered distribution of the selected marker is indicative of
prostate cancer in the subject.
5. The method according to claim 4, wherein one or more of an
increased level of RAB5 protein and/or mRNA, an increased secretion
of RAB5 protein, an increased level of APPL1 protein and/or mRNA,
an increased secretion of APPL1 protein, an increased level of EEA1
protein and/or mRNA, an increased secretion of EEA 1 protein, an
increased level of LIMP-2 protein and/or mRN A, an increased level
of TFR 1 protein and/or mRNA, an increased level of TFR2 protein
and/or mRNA, an increased level of RAB4 protein and/or mRNA, an
increased secretion of RAB4 protein, an increased level of APPL2
protein and/or mRNA, a decreased level of LAMP1 protein and/or
mRNA, an increased secretion of RAB11 protein, a decreased
secretion of RAB7 protein, a decreased level of CAPTHESIN B protein
or mRNA, a decreased level of CAPTHESIN D protein or mRNA, an
increased level of .alpha.-GALACTOSIDASE protein or mRNA, a
decreased level of STX7 protein or mRNA, a decreased level of STX12
protein or mRNA, an increased secretion of PDCD6IP protein, a
decreased secretion of SDCBP protein, a decreased secretion of
SORT1 protein, a decreased level of FGF1 protein or mRNA, a
decreased level of FGF2 protein or mRNA, an increased level of FGF3
protein or mRNA, a decreased level of FGFR1 protein or mRNA, a
decreased level of FGFR2 protein or mRNA, an increased level of
FGFR3 protein or mRNA, an increased level of NOX2 protein or mRNA,
and increased level of NOX4 protein or mRNA, an increased nuclear
and/or nucleoli level of APPL1 protein, an increased nuclear
membrane level of RAB7 protein, and art enlarged LIMP2 protein
positive vesicles, is indicative of prostate cancer in the
subject.
6. The method according to claims 4 or 5, wherein an altered
presence, altered level, altered expression, altered secretion and
altered distribution is as compared to one or more of non-malignant
tissue, prostatic intraepithelial neoplasia, primary prostate
cancer and metastatic prostate cancer.
7. The method according to claim 6, wherein LIMP2 protein or mRNA
is increased in prostatic intraepithelial neoplasia as compared
non-malignant prostate, LAMP1 protein or mRNA is decreased in
metastatic prostate cancer as compared prostatic intraepithelial
neoplasia, LAMP1 protein or mRNA is increased in primary prostate
cancer as compared to metastatic prostate cancer, CAPTHESIN B
protein or mRNA is decreased in primary prostate cancer as compared
to non-malignant tissue, CAPTHESIN B protein or mRNA is decreased
in metastatic prostate cancer as compared to non-malignant
prostate, ACID CERAMIDASE protein or mRNA is increased in prostatic
intraepithelial neoplasia as compared to non-malignant prostate,
ACID CERAMIDASE protein or mRNA is increased in prostatic
intraepithelial neoplasia as compared to metastatic prostate
cancer, ACID CERAMIDASE protein or mRNA is increased in primary
prostate cancer as compared to metastatic prostate cancer, APPL1
protein or mRNA is increased in primary prostate cancer as compared
to non-malignant tissue, APPL2 protein or snRNA is increased in
prostatic intraepithelial neoplasia as compared to non-malignant
prostate, APPL2 protein or mRNA is increased in primary prostate
cancer as compared to non-malignant tissue, APPL2 protein or mRNA
is increased in prostatic intraepithelial neoplasia as compared to
metastatic prostate cancer, APPL2 protein or mRNA is increased in
primary prostate cancer as compared to metastatic prostate cancer,
RAB5 protein or mRNA is decreased in metastatic prostate cancer as
compared to non-malignant prostate, EEA1 protein or mRNA is
decreased in metastatic prostate cancer as compared non-malignant
prostate, EEA1 protein or mRNA is decreased in prostatic
intraepithelial neoplasia as compared to metastatic prostate
cancer, RAB4A protein or mRNA is decreased in metastatic prostate
cancer as compared to non-malignant prostate, RAB4A, protein or
mRNA is decreased in prostatic intraepithelial neoplasia as
compared to metastatic prostate cancer, RAB4A protein or mRNA is
decreased in prostatic intraepithelial neoplasia as compared to
primary prostatic cancer, RAB4A protein or mRNA is decreased in
primary prostate cancer as compared to metastatic prostate cancer,
MYO1B protein or mRNA is increased in prostatic intraepithelial
neoplasia as compared to metastatic prostate cancer, MYO1B protein
or mRNA is decreased in metastatic prostate cancer as compared
prostatic intraepithelial neoplasia, MYO1 B protein or mRNA is
decreased in metastatic prostate cancer as compared to primary
prostate cancer, PDCD61P protein or mRNA is decreased in prostatic
intraepithelial neoplasia as compared to metastatic prostate
cancer, PDCD6IP protein or mRNA is decreased in prostatic
intraepithelial neoplasia as compared to non-malignant prostate,
SDCBP protein or mRNA is decreased in metastatic prostate cancer as
compared to primary prostate cancer. STX7 protein or mRNA is
decreased in metastatic prostate cancer as compared to primary
prostate cancer, FGFR1 protein or mRNA is increased in metastatic
prostate cancer as compared prostatic intraepithelial neoplasia,
FGFR2 protein or mRNA is decreased in primary prostate cancer as
compared to non-malignant tissue, NOX2 protein or mRNA is increased
in primary prostate cancer as compared to non-malignant tissue and
NOX4 protein or mRNA is increased in metastatic prostate cancer as
compared prostatic intraepithelial neoplasia.
8. The method according to any one of claims 1 to 7, wherein the
method comprises obtaining a biological sample from the subject and
processing the biological sample to allow detection of the selected
marker.
9. The method according to claim 8, wherein the biological sample
comprises one or more of a blood sample, a plasma sample, a serum
sample, a biopsy and a prostate tissue sample.
10. The method according to any one of claims 1 to 9, wherein the
detecting of the selected marker comprises polymerase chain
reaction.
11. The method according to any one of claims 1 to 10, wherein the
detecting of the selected marker comprises immunological
detection.
12. The method according to any one of claims 1 to 11, wherein the
method comprises detecting two or more selected markers.
13. The method according to claim 12, wherein the method comprises
determining the ratio of the level of one selected marker to
another selected marker.
14. The method according to claim 13, wherein an altered ratio as
compared to non-maliatant tissue is indicative of a prostate cancer
in the subject.
15. The method according to claim 14, wherein an increased ratio of
an early endosomal marker to a late endosomal marker as compared to
non-malignant tissue is indicative of a prostate cancer in the
subject.
16. The method according to any one of claims 1 to 15, wherein the
method comprises comparing the presence and/or level of the
selected marker with the presence and/or level of one or more other
markers associated with an altered risk of prostate cancer and/or
one or more other markers known to be indicative of the presence or
absence of prostate cancer in the subject.
17. The method according to claim 16, wherein the one or more other
markers comprises prostate specific antigen.
18. The method according to any one of claims 1 to 17, wherein the
method further comprises obtaining information relating to one or
more clinical characteristics of the subject and using the
information in combination with one or more of the presence, level,
secretion and distribution of the selected marker to detect
prostate cancer in the subject.
19. The method according to any one of claims 1 to 18, wherein the
method comprises using a computer processor means to process data
associated with one or more of the presence, level, secretion and
distribution of the selected marker, and optionally one or more
clinical characteristics, to generate a likelihood and/or risk of
the presence of prostate cancer in the subject.
20. The method according to any one of claims 1 to 19, wherein the
prostate cancer is selected from a prostatic intraepithelial
neoplasia, a primary prostate cancer, and a metastatic prostate
cancer.
21. The method according to any one of claims 1 to 20, wherein the
method is used to diagnose prostate cancer in the subject, to
screen for prostate cancer in the subject, for assessing prognosis,
to determine the metastatic potential of a prostate cancer, to
identify a subject suffering from prostate cancer, to identify a
subject susceptible to prostate cancer, to determine the rate of
relapse of prostate cancer in the subject, to determine the risk of
mortality from prostate cancer in the subject, to stratify the
prostate cancer, to discriminate between prostate cancer and not
having prostate cancer in the subject, to determine whether the
prostate cancer is an organ confined cancer, to discriminate
between prostate cancer and one or more of benign prostatic
hyperplasia, prostatitis and an inflammatory condition of the
prostate, to determine pathogenic progression, to assess whether
the prostate cancer is slowing growing, indolent, or aggressive, to
exclude the presence of prostate cancer in the subject to identity
a subject suitable for treatment and/or surgery for prostate
cancer, and to determine the likelihood or risk of a subject baying
prostate cancer.
22. A method of detecting a prostate cancer in a subject, the
method comprising: obtaining a biological sample from the subject;
processing the sample to allow detection of a marker selected from
an endosomal associated marker and/or a lysosomal associated
marker; detecting one or more of an altered presence, level,
secretion and distribution of the selected marker in the processed
sample; and identifying a prostate cancer in the subject.
23. A method of screening for a prostate cancer in a subject, the
method comprising detecting a. marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject.
24. The method according to claim 23, wherein the method is used to
identify a subject suffering from, or susceptible to, a prostate
cancer.
25. The method according to claim 23, wherein the method is used to
exclude a subject not suffering from, or not susceptible to, a
prostate cancer.
26. A method of identifying a subject suffering from, or
susceptible to, a prostate cancer, the method comprising detecting
a marker selected from an endosomal associated marker and/or a
lysosomal associated marker from the subject.
27. A method of diagnosis for detecting a prostate cancer in a
subject, the method comprising detecting a marker selected from an
endosomal associated marker and/or a lysosomal associated marker
from the subject.
28. A method of determining the likelihood and/or risk of a subject
suffering from, or being susceptible to, a prostate cancer, the
method comprising detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject.
29. A method for determining the progression of a prostate cancer
in a subject, the method comprising detecting a marker selected
from an endosomal associated marker and/or a lysosomal associated
marker from the subject.
30. The method according to claim 29, wherein the method comprises
determining biochemical recurrence of the cancer, relapse rate
and/or survival rate.
31. The method according to claim 30, wherein the level of the
marker is indicative of a reduced relapse rat and/or increased
survival rate.
32. The method according to claim 30, wherein the marker comprises
one or more of LIMP2, CATHEPSIN B, CAPTHESIN D,
.alpha.-GALACTOSIDASE, RAB5A, EEA1, RAB7A, M6PR, IGFR2, SORT1,
MYO1B, PDCD6IP, SDC1, STX12, FGF2, FGF3 and/or a mRNA encoding one
of the aforementioned, a fragment of one of the aforementioned, a
derivative of one of the aforementioned, and a processed form of
one of the aforementioned.
33. The method according to claim 32, wherein the marker comprises
one or more of decreased or lower LIMP2, increased or higher
CATHEPSIN B, increased or higher CAPTHESIN D, increased or higher
.alpha.-GALACTOSIDASE, decreased or lower RAB5A, decreased or lower
EEA1, increased or higher RAB7A, increased or higher M6PR,
decreased or lower IGFR2, decreased or lower SORT1, decreased or
lower MYO1B, increased or higher PDCD6IP, increased or higher
STX12, increased or higher FGF2, increased or higher FGF3 and/or a
mRNA encoding one of the aforementioned, a fragment of one of the
aforementioned, a derivative of one of the aforementioned, and a
processed form of one of the aforementioned.
34. The method according to any one of claims 29 to 33, wherein the
method further comprises identifying the level of PSA expression in
the subject and stratifying the expression of the marker on the
basis of the PSA expression level in the subject.
35. The method according to claim 34, wherein the PSA is a level
indicative of a low risk of prostate cancer.
36. The method according to claim 35, wherein the PSA level is less
than 10 ng/ml.
37. The method according to claim 35 or 36, wherein the PSA is a
level of 7.8 ng/ml or less.
38. The method according to any one of claims 35 to 27, wherein the
marker comprises one or more of LIMP2, CATHEPSIN B,
.alpha.-GALACTOSIDASE, RAB5A, EEA1, M6PR, IGFR2, SORT1, MYO1B,
PDCD6IP, SDC1, STX 12, FGF2, FGF3 and/or a mRNA encoding one of the
aforementioned, a fragment of one of the aforementioned, a
derivative of one of the aforementioned, and a processed form of
one of the aforementioned.
39. The method according to claim 38, wherein the marker comprises
one or more of decreased or lower LIMP2, increased or higher
CATHEPSIN B, increased or higher .alpha.-GALACTOSIDASE, decreased
or lower RAB5A, decreased or lower EEA1, increased or higher M6PR,
decreased or lower IGFR2, decreased or lower SORT1, decreased or
lower MYO1B, increased or higher PDCD6IP, increased or higher SDC1,
increased or higher STX12, increased or higher FGF2, increased or
higher FGF3 and or a mRNA encoding one of the aforementioned, a
fragment of one of the aforementioned, a derivative of one of the
aforementioned, and a processed form of one of the
aforementioned.
40. The method according to claim 30, wherein the level of the
marker is indicative of an increased relapse rat and/or decreased
survival rate.
41. The method according to claim 40, wherein the marker comprises
one or more of LIMP2, CATHEPSIN B, CAPTHESIN D,
.alpha.-GALACTOSIDASE, RAB5A, EEA1, RAB7A, M6PR, IGFR2, SORT1,
MYO1B, PDCD6IP, SDC1, STX12, FGF2, FGF3 and/or a mRNA encoding one
of the aforementioned, a fragment of one of the aforementioned, a
derivative of one of the aforementioned, and a processed form of
one of the aforementioned.
42. The method according to claim 40, wherein the marker comprises
one or more of increased or higher LIMP2, decreased or lower
CATHEPSIN B, decreased or lower CAPTHESIN D, decreased or lower
.alpha.-GALACTOSIDASE, increased or higher RAB5A, increased or
higher EEA1, decreased or lower RAB7A, decreased or lower M6PR,
increased or higher IGFR2, increased or higher SORT1, increased or
higher MYO1B, decreased or lower PDCD6IP, decreased or lower STX
12, decreased or lower FGF2, decreased or lower FGF3 and/or a mRNA
encoding one of the aforementioned, a fragment of one of the
aforementioned, a derivative of one of the aforementioned, and a
processed form of one of the aforementioned.
43. The method according to any one of claims 40 to 43, wherein the
method further comprises identifying the level of PSA expression in
the subject and stratifying the expression the marker on the basis
of the PSA expression level n the subject.
44. The method according to claim 43, wherein the PSA is a level
indicative of a low risk of prostate cancer.
45. The method according to claim 44, wherein the PSA level is less
than 10 ng/ml.
46. The method according to claim 44 or 45, wherein the PSA is a
level of 7.8 ng/ml or less.
47. The method according to any one of claims 44 to 46, wherein the
marker comprises one or more of LIMP2, CATHEPSIN B,
.alpha.-GALACTOSIDASE, RAB5A, EEA1, M6PR, IGFR2, SORT1, MYO1B,
PDCD6IP, SDC1, STX12, FGF2, FGF3 and/or a mRNA encoding one of the
aforementioned, a fragment of one of the aforementioned, a
derivative of one of the aforementioned, and a processed form of
one of the aforementioned.
48. The method according to claim 47, wherein the marker comprises
one or more of increased or higher LIMP2, decreased or lower
CATHEPSIN B, decreased or lower .alpha.-GALACTOSIDASE, increased or
higher RAB5A, increased or higher EEA1, decreased or lower M6PR,
increased or higher IGFR2, increased or higher SORT1, increased or
higher MYO1B, decreased or lower PDCD6IP, decreased or lower SDC1,
decreased or lower STX12, decreased or lower FGF2, decreased or
lower FGF3 and/or a mRNA encoding one of the aforementioned, a
fragment of one of the aforementioned, a derivative of one of the
aforementioned, and a processed form of one of the
aforementioned.
49. Akit for performing the method of any one of claims 1 to
48.
50. A method of treating a prostate cancer in a subject, the method
comprising: detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject; and treating the subject based on one or more of the
presence, level, secretion and distribution of the selected marker
detected.
51. An isolated or purified antibody raised to a polypeptide
comprising an amino acid sequence of one or more of
ASNDHDAAINRYSRLSKKRENDKVKYEVTEDVYT (SED ID NO. 1),
DEVASDPLYVPDPDPTKFPVNRNLTRKAGYLNARNKT (SEQ ID NO. 2) SEGQFVVLSS
SQSEESDLGE GGKKRESEA (SEQ ID NO. 3), an antigenic fragment of any
of the aforementioned amino acid sequences, and/or a variant of any
of the aforementioned amino acid sequences or an antigenic fragment
thereof.
52. An isolated and/or purified antibody binding to an epitope in
an amino acid sequence in the human APPL1 protein comprising one or
more of ASNDHDAAINRYSRLSKKRENDKVKYEVTEDVYT (SED ID NO. 1),
DEVASDPLYVPDPDPTKFPVNRNLTRKAGYLNARNKT (SEQ ID NO. 2), SEGQFVVLSS
SQSEESDLGE GGKKRESEA (SEQ ID NO. 3), and/or an equivalent region of
a homolog, ortholov, or paralog of the protein.
53. An isolated and/or purified antibody raised to a polypeptide
comprising an amino acid sequence of one or more of
PNTFKTLDSWRDEFLIQASPRDPENFPFVVLGNKI (SED ID NO. 4),
DPENFPFVVLGNKIDLENRQVATKRAQAWCYSKNN (SEQ ID NO. 5),
ALKQETEVELYNEFPEPIKLDKNDRAKASAESCSC (SEQ ID NO. 6), an antigenic
fragment of any of the aforementioned amino acid sequences, and/or
a variant of any of the aforementioned amino acid sequences or an
antigenic fragment thereof.
54. An isolated and/or purified antibody binding to an epitope in
an amino acid sequence in the human RAB7 protein comprising one or
more of PNTFKTLDSWRDEFLIQASPRDPENFPFVVLGNKI (SED ID NO. 4),
DPENFPFVVLGNKIDLENRQVATKRAQAWCYSKNN (SEQ ID NO. 5),
ALKQETEVELYNEFPEPIKLDKNDRAKASAESCSC (SEQ ID NO. 6), and/or an
equivalent region of a homolog, ortholog or paralog of the
protein.
55. A method of detecting an APPL1 protein or a fragment thereof,
the method comprising using an antibody according to claims 51 or
52 to detect the APPL1 protein or fragment thereof.
56. A method of detecting a RAB7 protein or a fragment thereof, the
method comprising using an antibody according to claims 53 or 54 to
detect the RAB7 protein or fragment thereof.
57. A method of detecting a prostate cancer in a subject, the
method comprising using an antibody according any one of claims 51
to 54 to detect an APPL1 or RAB7 protein, and/or a fragment,
derivative or a processed form thereof from the subject.
58. A method of identifying a selected marker for diagnosis and/or
prognosis of a prostate cancer, the method comprising: identifying
a marker selected from an endosomal associated marker and/or a
lysosomal associated marker: and determining the ability of the
selected marker to diagnose and/or propose a prostate cancer;
thereby identifying the marker as a selected marker for diagnosis
and/or prognosis of a prostate cancer.
Description
PRIORITY CLAIM
[0001] This application claims priority to Australian provisional
patent application number 2013902141 filed on 13 Jun. 2013, the
contents of which are hereby incorporated by reference.
FIELD
[0002] The present invention relates to methods for detecting
prostate cancer, methods for the diagnosis and/or prognosis of
prostate cancer, methods for determining the progression of
prostate cancer, methods for treating prostate cancer based on the
detection of markers, and antibodies useful for diagnosis and/or
prognosis of prostate cancer.
BACKGROUND
[0003] Prostate cancer is the most common form of cancer in males
from developed countries, and the incidence of this disease is
predicted to double globally by 2030. For example, in 2008 more
than twenty thousand Australian men were diagnosed with prostate
cancer, and there were nearly three thousand deaths, making this
disease one of the largest causes of cancer-related deaths.
[0004] The prostate-specific antigen (PSA) test is currently used
for prostate cancer screening, however, this assay suffers from a
number of disadvantages, including a high percentage of
false-positive results. PSA also cannot distinguish between
aggressive or more slow-growing cancers at the time of diagnosis,
resulting in over-treatment. Recently there have been
recommendations to abandon this procedure, particularly in older
men.
[0005] The digital rectal examination is an alternative procedure
to check the prostate for abnormalities, but this test is limited
by the inability to assess the whole gland and to some degree the
size of the tumour.
[0006] There is therefore an urgent need for more specific and/or
more accurate detection methods for prostate cancer, to assist in
early diagnosis and selection of the most appropriate therapeutic
interventions. Early detection significantly reduces mortality from
prostate cancer, making improved diagnostic and prognostic methods
an important objective.
[0007] Given the deficiencies associated with current techniques of
diagnosis and/or prognosis of prostate cancer, the ability to
utilise other biomarkers to assist in the detection of prostate
cancer would be highly advantageous. However, the identification of
clinically relevant biomarkers associated with prostate cancer
remains problematic.
[0008] Accordingly, there remains a need to identify alternative
biomarkers to detect prostate cancer and/or to provide one or more
advantages in the art.
SUMMARY
[0009] The present disclosure is based on the determination that
specific endosomal and/or lysosomal associated markers may be used
to detect prostate cancer in a subject.
[0010] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject,
thereby detecting a prostate cancer in the subject.
[0011] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject.
[0012] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject,
wherein one or more of an altered presence, level, secretion and
distribution of the selected marker is indicative of a prostate
cancer in the subject.
[0013] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising: [0014] obtaining a biological sample from the subject;
[0015] processing the sample to allow detection of a marker
selected from an endosomal associated marker and/or a lysosomal
associated marker; [0016] detecting one or more of an altered
presence, level, secretion and distribution of the selected marker
in the processed sample; and [0017] identifying a prostate cancer
in the subject.
[0018] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising: [0019] obtaining a biological sample from the subject;
[0020] processing the sample to allow detection of a marker
selected from an endosomal associated marker and/or a lysosomal
associated marker; [0021] detecting one or more of the presence,
level, secretion and distribution of the selected marker in the
processed sample; [0022] comparing one or more of the presence,
level, secretion and distribution of the selected marker with one
or more other markers known to be indicative of the presence or
absence of prostate cancer in the subject; and [0023] identifying
prostate cancer in the subject.
[0024] Certain embodiments of the present disclosure provide a
method of identifying a subject suffering from or susceptible to a
prostate cancer, the method comprising detecting a marker selected
from an endosomal associated marker and/or a lysosomal associated
marker from the subject.
[0025] Certain embodiments of the present disclosure provide a
method of identifying a subject suffering from or susceptible to a
prostate cancer, the method comprising detecting a marker selected
from an endosomal associated marker and/or a lysosomal associated
marker from the subject, wherein one or more of an altered
presence, level, secretion and distribution of the selected marker
is indicative that the subject is suffering from or susceptible to
a prostate cancer.
[0026] Certain embodiments of the present disclosure provide a
method of screening for a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject.
[0027] Certain embodiments of the present disclosure provide a
method of screening for a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject,
wherein one or more of an altered presence, level, secretion and
distribution of the selected marker is indicative of prostate
cancer in the subject.
[0028] Certain embodiments of the present disclosure provide method
of treating a prostate cancer in a subject, the method comprising:
[0029] detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject; and
[0030] treating the subject based on one or more of the presence,
level, secretion and distribution of the selected marker
detected.
[0031] Certain embodiments of the present disclosure provide a
method of diagnosis for detecting a prostate cancer in a subject,
the method comprising detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject.
[0032] Certain embodiments of the present disclosure provide a
method of determining the likelihood and/or risk of a subject
suffering from, or being susceptible to, a prostate cancer, the
method comprising detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject.
[0033] Certain embodiments of the present disclosure provide a
method for determining the progression of a prostate cancer in a
subject, the method comprising detecting a marker selected from an
endosomal associated marker and/or a lysosomal associated marker
from the subject.
[0034] Certain embodiments of the present disclosure provide a kit
for performing a method as described herein.
[0035] Certain embodiments of the present disclosure provide a
method of treating a prostate cancer in a subject, the method
comprising: [0036] detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject; and [0037] treating the subject based on one or more of
the presence, level, secretion and distribution of the selected
marker detected.
[0038] Certain embodiments of the present disclosure provide an
isolated or purified antibody raised to a polypeptide comprising an
amino acid sequence of one or more of
ASNDHDAAINRYSRLSKKRENDKVKYEVTEDVYT (SED ID NO. 1),
DEVASDPLYVPDPDPTKFPVNRNLTRKAGYLNARNKT (SEQ ID NO. 2),
SEGQFVVLSSSQSEESDLGEGGKKRESEA (SEQ ID NO. 3), an antigenic fragment
of any of the aforementioned amino acid sequences, and/or a variant
of any of the aforementioned amino acid sequences or an antigenic
fragment thereof.
[0039] Certain embodiments of the present disclosure provide an
isolated and/or purified antibody binding to an epitope in an amino
acid sequence in the human APPL1 protein comprising one or more of
ASNDHDAAINRYSRLSKKRENDKVKYEVTEDVYT (SED ID NO. 1),
DEVASDPLYVPDPDPTKFPVNRNLTRKAGYLNARNKT (SEQ ID NO. 2),
SEGQFVVLSSSQSEESDLGE GGKKRESEA (SEQ ID NO. 3), and/or an equivalent
region of a homolog, ortholog or paralog of the protein.
[0040] Certain embodiments of the present disclosure provide an
isolated and/or purified antibody raised to a polypeptide
comprising an amino acid sequence of one or more of
PNTFKTLDSWRDEFLIQASPRDPENFPFVVLGNKI (SED ID NO. 4),
DPENFPFVVLGNKIDLENRQVATKRAQAWCYSKNN (SEQ ID NO. 5),
ALKQETEVELYNEFPEPIKLDKNDRAKASAESCSC (SEQ ID NO. 6), an antigenic
fragment of any of the aforementioned amino acid sequences, and/or
a variant of any of the aforementioned amino acid sequences or an
antigenic fragment thereof.
[0041] Certain embodiments of the present disclosure provide an
isolated and/or purified antibody binding to an epitope in an amino
acid sequence in the human RAB7 protein comprising one or more of
PNTFKTLDSWRDEFLIQASPRDPENFPFVVLGNKI (SED ID NO. 4),
DPENFPFVVLGNKIDLENRQVATKRAQAWCYSKNN (SEQ ID NO. 5),
ALKQETEVELYNEFPEPIKLDKNDRAKASAESCSC (SEQ ID NO. 6), and/or an
equivalent region of a homolog, ortholog or paralog of the
protein.
[0042] Certain embodiments of the present disclosure provide a
method of detecting an APPL1 protein or a fragment thereof, the
method comprising using an APPL1 antibody as described herein.
[0043] Certain embodiments of the present disclosure provide a
method of detecting a RAB7 protein or a fragment thereof, the
method comprising using an RAB7 antibody as described herein.
[0044] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising using an APPL1 antibody and/or a RAB7 antibody as
described herein.
[0045] Certain embodiments of the present disclosure provide a
method of identifying a selected marker for diagnosis and/or
prognosis of a prostate cancer, the method comprising: [0046]
identifying a marker selected from an endosomal associated marker
and/or a lysosomal associated marker; and [0047] determining the
ability of the selected marker to diagnose and/or prognose a
prostate cancer; [0048] thereby identifying the marker as a
selected marker for diagnosis and/or prognosis of a prostate
cancer.
[0049] Other embodiments are disclosed herein.
BRIEF DESCRIPTION OF THE FIGURES
[0050] Certain embodiments are illustrated by the following
figures. It is to be understood that the following description is
for the purpose of describing particular embodiments only and is
not intended to be limiting with respect to the description.
[0051] FIG. 1 shows quantification of endosomal and lysosomal gene
expression in control and prostate cancer cell lines. Levels of
mRNA transcripts in non-malignant control cell lines (white bars)
and prostate cancer cell lines (black bars) were evaluated by qPCR
in triplicate experiments. Data was expressed relative to GAPDH
endogenous control and analysed by Kruskal-Wallis rank sum method.
Statistical significance (p<0.05) is represented by an
asterisk.
[0052] FIG. 2 shows detection and quantification of intracellular
lysosomal proteins in non-malignant control and prostate cancer
cell lines. (A) Representative images from Western blot analysis of
10 .mu.g whole cell lysate from non-malignant control cell lines
PNT 1 a and PNT2, and cancer cell lines 22RV1 and LNCaP, examined
in triplicate. (B) Protein amount was quantified by densitometry
relative to GAPDH endogenous control. Data was analysed by
Kruskal-Wallis rank sum method with statistical significance
(p<0.05) represented by an asterisk.
[0053] FIG. 3 shows confocal micrographs showing altered
localisation of endosomal markers in prostate cancer cell lines
compared to non-malignant control cell lines. Fixed cells were
probed for endosome markers (green) and counterstained with DAPI
nuclear stain (blue) and visualised by laser-scanning confocal
microscopy. Transmitted light illumination was captured for
visualisation of cell outline (white). Scale bars for all images
represent 10 .mu.m.
[0054] FIG. 4 shows confocal micrographs of
Lysotracker.RTM.-positive vesicles in prostate cell lines.
Non-malignant control prostate cell lines PNT1a and PNT2, and
prostate cancer cell lines 22RV1 and LNCaP were stained with
Lysotracker.RTM. (green). Cell outlines were visualised by TPMT and
membrane depicted by white blotted line. Scale bars for all images
represent 10 .mu.m.
[0055] FIG. 5 shows time-course of transferrin uptake in prostate
cell lines. Confocal micrographs showing increased uptake and
altered distribution of transferrin in prostate cancer cell lines
compared to non-malignant control cell lines; together with altered
actin staining. Cell cultures were incubated with transferrin Alexa
Fluor.RTM. 633 conjugate (red) for a period of 5, 15 and 30 minutes
prior to cell fixation and actin labelled with phalloidin Alexa
Fluor.RTM. 488 (green). Scale bars represent 10 .mu.m.
[0056] FIG. 6 shows transferrin and endosome/lysosome marker
co-fluorescence. Confocal micrographs showing transferrin (red) and
endosome/lysosome marker (green) in non-malignant control cell
lines PNT1a and PNT2, and prostate cancer cell lines 22RV1 and
LNCaP. Colocalisation is represented by yellow fluorescence.
[0057] FIG. 7 shows analysis of transferrin receptor expression and
cell localisation with transferrin. (A) Western blot analysis and
quantification of protein amount and gene expression of transferrin
receptor 1 (TFR1) and transferrin receptor 2 (TFR2). Quantification
of protein and gene expression was relative to GAPDH protein and
gene, respectively. Asterisk represents p<0.05. (B) Confocal
micrographs and enlargements showing transferrin (red) and
transferrin receptor (green) in non-malignant control cell lines
PNT1a and PNT2, and prostate cancer cell lines 22RV1 and LNCaP.
Colocalisation of transferrin receptor and transferrin is
represented by yellow fluorescence.
[0058] FIG. 8 shows AKT phosphorylation levels in non-malignant
control and cancer cell lines before and subsequent to transferrin
treatment.
[0059] FIG. 9 shows LAMP-1 and APPL1 expression in prostate tissue
isolated from different cases. LAMP-1 and APPL1 expression in
matched human non-malignant (A, D) and tumour (B, E) prostate
tissue. Both non-malignant and malignant tissue is stained for
LAMP-1 (C) and APPL1 (F). The arrows in D & F show APPL1
staining the basement membrane in non-malignant prostate tissue.
Scale bar=100 .mu.M in A, B & D and 200 .mu.M in C, E, F.
[0060] FIG. 10 shows detection and quantification of known prostate
cancer biomarkers in non-malignant and prostate cancer cell lines.
Western blots of biomarkers/GAPDH in cell extracts (A; 50 .mu.g of
whole cell lysate) and culture media (B; 3 mL culture media,
collected after 48 hours incubation with confluent cells) from the
non-malignant control cell lines RWPE 1, PNT1a and PNT2, and
prostate cancer cell lines 22RV1, CaHPV10, DU 145 and LNCaP.
Western blots are a representative of triplicates. The amount of
each intracellular (C) or secreted (D) protein was quantified from
the Western blots by densitometry relative to GAPDH (endogenous
control). Non-malignant cell lines are depicted by white bars and
prostate cancer cell lines by black bars and results represent the
mean .+-.SE (n=3).
[0061] FIG. 11 shows detection of lysosomal proteins in
non-malignant and prostate cancer cell lines. Western blots of
lysosomal proteins/GAPDH in cell extracts (A; 50 .mu.g of whole
cell lysate) and culture media (B; 3 mL culture media, collected
after 48 hours incubation with confluent cells) from the
non-malignant control cell lines RWPE 1, PNT1a and PNT2, and
prostate cancer cell lines 22RV1, CaHPV10, DU 145 and LNCaP.
Western blots are representative of triplicate experiments.
[0062] FIG. 12 shows quantification of lysosomal proteins in
non-malignant and prostate cancer cell lines. The amount of each
intracellular (A) or secreted (B) lysosomal protein was quantified
from the Western blots (FIG. 12.2) by densitometry relative to
GAPDH (endogenous control). Non-malignant cell lines are depicted
by white bars and prostate cancer cell lines by black bars and
results represent the mean .+-.SE (n=3).
[0063] FIG. 13 shows LIMP2 and LAMP1 gene expression is altered
independently of TFEB expression in prostate cancer. Expression
profiling data derived from Affymetrix Human Exon 1.0 ST arrays of
150 primary prostate cancers and 29 non-malignant tissues {Taylor,
2010 #976} were quantitated to show percentage change of gene
expression of TFEB, LIMP2 and LAMP1. Box-and-whisker graphs were
plotted with Tukey outliers (black points). Statistical
significance is represented by an asterisk (**P.ltoreq.0.01;
****P.ltoreq.0.0001).
[0064] FIG. 14 shows TFEB-regulated lysosomal genes with variable
expression in prostate cancer; cathepsin B had significantly
reduced expression whilst .alpha. glucosidase had increased
expression in prostate cancer. Expression profiling data derived
from Affymetrix Human Exon 1.0 ST arrays of 150 primary prostate
cancers and 29 non-malignant tissues {Taylor, 2010 #976} were
quantitated to show percentage change of expression of
lysosome-related genes. Box-and-whisker graphs were plotted with
Tukey outliers (black points). Statistical significance is
represented by an asterisk (*P.ltoreq.0.05;
****P.ltoreq.0.0001).
[0065] FIG. 15 shows APPL1 gene expression was significantly
increased in prostate cancer whilst other endosome-related markers
showed variable expression. Expression profiling data derived from
Affymetrix Human Exon 1.0 ST arrays of 150 primary prostate cancers
and 29 non-malignant tissues {Taylor, 2010 #976} were quantitated
to show percentage change of expression of endosome-related genes.
Box-and-whisker graphs were plotted with Tukey outliers (black
points). Statistical significance is represented by an asterisk
(*P.ltoreq.0.05).
[0066] FIG. 16 shows lysosome gene expression data from the cohort
by Tomlins et al. that mirror findings from the Taylor cohort.
Detailed analysis of prostate cancer tissue revealed altered
endosomal gene expression during prostate cancer progression.
Expression profiling data derived from the Chinnaiyan Human 20K Hs6
array of 27 nonmalignant tissues, 13 prostatic intraepithelial
neoplasia's, 32 primary prostate cancer and 22 metastatic cancer
tissue samples {Tomlins, 2007 #974} were quantitated to show
relative amount of expression of lysosome-related genes.
Statistical significance is represented by an asterisk
(*P.ltoreq.0.05; **P.ltoreq.0.01; ***P.ltoreq.0.001).
[0067] FIG. 17 shows expression of endosomal genes in the Tomlins
cohort with significant increases, while metastatic prostate cancer
tissue showed variable endosomal gene expression. Expression
profiling data derived from the Chinnaiyan Human 20K Hs6 array of
27 nonmalignant tissues, 13 prostatic intraepithelial neoplasia's,
32 primary prostate cancer and 22 metastatic cancer tissue samples
{Tomlins, 2007 #974} were quantitated to show relative amount of
endosome gene expression. Statistical significance is represented
by an asterisk (*P.ltoreq.0.05; **P.ltoreq.0.01;
***P.ltoreq.0.001)
[0068] FIG. 18 shows Kaplan-Meier analysis of lysosomal genes and
revealed cathepsin B as a candidate gene for patient stratification
based on BCR. Cathepsin B differentiated patients at risk of
relapse based on the amount of gene expression. Expression of
lysosomal genes that have previously been observed to be
significantly altered in primary cancer showed trends for
differences between high and low-risk patients, but were not
significant. Patients from the Glinsky cohort {Glinsky, 2004 #979}
were stratified into two groups by K means clustering based on gene
expression level (high--black line, low--grey line). Statistical
analysis was performed using Gehan-Breslow-Wilcoxon test.
[0069] FIG. 19 shows endosomal gene expression did not stratify
patients into significant prognostic groups. Patients from the
Glinsky cohort were stratified into two groups by K means
clustering based on amount (high--black line, low--grey line) of
APPL1, APPL2, RAB5A, EEA1, RAB4A and RAB7A gene expression.
Analysis was performed using Gehan-Breslow-Wilcoxon test; and no
statistical significance was observed between the expression
groups.
[0070] FIG. 20 shows Kaplan-Meier analysis of lysosomal gene
expression showed significant capability for prognosis in patients
expressing low PSA protein based on cathepsin B and a galactosidase
A expression. (A) Pre-operative PSA level of 7.8 ng/mL was used as
a cut-off discrimination level for patient's stratification into
poor- and good-prognosis subgroups. (B) From the good-prognosis
subgroup of PSA.ltoreq.7.8 ng/mL, patients were further stratified
into two groups by K-means clustering based on gene expression of
LIMP2, LAMP1, cathepsin B or .alpha. galactosidase A (high
expression--black line, low expression--grey line). Statistical
analysis was performed using Gehan-Breslow-Wilcoxon test.
Statistical significance is marked by an asterisk (*P.ltoreq.0.05;
**P.ltoreq.0.01). (C) Multivariate analysis of gene expression in
prostate cancer recurrence. BCR: biochemical recurrence; G-B-W:
Gehan-Breslow-Wilcoxon test; HR: hazard ratio; CI: confidence
interval.
[0071] FIG. 21 shows the combined gene signature of RAB5A, APPL1
and EEA1 could stratify low PSA-expressing patients into prognostic
subgroups based on BCR. (A) Patients from the Glinsky cohort
expressing PSA.ltoreq.7.8 mg/mL were stratified into groups by
K-means clustering based on RAB5A, APPL1 and EEA1 gene expression;
the three-gene combined signature of RAB5A, APPL1 and EEA1
stratified patients based on BCR (P.ltoreq.0.0221,
Gehan-Breslow-Wilcoxon test; high expression--black line, low
expression--grey line). (B) Multivariate analysis of gene
expression in prostate cancer recurrence. BCR: biochemical
recurrence; G-B-W: Gehan-Breslow-Wilcoxon test; HR: hazard ratio;
CI: confidence interval.
[0072] FIG. 22 shows detection and quantification of secreted
endosome-related proteins from non-malignant control and prostate
cancer cell lines. (A) Representative Western blots of protein
secreted from non-malignant control cell lines PNT 1 a and PNT2,
and prostate cancer cell lines 22RV1 and LNCaP. (B) The amount of
each protein in non-malignant control cells (white bars) and
prostate cancer cells (black bars) was quantified by densitometry
from triplicate Western blots and significant differences denoted
by an asterisk (*P.ltoreq.0.05; **p.ltoreq.0.01).
[0073] FIG. 23 shows rabbit anti-Appl1 polyclonal antibody
affinities to Appl 1 recombinant protein (Panel A) and rabbit
anti-Rab7 polyclonal antibody affinities to Rab7 recombinant
protein (Panel B).
[0074] FIG. 24 shows in Panel A standard curve for
europium-labelled rabbit anti-Rab7 polyclonal antibody (raised to
epitope #1) and Panel B shows standard curve for europium-labelled
rabbit anti-Appl1 polyclonal antibody (raised to epitope #3).
[0075] FIG. 25 shows vertical scatter plots of endosomal-lysosomal
gene expression data from the cohort by Tomlins et al.
[0076] FIG. 26 shows Kaplan-Meier analysis of
endosome/lysosome-related genes and patient stratification based on
biochemical recurrence (BCR).
[0077] FIG. 27 shows Kaplan-Meier survival and multivariate
analysis of endosomal-lysosomal gene expression for cancer patients
expressing .ltoreq.7.8 ng/mL PSA.
[0078] FIG. 28 shows (A) Box-and-whisker graphs showing percentage
change in gene expression of Syntaxin 7 and Syntaxin 12. (B)
Vertical scatter plots of gene expression data from the cohort by
Tomlins et al.
[0079] FIG. 29 shows Kaplan-Meier survival analysis of Syntaxin 7
and Syntaxin 12 gene expression.
[0080] FIG. 30 shows detection and quantification of intracellular
and secreted endosome-related proteins.
[0081] FIG. 31 shows box-and-whisker graphs showing percentage
change in gene expression of FGF1 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al.
[0082] FIG. 32 shows box-and-whisker graphs showing percentage
change in gene expression of FGF2 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al.
[0083] FIG. 33 shows box-and-whisker graphs showing percentage
change in gene expression of FGF3 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al.
[0084] FIG. 34 shows Kaplan-Meier survival analysis of FGF1, FGF2
and FGF3 gene expression.
[0085] FIG. 35 shows Kaplan-Meier survival analysis of FGF1, FGF2
and FGF3 gene expression.
[0086] FIG. 37 shows box-and-whisker graphs showing percentage
change in gene expression of FGFR1 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al. Statistical
significance is represented by an asterisk (****P.ltoreq.0.0001;
**P.ltoreq.0.01).
[0087] FIG. 38 shows box-and-whisker graphs showing percentage
change in gene expression of FGFR2 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al. Statistical
significance is represented by an asterisk (****P.ltoreq.0.0001;
***P.ltoreq.0.001).
[0088] FIG. 39 shows box-and-whisker graphs showing percentage
change in gene expression of FGFR3 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al.
[0089] FIG. 40 shows Kaplan-Meier survival analysis of FGFR1, FGFR2
and FGFR3 gene expression.
[0090] FIG. 41 shows Kaplan-Meier survival analysis of FGFR1, FGFR2
and FGFR3 gene expression.
[0091] FIG. 42 shows detection and quantification of NOX2 protein
(65 (56) & 30 kDa) from non-malignant control and prostate
cancer cell lines.
[0092] FIG. 43 shows box-and-whisker graphs showing percentage
change in gene expression of NOX2 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al. Statistical
significance is represented by an asterisk (****P.ltoreq.0.0001,
**P.ltoreq.0.01).
[0093] FIG. 44 shows detection and quantification of NOX4 protein
from non-malignant control and prostate cancer cell lines.
[0094] FIG. 45 shows box-and-whisker graphs showing percentage
change in gene expression of NOX4 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al. Statistical
significance is represented by an asterisk (****P.ltoreq.0.0001,
**P.ltoreq.0.01, *P.ltoreq.0.05).
[0095] FIG. 46 shows APPL1, Rab7 and LIMPII expression in prostate
tissue.
DETAILED DESCRIPTION
[0096] The present disclosure is based on the determination that
specific endosomal associated and/or lysosomal associated markers
may be used for the diagnosis and/or prognosis of prostate
cancer.
[0097] Certain embodiments of the present disclosure provide
methods for detecting a prostate cancer in a subject. Certain
embodiments of the present disclosure provide methods for
determining the progression of a prostate cancer in a subject.
Certain embodiments of the present disclosure provide methods of
treating prostate cancer in a subject based on use of selected
markers. Other embodiments are disclosed herein.
[0098] Certain disclosed embodiments have one or more combinations
of advantages. For example, some of the advantages of the
embodiments disclosed herein include one or more of the following:
the identification of a new class of markers for the diagnosis
and/or prognosis of prostate cancer; one or more markers that in
some instances may be used for both diagnosis and prognosis of
prostate cancer; one or more markers that are readily detectable in
a biological sample, such as in a tissue or biopsy sample, blood or
plasma; use of one or more protein based markers; use of one or
more nucleic acid based markers; one or more markers which may be
used in conjunction with other types of markers; use of a method
that is amenable to high throughput analysis of samples; use of a
method that assists in identifying subjects suitable for
pharmacological and/or surgical intervention to treat prostate
cancer; markers that are suitable for use in kits; to address one
or more problems in the art; to provide one or more advantages in
the art; or to provide a useful commercial choice. Other advantages
of certain embodiments are disclosed herein.
[0099] As described herein, the present disclosure is based on the
determination that specific endosomal associated and/or lysosomal
associated markers in a subject may be used to detect prostate
cancer.
[0100] Further, certain embodiments of the present disclosure are
based, at least in part, on the recognition that a unique change in
the cell biology of early and late endosomes occurs in prostate
cancer cells. Without being bound by theory, late endosomes
normally form intra-luminal vesicles by invagination of their outer
membrane (i.e. form small vesicles inside of the late endosome,
also called multi vesicular bodies). These small internal
.about.100 nm vesicles can be released from normal cells when the
late endosomes fuse with the cell surface, releasing their internal
contents from the cell, together with these exosome vesicles.
Because of the way that they are formed, exosomes can contain
cytosolic proteins as well as endosome vesicular machinery. In
normal controls early endosomal associated markers can therefore be
released from cells and detected in the extracellular
milieu/circulation. It has been found that there is a specific
reduction in the release of early endosomal associated markers from
prostate cancer cells when compared to control cells. More
importantly, it has been discovered that: the vesicular machinery
associated with different populations of early endosomes, are
released from prostate cancer cells, suggesting that early
endosomes may be able to form intra-luminal vesicles in prostate
cancer cells; early endosomes fuse with the plasma membrane of
prostate cancer cells and preferentially release these early
endosome derived exosome vesicles; the process of endosome
biogenesis is altered to form intra-luminal vesicles in early
rather than late endosomes. This provides a unique set of changes
to both early and late endosomes in prostate cancer cells and also
provides a basis for a ratio when detecting biomarkers from these
different endosome populations, which effectively identifies the
changes in prostate cancer when compared to control cells.
[0101] Certain embodiments of the present disclosure provide
methods and kits for detecting a prostate cancer in a subject.
[0102] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject.
[0103] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject,
thereby detecting the prostate cancer in the subject.
[0104] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject by detecting a
marker selected from an endosomal associated marker and/or a
lysosomal associated marker from the subject, thereby detecting the
prostate cancer on the basis of the endosomal associated marker
and/or the lysosomal associated marker so detected.
[0105] In certain embodiments, the prostate cancer is selected from
a prostatic intraepithelial neoplasia, a primary prostate cancer,
and a metastatic prostate cancer. Other forms and/or grades of
prostate cancer are contemplated.
[0106] In this regard, typically the Gleason Grading system is used
to evaluate a prostate cancer. A "score" is assigned to a prostate
cancer on the basis of the combination of a "Gleason" pattern
associated with various features of a tumor specimen and a
subsequent grade assigned to the patterns of the tumour specimen. A
Gleason score of 2-4 is considered to be a cancer of low
aggressiveness. A score of 5-6 is considered to be a cancer of
moderate aggressiveness. A score of 7 is considered to be a score
of intermediate aggressiveness. A score of 8-10 is considered to be
a cancer of high aggressiveness. In certain embodiments, the
prostate cancer is a cancer with a Gleason score of any of the
aforementioned scores.
[0107] Prostate cancers may also be categorised by stage, being a
measure of how far a cancer has developed. In Stage 1, the cancer
is small and contained within the prostate. In Stage 2, the cancer
is larger and may be in both lobes of the prostate, but is still
confined to the organ. In Stage 3, the cancer has spread beyond the
prostate and may have invaded the adjacent lymph glands or seminal
vesicles. In Stage 4, the cancer has spread to other organs, or to
bone. In certain embodiments, the prostate cancer is a cancer with
a staging of any of the aforementioned stages.
[0108] It will be appreciated that while the present disclosure is
described with reference to detecting a prostate cancer in a human
subject, the present disclosure contemplates detecting prostate
cancer in an animal subject, and accordingly veterinary
applications of the present disclosure are also contemplated.
[0109] In certain embodiments, the subject is suffering from a
prostate cancer. Examples of prostate cancers are as described
herein.
[0110] Certain embodiments of the present disclosure provide a
method of detecting a subject suffering from a prostate cancer, the
method comprising detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject.
[0111] In certain embodiments, the subject is a subject with an
increased likelihood or risk of suffering from a prostate cancer.
In certain embodiments, the subject is a subject susceptible to a
prostate cancer. In certain embodiments, the subject is a subject
with one or more risk factors associated with a prostate cancer. In
certain embodiments, the subject is a subject with an unknown
likelihood or risk of suffering prostate cancer.
[0112] In certain embodiments, the subject is a subject with a
measured or known PSA level. Examples of PSA levels are as
described herein, for example, as described in Example 8 herein. In
certain embodiments the subject is a subject with one or more of
the characteristics as described in one or more of the Figures
and/or Examples.
[0113] In certain embodiments, the subject is suitable for
treatment for a prostate cancer.
[0114] The term "associated marker" refers to a marker which is
enriched in one or more particular tissues, cells, organelles,
and/or cell compartments and as such can be used alone, or in
combination with other markers, to assist in the identification of
the tissue, cell, organelle, and/or cell compartment.
[0115] In certain embodiments, the endosomal associated marker
and/or the lysosomal associated marker comprises one or more of an
early endosomal marker, a late endosomal marker, a marker
associated with endosomal biogenesis, a marker associated with
endosomal trafficking and a marker associated with endosomal
recycling. Other types of endosomal and lysosomal markers are
contemplated. Methods for determining whether a marker is one of
the aforementioned markers are known in the art.
[0116] In certain embodiments, the selected marker as described
herein comprises a protein, a polypeptide, a fragment, a derivative
or a processed form of the aforementioned proteins or polypeptides,
a nucleic acid including a mRNA, a microRNA, a nuclear RNA, a rRNA,
or a fragment, a processed or unprocessed form of the
aforementioned RNAs, a lipid, a cell surface marker, a receptor, or
a cofactor. Other types of markers are contemplated.
[0117] In certain embodiments, the selected marker is a protein
marker, and/or a fragment, an antigenic fragment, a derivative or a
processed form thereof. Methods for detecting proteins are known in
the art and examples are also as described herein.
[0118] In certain embodiments, the selected marker is a RNA marker,
typically a mRNA, and/or a fragment, a derivative or a processed
form thereof. Methods for detecting mRNAs are known in the art and
examples are also as described herein.
[0119] As described herein, in certain embodiments, the detection
of a mRNA may require the production of a cDNA strand complementary
to the mRNA.
[0120] In certain embodiments, the selected marker may be useful as
a protein marker. In certain embodiments, the selected marker may
be useful as a mRNA marker. In certain embodiments, the selected
marker may be useful as a protein marker and a mRNA marker.
[0121] In certain embodiments, the selected marker comprises one or
more of CATHEPSIN B, CAPTHESIN D, .alpha.-GALACTOSIDASE, RAB7,
LIMP-1, LIMP-2, TFR1, TFR2, STAMP2, SORT1 (SORTILIN), APPL1, EEA-1,
LAMP-1, RAB4, APPL2, RAB5, RAB11, MPR, PAP, ACTIN, M6PR, IGFR2,
MYO1B, PDCD6IP, SDCBP, SDC1, STX7, STX12, FGF1, FGF2, FGF3, FGFR1,
FGFR2, FGFR3, NOX2, NOX4, and/or a mRNA encoding one of the
aforementioned, a fragment of one of the aforementioned, a
derivative of one of the aforementioned, and a processed form of
one of the aforementioned.
[0122] In certain embodiments, the selected marker comprises one or
more markers as described in any of the examples and/or
figures.
[0123] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising detecting a marker selected from one or more of
CATHEPSIN B, CAPTHESIN D, .alpha.-GALACTOSIDASE, RAB7, LIMP-1,
LIMP-2, TFR1, TFR2, STAMP2, SORT1 (SORTILIN), APPL1, EEA-1, LAMP-1,
RAB4, APPL2, RAB5, RAB11, MPR, PAP, ACTIN, M6PR, IGFR2, MYO1B,
PDCD6IP, SDCBP, SDC1, STX7, STX12, FGF1, FGF2, FGF3, FGFR1, FGFR2,
FGFR3, NOX2, NOX4, and/or a mRNA encoding one of the
aforementioned, a fragment of one of the aforementioned, a
derivative of one of the aforementioned, and a processed form of
one of the aforementioned.
[0124] It will be appreciated that the selected markers of the
present disclosure are referred to herein as the human forms of the
selected markers. However, it will be appreciated that the
detection and/or use of equivalent markers are also contemplated.
Equivalent markers in other species may be readily identified by a
person skilled in the art.
[0125] Methods for detecting markers are known in the art.
Typically, a marker present in a subject is detected in a sample,
or a processed form of a sample, taken from a subject. For example,
methods for detecting proteins and RNAs are known and may be
performed typically using commercially available products. General
methods, including methods for protein and RNA detection,
extraction and isolation are known, are as described in, for
example, Ausubel et al., Current Protocols of Molecular Biology,
John Wiley and Sons (1997), the entire contents of which is hereby
incorporated by reference.
[0126] Methods for detection of proteins markers are known and
include for example immunological detection methods such as
immunobinding, immunoblotting (eg Western analysis),
immunoprecipitation, immunoelectrophoresis, immunostaining,
immunohistochemistry, spectrophotometry, enzyme assays, mass
spectrometry, and microscopy. Other methods are contemplated.
[0127] Methods for detecting nucleic acids are known and include
microarray analysis, blotting (Northern, Southern), in situ
hybridization, RT-PCR, End-Point Stem-Loop Real-Time RT-PCR, miR-Q
RT-PCR, (A)-Tailed Universal Reverse Transcription, RNA
Amplification Profiling, cloning based methods, nanoparticle based
methods, splinted ligation methods, padlock-probes and
rolling-circle amplification, bead-based flow cytometric methods,
bioluminescence RNA detection methods, molecular beacon methods,
ribozyme methods, and quantitative LNA-ELF-FISH methods. Other
methods are contemplated.
[0128] In certain embodiments, the detecting of RNA markers
comprises reverse transcription. Methods for reverse transcribing
RNAs are known in the art. In certain embodiments, the detecting of
RNA markers comprises amplification of a nucleic acid. Methods for
nucleic acid amplification are known in the art. In certain
embodiments, the detecting of RNA markers comprises a polymerase
chain reaction. In certain embodiments, the polymerase chain
reaction comprises a quantitative polymerase chain reaction.
[0129] In certain embodiments, the detecting of RNA markers
comprises binding or hybridization of nucleic acids to one or more
target nucleic acids. In certain embodiments, the detecting of RNA
markers comprises binding of nucleic acids to one or more target
nucleic acids bound to a solid substrate, such as a chip. Methods
for binding nucleic acids to target nucleic acids, including
binding to nucleic acids bound to a solid substrate, are known.
[0130] In certain embodiments, the detecting of the selected marker
comprises a polymerase chain reaction. In certain embodiments, the
polymerase chain reaction comprises a quantitative polymerase chain
reaction.
[0131] In certain embodiments, the detecting of the selected marker
comprises immunological detection. In certain embodiments, the
immunological detection comprises ELISA, staining with an antibody,
immunohistochemistry, and/or flow cytometric detection. Methods
involving immunological detection are known in the art.
[0132] In certain embodiments, the methods as described herein
comprise detecting one or more of the presence, level, expression,
secretion and distribution of the selected marker.
[0133] In certain embodiments, one or more of an altered presence,
altered level, altered expression, altered secretion and altered
distribution of the selected marker is indicative of a prostate
cancer in the subject.
[0134] In certain embodiments, an increased level and/or an
increased secretion of an endosomal associated marker is indicative
of prostate cancer in the subject. In certain embodiments, a
decreased level and/or a decreased secretion of an endosomal
associated marker is indicative of prostate cancer in the
subject.
[0135] In certain embodiments, an increased level and/or an
increased secretion of a lysosomal associated marker is indicative
of prostate cancer in the subject. In certain embodiments, a
decreased level and/or a decreased secretion of a lysosomal
associated marker is indicative of prostate cancer in the
subject.
[0136] In certain embodiments, an increased level and/or an
increased secretion of a protein marker is indicative of a prostate
cancer in the subject. In certain embodiments, a decreased level
and/or a decreased secretion of a protein marker is indicative of a
prostate cancer in the subject.
[0137] In certain embodiments, an increased level of a mRNA marker
is indicative of prostate cancer in the subject. In certain
embodiments, a decreased level of a mRNA marker is indicative of
prostate cancer in the subject.
[0138] In certain embodiments, an increased level and/or an
increased secretion of an early endosomal marker is indicative of
prostate cancer in the subject.
[0139] In certain embodiments, one or more of an increased level of
RAB5 protein and/or mRNA, an increased secretion of RAB5 protein,
an increased level of APPL1 protein and/or mRNA, an increased
secretion of APPL1 protein, an increased level of EEA1 protein
and/or mRNA, an increased secretion of EEA1 protein, an increased
level of LIMP-2 protein and/or mRNA, an increased level of TFR1
protein and/or mRNA, an increased level of TFR2 protein and/or
mRNA, an increased level of RAB4 protein and/or mRNA, an increased
secretion of RAB4 protein, an increased level of APPL2 protein
and/or mRNA, a decreased level of LAMP1 protein and/or mRNA, an
increased secretion of RAB 11 protein, a decreased secretion of
RAB7 protein, a decreased level of CAPTHESIN B protein or mRNA, a
decreased level of CAPTHESIN D protein or mRNA, an increased level
of .alpha.-GALACTOSIDASE protein or mRNA, a decreased level of STX7
protein or mRNA, a decreased level of STX12 protein or mRNA, an
increased secretion of PDCD6IP protein, a decreased secretion of
SDCBP protein, a decreased secretion of SORT1 protein, a decreased
level of FGF1 protein or mRNA, a decreased level of FGF2 protein or
mRNA, an increased level of FGF3 protein or mRNA, a decreased level
of FGFR1 protein or mRNA, a decreased level of FGFR2 protein or
mRNA, an increased level of FGFR3 protein or mRNA, an increased
level of NOX2 protein or mRNA, and increased level of NOX4 protein
or mRNA, an increased nuclear and/or nucleoli level of APPL1
protein, an increased nuclear membrane level of RAB7 protein, and
an enlarged LIMP2 protein positive vesicles, is indicative of
prostate cancer in the subject.
[0140] In certain embodiments, an altered presence, altered level,
altered expression, altered secretion and altered distribution of
one or more markers is as compared to one or more of non-malignant
tissue, prostatic intraepithelial neoplasia, primary prostate
cancer and metastatic prostate cancer.
[0141] In certain embodiments, LIMP2 protein or mRNA is increased
in prostatic intraepithelial neoplasia as compared non-malignant
prostate, LAMP 1 protein or mRNA is decreased in metastatic
prostate cancer as compared prostatic intraepithelial neoplasia,
LAMP 1 protein or mRNA is increased in primary prostate cancer as
compared to metastatic prostate cancer, CAPTHESIN B protein or mRNA
is decreased in primary prostate cancer as compared to
non-malignant tissue, CAPTHESIN B protein or mRNA is decreased in
metastatic prostate cancer as compared to non-malignant prostate,
ACID CERAMIDASE protein or mRNA is increased in prostatic
intraepithelial neoplasia as compared to non-malignant prostate,
ACID CERAMIDASE protein or mRNA is increased in prostatic
intraepithelial neoplasia as compared to metastatic prostate
cancer, ACID CERAMIDASE protein or mRNA is increased in primary
prostate cancer as compared to metastatic prostate cancer, APPL1
protein or mRNA is increased in primary prostate cancer as compared
to non-malignant tissue, APPL2 protein or mRNA is increased in
prostatic intraepithelial neoplasia as compared to non-malignant
prostate, APPL2 protein or mRNA is increased in primary prostate
cancer as compared to non-malignant tissue, APPL2 protein or mRNA
is increased in prostatic intraepithelial neoplasia as compared to
metastatic prostate cancer, APPL2 protein or mRNA is increased in
primary prostate cancer as compared to metastatic prostate cancer,
RAB5 protein or mRNA is decreased in metastatic prostate cancer as
compared to non-malignant prostate, EEA1 protein or mRNA is
decreased in metastatic prostate cancer as compared non-malignant
prostate, EEA1 protein or mRNA is decreased in prostatic
intraepithelial neoplasia as compared to metastatic prostate
cancer, RAB4A protein or mRNA is decreased in metastatic prostate
cancer as compared to non-malignant prostate, RAB4A protein or mRNA
is decreased in prostatic intraepithelial neoplasia as compared to
metastatic prostate cancer, RAB4A protein or mRNA is decreased in
prostatic intraepithelial neoplasia as compared to primary
prostatic cancer, RAB4A protein or mRNA is decreased in primary
prostate cancer as compared to metastatic prostate cancer, MYO1B
protein or mRNA is increased in prostatic intraepithelial neoplasia
as compared to metastatic prostate cancer, MYO1B protein or mRNA is
decreased in metastatic prostate cancer as compared prostatic
intraepithelial neoplasia, MYO1B protein or mRNA is decreased in
metastatic prostate cancer as compared to primary prostate cancer,
PDCD6IP protein or mRNA is decreased in prostatic intraepithelial
neoplasia as compared to metastatic prostate cancer, PDCD6IP
protein or mRNA is decreased in prostatic intraepithelial neoplasia
as compared to non-malignant prostate, SDCBP protein or mRNA is
decreased in metastatic prostate cancer as compared to primary
prostate cancer, STX7 protein or mRNA is decreased in metastatic
prostate cancer as compared to primary prostate cancer, FGFR1
protein or mRNA is increased in metastatic prostate cancer as
compared prostatic intraepithelial neoplasia, FGFR2 protein or mRNA
is decreased in primary prostate cancer as compared to
non-malignant tissue, NOX2 protein or mRNA is increased in primary
prostate cancer as compared to non-malignant tissue and NOX4
protein or mRNA is increased in metastatic prostate cancer as
compared prostatic intraepithelial neoplasia.
[0142] In certain embodiments, the methods as described herein
comprise obtaining a biological sample from the subject.
[0143] In certain embodiments, the methods as described herein
comprise processing the biological sample to allow detection of the
selected marker. In certain embodiments, the methods as described
herein comprise processing a biological sample to allow detection
of a marker as described herein and detecting the marker in the
processed sample. In certain embodiments, the methods as described
herein comprise obtaining a biological sample from the subject and
processing the biological sample to allow detection of the selected
marker.
[0144] The term "biological sample" refers to a sample obtained
from the subject and/or a processed and/or treated form thereof.
For example, the biological sample may be untreated, diluted, a
derivative, an extract, a treated form, pre-cleared, filtered,
desalted, concentrated, diluted, buffered, centrifuged, induced,
pre-treated, processed to remove one or more components or
impurities from the sample, sliced, fixed, adhered to a slide, or
suitable combinations thereof. In certain embodiments, a selected
marker is detected in the sample directly. In certain embodiments,
a selected marker is detected in the sample after processing and/or
treating. In certain embodiments, the sample is processed and/or
treated prior to detecting the selected marker and/or concurrently
with detecting the selected marker.
[0145] Examples of biological samples include one or more
biological fluids, such as blood, plasma, urine, amniotic fluid,
tears, saliva, hair, skin, and one or more tissue samples or a
biopsy. Other types of biological samples are contemplated.
[0146] In certain embodiments, the biological sample comprises one
or more of a blood sample, a plasma sample, a serum sample, a
biopsy and a prostate tissue sample.
[0147] In certain embodiments, the biological sample comprises a
biopsy or a tissue sample. Certain embodiments provide detecting
the in situ level of a selected marker.
[0148] In certain embodiments, the selected marker comprises one or
more blood markers, plasma markers, and/or serum markers. Certain
embodiments provide detecting the circulating level of a selected
marker.
[0149] In certain embodiments, the detecting comprises a
qualitative determination. In certain embodiments, the detecting
comprises a qualitative determination of whether the selected
marker has one or more of an altered presence, an altered level, an
altered expression, an altered secretion and an altered
distribution. In certain embodiments, the detecting comprises a
quantitative determination of whether the selected marker has one
or more of an altered presence, an altered level, an altered
expression, an altered secretion and an altered distribution.
[0150] In certain embodiments, the detecting comprises a
qualitative determination. In certain embodiments, the detecting
comprises a qualitative determination of whether the selected
marker is present or absent. In certain embodiments, the detecting
comprises a quantitative assessment of the level of the selected
marker. For example, certain methods allow for the quantification
of the concentration of the selected marker. Methods for the
calculation or determination of the concentration of markers are
known in the art.
[0151] Certain embodiments of the present disclosure comprise
detecting two or more selected markers. Certain embodiments of the
present disclosure comprise detecting three or more selected
markers. Certain embodiments of the present disclosure comprise
detecting four or more selected markers.
[0152] In certain embodiments, the methods of the present
disclosure comprise detecting two or more of the selected markers.
In certain embodiments, the methods comprise detecting three or
more of the selected markers. In certain embodiments, the methods
comprise detecting four or more of the selected markers.
[0153] Certain embodiments of the present disclosure comprise
detecting two or more of the following selected markers: CATHEPSIN
B, CAPTHESIN D, .alpha.-GALACTOSIDASE, RAB7, LIMP-1, LIMP-2, TFR1,
TFR2, STAMP2, SORT1 (SORTILIN), APPL1, EEA-1, LAMP-1, RAB4, APPL2,
RAB5, RAB11, MPR, PAP, ACTIN, M6PR, IGFR2, MYO1B, PDCD6IP, SDCBP,
SDC1, STX7, STX12, FGF1, FGF2, FGF3, FGFR1, FGFR2, FGFR3, NOX2,
NOX4 and/or a mRNA encoding one of the aforementioned, a fragment
of one of the aforementioned, a derivative of one of the
aforementioned, and a processed form of one of the
aforementioned.
[0154] Certain embodiments of the present disclosure comprise
detecting three or more of the following selected markers:
CATHEPSIN B, CAPTHESIN D, .alpha.-GALACTOSIDASE, RAB7, LIMP-1,
LIMP-2, TFR1, TFR2, STAMP2, SORT1 (SORTILIN), APPL1, EEA-1, LAMP-1,
RAB4, APPL2, RAB5, RAB11, MPR, PAP, ACTIN, M6PR, IGFR2, MYO1B,
PDCD6IP, SDCBP, SDC1, STX7, STX12, FGF1, FGF2, FGF3, FGFR1, FGFR2,
FGFR3, NOX2, NOX4 and/or a mRNA encoding one of the aforementioned,
a fragment of one of the aforementioned, a derivative of one of the
aforementioned, and a processed form of one of the
aforementioned.
[0155] Certain embodiments of the present disclosure comprise
detecting four or more of the following selected markers: CATHEPSIN
B, CAPTHESIN D, .alpha.-GALACTOSIDASE, RAB7, LIMP-1, LIMP-2, TFR1,
TFR2, STAMP2, SORT1 (SORTILIN), APPL1, EEA-1, LAMP-1, RAB4, APPL2,
RAB5, RAB11, MPR, PAP, ACTIN, M6PR, IGFR2, MYO1B, PDCD6IP, SDCBP,
SDC1, STX7, STX12, FGF1, FGF2, FGF3, FGFR1, FGFR2, FGFR3, NOX2,
NOX4 and/or a mRNA encoding one of the aforementioned, a fragment
of one of the aforementioned, a derivative of one of the
aforementioned, and a processed form of one of the
aforementioned.
[0156] In certain embodiments, the methods as described herein
comprise determining the ratio of the level of one selected marker
to another selected marker.
[0157] In certain embodiments, an altered ratio is indicative of a
prostate cancer in the subject. In certain embodiments, an altered
ratio as compared to non-malignant tissue is indicative of a
prostate cancer in the subject. Other forms of comparison between
different types of prostate tissue are as described herein.
[0158] In certain embodiments, an increased ratio of an early
endosomal marker to a late endosomal marker is indicative of
prostate cancer in the subject. In certain embodiments, an
increased ratio of an early endosomal marker to a late endosomal
marker as compared to non-malignant tissue is indicative of a
prostate cancer in the subject.
[0159] In certain embodiments, the methods of the present
disclosure comprise detecting one or more other markers in addition
to the selected marker.
[0160] In certain embodiments, the methods of the present
disclosure provide use of one or more markers, control markers
and/or reference markers, as described herein.
[0161] An alteration in the presence, level, expression, secretion
and distribution of a marker is typically relative to the level of
one or more corresponding markers, for example one or more
corresponding proteins or mRNAs in one or more control subjects
and/or one or more subjects known to have prostate cancer.
[0162] In certain embodiments, the methods as described herein
comprise comparing the presence, level, expression, secretion and
distribution of the selected marker with one or more other markers
known to be indicative of a prostate cancer in a subject and/or
known to be indicative of the absence of a cancer.
[0163] In certain embodiments, the methods as described herein
comprise comparing the presence, level, expression, secretion and
distribution of the selected marker to one or more reference and/or
control markers.
[0164] In certain embodiments, the methods as described herein
comprise comparing the presence and/or level of the selected marker
with the presence and/or level of one or more other markers
associated with an altered risk of prostate cancer and/or one or
more other markers known to be indicative of the presence or
absence of prostate cancer in the subject.
[0165] In certain embodiments, the reference marker comprises an
endogenous marker. In certain embodiments, the reference marker
comprises an exogenous marker. For example, a sample may be spiked
with an exogenous reference marker.
[0166] In certain embodiments, the one or more other markers
comprises prostate specific antigen (PSA).
[0167] In certain embodiments, the methods of the present
disclosure comprise processing the biological sample to allow
detection of the selected markers. In certain embodiments, the
methods of the present disclosure comprise processing a biological
sample obtained from the subject to allow detection of the selected
marker. Subjects are as described herein.
[0168] In certain embodiments, the methods and kits as described
herein comprise use of one or more reagents for processing a sample
for analysis.
[0169] In certain embodiments, the methods as described herein
further comprise obtaining information relating to one or more
clinical characteristics of the subject and using the information
in combination with one or more of the presence, level, secretion
and distribution of the selected marker to detect prostate cancer
in the subject. In certain embodiments, the one or more clinical
characteristics comprise one or more of age, body mass index,
smoking, genetics and family history of cancer and/or prostate
cancer.
[0170] In certain embodiments, the methods as described herein
further comprise obtaining information relating to one or more
clinical characteristics of the subject and using the information
in combination with one or more of the presence, level, expression
secretion and distribution of the selected marker to detect
prostate cancer in the subject or the absence of prostate
cancer.
[0171] In certain embodiments, the methods as described herein
comprise using a computer processor means to process data
associated with one or more of the presence, level, secretion and
distribution of the selected marker to generate a likelihood and/or
risk of the presence of prostate cancer in the subject. Examples of
computer processor means are known.
[0172] In certain embodiments, the methods have a sensitivity of
detection of 0.60 or greater. In certain embodiments, the methods
have a sensitivity of detection of 0.70 or greater. In certain
embodiments, the methods have a sensitivity of detection of 0.80 or
greater. In certain embodiments, the methods have a sensitivity of
detection of 0.90 or greater. In certain embodiments, the methods
have a sensitivity of detection of 0.95 or greater.
[0173] In certain embodiments, the methods have a specificity of
detection of 0.60 or greater. In certain embodiments, the methods
have a specificity of detection of 0.70 or greater. In certain
embodiments, the methods have a specificity of detection of 0.80 or
greater. In certain embodiments, the methods have a specificity of
detection of 0.90 or greater. In certain embodiments, the methods
have a specificity of detection of 0.95 or greater.
[0174] In certain embodiments, the methods as described herein are
used to diagnose prostate cancer in the subject, to screen for
prostate cancer in the subject, for assessing prognosis, to
determine the metastatic potential of a prostate cancer, to
identify a subject suffering from prostate cancer, to identify a
subject susceptible to prostate cancer, to determine the rate of
relapse of prostate cancer in the subject, to determine the risk of
mortality from prostate cancer in the subject, to stratify the
prostate cancer, to discriminate between prostate cancer and not
having prostate cancer in the subject, to determine whether the
prostate cancer is an organ confined cancer, to discriminate
between prostate cancer and one or more of benign prostatic
hyperplasia, prostatitis and an inflammatory condition of the
prostate, to determine pathogenic progression, to assess whether
the prostate cancer is slow growing, indolent, or aggressive, to
exclude the presence of prostate cancer in the subject, to identify
a subject suitable for treatment and/or surgery for prostate
cancer, and to determine the likelihood or risk of a subject having
prostate cancer.
[0175] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising: [0176] obtaining a biological sample from the subject;
[0177] processing the sample to allow detection of a marker
selected from an endosomal associated marker and/or a lysosomal
associated marker; [0178] detecting one or more of an altered
presence, level, expression, secretion and distribution of the
selected marker in the processed sample; and [0179] identifying a
prostate cancer in the subject.
[0180] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising: [0181] obtaining a biological sample from the subject;
[0182] processing the sample to allow detection of a marker in the
sample; [0183] detecting one or more of CATHEPSIN B, CAPTHESIN D,
.alpha.-GALACTOSIDASE, RAB7, LIMP-1, LIMP-2, TFR1, TFR2, STAMP2,
SORT1 (SORTILIN), APPL1, EEA-1, LAMP-1, RAB4, APPL2, RAB5, RAB11,
MPR, PAP, ACTIN, M6PR, IGFR2, MYO1B, PDCD6IP, SDCBP, SDC1, STX7,
STX12, FGF1, FGF2, FGF3, FGFR1, FGFR2, FGFR3, NOX2, NOX4, and/or a
mRNA encoding one of the aforementioned, a fragment of one of the
aforementioned, a derivative of one of the aforementioned, and a
processed form of one of the aforementioned for an altered
presence, level, expression, secretion and distribution of the
selected marker in the processed sample; and [0184] identifying
prostate cancer in the subject.
[0185] Certain embodiments of the present disclosure provide a
method of detecting prostate cancer in a subject substantially as
described herein with reference to any of the accompanying examples
and/or figures.
[0186] Certain embodiments of the present disclosure provide a
method or kit for identifying a subject suffering from, or
susceptible to, a prostate cancer.
[0187] Certain embodiments of the present disclosure provide a
method of identifying a subject suffering from or susceptible to a
prostate cancer, the method comprising detecting a marker selected
from an endosomal associated marker and/or a lysosomal associated
marker from the subject.
[0188] Certain embodiments of the present disclosure provide a
method of identifying a subject suffering from, or susceptible to,
a prostate cancer, the method comprising detecting a marker
selected from an endosomal associated marker and/or a lysosomal
associated marker from the subject, wherein one or more of an
altered presence, level, expression, secretion and distribution of
the selected marker is indicative that the subject is suffering
from, or susceptible to, a prostate cancer.
[0189] Certain embodiments of the present disclosure provide a
method of identifying a subject suffering from or susceptible to a
prostate cancer in a subject, the method comprising detecting a
marker from the subject as hereinbefore described with reference to
any of the examples and/or figures.
[0190] Certain embodiments of the present disclosure provide
methods or kits for screening for a prostate cancer in a
subject.
[0191] Certain embodiments of the present disclosure provide a
method of screening for a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject.
[0192] In certain embodiments, the method is used to identify a
subject suffering from, or susceptible to, a prostate cancer.
[0193] In certain embodiments, the method is used to exclude a
subject not suffering from, or not susceptible to, a prostate
cancer.
[0194] Certain embodiments of the present disclosure provide a
method of screening for a prostate cancer in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject,
wherein one or more of an altered presence, level, expression,
secretion and distribution of the selected marker is indicative of
a prostate cancer in the subject.
[0195] Certain embodiments of the present disclosure provide a
method of screening for a prostate cancer in a subject, the method
comprising detecting a marker from the subject as hereinbefore
described with reference to any of the examples and/or figures.
[0196] Certain embodiments of the present disclosure provide a
method or kit for diagnosis of a prostate cancer in a subject.
[0197] Certain embodiments of the present disclosure provide a
method of diagnosis for detecting a prostate cancer in a subject,
the method comprising detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject.
[0198] Certain embodiments of the present disclosure provide a
method or kit for determining the likelihood and/or risk of a
subject suffering from, or being susceptible to, a prostate
cancer.
[0199] Certain embodiments of the present disclosure provide a
method of determining the likelihood and/or risk of a subject
suffering from, or being susceptible to, a prostate cancer, the
method comprising detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject.
[0200] Certain embodiments of the present disclosure provide a
method or kit for determining the progression of a prostate cancer
in a subject.
[0201] Certain embodiments of the present disclosure provide a
method for determining the progression of a prostate cancer in a
subject, the method comprising detecting a marker selected from an
endosomal associated marker and/or a lysosomal associated marker
from the subject.
[0202] In certain embodiments, the method comprises determining
biochemical recurrence of the cancer, relapse rate and/or survival
rate.
[0203] In certain embodiments, the level of the marker is
indicative of a reduced relapse rate and/or increased survival
rate. In certain embodiments, the marker comprises one or more of
LIMP2, CATHEPSIN B, CAPTHESIN D, .alpha.-GALACTOSIDASE, RAB5A,
EEA1, RAB7A, M6PR, IGFR2, SORT1, MYO1B, PDCD6IP, SDC1, STX12, FGF2,
FGF3 and/or a mRNA encoding one of the aforementioned, a fragment
of one of the aforementioned, a derivative of one of the
aforementioned, and a processed form of one of the
aforementioned.
[0204] In certain embodiments, the marker comprises one or more of
decreased or lower LIMP2, increased or higher CATHEPSIN B,
increased or higher CAPTHESIN D, increased or higher
.alpha.-GALACTOSIDASE, decreased or lower RAB5A, decreased or lower
EEA1, increased or higher RAB7A, increased or higher M6PR,
decreased or lower IGFR2, decreased or lower SORT1, decreased or
lower MYO1B, increased or higher PDCD6IP, increased or higher
STX12, increased or higher FGF2, increased or higher FGF3 and/or a
mRNA encoding one of the aforementioned, a fragment of one of the
aforementioned, a derivative of one of the aforementioned, and a
processed form of one of the aforementioned.
[0205] In certain embodiments, the method further comprises
identifying the level of PSA expression in the subject and
stratifying the expression of the marker on the basis of the PSA
expression level in the subject. PSA levels are as described
herein.
[0206] In certain embodiments, the PSA is a level indicative of a
low risk of a prostate cancer. In certain embodiments, the PSA
level is less than 10 ng/ml.
[0207] In certain embodiments, the PSA is a level of 7.8 ng/ml or
less. In certain embodiments, the marker comprises one or more of
LIMP2, CATHEPSIN B, .alpha.-GALACTOSIDASE, RAB5A, EEA1, M6PR,
IGFR2, SORT1, MYO1B, PDCD6IP, SDC1, STX12, FGF2, FGF3 and/or a mRNA
encoding one of the aforementioned, a fragment of one of the
aforementioned, a derivative of one of the aforementioned, and a
processed form of one of the aforementioned.
[0208] In certain embodiments, the marker comprises one or more of
decreased or lower LIMP2, increased or higher CATHEPSIN B,
increased or higher .alpha.-GALACTOSIDASE, decreased or lower
RAB5A, decreased or lower EEA1, increased or higher M6PR, decreased
or lower IGFR2, decreased or lower SORT1, decreased or lower MYO1B,
increased or higher PDCD6IP, increased or higher SDC1, increased or
higher STX12, increased or higher FGF2, increased or higher FGF3
and/or a mRNA encoding one of the aforementioned, a fragment of one
of the aforementioned, a derivative of one of the aforementioned,
and a processed form of one of the aforementioned.
[0209] In certain embodiments, the level of the marker is
indicative of an increased relapse rat and/or decreased survival
rate. In certain embodiments, the marker comprises one or more of
LIMP2, CATHEPSIN B, CAPTHESIN D, .alpha.-GALACTOSIDASE, RAB5A,
EEA1, RAB7A, M6PR, IGFR2, SORT1, MYO1B, PDCD6IP, SDC1, STX12, FGF2,
FGF3 and/or a mRNA encoding one of the aforementioned, a fragment
of one of the aforementioned, a derivative of one of the
aforementioned, and a processed form of one of the
aforementioned.
[0210] In certain embodiments, the marker comprises one or more of
increased or higher LIMP2, decreased or lower CATHEPSIN B,
decreased or lower CAPTHESIN D, decreased or lower
.alpha.-GALACTOSIDASE, increased or higher RAB5A, increased or
higher EEA1, decreased or lower RAB7A, decreased or lower M6PR,
increased or higher IGFR2, increased or higher SORT1, increased or
higher MYO1B, decreased or lower PDCD6IP, decreased or lower STX12,
decreased or lower FGF2, decreased or lower FGF3 and/or a mRNA
encoding one of the aforementioned, a fragment of one of the
aforementioned, a derivative of one of the aforementioned, and a
processed form of one of the aforementioned.
[0211] In certain embodiments, the method further comprises
identifying the level of PSA expression in the subject and
stratifying the expression of the marker on the basis of the PSA
expression level in the subject. In certain embodiments, the PSA is
a level indicative of a low risk of prostate cancer. In certain
embodiments, the PSA level is less than 10 ng/ml. In certain
embodiments, the PSA is a level of 7.8 ng/ml or less.
[0212] In certain embodiments, the marker comprises one or more of
LIMP2, CATHEPSIN B, .alpha.-GALACTOSIDASE, RAB5A, EEA1, M6PR,
IGFR2, SORT1, MYO1B, PDCD6IP, SDC1, STX12, FGF2, FGF3 and/or a mRNA
encoding one of the aforementioned, a fragment of one of the
aforementioned, a derivative of one of the aforementioned, and a
processed form of one of the aforementioned.
[0213] In certain embodiments, the marker comprises one or more of
increased or higher LIMP2, decreased or lower CATHEPSIN B,
decreased or lower .alpha.-GALACTOSIDASE, increased or higher
RAB5A, increased or higher EEA1, decreased or lower M6PR, increased
or higher IGFR2, increased or higher SORT1, increased or higher
MYO1B, decreased or lower PDCD6IP, decreased or lower SDC1,
decreased or lower STX12, decreased or lower FGF2, decreased or
lower FGF3 and/or a mRNA encoding one of the aforementioned, a
fragment of one of the aforementioned, a derivative of one of the
aforementioned, and a processed form of one of the
aforementioned.
[0214] Certain embodiments of the present disclosure provide a kit
for performing a method as described herein. The kits may comprise
one or more components, reagents, and/or instructions as described
herein.
[0215] In certain embodiments, the kit comprises one or more
reagents and/or instructions for determining the presence, level,
expression, secretion and distribution of a selected marker.
[0216] Certain embodiments of the present disclosure provide a
method of treating a prostate cancer.
[0217] The term "treating", and related terms such as "treatment"
and "treat", refer to obtaining a desired effect in terms of
improving the condition of the subject, ameliorating, arresting,
suppressing, relieving and/or slowing the progression of one or
more symptoms in the subject, a partial or complete stabilization
of the subject, a regression of the one or more symptoms, or a cure
of a disease, condition or state in the subject.
[0218] Certain embodiments of the present disclosure provide a
method of treating a prostate cancer in a subject, the method
comprising: [0219] detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject; and [0220] treating the subject based on one or more of
the presence, level, secretion and distribution of the selected
marker detected.
[0221] Certain embodiments of the present disclosure provide method
of treating a prostate cancer in a subject, the method comprising:
[0222] detecting a marker as described herein; and [0223] treating
the subject based on one or more of the presence, level, secretion
and distribution of the marker so detected.
[0224] In certain embodiments, the treating comprising one or more
of surgical intervention, radiation therapy and administration of a
therapeutic agent.
[0225] Certain embodiments of the present disclosure provide a
method of treating a prostate cancer by surgical intervention to a
subject based on one or more of the presence, level, expression,
secretion and distribution of the selected marker detected, as
described herein. Methods of surgical intervention for prostate
cancer are known in the art.
[0226] Certain embodiments of the present disclosure provide a
method of treating a prostate cancer by administering to a subject
an effective amount of a therapeutic agent based on one or more of
the presence, level, expression, secretion and distribution of the
selected marker detected, as described herein. Methods of
pharmacological intervention for prostate cancer are known in the
art.
[0227] Certain embodiments of the present disclosure provide a
method of treating a prostate cancer by radiation therapy based on
one or more of the presence, level, expression, secretion and
distribution of the selected marker detected, as described herein.
Methods of radiation therapy for prostate cancer are known in the
art.
[0228] In certain embodiments, the treatment occurs when one or
more of the presence, level, expression, secretion and distribution
presence of the selected marker is indicative of the presence of
prostate cancer and/or an increased likelihood or risk of prostate
cancer, as described herein.
[0229] In certain embodiments, one or more of an altered presence,
level, expression secretion and distribution level of the selected
marker is indicative that the subject is suitable for treatment.
Alterations in the presence, level, expression, secretion, and
distribution are as described herein.
[0230] In certain embodiments, an increased level of the selected
marker is indicative that the subject is suitable for treatment. In
certain embodiments, a decreased level of the selected marker is
indicative that the subject is suitable for treatment. In certain
embodiments, a down regulation of selected marker is indicative
that the subject is suitable for treatment. In certain embodiments,
an up regulation of the selected marker is indicative that the
subject is suitable for treatment. In certain embodiments, a down
regulation of one selected marker and/or an up-regulation of
another selected marker is indicative that the subject is suitable
for treatment.
[0231] As described herein, certain embodiments of the present
disclosure provide methods as follows: to diagnose prostate cancer
in the subject, to screen for prostate cancer in the subject, for
assessing prognosis, to determine the metastatic potential of a
prostate cancer, to identify a subject suffering from prostate
cancer, to identify a subject susceptible to prostate cancer, to
determine the rate of relapse of prostate cancer in the subject, to
determine the risk of mortality from prostate cancer in the
subject, to stratify the prostate cancer, to discriminate between
prostate cancer and not having prostate cancer in the subject, to
determine whether the prostate cancer is an organ confined cancer,
to discriminate between prostate cancer and one or more of benign
prostatic hyperplasia, prostatitis and an inflammatory condition of
the prostate, to determine pathogenic progression, to assess
whether the prostate cancer is slow growing, indolent, or
aggressive, to exclude the presence of prostate cancer in the
subject, to identify a subject suitable for treatment and/or
surgery for prostate cancer, and to determine the likelihood or
risk of a subject having prostate cancer. Endosomal associated
markers and/or lysosomal associated markers are as described
herein. Methods for detecting markers are as described herein.
[0232] Certain embodiments of the present disclosure provide a
method or kit for assessing prognosis of a subject susceptible to
or suffering from a prostate cancer, the method comprising
detecting a marker selected from an endosomal associated marker
and/or a lysosomal associated marker from the subject.
[0233] Certain embodiments of the present disclosure provide a
method or kit to determine the metastatic potential of a prostate
cancer in a subject, the method comprising detecting a marker
selected from an endosomal associated marker and/or a lysosomal
associated marker from the subject.
[0234] Certain embodiments of the present disclosure provide a
method or kit to determine the rate of relapse of a prostate cancer
in a subject, the method comprising detecting a marker selected
from an endosomal associated marker and/or a lysosomal associated
marker from the subject.
[0235] Certain embodiments of the present disclosure provide a
method or kit to determine the risk of mortality from a prostate
cancer in a subject, the method comprising detecting a marker
selected from an endosomal associated marker and/or a lysosomal
associated marker from the subject.
[0236] Certain embodiments of the present disclosure provide a
method or kit to stratify a prostate cancer in a subject, the
method comprising detecting a marker selected from an endosomal
associated marker and/or a lysosomal associated marker from the
subject.
[0237] Certain embodiments of the present disclosure provide a
method or kit to discriminate between a subject having a prostate
cancer and not having a prostate cancer, the method comprising
detecting a marker selected from an endosomal associated marker
and/or a lysosomal associated marker from the subject.
[0238] Certain embodiments of the present disclosure provide a
method or kit to determine whether a prostate cancer is an organ
confined cancer in a subject, the method comprising detecting a
marker selected from an endosomal associated marker and/or a
lysosomal associated marker from the subject.
[0239] Certain embodiments of the present disclosure provide a
method or kit to discriminate between a prostate cancer and one or
more of benign prostatic hyperplasia, prostatitis and an
inflammatory condition of the prostate in a subject, the method
comprising detecting a marker selected from an endosomal associated
marker and/or a lysosomal associated marker from the subject.
[0240] Certain embodiments of the present disclosure provide a
method or kit to determine pathogenic progression of a prostate
cancer in a subject, the method comprising detecting a marker
selected from an endosomal associated marker and/or a lysosomal
associated marker from the subject.
[0241] Certain embodiments of the present disclosure provide a
method or kit to assess whether a prostate cancer in a subject is
slow growing, indolent, or aggressive, the method comprising
detecting a marker selected from an endosomal associated marker
and/or a lysosomal associated marker from the subject.
[0242] Certain embodiments of the present disclosure provide a
method or kit to exclude the presence of a prostate cancer in a
subject, the method comprising detecting a marker selected from an
endosomal associated marker and/or a lysosomal associated marker
from the subject.
[0243] Certain embodiments of the present disclosure provide a
method or kit to identify a subject suitable for treatment and/or
surgery for prostate cancer, the method comprising detecting a
marker selected from an endosomal associated marker and/or a
lysosomal associated marker from the subject.
[0244] Certain embodiments of the present disclosure provide a
method or kit to determine the likelihood or risk of a subject
having a prostate cancer, the method comprising detecting a marker
selected from an endosomal associated marker and/or a lysosomal
associated marker from the subject.
[0245] Certain embodiments of the present disclosure provide a
method or kit for identifying a selected marker for diagnosis
and/or prognosis of a prostate cancer. Certain embodiments of the
present disclosure provide a method of screening for a selected
marker for diagnosis and/or prognosis of a prostate cancer.
[0246] Methods for identifying and/or screening markers are as
described herein.
[0247] Certain embodiments of the present disclosure provide a
method of identifying a selected marker for diagnosis and/or
prognosis of a prostate cancer, the method comprising: [0248]
identifying a marker selected from an endosomal associated marker
and/or a lysosomal associated marker; and [0249] determining the
ability of the selected marker to diagnose and/or prognose a
prostate cancer; [0250] thereby identifying the marker as a
selected marker for diagnosis and/or prognosis of a prostate
cancer.
[0251] Certain embodiments of the present disclosure provide
markers identified according to a method as described herein for
use in diagnosis and/or prognosis of a prostate cancer.
[0252] Certain embodiments of the present disclosure provide
isolated and/or purified antibodies, and/or antigen binding
fragments thereof. Antibodies and fragments thereof are as
described herein. Antibodies, and antigen binding fragments
thereof, may be used for example to detect a prostate cancer, such
as for use in kits as described herein.
[0253] The term "antibody" is to be understood to mean an
immunoglobulin molecule with the ability to bind an antigenic
region of another molecule, and includes monoclonal antibodies,
polyclonal antibodies, multivalent antibodies, chimeric antibodies,
multispecific antibodies, diabodies and fragments of an
immunoglobulin molecule or combinations thereof that have the
ability to bind to the antigenic region of another molecule with
the desired affinity including a Fab, Fab', F(ab')2, Fv, a
single-chain antibody (scFv) or a polypeptide that contains at
least a portion of an immunoglobulin (or a variant of an
immunoglobulin) that is sufficient to confer specific antigen
binding, such as a molecule including one or more Complementarity
Determining Regions (CDRs).
[0254] In certain embodiments, the antibody (or antigen binding
fragment thereof) comprises an affinity of at least
10.sup.6M.sup.-1, at least 10.sup.7M.sup.-1, at least
10.sup.8M.sup.-1, at least 10.sup.9M.sup.-1, at least
10.sup.10M.sup.-1, at least 10 .sup.11M.sup.-1, or at least
10.sup.12M.sup.-1 to the antigen.
[0255] Antibodies may be generated using known methods in the art.
For the production of antibodies, various hosts including goats,
rabbits, rats, mice, humans, and others, may be immunized by
injection with an appropriate antigen. Depending on the host
species, various adjuvants may be used to increase an immunological
response. Such standard adjuvants include Freund's adjuvant,
mineral gels such as aluminium hydroxide, and surface-active
substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole limpet hemocyanin, and
dinitrophenol.
[0256] In certain embodiments, the antibody is a polyclonal
antibody. Methods for producing and isolating polyclonal antibodies
are known. In general, polyclonal antibodies are produced from
B-lymphocytes. Typically polyclonal antibodies are obtained
directly from an immunized subject, such as an immunized animal.
Methods of immunization are known in the art.
[0257] In certain embodiments, the antibody is a monoclonal
antibody. Monoclonal antibodies may be prepared using a technique
that provides for the production of antibody molecules by
continuous isolated cells in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. Methods for the
preparation of monoclonal antibodies include for example Kohler et
al. (1975) Nature 256:495-497 (herein incorporated by reference);
Kozbor et al. (1985) J. Immunol. Methods 81:31-42 (herein
incorporated by reference); Cote et al. (1983) Proc. Natl. Acad.
Sci 80:2026-2030 (herein incorporated by reference); and Cole et
al. (1984) Mol. Cell Biol. 62: 109-120 (herein incorporated by
reference).
[0258] In certain embodiments, the antibody and/or an antigen
binding fragment thereof comprises an isolated antibody. In certain
embodiments, the antibody and/or an antigen binding fragment
thereof comprise a purified antibody. Methods for producing and
isolating polyclonal and monoclonal antibodies are known.
[0259] The term "isolated" refers to a species, such as a nucleic
acid, a polypeptide or an antibody, that has been separated from
its natural environment. Certain embodiments of the present
disclosure provide an isolated nucleic acid, polypeptide, protein
or antibody as described herein.
[0260] An isolated nucleic acid, polypeptide or antibody may be
partially or substantially purified. In some cases, the isolated
entity is in a substantially un-purified state, being associated
with a variety of other species. In some cases, the isolated entity
is in a substantially purified state, being substantially free of
other substances with which it is associated in nature or in vivo.
The term "purified" refers to a species that has undergone some
form of process to increase the proportion of a desired species.
Certain embodiments of the present disclosure provide a purified
nucleic acid, polypeptide, protein or antibody as described
herein.
[0261] In certain embodiments, the antibody has an isotype selected
from the group consisting of IgG1, IgG2a, IgG2b, IgG3, IgM and
IgA.
[0262] In certain embodiments, the antibody and/or an antigen
binding fragment thereof is a mouse antibody and/or an antigen
binding fragment thereof, a human antibody and/or an antigen
binding fragment thereof, or a humanized antibody and/or an antigen
binding fragment thereof. Other types of antibodies (or antigen
binding fragments thereof) are contemplated
[0263] Humanized antibodies, or antibodies adapted for
non-rejection by other mammals, may be produced by a suitable
method known in the art, including for example resurfacing or CDR
grafting. In resurfacing technology, molecular modeling,
statistical analysis and mutagenesis are combined to adjust the
non-CDR surfaces of variable regions to resemble the surfaces of
known antibodies of the target host. Strategies and methods for the
resurfacing of antibodies, and other methods for reducing
immunogenicity of antibodies within a different host are known, for
example as described in U.S. Pat. No. 5,639,641. Humanized forms of
the antibodies may also be made by CDR grafting, by substituting
the complementarity determining regions of, for example, a mouse
antibody, into a human framework domain.
[0264] Methods for humanizing antibodies are known. For example,
the antibody may be generated as described in U.S. Pat. No.
6,180,370 (herein incorporated by reference); WO 92/22653 (herein
incorporated by reference); Wright et al. (1992) Critical Rev. in
Immunol. 12(3,4): 125-168 (herein incorporated by reference); and
Gu et al. (1997) Thrombosis and Hematocyst 77(4):755-759) (herein
incorporated by reference).
[0265] Humanized antibodies typically have constant regions and
variable regions other than the complementarity determining regions
(CDRs) derived substantially or exclusively from a human antibody
and CDRs derived substantially or exclusively from the non-human
antibody of interest.
[0266] Techniques developed for the production of "chimeric
antibodies", for example the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity, may be performed by a suitable
method. For example, chimeric antibodies may be produced as
described in Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci
81:6851-6855 (herein incorporated by reference); Neuberger, M. S.
et al. (1984) Nature 312:604-608 (herein incorporated by
reference); and Takeda, S. et al. (1985) Nature 314:452-454 (herein
incorporated by reference).
[0267] Immunoassays may be used for screening to identify
antibodies and/or antigen binding fragments thereof having the
desired specificity.
[0268] Antibody molecules and antigen binding fragments thereof may
also be produced recombinantly by methods known in the art, for
example by expression in E. coli expression systems. For example, a
method for the production of recombinant antibodies is as described
in U.S. Pat. No. 4,816,567 (herein incorporated by reference).
Antigen binding fragments may also be produced, for example, by
phage display technologies or using peptide libraries, which are
known in the art.
[0269] Certain embodiments of the present disclosure provide an
isolated or purified antibody, or an antigen binding fragment
thereof, raised to a polypeptide as described herein.
[0270] Certain embodiments of the present disclosure provide an
isolated or purified antibody, or an antigen binding fragment
thereof, raised to a polypeptide comprising an amino acid sequence
of one or more of ASNDHDAAINRYSRLSKKRENDKVKYEVTEDVYT (SED ID NO.
1), DEVASDPLYVPDPDPTKFPVNRNLTRKAGYLNARNKT (SEQ ID NO. 2), and
SEGQFVVLSS SQSEESDLGE GGKKRESEA (SEQ ID NO. 3), an antigenic
fragment of any of the aforementioned amino acid sequences, and/or
a variant of any of the aforementioned amino acid sequences or an
antigenic fragment thereof. Certain embodiments of the present
disclosure provide an isolated or purified antibody, or an antigen
binding fragment thereof, raised to a polypeptide consisting of one
or more of the aforementioned amino acid sequences.
[0271] In certain embodiments, the antibody, or antigen binding
fragment thereof, is raised to one or more polypeptides consisting
of an amino acid sequence of ASNDHDAAINRYSRLSKKRENDKVKYEVTEDVYT
(SED ID NO. 1), DEVASDPLYVPDPDPTKFPVNRNLTRKAGYLNARNKT (SEQ ID NO.
2), and SEGQFVVLSS SQSEESDLGE GGKKRESEA (SEQ ID NO. 3),
NRYSRLSKKRENDKV (SEQ ID NO. 7), DPDPTKFPVNRNLTR (SEQ ID NO. 8), and
SQSEESDLGEGGKKR (SEQ ID NO. 9), an antigenic fragment of any of the
aforementioned amino acid sequences, and/or a variant of any of the
aforementioned amino acid sequences or antigenic fragment
thereof.
[0272] Certain embodiments of the present disclosure also provide
polypeptides or proteins as described herein.
[0273] Certain embodiments of the present disclosure provide a
polypeptide consisting of one or more of the following amino acid
sequences: ASNDHDAAINRYSRLSKKRENDKVKYEVTEDVYT (SED ID NO. 1),
DEVASDPLYVPDPDPTKFPVNRNLTRKAGYLNARNKT (SEQ ID NO. 2), SEGQFVVLSS
SQSEESDLGE GGKKRESEA (SEQ ID NO. 3), and NRYSRLSKKRENDKV (SEQ ID
NO. 7), DPDPTKFPVNRNLTR (SEQ ID NO. 8), SQSEESDLGEGGKKR (SEQ ID NO.
9), a fragment of any of the aforementioned amino sequences, an
antigenic fragment of any of the aforementioned amino acid
sequences, and/or a variant of any of the aforementioned amino acid
sequences or an antigenic fragment thereof. In certain embodiments,
the polypeptide is an isolated polypeptide. Such polypeptides may,
for example, be used to raise an antibody.
[0274] Certain embodiments of the present disclosure provide a
non-naturally occurring polypeptide comprising one or more of the
following amino acid sequences: ASNDHDAAINRYSRLSKKRENDKVKYEVTEDVYT
(SED ID NO. 1), DEVASDPLYVPDPDPTKFPVNRNLTRKAGYLNARNKT (SEQ ID NO.
2), SEGQFVVLSS SQSEESDLGE GGKKRESEA (SEQ ID NO. 3), and
NRYSRLSKKRENDKV (SEQ ID NO. 7), DPDPTKFPVNRNLTR (SEQ ID NO. 8),
SQSEESDLGEGGKKR (SEQ ID NO. 9), a fragment of any of the
aforementioned amino sequences, an antigenic fragment of any of the
aforementioned amino acid sequences, and/or a variant of any of the
aforementioned amino acid sequences or an antigenic fragment
thereof. In certain embodiments, the polypeptide is an isolated
polypeptide. Such polypeptides may, for example, be used to raise
an antibody.
[0275] Certain embodiments of the present disclosure provide an
isolated and/or purified antibody binding to an epitope in an amino
acid sequence in the human APPL1 protein comprising one or more of
ASNDHDAAINRYSRLSKKRENDKVKYEVTEDVYT (SED ID NO.1),
DEVASDPLYVPDPDPTKFPVNRNLTRKAGYLNARNKT (SEQ ID NO. 2), SEGQFVVLSS
SQSEESDLGE GGKKRESEA (SEQ ID NO. 3), and/or an equivalent region of
a homolog, ortholog or paralog of the protein. Methods for
identifying the equivalent binding regions of related targets are
known in the art.
[0276] In certain embodiments, the epitope comprises one or more of
the amino acid sequences NRYSRLSKKRENDKV (SEQ ID NO. 7),
DPDPTKFPVNRNLTR (SEQ ID NO. 8), and QSEESDLGEGGKKR (SEQ ID NO. 9),
and/or an equivalent region of a homolog, ortholog or paralog of
the APPL1 protein.
[0277] In certain embodiments, a polypeptide (or protein) as
described herein is an isolated polypeptide. In certain
embodiments, the polypeptide (or protein) as described herein is a
purified polypeptide. In certain embodiments, a polypeptide (or
protein) as described herein is a non-naturally occurring
polypeptide. In certain embodiments, a polypeptide (or protein) as
described herein is a recombinant polypeptide. In certain
embodiments, a polypeptide (or protein) as described herein is a
synthetic polypeptide. Other types of polypeptides are
contemplated.
[0278] The term "variant" of a polypeptide or of an amino acid
sequence includes, for example, one or more of amino acid insertion
variants, amino acid deletion variants, amino acid substitution
variants, and amino acid modification variants (natural and/or
synthetic).
[0279] For example, amino acid insertion variants may comprise
amino- and/or carboxy-terminal fusions (of any desired length) and
also insertions of single or two or more amino acids in a
particular amino acid sequence. In the case of amino acid sequence
variants having an insertion, one or more amino acid residues may
be inserted into a particular site in an amino acid sequence,
although random insertion with appropriate screening of the
resulting product is also possible.
[0280] Amino acid deletion variants are characterized by the
removal of one or more amino acids from the sequence. Amino acid
substitution variants are characterized by at least one residue in
the sequence being removed and another residue being inserted in
its place.
[0281] Amino acid changes in variants may be non-conservative
and/or conservative amino acid changes, i.e., substitutions of
similarly charged or uncharged amino acids. A conservative amino
acid change involves substitution of one of a family of amino acids
which are related in their side chains. Naturally occurring amino
acids are generally divided into four families: acidic (aspartate,
glutamate), basic (lysine, arginine, histidine), non-polar
(alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), and uncharged polar (glycine, asparagine,
glutamine, cystine, serine, threonine, tyrosine) amino acids.
Phenylalanine, tryptophan, and tyrosine are sometimes classified
jointly as aromatic amino acids.
[0282] The polypeptides and amino acid variants described herein
may be readily prepared with the aid of known peptide synthesis
techniques such as, for example, by solid phase synthesis and
similar methods or by recombinant DNA manipulation. The
manipulation of DNA sequences for preparing proteins and peptides
having substitutions, insertions or deletions, is described in
detail in Sambrook, J, Fritsch, E. F. and Maniatis, T. Molecular
Cloning: A Laboratory Manual 2nd. ed. Cold Spring Harbor Laboratory
Press, New York. (1989), herein incorporated by reference, and
Ausubel et al., Current Protocols in Molecular Biology (2011), John
Wiley & Sons, Inc., herein incorporated by reference.
[0283] The term "derivatives" refers to a modified form of a
species. For example, a derivative of a polypeptide or protein
refers to a modified form of a polypeptide or protein. Such
modifications include chemical modifications and comprise single or
multiple substitutions, deletions and/or additions of any molecules
associated with the protein or peptide, such as carbohydrates,
lipids and/or proteins or peptides. The term "derivative" also
extends to all functional chemical equivalents of said proteins and
peptides.
[0284] Methods for isolating and/or producing polypeptides and
protein are known, and are as described generally in Sambrook, J,
Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory
Manual 2nd. ed. Cold Spring Harbor Laboratory Press, New York.
(1989), herein incorporated by reference, and Ausubel et al.,
Current Protocols in Molecular Biology (2011), John Wiley &
Sons, Inc., herein incorporated by reference.
[0285] Certain embodiments of the present disclosure provide an
isolated and/or purified antibody raised to a polypeptide
comprising an amino acid sequence of one or more of
PNTFKTLDSWRDEFLIQASPRDPENFPFVVLGNKI (SED ID NO. 4),
DPENFPFVVLGNKIDLENRQVATKRAQAWCYSKNN (SEQ ID NO. 5),
ALKQETEVELYNEFPEPIKLDKNDRAKASAESCSC (SEQ ID NO. 6), an antigenic
fragment of any of the aforementioned amino acid sequences, and/or
a variant of any of the aforementioned amino acid sequences or an
antigenic fragment thereof. Certain embodiments of the present
disclosure provide an isolated or purified antibody, or an antigen
binding fragment thereof, raised to a polypeptide consisting of one
or more of the aforementioned amino acid sequences.
[0286] In certain embodiments the antibody is raised to a
polypeptide comprising an amino acid sequence of one or more of
RDEFLIQASPRDPEN (SEQ ID NO. 10), GNKIDLENRQVATKR (SEQ ID NO. 11)
and YNEFPEPIKLDKNDR (SEQ ID NO. 12), an antigenic fragment of any
of the aforementioned amino acid sequences, and/or a variant of any
of the aforementioned amino acid sequences or an antigenic fragment
thereof. Certain embodiments of the present disclosure provide an
isolated or purified antibody, or an antigen binding fragment
thereof, raised to a polypeptide consisting of one or more of the
aforementioned amino acid sequences.
[0287] Certain embodiments of the present disclosure provide a
polypeptide consisting of one or more of the following amino acid
sequences: PNTFKTLDSWRDEFLIQASPRDPENFPF VVLGNKI (SED ID NO. 4),
DPENFPFVVLGNKIDLENRQVATKRAQAWCYSKNN (SEQ ID NO. 5),
ALKQETEVELYNEFPEPIKLDKNDRAKA SAESCSC (SEQ ID NO. 6),
RDEFLIQASPRDPEN (SEQ ID NO. 10), GNKIDLENRQVATKR (SEQ ID NO. 11)
and YNEFPEPIKLDKNDR (SEQ ID NO. 12), a fragment of any of the
aforementioned amino sequences, an antigenic fragment of any of the
aforementioned amino acid sequences, and/or a variant of any of the
aforementioned amino acid sequences or an antigenic fragment
thereof. Such polypeptides may, for example, be used to raise an
antibody.
[0288] Certain embodiments of the present disclosure provide a
non-naturally occurring polypeptide comprising one or more of the
following amino acid sequences: PNTFKTLDSWRDEFLIQASPRDPENFPF
VVLGNKI (SED ID NO. 4), DPENFPFVVLGNKIDLENRQVATKRAQAWCYSKNN (SEQ ID
NO. 5), ALKQETEVELYNEFPEPIKLDKNDRAKA SAESCSC (SEQ ID NO. 6),
RDEFLIQASPRDPEN (SEQ ID NO. 10), GNKIDLENRQVATKR (SEQ ID NO. 11)
and YNEFPEPIKLDKNDR (SEQ ID NO. 12), a fragment of any of the
aforementioned amino sequences, an antigenic fragment of any of the
aforementioned amino acid sequences, and/or a variant of any of the
aforementioned amino acid sequences or an antigenic fragment
thereof. Such polypeptides may, for example, be used to raise an
antibody.
[0289] Certain embodiments of the present disclosure provide an
isolated and/or purified antibody binding to an epitope in an amino
acid sequence in the human RAB7 protein comprising one or more of
PNTFKTLDSWRDEFLIQASPRDPENFPF VVLGNKI (SED ID NO. 4),
DPENFPFVVLGNKIDLENRQVATKRAQAWCYSKNN (SEQ ID NO. 5),
ALKQETEVELYNEFPEPIKLDKNDRAKASAESCSC (SEQ ID NO. 6), and/or an
equivalent region of a homolog, ortholog or paralog of the
protein.
[0290] In certain embodiments, the epitope comprises one or more of
the amino acid sequences RDEFLIQASPRDPEN (SEQ ID NO. 10),
GNKIDLENRQVATKR (SEQ ID NO. 11) and YNEFPEPIKLDKNDR (SEQ ID NO. 12)
and/or an equivalent region of a homolog, ortholog or paralog of
the RAB7 protein.
[0291] Certain embodiments of the present disclosure provide an
isolated and/or purified antibody raised to a polypeptide
comprising an amino acid sequence of CKKLDDFVETGDIRTMVFP (SEQ ID
NO. 13), an antigenic fragment of any of the aforementioned amino
acid sequences, and/or a variant of any of the aforementioned amino
acid sequences or an antigenic fragment thereof. Certain
embodiments of the present disclosure provide an isolated or
purified antibody, or an antigen binding fragment thereof, raised
to a polypeptide consisting of the aforementioned amino acid
sequence.
[0292] Certain embodiments of the present disclosure provide an
isolated and/or purified antibody binding to an epitope in an amino
acid sequence in the human LIMP-2 protein comprising
CKKLDDFVETGDIRTMVFP (SEQ ID NO. 13) and/or an equivalent region of
a homolog, ortholog or paralog of the protein.
[0293] Certain embodiments of the present disclosure provide a
polypeptide consisting of the following amino acid sequence:
CKKLDDFVETGDIRTMVFP (SEQ ID NO. 13), a fragment of the
aforementioned amino sequence, an antigenic fragment of the
aforementioned amino acid sequence, and/or a variant of the
aforementioned amino acid sequence or an antigenic fragment
thereof. Such polypeptides may, for example, be used to raise an
antibody.
[0294] Certain embodiments of the present disclosure provide a
non-naturally occurring polypeptide comprising the following amino
acid sequence: CKKLDDFVETGDIRTMVFP (SEQ ID NO. 13), a fragment of
the aforementioned amino sequence, an antigenic fragment of the
aforementioned amino acid sequence, and/or a variant of the
aforementioned amino acid sequence or an antigenic fragment
thereof. Such polypeptides may, for example, be used to raise an
antibody.
[0295] Certain embodiments of the present disclosure provide a
method of detecting an APPL1 protein or a fragment thereof, the
method comprising using an antibody as described herein.
[0296] Certain embodiments of the present disclosure provide a
method of detecting a RAB7 protein or a fragment thereof, the
method comprising using an antibody as described herein.
[0297] Certain embodiments of the present disclosure provide a
method of detecting a LIMP2 protein or a fragment thereof, the
method comprising using an antibody as described herein.
[0298] Certain embodiments of the present disclosure provide a
method of detecting a prostate cancer in a subject, the method
comprising using an antibody as described herein to detect an
APPL1, RAB7 or LIMP2 protein, and/or a fragment, derivative or a
processed form thereof from the subject.
[0299] Certain embodiments of the present disclosure provide a kit
comprising an antibody as described herein. The kit may comprise
one or more other reagents as described herein.
[0300] Certain embodiments of the present disclosure provide a
hybridoma producing an antibody as described herein. Methods for
producing hybridomas and monoclonal antibodies are known in the
art.
[0301] A typical protocol for the production of a hybridoma is as
follows: Animals (e.g. mice) are first exposed to the selected
antigen. Usually this is done by a series of injections of the
antigen, over the course of several weeks. Once splenocytes are
isolated from the mammal's spleen, the B cells may be fused with
immortalised myeloma cells. The myeloma cells are generally
selected to ensure they are not secreting antibody themselves and
that they lack the hypoxanthine-guanine phosphoribosyltransferase
(HGPRT) gene, making them sensitive to HAT medium. The fusion may
be accomplished, for example, using polyethylene glycol or Sendai
virus.
[0302] Fused cells are incubated in HAT medium for roughly 10 to 14
days. Aminopterin blocks the pathway that allows for nucleotide
synthesis and unfused myeloma cells die, as they cannot produce
nucleotides by the de novo or salvage pathways, because they lack
HGPRT. Removal of the unfused myeloma cells is necessary because
they have the potential to outgrow other cells, especially weakly
established hybridomas. Unfused B cells die as they have a short
life span. In this way, only the B cell-myeloma hybrids survive,
since the HGPRT gene coming from the B cells is functional. These
cells produce antibodies and are immortal. The incubated medium is
then diluted into multi-well plates to such an extent that each
well contains only one cell. Since the antibodies in a well are
produced by the same B cell, they will be directed towards the same
epitope, and are thus monoclonal antibodies.
[0303] The next stage is a rapid primary screening process, which
identifies and selects only those hybridomas that produce
antibodies of appropriate specificity. The hybridoma culture
supernatant, secondary enzyme labeled conjugate, and chromogenic or
fluorescent substrate, are then incubated, and the formation of a
colored product indicates a positive hybridoma. Alternatively,
immunocytochemical screening or flow cytometry can also be
used.
[0304] The B cell that produces the desired antibodies can be
cloned to produce many identical daughter clones. Supplemental
media containing interleukin-6 are essential for this step. Once a
hybridoma colony is established, it will continually grow in
culture medium like RPMI-1640 (with antibiotics and fetal bovine
serum) and produce antibodies.
[0305] Multiwell plates are used initially to grow the hybridomas,
and after selection, are changed to larger tissue culture flasks.
This maintains the well-being of the hybridomas and provides enough
cells for cryopreservation and supernatant for subsequent
investigations. The culture supernatant can yield 1 to 60 .mu.g/ml
of monoclonal antibody, which is maintained at -20.degree. C. or
lower until required.
[0306] By using culture supernatant or a purified immunoglobulin
preparation, further analysis of a potential monoclonal antibody
producing hybridomas can be made in terms of reactivity,
specificity, and cross-reactivity.
[0307] Finally, standard techniques may be used for recombinant DNA
technology, oligonucleotide synthesis, antibody production, peptide
synthesis, tissue culture and transfection. Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods known
in the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification. See e.g., Sambrook et al. Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)), herein incorporated by
reference.
[0308] Exemplary embodiments are illustrated by the following
examples. It is to be understood that the following description is
for the purpose of describing particular embodiments only and is
not intended to be limiting with respect to the above
description.
EXAMPLE 1
Altered Endosome Biogenesis in Prostate Cancer
[0309] Materials and Methods
[0310] (i) Reagents
[0311] A LIMP-2 sheep polyclonal antibody was generated using the
peptide sequence CKKLDDFVETGDIRTMVFP (SEQ ID NO. 13) (Mimotopes Pty
Ltd., Victoria, Australia). Primary antibodies used in this study
included rabbit polyclonal antibodies against APPL1 (0.4 .mu.g/mL,
Abcam PLC, Cambridge United Kingdom, cat #ab95195), APPL2 (0.4
.mu.g/m, Abcam, cat #ab95196), Rab4 (1 .mu.g/mL, Abcam, cat
#ab13252), TGN46 (10 .mu.g/mL, Abcam, cat #ab50595), TfR1 (1
.mu.g/mL, Abcam, cat #ab108985), TfR2 (1 .mu.g/mL, Abcam, cat
#ab80194), Akt (1/1000, Cell Signaling Technology, Inc., MA, USA)
and Phospho-Akt (Thr308 1/1000, Cell Signaling Technology, Inc.).
Goat anti-Rab5 (1 .mu.g/mL, Santa Cruz Biotechnology, CA, USA),
Rab7 (1 .mu.g/mL, Santa Cruz Biotechnology) and EEA1 (1 .mu.g/mL,
Santa Cruz Biotechnology) polyclonal antibodies were also used.
LAMP-1 (1 .mu.g/mL) mouse monoclonal BB6 was provided by Umea
University, Sweden). HRP conjugated secondary antibodies for
Western blot analysis included anti-goat/sheep (1/2000, Merck
Millipore Pty. Ltd., Victoria, Australia), anti-rabbit (1/2000,
Sigma Aldrich Pty. Ltd., New South Wales, Australia) and anti-mouse
(1/2000 Sigma Aldrich Pty. Ltd.). HRP-conjugated anti-GAPDH
(1/20000 Sigma Aldrich Pty. Ltd.). The secondary and other antibody
conjugated fluorophores that were used included Alexa Fluor.RTM.
488 (1/250), Alexa Fluor.RTM. 633 (1/250), Transferrin-633
(1/1000), Phalloidin-488 (1/100), LysoTracker.RTM. (5 .mu.M), from
Life Technologies Pty Ltd., Victoria, Australia. Primers were
obtained from Geneworks Pty Ltd., Adelaide, Australia and the
sequences listed in Supporting Information Table 1.
[0312] (ii) Cell Lines and Culture Conditions
[0313] The cell lines PNT1a and PNT2, 22RV1 and LNCaP (clone FCG)
were obtained from the European Collection of Cell Cultures via
CellBank Australia (Children's Medical Research Institute, New
South Wales, Australia). These cell lines were absent from the list
of cross-contaminated or misidentified cell lines, version 6.8 (9
Mar. 2012). PNT1a and PNT2 were previously derived from Simian
vacuolating virus 40 (SV40) immortalised cell lines. The 22RV1
cancer cell line was previously derived from a xenograft, which had
been serially propagated in mice after castration-induced
regression and relapse of a parental, androgen-dependent xenograft.
This cell line expressed the androgen receptor, but its
proliferation was unresponsive to androgen stimulation. LNCaP was
previously derived from a lymph node metastasis of prostate
adenocarcinoma and is androgen responsive.
[0314] Cell lines were cultured in T75 tissue culture flasks and
maintained in Roswell Park Memorial Institute (RPMI) 1640 culture
medium (Gibco.RTM., Life Technologies Australia Pty Ltd., Victoria,
Australia), supplemented with 10% foetal calf serum (In Vitro
Technologies Pty Ltd., Victoria, Australia) and 2 mM L-glutamine
(Sigma Aldrich Pty Ltd., New South Wales, Australia). Cells were
incubated at 37.degree. C. with 5% CO.sub.2 in a Sanyo MCO-17AI
humidified incubator (Sanyo Electric Biomedical Co., Ltd., Osaka,
Japan). Cells at approximately 90% confluence were passaged by
washing with sterile PBS (Sigma Aldrich), trypsin treated
(Trypsin-EDTA solution containing 0.12% trypsin, 0.02% EDTA;
SAFC.RTM., Sigma Aldrich, New South Wales, Australia), for
dissociation from the culture surface and then suspended in
supplemented culture medium.
[0315] (iii) Cell Extract Preparation
[0316] The culture medium was aspirated from 80-90% confluent cell
cultures, the cells washed once with PBS, and then incubated with
800 .mu.L of a 20 mM Tris (pH 7.0), 500 mM sodium chloride and 2%
SDS solution. Cells were harvested and an extract prepared by
heating to 65.degree. C. and sonication for one minute. The lysate
was then passaged 6 times through a 25-guage needle. Total protein
in cell extracts was quantified using a bicinchoninic acid assay
according to the manufacturer's instructions (Micro BCA kit,
Pierce, Rockford, Ill., USA). Samples were quantified using a
Wallac Victor.TM. optical plate-reader and Workout software v2.0
(Perkin-Elmer Pty, Ltd., Victoria, Australia), using a 5-point
parameter standard curve. Cell extracts were stored at -20.degree.
C.
[0317] (iv) Gene Expression Analysis
[0318] Cell lines were cultured to 80-90% confluence (triplicate
T75 flasks), the culture medium aspirated and the cell layer washed
with PBS, before the addition of 1 mL TRI reagent.RTM. (Applied
Biosystems Pty Ltd., Victoria, Australia) for harvesting. Two
hundred microlitres of chloroform was added per millilitre of TRI
reagent.RTM. and samples shaken vigorously for one minute,
incubated at room temperature for three minutes and then
centrifuged for 15 minutes at 16,000 g at 4.degree. C. RNA
extraction was performed using an RNeasy.RTM. mini kit (Qiagen Pty
Ltd., Victoria, Australia) according to the manufacturer's
instructions. The concentration of extracted RNA was determined
using a NanoDrop.TM. 2000 spectrophotometer (Thermo Fisher
Scientific Australia Pty Ltd., Victoria, Australia) at 260 nm.
Ratios of 260/280 nm and 260/230 nm were assessed to ensure samples
were free from protein and DNA contamination; a ratio greater than
1.6 indicated a sample free of contamination.
[0319] Complementary DNA (cDNA) for qRT-PCR was prepared using a
High Capacity RNA-to-cDNA Kit (Life Technologies Pty Ltd.,
Victoria, Australia). Primer sequences were obtained from either
published literature, Harvard PrimerBank or designed using NCBI
Primer-BLAST. Primers were either selected or designed based on the
criteria of the final amplicon-size being less than 150 base-pairs,
a melting temperature near 60.degree. C. and where possible,
extension across an exon-exon junction. For quantitative RT-PCR 10
.mu.L of reaction mixture contained 5 .mu.L Power SYBR.RTM. Green
PCR Master Mix (Life Technologies Australia Pty Ltd., Victoria,
Australia), 0.5 .mu.L each of 10 nM forward and reverse primer, 2
.mu.L cDNA sample diluted to 1:25 with DEPC-treated H.sub.2O, and 2
.mu.L DEPC-treated H.sub.2O. Reactions were plated in triplicate
onto 96-well plates (Life Technologies Australia Pty Ltd.,
Victoria, Australia), with each plate containing serial dilutions
of a reference cDNA sample for the target-gene and endogenous-gene
standard curves, to control for reaction efficiency. qPCR was
performed using a 7500 Fast Real-Time PCR System (Life Technologies
Australia Pty Ltd., Victoria, Australia) using ABI 7500 software
v2.0.2.
[0320] Cycling conditions for all targets comprised; 50.degree. C.
for 2 minutes, 95.degree. C. for 10 minutes to activate the enzyme
and denature cDNA followed by 40 cycles of a 95.degree. C.
15-second denaturation step and a 60.degree. C. 60-second extension
and signal-acquisition step. Cycle threshold (C.sub.T) values were
derived at a threshold level of 0.35 in the exponential phase of
amplification and above baseline noise. The relative amount of gene
expression from each sample was derived by calculation of C.sub.T
values versus standard curves produced from serial-dilutions.
Reaction efficiencies, calculated from the slope of a linear
trend-line plotted from diluted standards, were between 90 and
110%. Mean gene expression was derived from the mRNA amount on each
replicate plate, with each single plate providing a mean C.sub.T
and mRNA level from replicate wells.
[0321] (v) Western Blotting
[0322] Ten .mu.g of cell lysate was heat-denatured (5 min at
100.degree. C. in NuPAGE.RTM. LDS Sample Buffer and reducing agent)
then electrophoresed at 120V for 1.5 hours using pre-cast gels in
an XCell SureLock Mini-Cell system (Life Technologies Australia Pty
Ltd., Victoria, Australia). Protein was then transferred to
polyvinylidene difluoride membranes (Polyscreen.RTM., PerkinElmer,
Victoria, Australia) at 35 V for one hour. The transfer membranes
were blocked for 1 hour at RT using 5% (w/v) skim milk solution in
TBS-tween (0.1%; block) and incubated with primary antibody
overnight at 4.degree. C. The membranes were washed 3.times.5 min
in TBS-tween (0.1%) and then incubated with the appropriate
horseradish-peroxidase conjugated secondary antibody diluted 1/2000
in block solution. The membranes were developed using aNovex.RTM.
ECL chemiluminescent substrate reagent kit (Life Technologies
Australia Pty Ltd., Victoria, Australia) and proteins visualised
using an ImageQuant.TM. LAS 4000 imager, software version 1.2.0.101
(GE Healthcare Bio-Sciences Pty Ltd., New South Wales, Australia).
Triplicate samples were analysed and images quantified relative to
a reference GAPDH loading control using AlphaViewSA.TM. software
v3.0.0.0 (ProteinSimple, Santa Clara, Calif.). Kruskal-Wallis rank
sum statistical analyses using Stata/SE v11.2 (StataCorp LP, Texas,
U.S.A) was performed to determine significance between
non-malignant control and cancer cell line groups (95% confidence
limit; p<0.05).
[0323] (vi) Confocal Microscopy
[0324] Cells were cultured on 22 mm glass coverslips, fixed with 4%
(v/v) formaldehyde in PBS for 20 minutes at room temperature, then
permeabilised with 0.1% Triton-X (v/v) in PBS for 10 minutes.
Non-specific antibody reactivity was blocked by incubation with 5%
(w/v) bovine serum albumin in PBS for two hours at RT. Cells were
then incubated with primary antibody in 5% BSA for two hours at
room temperature, then secondary antibody for one hour at RT.
Unbound antibody was removed by three PBS washes and coverslips
mounted in ProLong.RTM. Gold Antifade Reagent containing DAPI
nuclear stain (Life Technologies Australia Pty Ltd., Victoria,
Australia). Confocal microscopy was performed using a Zeiss LSM 710
META NLO laser scanning microscope and associated Carl Zeiss Zen
2009 software. Laser lines of 370, 488, 543 and 633 nm were
utilised for DAPI, Alexa Fluor.RTM. 488, Cy3 and Alexa Fluor.RTM.
633 fluorescence, respectively. Images were exported as greyscale
16-bit TIFF files and processed using Adobe.RTM. Photoshop.RTM. CS5
(Adobe Systems Inc., San Jose, Calif., U.S.A).
[0325] (vii) Immunohistochemistry
[0326] Matched human non-malignant and tumour prostate tissue
sections (3 .mu.m) were mounted on Superfrost Ultra Plus.RTM.
slides (Menzel-Glaser) and heated overnight at 50.degree. C.
Sections were then dewaxed in xylene, rehydrated in ethanol and
incubated in 0.3% H.sub.2O.sub.2 in PBS for 15 min at RT. HIER was
carried out using 10 mM citrate buffer (pH 6.5) in a Decloaking
Chamber (Biocare Medical) for 5 min at 125.degree. C. Slides were
blocked first using an Invitrogen Avidin/Biotin Kit (as per
manufacturer's instructions) and then in 5% blocking serum (SIGMA)
for 30 min at RT in a humid chamber. Sections were then incubated
with primary antibody overnight at 4.degree. C. in a humid chamber,
followed by incubation with the appropriate biotinylated secondary
antibody (1/400; DAKO) for 1 hour at RT in a humid chamber, then
streptavidin-horseradish peroxidise (1/500; DAKO) at RT for 1 hour
in a humid chamber and finally with DAB/H.sub.2O.sub.2. The tissue
sections were then counterstained with Lillie-Mayer's haemotoxylin,
rinsed in water, rehydrated and mounted on slides with DPX. Images
were obtained by scanning slides using a Nanozoomer (Hamamatsu)
[0327] Results
[0328] (i) Increased Endosome Related Gene and Protein Expression
in Prostate Cancer Cells
[0329] The expression of endosome and lysosome related genes was
quantified by qRT-PCR in control and prostate cancer cells and
normalised to the expression of GAPDH mRNA. The amounts of APPL1,
APPL2, EEA1, RAB5A, RAB4A and LIMP2 mRNA were significantly
increased in prostate cancer when compared to non-malignant control
cell lines (p<0.05; FIG. 1). In each case there was an
approximately 2-3 fold increase in mRNA expression. There was not a
significant difference in the amount of either RAB7A or LAMP1 mRNA
detected in prostate cancer cells compared to non-malignant
controls. Western analysis demonstrated significant increases in
the amount of APPL1, APPL2, EEA1, Rab4 and LIMP-2 protein in
extracts from prostate cancer cells when compared to non-malignant
control cells (p<0.05; FIG. 2). Moreover, for both LIMP-2 and
Rab4 the increase was approximately 2-4 fold for prostate cancer
when compared to non-malignant control cells (FIG. 2). There was
not a significant difference in the amount of Rab5 A, Rab7 and
LAMP-1 protein detected in non-malignant compared to prostate
cancer cells (FIG. 2).
[0330] (ii) Altered Distribution of Endosomes and Lysosomes in
Prostate Cancer Cells
[0331] Representative confocal images for the distribution of
endosomes and lysosomes (FIG. 3), show increased staining and
altered compartment distribution in prostate cancer compared to the
non-malignant controls. APPL1 positive endosomes were detected
mainly in the perinuclear region of non-malignant control cells,
whereas in prostate cancer cells these compartments were
distributed towards the cell periphery and tended to be more
concentrated near the plasma membrane in cellular
extensions/pseudopodia. Rab5A displayed a similar distribution to
its effector EEA1 in both non-malignant control and prostate cancer
cells. However, in non-malignant control cells both Rab5A and EEA1
were concentrated in the perinuclear region, while in prostate
cancer cells these endosomal compartments were found throughout the
cytoplasm, with some compartments located towards the cell
periphery in cellular extensions. Rab7 positive endosomes were
located mainly in the perinuclear region of both non-malignant
control and prostate cancer cells. In non-malignant control cells
LIMP-2 was concentrated in the perinuclear region, with some
tubular and punctuate vesicular staining in the remainder of the
cytoplasm and near the cell surface. In contrast, prostate cancer
cells displayed relatively smaller LIMP-2 compartments, which had
an even distribution throughout the cytoplasm. In non-malignant
control cells, LAMP-1 was detected on compartments that were
concentrated in the perinuclear region, whereas in prostate cancer
cells the LAMP-1 compartments were distributed away from the
perinuclear region, concentrated in cellular extensions. Consistent
with the LAMP-1 staining, LysoTracker.TM. positive acidic
compartments were concentrated mainly in the perinuclear region of
non-malignant control cells, whereas in prostate cancer cells,
these compartments were detected in both the perinuclear region and
in cytoplasmic extensions (FIG. 4).
[0332] (iii) Altered Distribution of Endocytosed Transferrin in
Prostate Cancer Cells
[0333] Previous studies have reported increased uptake of
transferrin in prostate cancer cells, prompting the investigation
of receptor expression and transferrin endocytosis in relation to
the observed increase in endosome protein expression and altered
endosome distribution. In non-malignant control cells, endocytosed
transferrin was observed in punctuate intracellular structures
after 5 minutes and in the perinuclear region at 15 and 30 minutes
(FIG. 5). The prostate cancer cells endocytosed more transferrin
than the non-malignant control cell lines and the internalised
transferrin was not as concentrated in the perinuclear region of
prostate cancer cells, with more in the cell periphery, when
compared to the non-malignant controls (FIG. 5). There was also a
dramatic reduction in actin staining for the prostate cancer
compared to the non-malignant control cell lines (FIG. 5). In the
non-malignant control cells, transferrin was clustered in LIMP-2
and Rab7 positive endosomes localised in the perinuclear region
(FIG. 6). While the prostate cancer cells had some LIMP-2 positive
staining in the perinuclear region and some co-localisation with
the Golgi marker TGN46, the majority of transferrin was localised
in different endosomal compartments (i.e. Appl 1, Rab5A, EEA1)
distributed throughout the cytoplasm and in cellular extensions
(FIG. 6). The Rab4 recycling endosomes and LAMP-1 positive
lysosomes had similar patterns of transferrin staining for the
prostate cancer and non-malignant control cell lines (FIG. 6).
Further analysis of the transferrin receptors revealed variable
gene and protein expression for TfR1 and TfR2 (FIG. 7). There was a
significant increase in TFR1 gene expression in prostate cancer
cells when compared to non-malignant controls, but only a
qualitative increase in TFR1 protein in the prostate cancer cell
line 22RV1 and not for LNCaP (FIG. 7A). While there was
significantly more TFR2 protein detected in prostate cancer cells
when compared to the non-malignant controls, there was only and
increase in TFR2 gene expression observed in LNCaP (FIG. 7A). Thus,
while there was an increased amount of TFR protein in prostate
cancer compared to non-malignant control cells, there was
relatively more TFR1 in 22RV1 and more TFR2 in LNCaP cells.
[0334] (iv) Altered Akt Signalling in Prostate Cancer Cells
[0335] The total amount of Akt protein detected in non-malignant
control cells was similar to that detected in prostate cancer cells
(FIG. 8). There were, however, differences in the amount of
phosphorylated Akt in the prostate cancer lines, with 22RV1 showing
a marked reduction in the amount of phosphorylated Akt whereas
LNCaP had increased amount of phosphorylated Akt (FIG. 8). More
importantly, following the addition of transferrin, there was a
significant increase in the amount of phosphorylated Akt in
non-malignant control cells, but no change in the amount of
phosphorylated Akt in either of the cancer cells (FIG. 8B).
Interestingly, the increased amount of phosphorylated Akt observed
in the LNCaP cells (that had not been treated with transferrin),
correlated with the amount of TfR2 detected in this cell line (FIG.
7A and FIG. 8).
[0336] (v) Distribution of LAMP-1 and APPL1 in Non-Malignant and
Malignant Human Prostate Tissue.
[0337] Immunohistochemistry was used to investigate the
distribution of LAMP-1 and APPL1 in prostate cancer patient tissue
samples. The lysosomal marker LAMP-1 showed some tumour specific
staining in some patient samples (FIG. 9), but consistent with
previous studies, there was variable results and some patient
samples had little or no LAMP-1 staining (data not shown). APPL1,
however, specifically delineated the cancer margins and showed
dramatically increased staining within the tumour mass (FIG. 9).
This APPL1 staining pattern was consistent for each of the 6
different patient samples examined.
[0338] Discussion
[0339] Prostate cancer is one of the most frequently diagnosed
cancers in men and a leading cause of cancer related deaths
world-wide, particularly in the United States and Australasian
populations. The prostate specific antigen is still a commonly used
test to detect prostate cancer, but has significant problems in
terms of miss-diagnosis and prognostic prediction. Some promising
adjunct tests have recently been developed including prostate
cancer antigen 3 (PCA3), the analysis of cholesterol sulphate and a
novel sequence of the gene protein kinase C-zeta (PRKCZ) which is
translated to the protein PRKC-.zeta.-.sub.PrC. However, these
biomarkers have a number of deficiencies that do not provide a
useful method for the early and accurate detection of prostate
cancer to enable appropriate therapeutic intervention.
[0340] There have been extensive protein and proteomic studies to
delineate potential new prostate cancer biomarkers, but despite
this, suitable markers have yet to be identified.
[0341] Here we observed altered distribution of endosome and
lysosome vesicles into the cellular periphery of prostate cancer
cells. Increases in Na.sup.+/H.sup.+exchange activity
(acidification), RhoA GTPase activity and PI3K activation have been
shown to result in exocytosis from prostate cancer cells. The
increased endosomal associated gene and protein expression,
suggested that endosome related proteins may provide an important
new focus for prostate cancer disease marker studies.
[0342] We observed increased gene and protein expression of the
endosomal protein LIMP-2 in prostate cancer cell lines, prompting
us to investigate endosomal biogenesis in prostate cancer cells.
The early endosome associated proteins EEA1, APPL1, APPL2 and
recycling endosome protein Rab4 were significantly upregulated in
prostate cancer cells, supporting the hypothesis of altered
endosome biogenesis in prostate cancer. Furthermore, the EEA1,
APPL1 and APPL2 endosome sub-populations each displayed altered
intracellular distribution consistent with altered endosome traffic
and potentially function.
[0343] The significant changes that we observed in endosome
associated gene and protein expression, together with the altered
distribution of endosome populations prompted us to investigate
transferrin receptor expression together with transferrin
endocytosis, sorting and Akt signalling as measures of endosome
function. Akt signalling is also essential for regulating cell
growth and survival; and this controls the cell surface expression
of transferrin and growth factor receptors. The transferrin
receptor has previously been observed to colocalise with Rab5 and
the motor protein myosin VI; the latter of which is involved in
retrograde transport to the plasma membrane. This was consistent
with our observations of endosome populations co-staining with
labelled transferrin in the cellular periphery of prostate cancer
cells. There also appeared to be a deregulation of Akt signalling
in the prostate cancer cells, which may have altered the
intracellular location of the transferrin receptor, routing it into
the different populations of APPL1, APPL2 and Rab5 endosomes.
[0344] APPL1 has been shown to be directly involved in insulin
signalling and the translocation of the glucose transporter GLUT-4,
which is mediated by direct binding of APPL1 to PI3K and Akt. The
increased gene and protein expression of APPL1 that we observed in
prostate cancer cells might be expected to cause increased glucose
uptake, due to its effect on GLUT-4 and this could have
implications for energy metabolism in these cancer cells. Indeed,
APPL1 also regulates other aspects of both lipid and glucose
metabolism, activating AMP-activated kinase, p38 MAP kinase (MAPK)
and PPAR.alpha.. APPL2, a relation of APPL1, has been shown to
function as a negative regulator of adiponectin signalling, by
competitive binding with APPL1 for interaction with the adiponectin
receptor, again regulating energy metabolism. The increased
expression of both APPL1 and APPL2 could therefore impact heavily
on prostate cancer cell metabolism, particularly as these proteins
were not observed together, but rather on separate populations of
endosomes. This could have direct significance for increased energy
utilisation and prostate cancer cell survival. The altered APPL1
expression and effect on Akt signalling in prostate cancer cells
would be expected to also have significant consequence for other
aspects of prostate cancer biology, due to the importance of the
APPL1/PI3K/Akt signalling pathway in leading cell adhesion and cell
migration. Notably, APPL1 also acts as a mediator of other
signalling pathways, by interaction with the cytosolic face of
integral or membrane associated proteins either at the cell surface
or in the endosome pathway; where it is directly involved in
endosome traffic.
[0345] Rab GTPases are integrally involved in the control of
endosome traffic, cycling between the cytoplasmic GDP bound state
and the active membrane associated GTP bound state. Rab5 and Rab7
respectively define early and late endosome compartments and during
endosome maturation Rab5 recruits the HOPS complex as a mechanism
to activate and be replaced by Rab7. mVps39 is known to be a
guanine nucleotide exchange factor (GEF), which promotes the GTP
bound state on endosomal Rabs, while TBC-2/TBC.sub.1D.sub.2 is a
Rab GTPase activating protein (GAP) that promotes the GDP bound
state; which in combination is used to regulate the membrane
localisation of Rab proteins. TBC-2/TBC.sub.1D.sub.2 is thought to
act as a regulator of endosome to lysosome traffic and is required
to maintain the correct size and distribution of endosomes. The
altered distribution of endosome populations that we observed in
prostate cancer cells suggests that TBC-2/TBC.sub.1D.sub.2 (GAP)
and or mVps39 (GEF) might be functionally impaired. Interestingly,
microarray analysis has detected increased expression of TBC-2/TBC
.sub.1D.sub.2 and reduced Vps39 mRNA.
[0346] The Gleason grading system to define histological
differentiation is used in conjunction with marker analysis to
predict the course of disease in prostate cancer patients. We
observed increased amounts of APPL1 protein in tissue biopsies from
prostate cancer patients, confirming the increased gene and protein
expression of APPL1 in prostate cancer cell lines.
[0347] We have demonstrated increased expression of early endosome
markers and altered localisation of endosome and lysosome
compartments in prostate cancer cells, which is associated with
altered endocytosis and recycling of the transferrin receptor. We
concluded that endosome biogenesis and function is altered in
prostate cancer cells, opening a potentially new avenue of
investigation, to develop markers that aid in the diagnosis and
prognosis of prostate cancer.
EXAMPLE 2
Lysosomal Enzymes as Potential Prostate Cancer Biomarkers
[0348] Materials and Methods
[0349] (i) Antibody Reagents
[0350] The primary antibodies used in this study included rabbit
polyclonal antibodies against hK2 (1 .mu.g/mL, Abcam PLC,
Cambridge, United Kingdom, cat #ab40948), hK3 (1 .mu.g/mL, Abcam,
cat #ab40949), hK4 (1 .mu.g/mL, Abcam, cat #ab40950), hKl5 (1
.mu.g/mL, Abcam, cat #ab40961), acid ceramidase (1 .mu.g/mL, Abcam,
cat #ab74469). Mouse monoclonal antibodies against prostatic acid
phosphatase (1 .mu.g/mL, Abcam, cat #ab75704), cathepsin B (0.25
.mu.g/mL, Abcam, cat #ab58802), cathepsin D (5 .mu.g/mL, Abcam, cat
#ab6313). Sheep polyclonal antibodies against .alpha.-glucosidase
(1 .mu.g/mL), .beta.-glucosidase (1 .mu.g/mL) and
.alpha.-galactosidase A (1 .mu.g/mL) were a kind gift from the
Lysosomal Diseases Research Unit (Women's and Children's Hospital,
SA Pathology Services, Adelaide, South Australia); and the LAMP-1
(1 .mu.g/mL) mouse monoclonal BB6 was from Umea University, Umea,
Sweden. The horseradish-peroxidase (HRP) conjugated secondary
antibodies for Western blotting included anti-goat/sheep
immunoglobulin (1/2000, Merck Millipore Pty. Ltd., Victoria,
Australia), anti-rabbit immunoglobulin (1/2000, Sigma Aldrich Pty.
Ltd., New South Wales, Australia), anti-mouse immunoglobulin
(1/2000 Sigma Aldrich Pty. Ltd.), and HRP-conjugated anti-GAPDH
(1/20000 Sigma Aldrich Pty. Ltd.).
[0351] (ii) Cell Lines and Culture Conditions
[0352] The cell lines PNT1a and PNT2, 22RV1 and LNCaP clone FCG
were obtained from the European Collection of Cell Cultures via
CellBank Australia (Children's Medical Research Institute, New
South Wales, Australia). Cell lines RWPE-1, CaHPV10 and DU-145,
were obtained from the American Tissue Culture Collection via
Cryosite (Cryosite Ltd., New South Wales, Australia). These cell
lines are absent from the list of cross-contaminated or
misidentified cell lines, version 6.8 (9 Mar. 2012). Cell lines
were maintained in culture media recommended by the ATCC and ECCC.
PNT1 a, PNT2 and 22RV1 cell lines were cultured in Roswell Park
Memorial Institute (RPMI) 1640 media (Gibco.RTM., Life Technologies
Australia Pty Ltd., Victoria, Australia), supplemented with 10%
foetal calf serum (FCS; In Vitro Technologies Pty Ltd., Victoria,
Australia) and 2 mM L-glutamine (Sigma Aldrich Pty Ltd., New South
Wales, Australia). The RWPE-1 and CaHPV10 cell lines were cultured
in Keratinocyte Serum-Free Media (K-SFM) containing L-glutamine
(Gibco.RTM.), supplemented with the supplied human recombinant
epidermal growth factor 1-53 (EGF 1-53) and bovine pituitary
extract (BPE). The DU-145 cell line was cultured in minimum
essential medium (MEM) (Gibco.RTM.) and supplemented with 2 mM
L-glutamine and 10% FCS. LNCaP was cultured in RPMI-1640 media
supplemented with 2 mM L-glutamine, 10% FCS, 10 mM HEPES and 1 mM
sodium pyruvate. Cells were incubated in a humidified incubator at
37.degree. C. with 5% CO.sub.2.
[0353] (iii) Cell Extract Preparation
[0354] The culture medium was aspirated from 80-90% confluent cell
cultures, the cells washed once with PBS, and then incubated with
800 .mu.L of a 20 mM Tris (pH 7.0), 500 mM sodium chloride and 2%
SDS solution. Cells were harvested and a cell extract prepared by
heating to 65.degree. C. and sonication for one minute. The
resulting lysate was then passaged 6 times through a 25-guage
needle. Total protein in the cell extracts was quantified using a
bicinchoninic acid assay, according to the manufacturer's
instructions (Micro BCA kit, Pierce, Rockford, Ill., USA).
[0355] (iv) Western Blotting
[0356] Ten micrograms of cell lysate was heat-denatured (5 minutes
at 100.degree. C. in NuPAGE.RTM. LDS Sample Buffer and reducing
agent) then electrophoresed at 120 V for 1.5 hours using pre-cast
gels in an XCell SureLock Mini-Cell system (Life Technologies
Australia Pty Ltd., Victoria, Australia). The protein was then
transferred to polyvinylidene difluoride membranes
(Polyscreen.RTM., PerkinElmer, Victoria, Australia). The transfer
membranes were blocked for 1 hour at room temperature using 5%
(w/v) skim milk solution in 0.1% (v/v) TBS-tween and incubated with
primary antibody overnight at 4.degree. C. The membranes were
washed 3.times.5 min in 0.1% (v/v) TBS-tween and then incubated
with the appropriate HRP-conjugated secondary antibody diluted
1/2000 in block solution. The membranes were developed using a
Novex.RTM. ECL chemiluminescent substrate reagent kit (Life
Technologies Australia Pty Ltd., Victoria, Australia),and proteins
visualised using an ImageQuant.TM. LAS 4000 imager (GE Healthcare
Bio-Sciences Pty Ltd., New South Wales, Australia). Triplicate
samples were analysed and images quantified relative to a reference
GAPDH loading control using AlphaViewSA.TM. software v3.0.0.0
(ProteinSimple, Santa Clara, Calif.). Kruskal-Wallis rank sum
statistical analyses were performed using Stata/SE v11.2 (StataCorp
LP, Texas, U.S.A) to determine significance between non-malignant
control and cancer cell line groups (95% confidence limit;
p<0.05).
[0357] Results
[0358] (i) Detection of Current Prostate Cancer Biomarkers in
Prostate Cancer Cell Extracts and Culture Media.
[0359] Western blotting was used to define the amount of prostatic
acid phosphatase (PAP), prostate-specific antigen (hK3) and other
kallikreins (hK2, hK4 and hK15) in cell lysates and culture media
from the non-malignant prostate cell lines (RWPE-1, PNT1a and PNT2)
and the prostate cancer cell lines (22RV1, LNCaP, CaHPV10 and
DU-145; FIG. 10). While the intracellular amount of PAP was higher
in the prostate cancer cell lines 22RV1 and LNCaP, when compared to
the non-malignant control cell lines, little or no protein was
detected in the CaHPV10 and DU-145 prostate cancer cell lines.
Similarly, hK2, hK3 and hK4 showed increased amounts of
intracellular protein in LNCaP when compared to the non-malignant
control cell lines, but minimal amounts were detected in the other
prostate cancer cell lines. The amounts of intracellular hK15 were
similar for all of the prostate cancer cell lines, as well as the
non-malignant control cell lines RWPE-1 and PNT2, albeit with a
lower amount detected in PNT1a. PAP, hK3, hK2, and hK4 were
secreted from LNCaP cells and the amount was increased when
compared to the other prostate cancer and non-malignant control
cell lines. Notably, the secretion of hK15 from 22RV1 and LNCaP was
elevated when compared to the other prostate cancer and
non-malignant control cell lines. These findings indicated variable
protein content, and PAP/kallikrein secretion for the prostate
cancer cell lines and an inability of these markers to distinguish
between a number of prostate cancer and non-malignant control cell
lines.
[0360] The detection of hK2, hK4 and hK15 in cell extracts revealed
single molecular forms that could not be used to discriminate
between non-malignant control and prostate cancer cell lines.
Prostatic acid phosphatase was variably processed in the 22RV1 and
LNCaP prostate cancer cell lines (40 and 38 kDa molecular forms)
compared to the non-malignant cell line RWPE-1 (38 kDa form), but
was absent from the other cell lines. Similarly, two molecular
forms of hK3 were detected in 22RV1 and LNCaP prostate cancer cell
lines (29 and 25 kDa), while the other control and cancer cell
lines only expressed the 29 kDa molecular form. The detection of
secreted hK2 and hK15 revealed molecular forms (27 kDa for hK2; 21
kDa and 25 kDa for hK15) that were similar to PNT 1 a but these
molecular forms were not detected in the other non-malignant
control and cancer cell lines. This indicated variable proteolytic
processing in some prostate cancer cell lines when compared to the
non-malignant control cell lines.
[0361] The secretion of kallikreins appeared to correlate with the
androgen-receptor status of prostate cancer cell lines and the
amounts secreted generally appeared to be independent of the
amounts detected in the cell extracts. An increased secretion of
hK3, hK2 and hK15 was observed from 22RV1 and LNCaP prostate cancer
cell lines. However, the amount of hK2 was elevated in cell
extracts from the RWPE-1 non-malignant cells when compared to
22RV1; while there was increased secretion of this marker from the
22RV 1 cancer cell line compared to the RWPE-1 non-malignant
control cell line. Elevated amounts of kallikreins are expected in
cell lines that are responsive to androgen, or possessing activated
androgen receptor, thereby activating genes containing
androgen-response elements (AREs).
[0362] (ii) Detection of Lysosomal Proteins in Non-Malignant
Control and Prostate Cancer Cell Extracts and Culture Media.
[0363] Western blotting was used to define the amount of different
lysosomal proteins in cell lysates and culture media from
non-malignant control and prostate cancer cell lines (FIG. 11, FIG.
12). The amounts of intracellular .alpha.-glucosidase
(.alpha.-Gluc), .beta.-glucosidase (.beta.-Gluc),
.alpha.-galactosidase A (.alpha.-Gal A), LAMP-1 and cathepsin D (26
kDa) was variable between the cell lines and could not
differentiate non-malignant controls from the prostate cancer cell
lines. The amount of pro-cathepsin D in cell extracts was similar
for non-malignant control, CaHPV10 and DU-145 cancer cell lines,
however there was no detectable protein in the cell extract of
22RV1 and LNCaP prostate cancer cell lines. The secretion of
0-glucosidase, .alpha.-galactosidase, pro-cathepsin B and D was
elevated in RWPE-1 when compared to the other non-malignant cell
lines PNT1a and PNT2. The amount of .alpha.-galactosidase secreted
from RWPE-1 was similar to that from the CaHPV10 prostate cancer
cell line. The amount of LAMP-1 and cathepsin D (26 kDa) secretion
was similar for each of the non-malignant control and prostate
cancer cell lines. The 22RV1 and DU-145 prostate cancer cell lines
had elevated secretion of .alpha.-glucosidase and
.beta.-glucosidase when compared to the other prostate cancer and
non-malignant control cell lines. Limited amounts of Cathepsin B
(22 kDa) was detected in the 22RV 1 prostate cancer cell line when
compared to the other prostate cancer and non-malignant control
cell lines. The amount of pro-cathepsin B was elevated in LNCaP
when compared to the other prostate cancer cell lines. RWPE-1 had
an increased amount of pro-cathepsin B when compared to the PNT 1 a
and PNT2 non-malignant control cell lines. The secretion of
cathepsin B (22 kDa) was higher for CaHPV10, DU-145 and LNCaP
prostate cancer cell lines compared to the non-malignant control
cell lines and the 22RV 1 prostate cancer cell line. Minimal
amounts of acid ceramidase were detected in the non-malignant
control cell lines and the prostate cancer cell lines 22RV1,
CaHPV10 and DU-145. In contrast, large amounts of acid ceramidase
were detected in cell extracts from the LNCaP cancer cell line.
Acid ceramidase (10-15 kDa) was not secreted from the non-malignant
control cell lines, but elevated amounts were secreted from the
22RV1, DU-145 and LNCaP prostate cancer cell lines when compared to
CaHPV10 and the non-malignant control cell lines. These findings
indicated variable intracellular content and secretion of lysosomal
proteins for non-malignant control and prostate cancer cell lines,
with a potential for cathepsin B and acid ceramidase to distinguish
between non-malignant control and prostate cancer cell lines. The
secretion of acid ceramidase and cathepsin B may therefore afford
some capacity to distinguish between non-malignant and cancer cell
lines.
[0364] Multiple molecular forms of .alpha.-glucosidase were
detected in the cell extracts of non-malignant and prostate cancer
cell lines. A 110 kDa molecular form of .alpha.-glucosidase was
detected in 22RV1 and LNCaP cancer cell lines, but only minimal
amounts were detected in the other prostate cancer and
non-malignant control cell lines. The molecular weight of
.beta.-glucosidase and LAMP-1 varied between prostate cancer and
non-malignant cell lines and was unable to discriminate between
these types of cell lines. Two molecular forms of
.alpha.-galactosidase A (42 and 43 kDa) were present in both
CaHPV10 and RWPE-1 cell lines, but not in other prostate cancer or
non-malignant cell lines. Both CaHPV10 and RWPE-1 displayed low
amounts of the 70 kDa molecular form of .alpha.-glucosidase when
compared to the other non-malignant and prostate cancer cell lines.
Interestingly, in both RWPE-1 and CaHPV10 cell lines there was
similar protein processing for .alpha.-glucosidase and
.alpha.-galactosidase A, elevated molecular weight of
.beta.-glucosidase and elevated secretion amounts of
.alpha.-galactosidase A, which differed from the other
non-malignant and prostate cancer cell lines; and this correlated
with the different technique used to immortalise these two cell
lines. Two molecular forms of pro-cathepsin B (37 and 39 kDa) were
detected in the cell extract of CaHPV10, DU-145 and LNCaP prostate
cancer and RWPE-1 non-malignant control cell lines. A 60 kDa
molecular form of .beta.-glucosidase was secreted from RWPE-1 and
CaHPV10 cells, which was more diffuse from the other cell lines and
suggested variable glycosylation. The molecular weight of the
secreted form of .alpha.-galactosidase A was lower in the prostate
cancer cell lines 22RV1, DU-145 and LNCaP when compared to the
non-malignant control cell lines and CaHPV10 prostate cancer cell
line. The variable processing and increased secretion of some
lysosomal proteins may have some capacity to distinguish between
prostate cancer and non-malignant cell lines.
EXAMPLE 3
Microarray Expression Profiling of Endosome and Lysosome Genes in
vivo
[0365] The observations of altered endosome biology in prostate
cancer cells suggested that the increased expression of specific
endosomal gene and proteins might critically influence the
development and or progression of prostate cancer. Altered endosome
biogenesis may therefore provide a new avenue for the investigation
of biomarkers that can be used for prostate cancer diagnosis and
prognosis. However, it was important to correlate the in vitro
observations with patient data, to establish that the novel changes
are biologically relevant. Verifying that there was altered
endosome-related gene expression in prostate cancer microarray
databases was viewed as an important first step in this
process.
[0366] Gene microarray databases enable the investigation of
biomarker expression in patients and to determine any relation to
known clinical parameters, which may then be used to predict
clinical outcome.
[0367] To analyse endosome-lysosome gene expression in relation to
patient outcome a range of prostate cancer microarray databases
were selected for investigation. Multiple microarray cohorts are
available for analysis from Oncomine (Life Technologies Pty., Ltd.)
or the Gene Expression Omnibus. A recent cohort of patients treated
by radical prostatectomy at the Memorial Sloan-Kettering Cancer
Center (MSKCC) was also selected and herein referred to as the
Taylor cohort. This cohort comprised 150 primary prostate cancer
and 29 matched non-malignant control samples. For the Taylor
cohort, specimens were collected and snap-frozen prior to the
identification of cancer regions; RNA extraction was performed on
samples containing greater than 70% cancer-cell content, based on
histological assessment. Analysis was performed using an Affymetrix
Human Exon 1.0 ST array with the resulting microarray data obtained
from the cBio Cancer Genomics Pathway Portal. This cohort was also
selected because it contained expression data for all of the
relevant endosome and lysosome genes that were analysed in vitro.
Further, the Affymetrix GeneChip.RTM. used in this cohort was
created from sequences derived from the RefSeq database build 34
(July 2003), which provides greater sequence accuracy than previous
microarrays using the Affymetrix GeneChip.RTM. HGU95. An additional
microarray by Tomlins et al. was also analysed and herein referred
to as the Tomlins cohort, which was generated using alternative
microarray technology; specifically, a non-commercial custom-made
array. This cohort comprised of non-malignant prostate tissue
(n=27), prostatic intraepithelial neoplasia (PIN; n=13), primary
cancer (n=32) and metastatic cancer tissue (n=20). Thus, in
addition to confirming the results obtained for the Taylor cohort,
the Tomlins cohort enabled the assessment of gene expression during
disease progression from PIN through to metastatic cancer, which
may be diagnostically relevant. Prostate samples for the Tomlins
cohort were collected using laser-capture micro-dissection (LCM)
with approximately 10,000 cells collected from each sample. This
method of sample collection was advantageous since specificity to
specific sample type can be increased, with tissue surrounding the
cancer excluded from capture, leading to cleaner RNA preparation
and analysis. The prostate cancer patient tissue analysed by
Glinsky et al. included cancer patients that were monitored over
the course of five years from initial cancer detection, to
determine recurrence based on PSA levels post-therapy; and
therefore provided a capacity to determine the prognostic
capability of endosome or lysosome genes, by analysing expression
during cancer progression. The analysis of these different
microarray databases aimed to verify the altered gene expression,
observed in vitro using cultured cells, as well as to provide some
indication of the consistency for marker expression in patient
samples.
[0368] Results
[0369] (i) Expression of Lysosome-Related Genes in Prostate Cancer
are Altered Independently of TFEB Expression.
[0370] The transcription of lysosome-related genes such as LIMP2
that influence endosome and lysosome biogenesis is regulated by
transcription factor EB (TFEB). Thus, TFEB gene expression may
provide evidence of altered endosome and lysosome biology in vivo.
Expression of TFEB was unchanged in prostate cancer compared to
non-malignant control prostate tissue (FIG. 13), whilst expression
of LIMP2 was significantly elevated in prostate cancer compared to
non-malignant tissue (P.ltoreq.0.0001). Conversely, LAMP1, which is
also a target of TFEB, had significantly reduced expression in
prostate cancer compared to non-malignant control tissue
(P.ltoreq.0.01). Other TFEB-regulated lysosomal genes were analysed
to determine if their expression was altered independently of this
transcription factor (FIG. 14). Cathepsin B (CTSB) showed
significantly reduced expression in prostate cancer tissue when
compared to non-malignant control tissue using the Taylor cohort
(P.ltoreq.0.0001), whilst .alpha. Glucosidase (GAA) had
significantly elevated expression in prostate cancer compared to
non-malignant control tissue (P.ltoreq.0.05). The expression of
acid ceramidase, .beta. glucosidase (GBA) and .alpha. galactosidase
A (GLA) were not significantly altered in prostate cancer tissue in
the Taylor cohort when compared to non-malignant control
tissue.
[0371] (ii) APPL1 had Significantly Elevated Expression in Primary
Prostate Cancer Tissue.
[0372] APPL1 gene expression was significantly elevated in prostate
cancer tissue from the Taylor cohort when compared to non-malignant
control tissue (P.ltoreq.0.05; FIG. 15). However, while elevated
expression of APPL2, RAB5A, EEA1, RAB4A and RAB7A were observed in
vitro, there was no significant difference in expression of these
endosome related genes in primary prostate cancer compared to
non-malignant prostate tissue (FIG. 15).
[0373] (iv) Lysosomal Gene Expression (FIG. 16) and Early
Endosome-Related Gene Expression (FIG. 17) was Altered During
Cancer Progression.
[0374] The expression of endosome and lysosome related mRNA
transcripts was also analysed from the Tomlins cohort enabling the
analysis of prostatic intraepithelial neoplasia (PIN) and
metastatic cancer tissue in addition to primary cancer and
non-malignant tissue. The expression data for some genes such as
TFEB was unavailable due to the custom microarray technology used
and more limited probe-set compared to the commercial microarray
used by Taylor et al. The gene expression of LIMP2 and LAMP1 in the
Tomlins cohort showed no significant change between non-malignant
control and primary prostate cancer tissue (FIG. 16). Other
TFEB-regulated genes displayed differential expression in the
prostate cancer tissue when compared to non-malignant control
tissue; cathepsin B was significantly reduced in primary cancer
tissue compared to non-malignant tissue (P.ltoreq.0.05), however
there was no significant change in the expression of cathepsin D,
acid ceramidase, .alpha. glucosidase or .alpha. galactosidase A
between non-malignant tissue and primary cancer tissue from the
Tomlins microarray (FIG. 16). No expression data was available for
.beta. glucosidase in this cohort.
[0375] Analysis of gene expression through the progressive disease
states revealed a significant increase in LIMP2 expression in
prostatic intraepithelial neoplasia (PIN) (P.ltoreq.0.05; FIG. 16);
variations of LIMP2 expression were observed in metastatic tissue,
however the mean reduction was not statistically significant. LAMP
1 expression was elevated in PIN and primary cancer compared to
non-malignant tissue, with expression in metastatic tissue
significantly reduced when compared with PIN (P.ltoreq.0.05) and
primary prostate cancer tissue (P.ltoreq.0.001). Cathepsin B
expression in primary prostate cancer and metastatic tissue was
significantly downregulated when compared to non-malignant control
tissue (P.ltoreq.0.05 and P.ltoreq.0.001 respectively). Cathepsin D
expression showed some reduction through PIN, primary cancer and
metastatic disease states, however this was not statistically
significant when compared with non-malignant control tissue. In
primary cancer tissue acid ceramidase displayed similar expression
to that observed in non-malignant control tissue; however, PIN
tissue expression was significantly elevated when compared to
non-malignant control and primary cancer tissue (P.ltoreq.0.01 and
P.ltoreq.0.05 respectively). The expression of ASAH1 was
significantly reduced in metastatic prostate tissue when compared
to non-malignant control (P.ltoreq.0.01), PIN (P.ltoreq.0.001) and
primary cancer tissue (P.ltoreq.0.001).
[0376] APPL1 expression was significantly increased in primary
prostate cancer compared to non-malignant control tissue
(P.ltoreq.0.05; FIG. 17); although there was no significant change
in expression of APPL1 between metastatic prostate tissue and
non-malignant tissue. The expression of APPL2 was significantly
elevated in PIN and primary prostate cancer compared to
non-malignant control tissue (P.ltoreq.0.05 and P.ltoreq.0.01
respectively); while metastatic tissue expressed similar amounts of
APPL2 to non-malignant tissue, and this was significantly reduced
when compared to PIN and primary prostate cancer tissue
(P.ltoreq.0.01 and P.ltoreq.0.001 respectively). The expression of
RAB5A was significantly reduced in metastatic prostate tissue when
compared to primary prostate cancer tissue (P.ltoreq.0.05), however
there was no significant change in expression between
non-malignant, PIN or primary prostate cancer tissue. The
expression of EEA1 was significantly elevated in primary cancer
compared to non-malignant tissue (P.ltoreq.0.01); whereas a
significant reduction was observed in EEA1 expression for
metastatic tissue compared to primary prostate cancer
(P.ltoreq.0.001) and PIN tissue (P.ltoreq.0.05). The expression of
RAB4A was similar in primary prostate cancer compared to
non-malignant control tissue; and the expression of RAB4A in PIN
tissue was significantly elevated when compared to primary prostate
cancer tissue (P.ltoreq.0.05). There was a significant reduction in
the expression of RAB4A in metastatic prostate tissue when compared
to non-malignant control (P.ltoreq.0.001), PIN (P.ltoreq.0.001) and
primary prostate cancer tissue (P.ltoreq.0.01). There was no
significant change in the expression of RAB7A observed in PIN,
primary cancer or metastatic cancer from the Tomlins microarray
when compared to non-malignant control tissue.
[0377] (v) Cathepsin B Appeared to Show Some Prognostic Value in
Determining Survival Outcome for Prostate Cancer Patients.
[0378] The quantification of gene expression may be a valuable tool
for prostate cancer prognosis, as expression of some lysosome and
endosome genes vary through PIN, primary cancer and metastatic
cancer progression. To assess the prognostic potential of lysosomal
genes, we classified patients from the Glinsky cohort into two
groups using K means clustering based on gene expression. The
Glinsky cohort microarray provides data of initial PSA levels
detected upon biopsy, and information on relapse subsequent to
therapy. Clustering of high or low cathepsin B expression, revealed
patients with low amounts of cathepsin B expression who had
significantly increased risk of biochemical recurrence (BCR). (FIG.
18). The grouping by high or low expression for LIMP2, LAMP1,
cathepsin D, acid ceramidase or .alpha. galactosidase A genes did
not stratify patients significantly into prognostic groups, however
there was a trend observed for .alpha. galactosidase A (P=0.077;
FIG. 18). From the increased expression of early endosome-related
genes observed in the Taylor and Tomlins cohorts, we analysed the
potential of these early endosome-related genes to stratify
patients into prognostic groups (FIG. 19). However, classifying
patients into two groups based on expression of individual early
endosome genes by K means clustering did not significantly separate
patients into prognostic groups.
[0379] (vi) Kaplan-Meier Analysis of Lysosome and Endosome Gene
Expression Showed Significant Prognostic Value in Cancer Patients
Expressing Low PSA Protein Levels.
[0380] Patients expressing PSA protein greater than 7.9 ng/mL at
the time of diagnosis had a significantly increased risk of BCR
when compared to the low-expression group (HR 2.422, P=0.0087; FIG.
20); however there was a proportion of patients (34%) with BCR that
expressed low PSA that would benefit from improved diagnostic and
prognostic assays. Stratification of patients with PSA.ltoreq.7.8
ng/mL into high and low gene expression groups revealed a trend of
high LIMP2 expression to result in increased BCR (FIG. 20B). The
expression of LAMP1 did not appear to show any prognostic value.
Clustering by cathepsin B expression indicated that patients in the
low expression group were at significant risk of BCR when compared
to those in the high expression group (HR 0.2752, P=0.019; FIG.
20B). Low expression of .alpha. galactosidase A was also able to
stratify patients into a group with higher risk of BCR (HR 0.3859,
P=0.0396; FIG. 20B).
[0381] Individual RAB5A, APPL1 and EEA1 gene expression was not
able to stratify patients into statistically significant risk
groups (FIG. 21A). However, stratification of patients based on
expression of a three-gene signature using K means clustering
methodology robustly separated patients into two groups with low or
high expression of combined RAB5A, APPL1 and EEA1 (FIG. 21A).
Kaplan-Meier survival analysis indicated patients in the high
expression group of the three-gene signature were at significantly
higher risk of BCR when compared to those in the lower-expression
group (HR 4.138, P=0.0221, 95% CI 1.3950-2.270; FIG. 21B).
[0382] Discussion
[0383] Endosomal and lysosomal biogenesis is normally regulated by
the transcription factor "EB" (TFEB), via control over the
transcription factor of lysosomal genes containing "CLEAR
elements". Target genes of TFEB include LIMP2 and LAMP1, which had
differential expression in prostate cancer cell lines. Whilst we
also observed elevated LIMP2 and reduced LAMP1 gene expression in
prostate cancer tissue from these microarrays, there was no change
in TFEB expression, suggesting that the normal action of TFEB may
be altered in prostate cancer.
[0384] Altered gene expression during cancer development may be a
valuable prognostic indicator. Increased expression of acid
ceramidase (ASAH1) may be effective for the diagnosis of
pre-cancerous PIN lesions and may be associated with improved
prognosis following intervention.
[0385] Multivariate risk analysis and stratification of patients
into low- and high-risk groups may provide a more individualised
approach to prostate cancer detection and therapy, reducing
over-diagnosis and over-treatment and thereby reducing patient
morbidity and mortality. Active-surveillance is often used for
patients deemed to have low-risk prostate cancer (e.g. clinical
category T1c, Gleason score .ltoreq.6, and PSA.ltoreq.10 ng/mL),
however older men are at an increased risk of mortality from
prostate cancer despite these low scores and PSA serum levels.
Examination of the survival curves of prostate cancer patients
expressing low PSA, that represent a watchful-waiting group,
reveals prostate cancers that are aggressive and result in more
rapid recurrence, however additional factors such as Gleason score
can improve patient stratification. Through quantitation of a gene
signature of the three endosome genes, APPL1, EEA1 and Rab5A, in
prostate cancer patients expressing PSA.ltoreq.7.8 ng/mL, led to
patient stratification into high and low-risk recurrence groups.
The capacity of these endosomal genes to stratify patients into
prognostic risk groups suggested that they play an important role
in the development and progression of prostate cancer and may
therefore provide a new therapeutic target.
EXAMPLE 4
Evaluation of Endosome Marker Secretion for the Detection of
Prostate Cancer
[0386] Results
[0387] (i) Extracellular endosome proteins differentiate
non-malignant and prostate cancer cell lines (FIG. 2). There was a
significant increase in the amount of APPL1 and APPL2 detected in
the culture media from prostate cancer compared to non-malignant
control cell lines (P.ltoreq.0.01). There was also a significant
increase in the amount of Rab5A (approximately 20%; P.ltoreq.0.05)
detected in the culture media from prostate cancer cell lines when
compared to non-malignant controls. EEA1 was detected in the
culture media from LNCaP and 22RV1 prostate cancer cell lines, but
was not detected in the culture media from the non-malignant
control cell lines PNT1a or PNT2 (P.ltoreq.0.01). There was a
significant increase in the amount of Rab4 detected in the culture
media from prostate cancer compared to non-malignant control cell
lines (P.ltoreq.0.05), although the amount detected in the latter,
PNT1a and PNT2 cell lines, was variable. There was a significant
reduction in the amount of Rab7 detected in the culture media from
the prostate cancer compared to the non-malignant control cell
lines (P.ltoreq.0.05).
[0388] Discussion
[0389] Currently the prostate specific antigen (PSA) is used as a
diagnostic marker, but this has reliability issues and still
requires an invasive biopsy to further determine the expected
course for the cancer development. There is therefore an urgent
need for new biomarkers to enable accurate detection and prognosis
of prostate cancer and to avoid unnecessary, invasive and costly
procedures. We have observed significant increases in the amount of
early endosome markers detected in the culture media from prostate
cancer cell lines. In particular, the vesicular machinery
associated with early endosomes, including APPL1, APPL2, Rab5A and
EEA1 and recycling endosome protein Rab4, were markedly elevated in
the culture media from prostate cancer cell lines, whilst the late
endosome protein Rab7 showed a significant reduction in prostate
cancer cells, when compared to non-malignant controls.
[0390] The secretion of early endosome markers APPL1, APPL2 and
EEA1, but not late endosome marker Rab7 from prostate cancer cell
lines implied that the maturation of endosome compartments into
multivesicular bodies and exosome vesicles is altered in prostate
cancer and may therefore be used to distinguish prostate cancer
from non-malignant tissue.
[0391] A possible mechanism by which exosome formation may be
disturbed in prostate cancer involves the vesicular GTPase Rab5.
The over-expression of Rab5 allows simultaneous exposure of PI3P
and APPL binding domains on the Rab5 protein, which would
effectively reduce APPL1 dissociation and enable concurrent binding
of EEA1. This could therefore result in endosomes that are Rab5-,
EEA1- and APPL1-positive. Initiation of endosome membrane collapse
and invagination to form exosomes is initiated by PI3P and the
recruitment of Hrs and ESCRT complexes. The unusual composition of
endosome vesicular machinery on prostate cancer endosomes may
predispose exosome formation in early endosomes. The release of
these exosomes from early endosomes would then account for the
apparent secretion of early endosome vesicular machinery observed
from the prostate cancer cell lines. This Rab5, EEA1 and APPL1
secretion may provide the basis of an assay to enable the detection
of prostate cancer.
[0392] Blood and urine are ideal patient samples for the detection
of vesicular machinery secreted in exosomes, as they represent
potential analytes for a non-invasive diagnostic assay.
[0393] The altered secretion of early endosome and late endosome
markers observed when comparing non-malignant control and prostate
cancer cell lines provides a new avenue for investigation of
diagnostic and prognostic markers that may lead to more accurate
detection and prognosis of prostate cancer. The data here supports
the notion that endosome biology is disturbed in prostate cancer,
resulting in increased secretion of early-endosome markers and
reduced secretion of late endosome markers from prostate cancer
cell lines. Taken together, these results suggest that alterations
in the secretion of early versus late endosome markers may provide
the discrimination required for the early detection of prostate
cancer.
EXAMPLE 5
General Materials and Methods
[0394] Materials
[0395] Antibodies and Fluorescent Probes
[0396] Table 1 below details the properties of
fluorescent-conjugate antibodies used in confocal microscopy
analysis. Where dual labelling is performed using two primary
antibodies of the same host-species, Zenon.RTM. IgG labelling kits
(Life Technologies Australia Pty Ltd., Victoria, Australia) were
used to label the primary antibodies directly, with the labelling
protocol carried out as per manufacturers' instructions. Specific
concentrations of primary antibodies and secondary conjugates used
for Western blotting and immune fluorescence are detailed in the
Tables below.
TABLE-US-00001 TABLE 1 Fluorescent probes used in laser-scanning
confocal microscopy Excitation Emission Detection Fluorophore
maxima (.lamda.) maxima (.lamda.) spectrum (nm) DAPI 358 461
407-471 Phalloidin 495 518 500-560 Alexa Fluor .RTM. 488 Alexa
Fluor .RTM. 488 495 519 500-550 Zenon .RTM. 488 495 519 500-550
LysoTracker .RTM. Green 504 511 505-560 DND-26 LysoTracker .RTM.
Red 577 590 590-700 DND-99 Zenon .RTM. 568 578 603 570-630 Cy3 550
570 570-630 Alexa Fluor .RTM. 633 632 647 650-730 Transferrin 632
647 650-730 Alexa Fluor .RTM. 633
TABLE-US-00002 TABLE 2 Primary and secondary antibodies used in
Western blot analysis Working Antibody concentration Source cat#
M-Cathepsin B 0.25 .mu.g/mL ab58802 M-Cathepsin D 5 .mu.g/mL ab6313
Rb-Acid ceramidase 1 .mu.g/mL ab74469 Sh-.alpha.-Glucosidase 1
.mu.g/mL In-house Sh-.beta.-Glucosidase 1 .mu.g/mL In-house
Sh-.alpha.-Galactosidase A 1 .mu.g/mL In-house M-Prostatic 1
.mu.g/mL ab75704 acid phosphatase Rb-KLK2 1 .mu.g/mL ab40948
Rb-KLK3 1 .mu.g/mL ab40949 Rb-KLK4 1 .mu.g/mL ab40950 Rb-KLK15 1
.mu.g/mL ab40961 M-TFEB 1 .mu.g/mL ab56330 Rb-TfR1 1 .mu.g/mL
ab108985 Rb-TfR2 1 .mu.g/mL ab80194 Sh-LIMP2 5 .mu.g/mL In-house
Rb-APPL1 0.4 .mu.g/mL ab95195 Rb-APPL2 0.4 .mu.g/mL ab95196 G-RAB5A
1 .mu.g/mL R9704 G-EEA1 1 .mu.g/mL sc-6415 Rb-RAB4 1 .mu.g/mL
ab13252 G-RAB7A 1 .mu.g/mL Sc-6563 M-LAMP1 BB6 1 .mu.g/mL In-house
Rb-AKT 2 .mu.g/mL In-house Rb-AKT-P 2 .mu.g/mL In-house M-GAPDH HRP
1/20000 G9295 G-.alpha.-Rabbit HRP 1/2000 A6154 G-.alpha.-Mouse HRP
1/2000 A4416 D-.alpha.-Sheep/Goat HRP 1/2000 AB324P
TABLE-US-00003 TABLE 3 Primary antibodies and fluorophores used in
immune fluorescence assays Working Antibody concentration Source
cat# Rb-TfR1 5 .mu.g/mL ab108985 Rb-TtR2 5 .mu.g/mL ab80194
Sh-LIMP2 10 .mu.g/mL In-house Rb-APPL1 2 .mu.g/mL ab95195 G-RAB5A 4
.mu.g/mL R9704 G-EEA1 4 .mu.g/mL sc-6415 Rb-RAB4 4 .mu.g/mL ab13252
G-RAB7A 4 .mu.g/mL Sc-6563 M-LAMP1 BB6 5 .mu.g/mL In-house Rb-TGN46
10 .mu.g/mL ab50595 LysoTraeker .RTM. 488 5 .mu.M (1/200) L7535
Transferrin 633 5 .mu.g/mL T23362 Phalloidin 488 1/100 A12379
D-.alpha.-Mouse 1/250 A11055 Alexa fluor .RTM. 488 D-.alpha.-Sheep
1/250 A11015 Alexa fluor .RTM. 488 D-.alpha.-Goat Cy3 1/250
AP180C
[0397] Cell Culture and Extract Preparation
[0398] Cell Lines
[0399] The cell lines PNT1a and PNT2, 22RV I and LNCaP clone FCG
were obtained from the European Collection of Cell Cultures via
CellBank Australia (Children's Medical Research Institute, New
South Wales, Australia). Cell lines RWPE 1, CaHPV10 and DU 145,
were obtained from the American Tissue Culture Collection via
Cryosite (Cryosite Ltd., New South Wales, Australia). These cell
lines are absent from the List of Cross-Contaminated or
Misidentified Cell Lines, version 6.8 (9 Mar. 2012).
[0400] Any study of prostate cancer should equally be studied on
normal non-cancerous prostate. Analysing cell lines derived from
primary or metastatic prostate cancer results in a need for cell
lines derived from healthy prostate tissue. This presents a problem
for cell culture regarding the normal division and replication
cycle of a healthy cell. Unlike the majority of cancer cell lines,
which will multiply at a regular rate, to generate a
n.on-carcinorna cell line, a healthy cell must be developed to
invoke a constant cell cycle and rate of division.
[0401] As such, the cell lines RWPE 1, PNT1a and PNT2 used in this
study to represent normal epithelial-cell phenotypes, have been
adapted and immortalised with viruses. Specifically, human
papilloma virus-18 (HPV 18) was used to immortalise the RWPE 1 cell
line, and Simian vacuolating virus 40 (SV40) to immortalise PNT1a
and PNT2 cell lines. The cancer cell line CaHPVIO was derived from
a primary adenocarcinoma of the prostate immortalised with HPV 18.
This cell line is not androgen responsive.
[0402] The 22RV1 cancer cell line was derived from a xenograft that
had been serially propagated in mice after castration-induced
regression and relapse of a parental, androgen-dependent xenograft.
The cell line expresses androgen receptor however its proliferation
is unresponsive to androgen stimulation.
[0403] The cancer cell line LNCaP was previously derived from a
lymph node metastasis of prostate adenocarcinoma. The LNCaP cell
line is androgen responsive.
[0404] Cancer cell line DU 145 was previously derived from a
moderately-differentiated brain metastasis of prostate
adenocarcinoma. They are epithelial cells, and unresponsive to
androgen stimulation.
[0405] Culture Conditions
[0406] PNT1 a, PNT2 and 22RV1 cell lines were maintained in Roswell
Park Memorial Institute (RPMI) 1640 media (Gibco.RTM., Life
Technologies), supplemented with 10% foetal calf se-rum (FCS) (In
Vitro Technologies Pty Ltd., Victoria, Australia) and 2 mM
L-glutamine (Sigma Aldrich Pty Ltd., New South Wales, Australia).
RWPE 1 and CaHPV10 cell lines were maintained in Keratinocyte
Serum-Free Media containing L-glutamine (K-SFM) (Gibco.RTM.),
supplemented with the supplied human recombinant Epidermal Growth
Factor 1 53 (EGF 1 53) and Bovine Pituitary Extract (BPE). The DU
145 cell line was maintained in Minimum Essential Medium (MEM)
(Gibco.RTM.) and supplemented with 2 mM L-glutamine and 10% FCS.
Cancer cell line LNCaP was maintained in RPMI 1640 media
supplemented with 2 mM L-glutamine, 10% FCS, 10 mM HEPES (Life
Technologies) and 1 mM sodium pyruvate (Sigma Aldrich). Cells were
incubated in a humidified incubator at 37.degree. C. with 5%
CO.sub.2 in a Sanyo MCO-17AI CO.sub.2 Incubator (Sanyo Electric
Biomedical Co., Ltd., Osaka, Japan).
[0407] Cells were passaged at approximately 90% confluence, washed
with sterile dPBS (Sigma Aldrich) and trypsinised using 1.times.
Trypsin-EDTA solution containing 0.12% trypsin, 0.02% EDTA
(SAFC.RTM., Sigma Aldrich) until dissociated from the culture flask
surface. Trypsin was neutralised with growth media containing 10%
RWPE 1 and CaHPV10 cell lines cultured in serum-free media were
pelleted at 250 g for five minutes and washed with dPBS to remove
foetal calf serum. Cells were passaged at a 1:10 to 1:20 ratio for
general maintenance.
[0408] For long-term storage, cell lines were stored in liquid
nitrogen. For cryopreservation, cell cultures were passaged as
normal. After trypsin inactivation using 10% FCS, cells were
pelleted by centrifugation at 250 g for five minutes and
resuspended in their respective culture media containing 10% FCS
and 10% DMSO (Sigma Aldrich). 1 mL aliquots of cell suspension were
stored at -80.degree. C. overnight in an isopropanol freezing
container (Nalgene, Thermo Fisher Scientific Australia Pty Ltd.,
Victoria, Australia) prior to long-term storage in liquid
nitrogen.
[0409] Sample Preparation
[0410] Cell Extract Preparation for Immune Blotting
[0411] Cell lines were seeded in T75 flasks and incubated at
37.degree. C. and 5% CO2. At a confluence of 80-90%, culture media
was aspirated, cells washed once with dPBS, and cells incubated for
48 hours with serum-free media.
[0412] Total cell protein was extracted using a TNS buffer
containing 20 mM Tris (pH 7.0), 500 mM sodium chloride and 2% SDS
solution, diluted in Milli-Q H2O. A solution containing Tris and
sodium chloride was prepared to 80% of the final volume, with the
remaining 20% volume comprising 10% SDS, added within 30 minutes
prior to use. Following 48 hours of incubation, cell culture media
was aspirated and cells washed twice with room-temperature dPBS.
800 .mu.L of TNS buffer was added to the culture flask and rotated
to cover cell-layer in its entirety. Cells were harvested by using
a rubber cell scraper, and the resulting extract transferred to a
1.5 mL tube. Extract was immediately heated to 65.degree. C. for
five minutes and sonicated at high-frequency for one minute.
[0413] To obtain a more homogenous lysate, cell material was passed
through a 25-guage (0.65 mm) syringe needle, a total of 6 times.
Additional TNS buffer was added, where required, to produce a less
viscous lysate. Samples were stored at -20.degree. C.
[0414] Total protein was quantified using a bicinchoninic acid
assay (Micro BCA kit, Pierce, Thermo Scientific Pty Ltd., Rockford,
Ill., USA) according to the manufacturer's instructions. Samples
were quantified using a Wallac Victor.TM. optical plate-reader and
Workout software v2.0 (Perkin-Elmer Pty Ltd., Victoria, Australia)
using a 5-point parameter standard-fit curve.
[0415] Conditioned media collected at time of cell harvesting and
previously stored at -80.degree. C. was thawed, and 33 mL aliquots
centrifuged at 1000 g for 10 minutes at 4.degree. C. to remove any
cell debris. 30 mL of supernatant was transferred to a 50 mL
conical tube and sodium deoxycholate (DOC) (Sigma Aldrich) added to
a final 0.02% v/v concentration. Following a brief vortex, the
samples were kept on ice for 30 minutes.
[0416] A 100% w/v solution of trichloroacetic acid (TCA) (Sigma
Aldrich) was added to a final concentration of 15% v/v, agitated by
vortex for 30 seconds, and incubated on ice for two hours to
precipitate protein from the culture media. Precipitated protein
was collected by centrifugation of samples at 5,500 g for 30
minutes, with supernatant containing TCA and contaminant waste
aspirated. The protein pellet was washed with 4 mL ice-cold
(-20.degree. C.) acetone by vortex and incubated at room
temperature for five minutes. The sample underwent a further
centrifugation at 5,500 g for 30 minutes and the acetone wash
repeated. After a final centrifuge step, the supernatant was
aspirated, and conical tube containing protein pellet was dried
under a slow stream of nitrogen gas. Each protein pellet, produced
from 30 mL media, was resuspended in a 135 .mu.L 1.times.SDS-sample
buffer/PBS solution, and stored at -20.degree. C. for use at a
later time in Western blot assays.
[0417] Cell Extract Preparation for Quantitative Analysis of Gene
Expression
[0418] Cell lines were cultured in triplicate in 75 cm.sup.2 flasks
as described for Western blotting, with serum deprivation for 48
hours. Media was aspirated and flasks washed with dPBS. 1 mL TRI
reagent.RTM. (Applied Biosystems Pty Ltd., Victoria, Australia) was
added to each T75 flask and scraped using a rubber cell-scraper and
transferred to a 1.5 mL flip-top tube. Samples were stored at
-80.degree. C. until further required.
[0419] Cell Culture Preparation for Immune Fluorescence Analysis by
Confocal Microscopy
[0420] Cell cultures were passaged as normal, seeded at low density
on 22 mm, "Number 1", glass coverslips (Menzel-Glaser, Thermo
Fisher Scientific Australia Pty Ltd., Victoria, Australia) and
incubated in culture media for 48 hours at 37.degree. C. and
atmosphere of 5% CO.sub.2. After 48 hours, culture media was
aspirated and cells fixed using 4% formaldehyde (v/v) PBS solution
for 20 minutes at room temperature. Fixed cells were further
permeabilised with 0.1% Triton-X (v/v) in PBS for 10 minutes.
[0421] Quantitative Analysis of Protein Expression
[0422] SDS-PAGE and Transfer
[0423] 10 .mu.g of total protein from cell lysates were
heat-denatured for five minutes at 100.degree. C. in sample buffer
(NuPAGE.RTM. LDS Sample Buffer (4.times.), Life Technologies) and
reducing agent containing 50 mM dithiothreitol (NuPAGE.RTM. Sample
Reducing Agent (10.times.)). Prepared samples were separated using
a 10% 15-well, or 12% 17-well SDS-PAGE pre-cast gel in an XCell
SureLock.TM. Mini-Cell electrophoresis system, buffered with
SDS-PAGE running-buffer (Life Technologies) at 120 V (fixed, with
variable current), for 1.5 hours. Following electrophoresis,
separated protein was transferred to polyvinylidene difluoride
(PVDF) membranes (Polyscreen.RTM., Perkin-Elmer) at 35 V for one
hour.
[0424] Immunoblotting and Detection
[0425] Membranes were blocked for 1 hour at room temperature using
5% w/v skim-milk solution in TBS-tween (0.1%) and incubated with
the appropriate antibody (Table 2.2) diluted in 5% skim-milk
overnight at 4.degree. C. with gentle agitation. Following
incubation with the primary antibody, membranes were washed thrice
with TBS-tween for five minutes at room temperature. A
horseradish-peroxidase conjugate secondary antibody (Table 2.2),
diluted to 1/2000 in 5% skim-milk solution, was incubated with the
PVDF membrane for one hour at room temperature with gentle
agitation.
[0426] Membranes were washed three times with TBS-tween for five
minutes, followed by antibody detection using electro
chemiluminescence (ECL). Membranes were incubated for one minute in
the dark using Novex.RTM. ECL chemiluminescent substrate reagent
kit (Life Technologies), and visualised using an ImageQuant.TM. LAS
4000 imager, software version 1.2.0.101 (GE Healthcare Bio-Sciences
Pty Ltd., New South Wales, Australia). GAPDH loading control was
detected using a goat a GAPDH HRP conjugate (Sigma Aldrich) at a
1:20,000 dilution in 5% skim-milk Detection of GAPDH was performed
concurrently, where molecular weights would allow for detection,
with secondary-HRP detection of primary antibody, at room
temperature for one hour. Where molecular weights of GAPDH and
marker protein would potentially conflict, PVDF membranes were
stripped of primary antibody and re-probed with a GAPDH HRP
conjugate.
[0427] Blots were performed in triplicate and images quantified
using AlphaViewSA.TM. software v3.0.0.0 (ProteinSimple, Santa
Clara, Calif.). Lane-analysis was performed using the software to
determine molecular weights of detected protein. The amount of
protein was quantified based on signal density and size of band and
normalised to GAPDH signal that was derived from band analysis.
Clustered linear regression or Kruskal-Wallis rank sum statistical
methods were used depending on sample and group sizes to determine
significance of detected protein amounts between control and cancer
cell lines. Tests were performed using Stata/SE v11.2 (StataCorp
LP, Texas, U.S.A). Significant results were greater than 95%
confidence (P.ltoreq.0.05).
[0428] Membrane Stripping
[0429] Proceeding primary antibody detection, PVDF membranes were
stripped and probed for GAPDH, used as a loading control for total
protein amount. PVDF membranes were washed twice in a stripping
buffer containing 0.02 M Tris, 0.5 M NaCl, 2% SDS and 100 mM .beta.
mercaptoethanol in Milli-Q H2O. Membranes were agitated vigorously
with the buffer at 55.degree. C. for 30 minutes and further washed
with vigorous agitation three times with TBS-tween for 15, 20 and
30 minutes at room temperature. The PVDF membranes were blocked
with 5% skim-milk in TBS-tween for 1 hour at room temperature and
detection of GAPDH performed using 1:20,000 dilution of
HRP-conjugated a GAPDH antibody.
[0430] Quantitative Analysis of Gene Expression
[0431] RNA Extraction
[0432] Cells frozen with TRI reagent.RTM. were thawed and incubated
at room temperature for five minutes. 200 .mu.L chloroform was
added to each millilitre of TRI reagent.RTM. used in cell
harvesting. Samples were shaken vigorously for one minute, and
stood at room temperature for three minutes. Samples were then
centrifuged for 15 minutes at 16,000 g, at 4.degree. C.
[0433] RNA extraction was performed using an RNeasy.RTM. mini kit
(Qiagen Pty Ltd., Victoria, Australia) following manufacturer's
instructions. The upper aqueous phase was transferred to a fresh
tube and mixed in an equal volume with 70% ethanol, diluted in
RNase-free H.sub.2O. 700 .mu.L of sample was transferred to an
RNeasy.RTM. purification column (Qiagen) and centrifuged for 20
seconds at 16,000 g. After centrifugation, flow-through was
discarded and the remaining sample/ethanol mix loaded onto the
column and centrifuged at 16,000 g for 20 seconds. 350 .mu.L RW1
buffer was pipetted onto the spin-column membrane and column
centrifuged for 15 seconds at 16,000 g.
[0434] Samples were treated with DNAse I to remove genomic DNA
following manufacturer's instructions (Life Sciences Pty Ltd.,
Victoria, Australia). DNAse I stock solution was diluted 1:7 with
buffer RDD of which 80 .mu.L DNAse I solution was then directly
applied to the spin-column membrane and incubated for 15 minutes at
room temperature.
[0435] A further 350 .mu.L buffer RW1 was added onto the column and
centrifuged for 15 seconds at 16,000 g followed by two washes using
500 .mu.L RPE buffer. To remove any remaining buffer, the column
was centrifuged for 2 minutes at 16,000 g followed by a further one
minute at 16,000 g. Elution of each sample was achieved by
application of 50 .mu.L of RNase-free H2O directly to the
spin-column membrane, followed by centrifugation for one minute at
16,000 g. Eluted RNA samples were stored at -80.degree. C.
[0436] The concentration of extracted RNA was determined using a
NanoDrop.TM. 2000 spectrophotometer (Thermo Fisher Scientific
Australia Pty Ltd., Victoria, Australia) at 260 nm. 260/280 nm and
260/230 nm ratios were assessed to ensure samples were free from
protein and DNA contamination, where a ratio greater than 1.6
indicates a sample free of contamination.
[0437] cDNA Synthesis
[0438] Complementary DNA (cDNA) for use in qRT-PCR was prepared
using the High Capacity RNA-to-cDNA Kit (Life Technologies)
following manufacturer's instructions. For a 20 .mu.L reaction
containing 2 .mu.g of RNA, 10 .mu.L master-mix buffer and 1 .mu.L
of reverse transcriptase enzyme were added, with the volume made to
20 .mu.L with RNase-free H.sub.2O. Amplification was performed
using a DNA Engine.RTM. Peltier Thermal Cycler (Bio-Rad
Laboratories Pty, Ltd., New South Wales, Australia), with
incubation of reaction mixes for one hour at 37.degree. C. followed
by 95.degree. C. for five minutes and then chilled to 4.degree. C.
cDNA samples were stored at -20.degree. C. until required.
[0439] Primer Design
[0440] Primer sequences, detailed in Table 2.4 below, were obtained
from either published literature, Harvard PrimerBank, or designed
using NCBI Primer BLAST. Primers selected from the Harvard
PrimerBank were chosen based on criteria of the final amplicon size
being less than 150 base-pairs, that their melting temperature be
near 60.degree. C. and that, where possible, they extended across
an exon-exon junction. Primers designed using Primer-BLAST also had
these criteria. Oligonucleotides were purchased from GeneWorks Pty
Ltd, South Australia.
[0441] Quantitative RT-PCR
[0442] 10 .mu.L reaction mixtures were set up containing 5 .mu.L
Power SYBR.RTM. Green PCR Master Mix (Life Technologies), 0.5 .mu.L
each of 10 nM forward and reverse primer, 2 .mu.L cDNA sample
diluted to 1:25 with DEPC-treated H2O, and made up to 10 .mu.L with
2 .mu.L DEPC-treated H2O. Reactions were plated onto 96-well plates
(Life Technologies) in triplicate, with each plate containing
serial dilutions of a reference cDNA sample (from the LNCaP cell
line) for target gene and endogenous gene standard curves, to
control for reaction efficiency.
[0443] qPCR was performed using a 7500 Fast Real-Time PCR System
(Life Technologies) using ABI 7500 software v2.0.2.
[0444] Cycling conditions for all targets comprised; 50.degree. C.
for 2 minutes, 95.degree. C. for 10 minutes to activate enzyme and
denature cDNA, followed by 40 cycles of a 95.degree. C. 15-second
denaturation step and a 60.degree. C. 60-second extension and
signal acquisition step. Melt curves were produced following each
run to confirm absence of primer-dimers or product and other
contaminants.
[0445] Cycle threshold (CT) values were derived at a threshold
level of 0.35, in the exponential phase of amplification and above
baseline noise. The relative amount of gene expression from each
sample was derived by calculation of CT values vs. standard curves
produced from serially diluted standard material. Reaction
efficiencies, calculated from the slope of a linear trend line
plotted from diluted standards, were between 90 and 110%. Each
target was assessed in triplicate on a single plate, with
triplicate biological replicates run independently. Mean gene
expression was derived from the mRNA amount on each replicate
plate, with each single plate providing a mean CT and mRNA level
from replicate wells.
TABLE-US-00004 TABLE 5 Primer sequences used in qPCR GeneBank ID
Primer Gene Sequence (5'-3') (NM_) position Source GAPDH F
TGCACCACCAACTGCTTAGC 002046.3 556-575 {Sardiello, (SEQ ID NO. 14)
2009} R GGCATGGACTGTGGTCATGAG 642-622 (SEQ ID NO. 15) LAMP1 F
ACGTTACAGCGTCCAGCTCAT 005561.3 608-628 {Sardiello, (SEQ ID NO. 16)
2009} R TCTTTGGAGCTCGCATTGG 685-667 (SEQ ID NO. 17) SCARB2 F
AAAGCAGCCAAGAGGTTCC 005506.3 1488-1506 Primer- (LIMP-2) (SEQ ID NO.
18) BLAST R GTCTCCCGTTTCAACAAAGTC 1556-1536 (SEQ ID NO. 19) TFRC F
GGCTACTTGGGCTATTGTAAAGG 003234.2 250-272 PrimerBank (SEQ ID NO. 20)
R CAGTTTCTCCGACAACTTTCTCT 405-383 (SEQ ID NO. 21) TFR2 F
CGTGCGGAGACTCTGTGTT 003227.3 329-347 PrimerBank (SEQ ID NO. 22) R
ATCCAGGTCAGGCTCATAGTT (SEQ ID NO. 23) APPL1 F ACTTGGGTACATGCAAGCTCA
012096.2 747-767 PrimerBank (SEQ ID NO. 24) R TCCCTGCGAACATTCTGAACG
863-843 (SEQ ID NO. 25) APPL2 F AGCTGATCGCGCCTGGAACG 018171.3
1543-1562 Primer- (SEQ ID NO. 26) BLAST R GGGTTGGTACGCCTGCTCCCT
1636-1616 (SEQ ID NO. 27) EEA1 F CCCAACTTGCTACTGAAATTGC 003566.3
497-518 PrimerBank (SEQ ID NO. 28) R TGTCAGACGTGTCACTTTTTGT 591-570
(SEQ ID NO. 29) RAB5A F AGACCCAACGGGCCAAATAC 004162.4 22-41
PrimerBank (SEQ ID NO. 30) R GCCCCAATGGTACTCTCTTGAA 164-143 (SEQ ID
NO. 31) RABEP1 F ATTAAGGCGATTGCCACAGTC 004703.4 486-506 PrimerBank
(Rabaptin5) (SEQ ID NO. 32) R TGGTGCTCATAGTCACGAACT 616-596 (SEQ ID
NO. 33) RAB4A F GGGGCTCTCCTCGTCTATGAT 004578.2 470-490 PrimerBank
(SEQ ID NO. 34) R AGCGCATTGTAGGTTTCTCGG 519-499 (SEQ ID NO. 35)
RAB11 F CAACAAGAAGCATCCAGGTTGA 004663.4 146-167 PrimerBank (SEQ ID
NO. 36) R GCACCTACAGCTCCACGATAAT 260-239 (SEQ ID NO. 37) RAB25 F
TCGGCGAATCAGGTGTGGGGA 020387.2 279-299 Primer- (SEQ ID NO. 38)
BLAST R ATGGTGGTGCGGCTGTCGTG 360-341 (SEQ ID NO. 39) RAB7A F
GTGTTGCTGAAGGTTATCATCCT 004637.5 19-41 PrimerBank (SEQ ID NO. 40) R
GCTCCTATTGTGGCTTTGTACTG 128-106 (SEQ ID NO. 41) RAB9 F
CCTCATTGCGCCCAGACGGG 004251.4 108-127 Primer- (SEQ ID NO. 42) BLAST
R AGTGCAAGAGTGTCTCGCGGC 181-161 (SEQ ID NO. 43)
[0446] Confocal Microscopy
[0447] Immune Fluorescence Labelling
[0448] Non-specific antibody reactivity was reduced via incubation
of fixed cells in a 5% (w/v) bovine serum albumin (BSA) PBS
solution for two hours at room temperature with slow agitation.
Cells were then incubated with the appropriate concentration of
primary antibody (Table 2.3) diluted in 5% BSA for two hours at
room temperature followed by secondary antibody incubation for one
hour at room temperature. Unbound antibody was removed by three PBS
washes and coverslips mounted on 1 mm microscope slides with
ProLong.RTM. Gold Antifade Reagent containing DAPI nuclear stain
(Life Technologies).
[0449] Image Acquisition and Processing
[0450] Confocal microscopy was performed using a Zeiss LSM 710 META
NLO laser scanning microscope (University of South Australia,
Australia) and associated Carl Zeiss Zen 2009 software. Laser lines
of 370, 488, 543 and 633 nm were utilised for DAPI, Alexa
Fluor.RTM. 488, Cy3 and Alexa Fluor.RTM. 633 fluorescence. Laser
intensities were set between 5% and 25% depending on fluorescent
target. To eliminate signal cross over between fluorophores,
emission fluorescence was filtered with a 20 nm gap between each
channel. Each channel was captured by between frame sequential
scanning with a 4-line average with a pixel-dwell of 3.15 vs.
Pinhole diameters for each laser were set to 1 Airy unit. Cells
were observed under a 1.4-aperture 63x oil-immersion objective
lens, with 2.times. optical zoom applied. Images were captured at a
resolution of 1024 p.times.2 with a 16 bit greyscale depth. Based
on image capture resolution of 1024 pixels and lens and zoom
factor, each pixel is equivalent to 0.066 .mu.M in dimension. At
least four cells were randomly selected on each coverslip, and a
representative image processed for creation of figures. Images were
exported as grayscale 16 bit TIFF files, and further processing
performed using Adobe.RTM. Photoshop .RTM. CS5 (Adobe Systems Inc.,
San Jose, Calif., U.S.A).
[0451] Visualisation of Intracellular Trafficking and
Endocytosis
[0452] LysoTracker.RTM. positive vesicles were visualised by live
cell confocal microscopy to prevent leakage of LysoTracker.RTM.
that can occur in fixed cells. Cells were cultured in standard
conditions in DKSH hydrophobic chamber slides (DKSH Australia Pty
Ltd., Victoria, Australia). Subsequently, LysoTracker.RTM. was
diluted 1/200 in culture media and added to cells. At five minutes
of incubation at 37.degree. C., cell cultures were visualised.
[0453] Transferrin endocytosis assays was performed on cells
cultured in normal conditions detailed herein. At 48 hours, culture
media was replaced with fresh media containing a 1/1000 dilution of
transferrin-633 conjugate. Cells were incubated for a period of 5,
15 or 30 minutes, following which the media was aspirated, cells
washed briefly in dPBS, and fixed with 4% PFA. Fixed cells were
blocked for two hours with 5% BSA before a 90 minute incubation
with phalloidin-488 conjugate. Unbound antibody was removed by
three PBS washes and coverslips mounted on 1 mm microscope slides
with ProLong.RTM. Gold Antifade Reagent containing DAPI nuclear
stain (Life Technologies).
[0454] Co-Fluorescence of Transferrin and Endosome Markers.
[0455] Cells were incubated in normal conditions as detailed
herein. At 48 hours, culture media was replaced with fresh media
containing a 1/1000 dilution of transferrin-633 conjugate. Cells
were incubated with transferrin-633 media for a period of 20
minutes. Subsequently, media was aspirated, washed briefly with
dPBS, fixed and permeabilised. Labelling with primary and secondary
antibodies proceeded as detailed herein.
[0456] Public Prostate Cancer Cohorts
[0457] The Taylor cohort consisted of patients treated by radical
prostatectomy at Memorial Sloan-Kettering Cancer Center (MSKCC),
profiling 150 prostate cancer and 29 nonmalignant tissues was
performed using Affymetrix Human Exon 1.0 ST arrays. Data was
obtained from the cBio Cancer Genomics Pathway Portal. The Chandran
cohort is composed of 18 normal tissues, 65 primary, 25 metastatic
and 63 normal adjacent non-malignant tissues. Expression profiling
of these tissues was carried out using Affymetrix U95Av2 human gene
arrays. Data was retrieved from NCBI GEO (accession number
GSE6919). A cohort by Tomlins was retrieved from NCBI GEO
(accession number GSE6099) and is comprised of 32 primary, 20
metastatic, 13 prostatic intraepithelial neoplasia (PIN), and 27
normal tissue samples. Analysis of samples was previously performed
using a Chinnaiyan Human 20K Hs6 array. An additional cohort from
MSKCC, the Glinsky cohort was composed of 79 malignant prostate
tissues with clinical follow-up data to 106 months that was used to
assess for biochemical recurrence of prostate cancer (BCR). This
comprised 29 patients with BCR as defined by a PSA level
.gtoreq.0.2 ng/mL and 50 with no progression.
[0458] Statistical analysis between normal and prostate cancer
tissue was performed using a two-tailed unpaired t test with
Welch's correction. To evaluate differences in expression between
multiple groups in the Chandran and Tomlins cohort, Kruskal-Wallis
with Dunn's multiple comparison test was performed. Data from the
Glinsky cohort was analysed using a two-tailed unpaired t test with
Welch's correction to determine differences between Gleason grade 3
and 4 groups. K means clustering was implemented using Stata/SE
v11.2 (Stata Corp LP, Texas, U.S.A) to determine high and low gene
expression groups and evaluated using Kaplan-Meier survival curves
with Gehan-Breslow-Wilcoxon test to determine difference between
the curves. All statistical tests were performed using GraphPad
Prism 5.03 (GraphPad Software Inc., California, U.S.A).
EXAMPLE 6
Generation of Antibodies to APPL1 and RAB7
[0459] Epitopes with a length of 15 amino acids were selected from
the protein sequence of human APPL1 or RAB7 that were predicted to
have strong antigenic properties suitable for detection following
synthesis of antibodies to these specific sequences:
TABLE-US-00005 APPL1: Epitope #1 Position 145-159 (human): (SEQ ID
NO. 7) NRYSRLSKKRENDKV. Epitope #2 Position 265-279 (human): (SEQ
ID NO. 8) DPDPTKFPVNRNLTR. Epitope #3 Position 691-705 (human):
(SEQ ID NO. 9) SQSEESDLGEGGKKR. RAB7: Epitope #1 Position 103-117
(human): (SEQ ID NO. 10) RDEFLIQASPRDPEN. Epitope #2 Position
124-138 (human): (SEQ ID NO. 11) GNKIDLENRQVATKR. Epitope #3
Position 183-197 (human): (SEQ ID NO. 12) YNEFPEPIKLDKNDR.
[0460] Peptides were synthesized and used to immunise rabbits to
generate polyclonal antibodies.
[0461] FIG. 23A shows rabbit anti-Appl 1 polyclonal antibody
affinities to Appl 1 recombinant protein. FIG. 23B shows rabbit
anti-Rab7 polyclonal antibody affinities to Rab7 recombinant
protein. The affinity of the polyclonal antibodies produced to the
peptides detailed above were measured using surface-plasmon
resonance (BiaCore T100) to purified APPL1 or RAB7 protein. The
results are shown in FIG. 23. The Affinity of Appl 1 #2 (FIG. 23A)
and Rab7#3 (FIG. 23B) were highest, with greatest binding and
affinity post-wash, suggesting that these would bind more of their
respective protein in downstream assays and be suitable as a
capture antibody for DELFIA and sandwich ELISAs. Appl1#3 and Rab7#1
showed good affinities to the purified protein and would be
suitable for Europium-labelling to use in DELFIA assays for
detection of their respective target proteins.
[0462] FIG. 24A shows a standard curve for europium-labelled rabbit
anti-Rab7 polyclonal antibody (raised to epitope #1) for Rab7
protein in a direct DELFIA assay. 3.9 mL labelled antibody was
previously obtained at 0.13 mg/mL (0.51 mg total Ab), with an
approximate Europium-label yield of 1.6. FIG. 24B shows a standard
curve for europium-labelled rabbit anti-Appl 1 polyclonal antibody
(raised to epitope #3) for Appl 1 protein in a direct DELFIA assay.
4.8 mL labelled antibody was previously obtained at 0.11 mg/mL
(0.53 mg total Ab), with an approximate Europium-label yield of
3.8.
EXAMPLE 7
Diagnostic and Prognostic Value of Additional Markers
[0463] (i) Altered Endosome-Related Gene Expression During Cancer
Progression
[0464] The expression of endosome-related mRNA transcripts was
analysed from the Tomlins cohort enabling the analysis of prostatic
intraepithelial neoplasia (PIN) and metastatic cancer tissue in
addition to primary cancer and non malignant tissue.
[0465] The data is shown in FIG. 25. Vertical scatter plots of
lysosomal gene expression data from the cohort by Tomlins et al.
Analysis of prostate cancer tissue revealed altered gene expression
during prostate cancer progression. Expression profiling data
derived from the Chinnaiyan Human 20K Hs6 array of 27 non-malignant
tissues, 13 prostatic intraepithelial neoplasias, 32 primary
prostate cancer and 22 metastatic cancer tissue samples (Tomlins et
al. 2007) were quantitated to show relative expression of
endosome-related genes. Statistical significance is represented by
an asterisk (**P.ltoreq.0.01; ***P.ltoreq.0.001).
[0466] The gene expression of M6PR and IGF2R and STEAP4 in the
Tomlins cohort showed no significant change between normal, PIN,
primary or metastatic cancer tissue. MYO1B, ALIX and SDCBP
displayed differential expression in the prostate cancer tissue
when compared to metastatic tissue (P.ltoreq.0.01). Significant
reductions in expression were observed between PIN and metastatic
tissue for MYO1B (P.ltoreq.0.001) and PDCD6IP (P.ltoreq.0.01) but
not for SDCBP. Expression of MYO1B was significantly increased in
PIN tissue compared to non malignant prostate tissue
(P.ltoreq.0.01).
[0467] (ii) Prognostic Potential for Endosome-Related Genes to
Stratify Patients at Risk of Biochemical Recurrence
[0468] To assess the prognostic potential of expression of
endosome-related genes, we classified patients from the Glinsky
cohort into two groups using K means clustering based on gene
expression. The Glinsky cohort microarray provides data of initial
PSA levels detected upon biopsy, and information on relapse
subsequent to therapy.
[0469] FIG. 26 shows Kaplan-Meier analysis of
endosome/lysosome-related genes and patient stratification based on
biochemical recurrence (BCR). Gene expression of SORT1, MYO1B and
PDCD61P stratified patients into prognostic risk groups. Patients
from the Glinsky cohort (Glinsky et al. 2004) were stratified into
two groups by K means clustering based on amount (high--black line,
low --grey line) of M6PR, IGF2R, SORT1, STEAP4, MYO1B, PDCD61P,
SDC1 and SDCBP gene expression. Analysis was performed using
Gehan-Breslow-Wilcoxon test.). BCR: biochemical recurrence; HR:
hazard ratio; CI: confidence interval.
[0470] Clustering of high (black line) or low (grey line) SORT1 and
MYO1B gene expression revealed patients with high expression had
significantly increased risk of biochemical recurrence (BCR)
(P.ltoreq.0.01 and P.ltoreq.0.05, respectively). Patients
expressing lower amounts of PDCD6IP were at a significantly higher
risk of BCR (P.ltoreq.0.05). The grouping by high or low expression
for M6PR, STEAP4, SDC 1 and SDCBP genes did not stratify patients
significantly into prognostic groups, however there was a trend
observed for IGF2R (P=0.096). The associated Table shows
Kaplan-Meier analysis of endosome/lysosome-related genes and
patient stratification based on biochemical recurrence (BCR). Gene
expression of SORT1, MYO1B and PDCD61P stratified patients into
prognostic risk groups. Patients from the Glinsky cohort (Glinsky
et al. 2004) were stratified into two groups by K means clustering
based on amount (high--black line, low--grey line) of M6PR, IGF2R,
SORT1, STEAP4, MYO1B, PDCD61P, SDC 1 and SDCBP gene expression.
Analysis was performed using Gehan-Breslow-Wilcoxon test).
[0471] (iii) Prognostic Potential for Endosome-Related Genes in
Patients Expressing PSA.ltoreq.7.8 ng/.
[0472] FIG. 27 shows Kaplan-Meier survival and multivariate
analysis of lysosomal gene expression for cancer patients
expressing .ltoreq.7.8 ng/mL PSA. Kaplan-Meier analysis of
lysosome-related gene expression showed significant capability for
prognosis in patients expressing .ltoreq.7.8 ng/mL PSA based on
IGF2R, SORT1 or PDCD61P expression. From the good prognosis
subgroup of PSA.ltoreq.7.8 ng/mL, patients were further stratified
into two groups by K-means clustering based on gene expression of
M6PR, IGF2R, SORT1, STEAP4, MYO1B, PDCD61P, SDC1 and SDCBP (high
expression--black line, low expression--grey line). Statistical
analysis was performed using Gehan-Breslow-Wilcoxon test. BCR:
biochemical recurrence; HR: hazard ratio; CI: confidence
interval.
[0473] Stratification of patients with PSA.ltoreq.7.8 ng/mL into
high and low gene expression groups revealed that increased
expression of IGF2R (P.ltoreq.0.01), SORT1 (P.ltoreq.0.01) or ALIX
(P.ltoreq.0.05) resulted in a significantly increased risk of BCR
(FIG. 27). There was a trend for low expression of M6PR and SDC1,
and higher expression of MYO1B to stratify at-risk patients who
expressed low PSA. The expression of STEAP4 or SDCBP did not appear
to show any prognostic potential for those patients expressing low
levels of PSA.
[0474] (iv) Gene Expression of Syntaxin 7 (STX7) and Syntaxin 12
(STX12) is Reduced Significantly in Prostate Cancer Tissue.
[0475] The expression of Syntaxin 7 and Syntaxin 12 mRNA
transcripts was analysed from the Taylor cohort in prostate cancer
tissue compared to non malignant tissue.
[0476] FIG. 28 in Panel A shows box-and-whisker graphs showing
percentage change in gene expression of Syntaxin 7 and Syntaxin 12.
Panel B shows vertical scatter plots of gene expression data from
the cohort by Tomlins et al. Panel A shows expression profiling
data derived from Affymetrix Human Exon 1.0 ST arrays of 150
primary prostate cancers and 29 non-malignant tissues (Taylor 2010)
were quantitated to show percentage change of gene expression of
STX7 and STX12. Box-and-whisker graphs were plotted with Tukey
outliers (black points). Statistical significance is represented by
an asterisk (**P.ltoreq.0.01; ****P.ltoreq.0.0001). Panel B shows
analysis of prostate cancer tissue revealed altered STX7 gene
expression during prostate cancer progression. Expression profiling
data derived from the Chinnaiyan Human 20K Hs6 array of 27
non-malignant tissues, 13 prostatic intraepithelial neoplasias, 32
primary prostate cancer and 22 metastatic cancer tissue samples
(Tomlins et al. 2007) were quantitated to show relative expression
of endosome-related genes. Statistical significance is represented
by an asterisk (*P.ltoreq.0.05).
[0477] There was a statistically significant reduction in the
expression of STX7 (P.ltoreq.0.01) and STX12 (P.ltoreq.0.0001; FIG.
28A) in primary cancer tissue compared to non malignant tissue.
Analysis of STX7 gene expression during prostate cancer progression
from the Tomlins cohort showed a significant reduction in gene
expression of metastatic tissue compared to primary cancer tissue
(P.ltoreq.0.05; FIG. 28B).
[0478] (v) Prognostic Potential of Syntaxin 7 And 12 to Stratify
Patients at Risk of Biochemical Recurrence.
[0479] To assess the prognostic potential of expression of Syntaxin
7 and 12 gene expression, we classified patients from the Glinsky
cohort into two groups using K means clustering based on gene
expression.
[0480] FIG. 29 shows Kaplan-Meier survival analysis of Syntaxin 7
and Syntaxin 12 gene expression. (A) Kaplan-Meier analysis of
lysosome-related gene expression showed significant capability for
prognosis based on STX12 expression. (B) From the "good-prognosis"
subgroup of PSA.ltoreq.7.8 ng/mL, patients were further stratified
into two groups by K-means clustering based on gene expression of
STX7 and STX12 (high expression--black line, low expression--grey
line). Statistical analysis was performed using
Gehan-Breslow-Wilcoxon test. BCR: biochemical recurrence; HR:
hazard ratio; CI: confidence interval.
[0481] Clustering of high (black line) or low (grey line) STX12
gene expression revealed patients with low expression had
significantly increased risk of biochemical recurrence (BCR)
(P.ltoreq.0.01), however there was no prognostic capacity of STX7
(FIG. 29A). Stratification of patients with PSA.ltoreq.7.8 ng/mL
into high and low gene expression groups revealed that decreased
expression of STX12 resulted in a significantly increased risk
(P.ltoreq.0.05) of BCR (FIG. 29B), whilst there was no capacity of
STX7 to stratify patients.
[0482] (vi) Secreted, but not Intracellular, Amounts of
Endosome-Related Machinery are Significantly Altered in Prostate
Cancer Cells.
[0483] Western blotting was used to define the amount of ALIX,
Syndecan-1 and Sortilin-1 in cell lysates and culture media from
the non-malignant prostate cell lines (PNT1a and PNT2) and the
prostate cancer cell lines (22RV1, DU-145 and LNCaP). FIG. 30 shows
detection and quantification of intracellular and secreted
endosome-related proteins. (A) Representative images from Western
blot analysis of endosome-related proteins/GAPDH in cell extract
(A; 10 .mu.g whole cell lysate) and culture media (B; 3 mL culture
collected after 48 hours incubation with confluent cells and
corrected for cell number) from non-malignant control cell lines
PNT1a and PNT2, and cancer cell lines 22RV1, DU-145 and LNCaP,
examined in triplicate.
[0484] Western blotting was used to define the amount of ALIX,
Syndecan-1 and Sortilin-1 in cell lysates and culture media from
the non-malignant prostate cell lines (PNT1a and PNT2) and the
prostate cancer cell lines (22RV1, DU-145 and LNCaP). While the
intracellular amounts of these proteins was unaltered in cancer
cells compared to nonmalignant cells (FIG. 30A, C), there was a
significant increase in the amount of ALIX detected in the culture
media from prostate cancer compared to nonmalignant control cell
lines (P.ltoreq.0.01; FIG. 30C, D). Syndecan-1 and Sortilin-1 was
detected in the culture media from prostate cancer cell lines at a
significantly reduced level (P.ltoreq.0.01) compared to non
malignant cells.
[0485] (vii) FGF1 Gene Expression is Reduced in Prostate Cancer
Tissue.
[0486] FIG. 31 shows box-and-whisker graphs showing percentage
change in gene expression of FGF1 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al.
Box-and-whisker graphs were plotted with Tukey outliers (black
points). Statistical significance is represented by an asterisk
(**P.ltoreq.0.01).
[0487] Analysis of FGF1 gene expression in microarrays showed a
reduction that was statistically significant in the Grasso cohort
(P.ltoreq.0.01). There was evidence of a reduction in the Taylor
cohort however this was not statistically significant. Expression
of FGF1 from the Tomlins cohort showed variability between tissue
disease stages.
[0488] (viii) FGF2 Gene Expression is Reduced in Prostate Cancer
Tissue.
[0489] FIG. 32 shows box-and-whisker graphs showing percentage
change in gene expression of FGF2 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al.
Box-and-whisker graphs were plotted with Tukey outliers (black
points). Statistical significance is represented by an asterisk
(****P.ltoreq.0.0001).
[0490] Analysis of FGF2 gene expression in microarrays showed a
reduction that was statistically significant in both the Taylor and
Grasso cohort (P.ltoreq.0.0001). There was variability in tissue
types from the Tomlins cohort that may be attributed to the limited
data available for this gene in the cohort.
[0491] (ix) FGF3 Gene Expression is Reduced in Prostate Cancer
Tissue.
[0492] FIG. 33 shows box-and-whisker graphs showing percentage
change in gene expression of FGF3 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al.
Box-and-whisker graphs were plotted with Tukey outliers (black
points). Statistical significance is represented by an asterisk
(*P.ltoreq.0.05).
[0493] Analysis of FGF3 gene expression in microarrays showed a
significant increase in the Taylor cohort (P.ltoreq.0.05), however
there was not a significant increase in FGF3 expression in the
Grasso cohort.
[0494] (x) Kaplan-Meier Analysis of FGF2 Suggests a Trend for
Increased Risk of Relapse in Patients with Lower FGF2
Expression.
[0495] FIG. 34 shows Kaplan-Meier survival analysis of FGF1, FGF2
and FGF3 gene expression. The Kaplan-Meier analysis of FGF-related
gene expression showed some potential capability for prognosis
based on FGF2 expression.
[0496] Kaplan-Meier analysis of FGF-related gene expression showed
some capability for prognosis based on FGF2 expression however this
was not statistically significant. Patients were not stratified
into `high` or `low` FGF1 or FGF3 groups.
[0497] (xi) Kaplan-Meier Analysis of FGF-Related Genes Showed
Prognostic Value in Patients Expressing PSA.ltoreq.7.8 ng/ml
[0498] FIG. 35 shows Kaplan-Meier survival analysis of FGF1, FGF2
and FGF3 gene expression. From the "good-prognosis" subgroup of
PSA.ltoreq.7.8 ng/mL, patients were further stratified into two
groups by K-means clustering based on gene expression of FGF1, FGF2
and FGF3 (high expression--black line, low expression--grey line).
Statistical analysis was performed using Gehan-Breslow-Wilcoxon
test. BCR: biochemical recurrence; HR: hazard ratio; CI: confidence
interval.
[0499] Clustering of high (black line) or low (grey line) gene
expression of FGF genes revealed patients that expressed low PSA
protein that had low expression of FGF2 or FGF3 had significantly
increased risk of biochemical recurrence (P.ltoreq.0.05), however
there was no apparent prognostic capacity for FGF1.
[0500] (xii) FGFR1 Gene Expression is Reduced in Prostate Cancer
Tissue.
[0501] FIG. 36 shows a box-and-whisker graphs showing percentage
change in gene expression of FGFR1 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al. Statistical
significance is represented by an asterisk (****P.ltoreq.0.0001;
**P.ltoreq.0.01).
[0502] Analysis of FGFR1 gene expression in microarrays showed a
reduction in prostate cancer tissue that was statistically
significant in the Taylor and Grasso cohorts (P.ltoreq.0.0001).
Across tissue types, there was a reduction in PIN tissue compared
to nonmalignant tissue and a statistically significant increase
(P.ltoreq.0.01) in gene expression between PIN and metastatic
tissue.
[0503] (xiii) FGFR2 Gene Expression is Significantly Reduced in
Prostate Cancer Tissue.
[0504] FIG. 37 shows box-and-whisker graphs showing percentage
change in gene expression of FGFR2 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al. Statistical
significance is represented by an asterisk (****P.ltoreq.0.0001;
***P.ltoreq.0.001).
[0505] Analysis of FGFR2 gene expression in microarrays showed a
reduction in prostate cancer tissue that was statistically
significant in the Taylor and Grasso cohorts (P.ltoreq.0.0001).
Across tissue types, there was a reduction in PIN tissue and
metastatic tissue compared to nonmalignant tissue, and a
statistically significant reduction (P.ltoreq.0.001) in gene
expression in primary cancer tissue compared to nonmalignant tissue
in the Tomlins cohort.
[0506] (xiv) FGFR3 Gene Expression is Significantly Reduced in
Prostate Cancer Tissue.
[0507] FIG. 38 shows box-and-whisker graphs showing percentage
change in gene expression of FGFR3 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al.
[0508] Analysis of FGFR3 gene expression in microarrays showed no
significant change in FGFR3 expression in prostate cancer tissue
compared to nonmalignant prostate tissue. The expression of FGFR3
showed evidence of a reduction in each disease state compared to
normal tissue in the Tomlins cohort however this was not
statistically significant.
[0509] (xv) Kaplan-Meier Analysis of FGFR-Related Genes Showed
Prognostic Value in Patients Expressing.
[0510] FIG. 39 shows Kaplan-Meier survival analysis of FGFR1, FGFR2
and FGFR3 gene expression. The Kaplan-Meier analysis of
FGFR-related gene expression from the Glinksy cohort showed some
potential capability for prognosis based on FGFR2 or FGFR3
expression.
[0511] Clustering of high (black line) or low (grey line) gene
expression of FGFR genes revealed patients that had lower FGFR2 or
FGFR3 gene expression were at a significantly greater risk of
biochemical recurrence (P.ltoreq.0.05) compared to those patients
expressing a higher amount of FGFR2 or FGFR3. There was no
significant stratification of patients expressing FGFR1 for this
dataset.
[0512] (xvi) Kaplan-Meier Analysis of FGFR3 Showed Prognostic Value
in Patients Expressing PSA.ltoreq.7.8 ng/mL
[0513] FIG. 40 shows Kaplan-Meier survival analysis of FGFR1, FGFR2
and FGFR3 gene expression. From the "good-prognosis" subgroup of
PSA.ltoreq.7.8 ng/mL, patients were further stratified into two
groups by K-means clustering based on gene expression of FGFR1,
FGFR2 and FGFR3 (high expression--black line, low expression--grey
line). Statistical analysis was performed using
Gehan-Breslow-Wilcoxon test. BCR: biochemical recurrence; HR:
hazard ratio; CI: confidence interval.
[0514] Clustering of high (black line) or low (grey line) gene
expression of FGFR genes revealed patients that expressed low PSA
protein that had low expression of FGFR3 had a significantly
increased risk of biochemical recurrence (P.ltoreq.0.001). There
was a trend for lower expression of FGFR2 to suggest increased risk
of biochemical recurrence. No stratification of patients was
achieved clustering groups into high or low expression of
FGFR1.
[0515] (xvii) NOX2 Protein is Significantly Increased in Prostate
Cancer Cells.
[0516] FIG. 41 shows detection and quantification of NOX2 protein
(65 (56) & 30 kDa) from non-malignant control and prostate
cancer cell lines. Representative images from western blot analysis
of (A) 10 .mu.g whole cell lysate and (B) secreted NOX2 protein
from non-malignant control cell lines PNT1a and PNT2, and cancer
cell lines 22RV1, DU-145 and LNCaP, examined in triplicate. (C) The
amount of intracellular NOX2 was quantified by densitometry
relative to a GAPDH endogenous control. (D) Quantification of
secreted NOX2 protein was normalised to cell count at time of
protein collection. Data was analysed by clustered linear
regression with statistical significance (P.ltoreq.0.05)
represented by an asterisk.
[0517] Western blotting was used to define the amount of NOX2 in
cell lysates and culture media from the non malignant prostate cell
lines (PNT1a and PNT2) and the prostate cancer cell lines (22RV1,
DU-145 and LNCaP). The intracellular amount of NOX2 (56 kDa) was
significantly increased in cancer cells compared to non malignant
cells (FIG. 41A, C). An isoform of NOX2 (30 kDa)was expressed in
non malignant and prostate cancer cells however this was not
significantly altered. There was no secretion of a 56 kDa form of
NOX2 however there was evidence of secretion of a 30 kDa isoform
from prostate cancer and nonmalignant cells (FIG. 41B, D). NOX2
Antibody--Rabbit Polyclonal from Abeam, cat #ab31092 used at 1/2000
dilution (0.5 .mu.g/mL)
[0518] (xviii) Altered NOX2 Gene Expression in Prostate Cancer
Tissue.
[0519] FIG. 42 shows box-and-whisker graphs showing percentage
change in gene expression of NOX2 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al. Statistical
significance is represented by an asterisk (****P.ltoreq.0.0001,
**P.ltoreq.0.01).
[0520] The expression of NOX2 transcript was analysed from the
Tomlins, Taylor and Grasso cohorts. The gene expression of NOX2
showed a significant increase in cancer tissue of the Grasso cohort
(P.ltoreq.0.0001) compared to non malignant tissue. Analysis of
disease types of the Tomlins cohort revealed altered expression
across disease stages, and a significant increase (P.ltoreq.0.01)
of NOX2 expression in primary cancer tissue compared to
nonmalignant normal prostate tissue.
[0521] (xix) NOX4 is Unaltered in Prostate Cancer Cells Compared to
Non Malignant Cells.
[0522] FIG. 43 shows detection and quantification of NOX4 protein
from non-malignant control and prostate cancer cell lines. (A)
Representative image from western blot analysis of 10 .mu.g whole
cell lysate from non-malignant control cell lines PNT 1 a and PNT2,
and cancer cell lines 22RV1, DU-145 and LNCaP, examined in
triplicate. (B) The amount of intracellular NOX4 protein was
quantified by densitometry relative to a GAPDH endogenous control.
Data was analysed by clustered linear regression and no statistical
significance was found between non-malignant and cancer cell line
groups. Secreted protein from these cell lines was analysed (see
NOX2), however no signal was detected for NOX4.
[0523] Western blotting was used to define the amount of NOX4 in
cell lysates and culture media from the non-malignant prostate cell
lines (PNT1 a and PNT2) and the prostate cancer cell lines (22RV1,
DU-145 and LNCaP). The intracellular amount of NOX4 (63 kDa) was
unaltered in cancer cells compared to non malignant cells (FIG.
43A, B). No secreted NOX4 protein was detectable (data not
shown).
[0524] (xx) Altered NOX4 Gene Expression in Prostate Cancer
Tissue.
[0525] FIG. 44 shows box-and-whisker graphs showing percentage
change in gene expression of NOX4 (Taylor cohort) and Log 2
median-centred ratio (Grasso cohort) and vertical scatter plot of
gene expression data from the cohort by Tomlins et al. Statistical
significance is represented by an asterisk (****P.ltoreq.0.0001,
**P.ltoreq.0.01, *P.ltoreq.0.05).
[0526] The expression of NOX4 transcript was analysed from the
Tomlins, Taylor and Grasso cohorts. The gene expression of NOX4
showed a significant increase in cancer tissue of the Taylor and
Grasso cohorts (P.ltoreq.0.01 and P.ltoreq.0.0001, respectively)
compared to nonmalignant tissue. Analysis of disease types of the
Tomlins cohort showed a significant increase (P.ltoreq.0.05) of
NOX4 expression in metastatic tissue compared to PIN tissue. NOX4
antibody--Rabbit monoclonal from Abeam, cat #ab133303 used at
1/2000 dilution (0.5 .mu.g/mL)
[0527] (xxi) Altered APPL1, Rab7 and LIMPII expression in prostate
tissue (FIG. 49).
[0528] FIG. 45 shows APPL1, Rab7 and LIMPII expression in prostate
tissue. APPL1, Rab7 and LIMPII in matched human non-malignant (A,
B, C) and malignant (D, E, F) prostate tissue. The arrow in A shows
APPL1 staining the basement membrane in non-malignant prostate
tissue. In malignant prostate tissue: (D) dashed arrows show APPL1
staining both nuclei and nucleoli and solid arrow shows apparent
nuclear membrane staining; (E) arrow shows apparent Rab7 nuclear
membrane staining (F) arrows show enlarged LIMPII positive
vesicles. Scale bar=100 .mu.M in A, B & C and 200 .mu.M in D,
E, F. Antibodies used: rabbit anti APPL1 #3 (Detection Ab), rabbit
anti Rab7 #1 (Detection Ab), sheep anti LIMPII Ab.
[0529] Immunohistochemistry was used to investigate the
distribution of APPL1, the small GTPase Rab7 and LIMPII in prostate
cancer tissue. The endosomal marker APPL1 showed significant
basement membrane staining in non-malignant prostate tissue. In
malignant tissue, there appeared to be increased staining
associated with tumour mass and excitingly very specific nuclear
and nucleoli staining in tumour cells. This staining was not seen
in non-malignant tissue. For Rab7 specific cytoplasmic staining was
observed in the stroma and glandular structures in non-malignant
tissue. In contrast clear nuclear membrane staining was observed in
malignant tissue with what appeared to be a decrease in the amount
of cytoplasmic staining when compared to normal tissue. LIMPII
staining appeared similar in both malignant and non-malignant
tissue, however enlarged LIMPII positive vesicular structures were
observed in malignant tissue when compared to control tissue.
EXAMPLE 8
Diagnosis of Prostate Cancer and Treatment Options on the Basis of
the Diagnostic/Prognostic Potential of the Marker(s)
[0530] A diagnosis of the present of prostate cancer may be made
upon the basis of one or more of the level of mRNA expression of
one or more of the mRNAs for any of the markers as described
herein, the level of the marker proteins as described herein, the
secretion of the marker proteins as described herein, the presence
of the marker proteins in a biological fluid as described herein,
or on the basis of immunohistology on tissue or biopsy samples of
any of the marker proteins as described herein.
[0531] Examples of selected markers that may used include one or
more of the following proteins or their mRNAs: CATHEPSIN B,
CAPTHESIN D, .alpha.-GALACTOSIDASE, RAB7, LIMP-1, LIMP-2, TFR1,
TFR2, STAMP2, SORT1 (SORTILIN), APPL1, EEA-1, LAMP-1, RAB4, APPL2,
RAB5, RAB11, MPR, PAP, ACTIN, M6PR, IGFR2, MYO1B, PDCD6IP, SDCBP,
SDC1, STX7, STX12, FGF1, FGF2, FGF3, FGFR1, FGFR2, FGFR3, NOX2, and
NOX4.
[0532] For example, a cylindrical sample (biopsy) of prostate
tissue may be removed through the rectum, using hollow needles, and
a portion of the sample prepared for histology and
immunohistochemistry. If the prostate is surgically removed, a
pathologist may prepare a slice the prostate for analysis.
[0533] APPL1 may be selected as a suitable marker and analysis
conducted as described in Example 1 using immunohistochemistry to
determine the distribution of APPL1 using an APPL1 specific
antibody. APPL1 delineates the cancer margins and shows
dramatically increased staining within the tumour mass. Such
staining would be indicative of the presence of prostate
cancer.
[0534] On the basis of the detection using a selected marker as
described herein, a variety of treatment options are available,
dependent upon the diagnosis and/or prognosis and the extent of
recurrence of the cancer, in addition to, or in conjunction with,
the prognostic value of the selected markers described herein:
[0535] (i) Low Risk of Recurrence:
[0536] Treatment for patients with clinical stage T1-T2a, Gleason
score 2-6, PSA<10 ng/mL, with a life expectancy <10 y,
includes active surveillance
[0537] Treatment for patients with a life expectancy .gtoreq.10 y
includes active surveillance, or radical prostatectomy (RP) with or
without pelvic lymph node dissection (PLND) if predicted
probability of lymph node metastases .ltoreq.2%; RP being a
standard therapy for localized prostate cancer, involving the
removal of the prostate and seminal vesicles with or without pelvic
lymph nodes; this may be done using either open or laparoscopic
(robotic-assisted) technique; or
[0538] Radiation therapy for patients with localized disease, and
3-dimensional (3D) techniques such as 3D conformal radiation
treatment (3D-CRT), which offer benefits such as reduced toxicity
and the use of higher doses; second-generation techniques,
including intensity-modulated radiation therapy (IMRT), may also be
required, especially if doses .gtoreq.78Gy are administered.
[0539] Radiation therapy doses of 75.6-79Gy in conventional 36-41
fractions to the prostate with 3D-CRT/IMRT with daily image-guided
radiotherapy (IGRT) or brachytherapy (recommended dose rate: 145Gy
for iodine-125 and 125Gy for palladium-103).
[0540] Patients with low-risk cancer are typically not candidates
for pelvic lymph node irradiation or androgen deprivation therapy
(ADT).
[0541] (ii) Intermediate Risk of Recurrence:
[0542] Treatment for patients with clinical stage T2b-T2c, Gleason
score 7, PSA 10-20 ng/mL, who have a life expectancy <10 y,
include active surveillance; or
[0543] Radiation therapy (doses of 78-80+ Gy) with 3D-CRT/IMRT with
daily IGRT with or without short-term
neoadjuvant/concomitant/adjuvant ADT for 4-6 months with or without
brachytherapy (recommended dose rate: 145Gy for iodine-125 and
125Gy for palladium-103).
[0544] Treatment recommendations for patients with a life
expectancy .gtoreq.10 y includes RP with PLND if predicted
probability of lymph node metastasis .gtoreq.2% or radiation
therapy (doses of 78-80+ Gy) with 3D-CRT/IMRT with daily IGRT with
or without short-term neoadjuvant/concomitant/adjuvant ADT for 4-6
months with or without brachytherapy (recommended dose rate: 145Gy
for iodine-125 and 125Gy for palladium-103).
[0545] Intermediate-risk cancers consider combining brachytherapy
(recommended dose rate: 145Gy for iodine-125 and 125Gy for
palladium-103) with EBRT (40-50Gy) with or without 4-6 mo
neoadjuvant/concomitant/adjuvant ADT.
[0546] Administering ADT before, during, and after radiation
prolongs survival in patients.
[0547] (iii) High Risk of Recurrence:
[0548] Clinical Stage T3a, Gleason Score 8-10, PSA>20 ng/mL
[0549] Treatment options include radiation therapy (doses of 78-80+
Gy) with 3D-CRT/IMRT plus long-term
neoadjuvant/concomitant/adjuvant ADT for 2-3 y, or radiation
therapy (doses of 78-80+ Gy) with 3D-CRT/IMRT with daily IGRT plus
brachytherapy (recommended dose rate: 145Gy for iodine-125 and
125Gy for palladium-103) with or without short-term
neoadjuvant/concomitant/adjuvant ADT for 4-6 months, or RP plus
PLND for selected patients with no fixation.
[0550] High-risk cancers may be treated with combination EBRT
(40-50Gy) and brachytherapy with or without 4-6 months
neoadjuvant/concomitant/adjuvant ADT.
[0551] Reference to any prior art in this specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this prior art forms part of the common general knowledge in
any country.
[0552] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element or integer or group of elements or integers but not
the exclusion of any other element or integer or group of elements
or integers.
[0553] As used herein, the singular forms "a", "an" and "the"
include plural aspects unless the context already dictates
otherwise.
[0554] All methods described herein can be performed in any
suitable order unless indicated otherwise herein or clearly
contradicted by context. The use of any and all examples, or
exemplary language (e.g., "such as") provided herein, is intended
merely to better illuminate the example embodiments and does not
pose a limitation on the scope of the claimed invention unless
otherwise claimed. No language in the specification should be
construed as indicating any non-claimed element as essential.
[0555] The description provided herein is in relation to several
embodiments which may share common characteristics and features. It
is to be understood that one or more features of one embodiment may
be combinable with one or more features of the other embodiments.
In addition, a single feature or combination of features of the
embodiments may constitute additional embodiments.
[0556] The subject headings used herein are included only for the
ease of reference of the reader and should not be used to limit the
subject matter found throughout the disclosure or the claims. The
subject headings should not be used in construing the scope of the
claims or the claim limitations.
[0557] Future patent applications may be filed on the basis of the
present application, for example by claiming priority from the
present application, by claiming a divisional status and/or by
claiming a continuation status. It is to be understood that the
following claims are provided by way of example only, and are not
intended to limit the scope of what may be claimed in any such
future application. Nor should the claims be considered to limit
the understanding of (or exclude other understandings of) the
present disclosure. Features may be added to or omitted from the
example claims at a later date.
[0558] Although the present disclosure has been described with
reference to particular examples, it will be appreciated by those
skilled in the art that the disclosure may be embodied in many
other forms.
Sequence CWU 1
1
43134PRTHomo sapiens 1Ala Ser Asn Asp His Asp Ala Ala Ile Asn Arg
Tyr Ser Arg Leu Ser 1 5 10 15 Lys Lys Arg Glu Asn Asp Lys Val Lys
Tyr Glu Val Thr Glu Asp Val 20 25 30 Tyr Thr 237PRTHomo sapiens
2Asp Glu Val Ala Ser Asp Pro Leu Tyr Val Pro Asp Pro Asp Pro Thr 1
5 10 15 Lys Phe Pro Val Asn Arg Asn Leu Thr Arg Lys Ala Gly Tyr Leu
Asn 20 25 30 Ala Arg Asn Lys Thr 35 329PRTHomo sapiens 3Ser Glu Gly
Gln Phe Val Val Leu Ser Ser Ser Gln Ser Glu Glu Ser 1 5 10 15 Asp
Leu Gly Glu Gly Gly Lys Lys Arg Glu Ser Glu Ala 20 25 435PRTHomo
sapiens 4Pro Asn Thr Phe Lys Thr Leu Asp Ser Trp Arg Asp Glu Phe
Leu Ile 1 5 10 15 Gln Ala Ser Pro Arg Asp Pro Glu Asn Phe Pro Phe
Val Val Leu Gly 20 25 30 Asn Lys Ile 35 535PRTHomo sapiens 5Asp Pro
Glu Asn Phe Pro Phe Val Val Leu Gly Asn Lys Ile Asp Leu 1 5 10 15
Glu Asn Arg Gln Val Ala Thr Lys Arg Ala Gln Ala Trp Cys Tyr Ser 20
25 30 Lys Asn Asn 35 635PRTHomo sapiens 6Ala Leu Lys Gln Glu Thr
Glu Val Glu Leu Tyr Asn Glu Phe Pro Glu 1 5 10 15 Pro Ile Lys Leu
Asp Lys Asn Asp Arg Ala Lys Ala Ser Ala Glu Ser 20 25 30 Cys Ser
Cys 35 715PRTHomo sapiens 7Asn Arg Tyr Ser Arg Leu Ser Lys Lys Arg
Glu Asn Asp Lys Val 1 5 10 15 815PRTHomo sapiens 8Asp Pro Asp Pro
Thr Lys Phe Pro Val Asn Arg Asn Leu Thr Arg 1 5 10 15 914PRTHomo
sapiens 9Gln Ser Glu Glu Ser Asp Leu Gly Glu Gly Gly Lys Lys Arg 1
5 10 1015PRTHomo sapiens 10Arg Asp Glu Phe Leu Ile Gln Ala Ser Pro
Arg Asp Pro Glu Asn 1 5 10 15 1115PRTHomo sapiens 11Gly Asn Lys Ile
Asp Leu Glu Asn Arg Gln Val Ala Thr Lys Arg 1 5 10 15 1215PRTHomo
sapiens 12Tyr Asn Glu Phe Pro Glu Pro Ile Lys Leu Asp Lys Asn Asp
Arg 1 5 10 15 1319PRTHomo sapiens 13Cys Lys Lys Leu Asp Asp Phe Val
Glu Thr Gly Asp Ile Arg Thr Met 1 5 10 15 Val Phe Pro
1420DNAArtificial SequencePrimer Sequence 14tgcaccacca actgcttagc
201521DNAArtificial SequencePrimer Sequence 15ggcatggact gtggtcatga
g 211621DNAArtificial SequencePrimer Sequence 16acgttacagc
gtccagctca t 211719DNAArtificial SequencePrimer Sequence
17tctttggagc tcgcattgg 191819DNAArtificial SequencePrimer Sequence
18aaagcagcca agaggttcc 191921DNAArtificial SequencePrimer Sequence
19gtctcccgtt tcaacaaagt c 212023DNAArtificial SequencePrimer
Sequence 20ggctacttgg gctattgtaa agg 232123DNAArtificial
SequencePrimer Sequence 21cagtttctcc gacaactttc tct
232219DNAArtificial SequencePrimer Sequence 22cgtgcggaga ctctgtgtt
192321DNAArtificial SequencePrimer Sequence 23atccaggtca ggctcatagt
t 212421DNAArtificial SequencePrimer Sequence 24acttgggtac
atgcaagctc a 212521DNAArtificial SequencePrimer Sequence
25tccctgcgaa cattctgaac g 212620DNAArtificial SequencePrimer
Sequence 26agctgatcgc gcctggaacg 202721DNAArtificial SequencePrimer
Sequence 27gggttggtac gcctgctccc t 212822DNAArtificial
SequencePrimer Sequence 28cccaacttgc tactgaaatt gc
222922DNAArtificial SequencePrimer Sequence 29tgtcagacgt gtcacttttt
gt 223020DNAArtificial SequencePrimer Sequence 30agacccaacg
ggccaaatac 203122DNAArtificial SequencePrimer Sequence 31gccccaatgg
tactctcttg aa 223221DNAArtificial SequencePrimer Sequence
32attaaggcga ttgccacagt c 213321DNAArtificial SequencePrimer
Sequence 33tggtgctcat agtcacgaac t 213421DNAArtificial
SequencePrimer Sequence 34ggggctctcc tcgtctatga t
213521DNAArtificial SequencePrimer Sequence 35agcgcattgt aggtttctcg
g 213622DNAArtificial SequencePrimer Sequence 36caacaagaag
catccaggtt ga 223722DNAArtificial SequencePrimer Sequence
37gcacctacag ctccacgata at 223821DNAArtificial SequencePrimer
Sequence 38tcggcgaatc aggtgtgggg a 213920DNAArtificial
SequencePrimer Sequence 39atggtggtgc ggctgtcgtg 204023DNAArtificial
SequencePrimer Sequence 40gtgttgctga aggttatcat cct
234123DNAArtificial SequencePrimer Sequence 41gctcctattg tggctttgta
ctg 234220DNAArtificial SequencePrimer Sequence 42cctcattgcg
cccagacggg 204321DNAArtificial SequencePrimer Sequence 43agtgcaagag
tgtctcgcgg c 21
* * * * *