U.S. patent application number 14/929619 was filed with the patent office on 2016-03-03 for identification and treatment of cancer subsets.
This patent application is currently assigned to MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH. The applicant listed for this patent is Mayo Foundation for Medical Education and Research, The Translational Genomics Research Institute. Invention is credited to Mohammad R. Abbaszadegan, Michael Bittner, Aleksandar Sekulic, Jeffrey M. Trent.
Application Number | 20160058777 14/929619 |
Document ID | / |
Family ID | 45938736 |
Filed Date | 2016-03-03 |
United States Patent
Application |
20160058777 |
Kind Code |
A1 |
Bittner; Michael ; et
al. |
March 3, 2016 |
IDENTIFICATION AND TREATMENT OF CANCER SUBSETS
Abstract
Methods of predicting whether or not a tumor will be responsive
to IP6 treatment, methods of treating patients with cancer using
IP6, methods of predicting the progression of a disease, and kits
that facilitate these methods are disclosed.
Inventors: |
Bittner; Michael; (PHOENIX,
AZ) ; Trent; Jeffrey M.; (Paradise Valley, AZ)
; Sekulic; Aleksandar; (Scottsdale, AZ) ;
Abbaszadegan; Mohammad R.; (Tempe, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Translational Genomics Research Institute
Mayo Foundation for Medical Education and Research |
Phoenix
Rochester |
AZ
MN |
US
US |
|
|
Assignee: |
MAYO FOUNDATION FOR MEDICAL
EDUCATION AND RESEARCH
Rochester
MN
THE TRANSLATIONAL GENOMICS RESEARCH INSTITUTE
Phoenix
AZ
|
Family ID: |
45938736 |
Appl. No.: |
14/929619 |
Filed: |
November 2, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13274058 |
Oct 14, 2011 |
9180136 |
|
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14929619 |
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61393065 |
Oct 14, 2010 |
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Current U.S.
Class: |
514/102 |
Current CPC
Class: |
C12Q 2600/118 20130101;
C12Q 2600/106 20130101; A61P 35/00 20180101; G01N 33/57492
20130101; C12Q 1/6886 20130101; A61K 9/0014 20130101; A61K 31/6615
20130101; C12Q 2600/158 20130101 |
International
Class: |
A61K 31/6615 20060101
A61K031/6615; A61K 9/00 20060101 A61K009/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
CA27502 awarded by the National Institutes of Health. The
government has certain rights in this invention.
Claims
1. A method of treating a tumor, the method comprising the step of:
contacting the tumor with an effective therapeutic amount of a
pharmaceutical composition comprising inositol hexaphosphate (IP6)
or a pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein tumor is associated with skin
cancer.
3. The method of claim 1, wherein the tumor is squamous cell
carcinoma, squamous cell carcinoma in situ, or actinic
keratosis.
4. The method of claim 1, wherein the tumor is suspect to be
squamous cell carcinoma, squamous cell carcinoma in situ, or
actinic keratosis.
5. The method of claim 4, wherein the tumor has an INPP5
deletion.
6. The method of claim 1, wherein the pharmaceutical composition
comprises at least one excipient.
7. The method of claim 1, wherein the pharmaceutical composition is
formulated for topical administration.
8. The method of claim 7, wherein contacting the tumor comprises
topically administering the pharmaceutical composition to a subject
with the tumor.
9. A method of treating skin cancer, the method comprising the step
of: administering to a subject suspect of having skin cancer an
effective therapeutic amount of a pharmaceutical composition
comprising IP6 or a pharmaceutically acceptable salt thereof.
10. The method of claim 9, wherein the skin cancer is selected from
the group consisting of squamous cell carcinoma or squamous cell
carcinoma in situ.
11. The method of claim 10, wherein cells of the skin cancer have
an INPP5 deletion.
12. The method of claim 9, wherein the pharmaceutical composition
is formulated for topical administration.
13. The method of claim 12, wherein the pharmaceutical composition
is formulated as at least one of a liquid solution, cream, paste,
lotion, shake lotion, powder, emulsion, ointment, gel base, and a
transdermal patch.
14. The method of claim 9, wherein the pharmaceutical composition
comprises at least one excipient.
15. The method of claim 14, wherein the at least one excipient is
selected from the list consisting of petrolatum, lanolin,
polyethylene glycols, beeswax, mineral oil, diluents such as water
and alcohol, and emulsifiers and stabilizers, and one or more
thickening agents.
16. A method of treating a subject with skin cancer or a condition
that may progress to skin cancer, the method comprising the step
of: administering to the subject an effective therapeutic amount of
a pharmaceutical composition comprising IP6 or a pharmaceutically
acceptable salt thereof.
17. The method of claim 16, wherein the pharmaceutical composition
comprises at least one excipient selected from the list consisting
of petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil,
diluents such as water and alcohol, and emulsifiers and
stabilizers, and one or more thickening agents.
18. The method of claim 16, wherein the pharmaceutical composition
is formulated for topical administration.
19. The method of claim 18, wherein the pharmaceutical composition
is formulated as at least one of a liquid solution, cream, paste,
lotion, shake lotion, powder, emulsion, ointment, gel base, and a
transdermal patch.
20. The method of claim 16, wherein the skin cancer or condition
that may progress to skin cancer is selected from the group
consisting of squamous cell carcinoma, squamous cell carcinoma in
situ, or actinic keratosis, and further where in the skin cancer or
condition that may progress to skin cancer is associated with cells
that have an INPP5 deletion.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/274,058, filed Oct. 14, 2011 entitled
IDENTIFICATION AND TREATMENT OF CANCER SUBSETS, which claims
priority to U.S. provisional application entitled IDENTIFICATION
AND TREATMENT OF CANCER SUBSETS, with application No. 61/393,065,
filed on 14 Oct. 2010, the contents of these applications are
hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention is related to skin cancer, and more
specifically, methods and kits for skin squamous cell carcinoma
prognosis, diagnosis, theranosis and treatment using INPP5A
expression as an indication.
BACKGROUND OF THE INVENTION
[0004] Over 1,000,000 non-melanoma skin cancers are diagnosed
annually in the US, making these the most common type of cancer and
the fifth most costly cancer type in the Medicare population.
Approximately eighty percent of nonmelanoma skin cancers are
basal-cell carcinomas, and twenty percent are squamous-cell
carcinomas (SCC). Unlike almost all basal-cell carcinomas,
cutaneous squamous-cell carcinomas are associated with a
substantial risk of metastasis. The principal precursor of
cutaneous squamous-cell carcinoma is actinic keratosis (AK). AK has
been described as a type of carcinoma in situ or SCCIS, in which
carcinoma involves only the epidermis. Some of AK may evolve into
invasive squamous-cell carcinoma. Options for treating AK include
cryosurgery, electrodesiccation and curettage, topical
fluorouracil, dermabrasion, and laser resurfacing.
[0005] On histological examination actinic keratoses and invasive
squamous-cell carcinomas exhibit a spectrum of neoplastic changes.
From a therapeutic standpoint, it is impractical and unnecessary to
treat each individual keratotic lesion. Only patients with many
lesions are followed closely, and thus, many events or tumor
progression are undetected until at an advanced stage. Therefore,
accurate prognostic markers and targeted therapies as well as more
effective early chemopreventive strategies are necessary so that
evolving squamous-cell carcinomas can be detected and treated
expeditiously.
BRIEF SUMMARY OF THE INVENTION
[0006] Provided herein is a method of identifying a tumor
responding to INPP5A metabolites. The method generally comprises
obtaining a sample of the tumor; adding a first reagent to a
mixture comprising the sample, wherein the first reagent is capable
of detecting a marker comprising sequence selected from the group
consisting of SEQ ID NO. 1 and SEQ ID NO. 2; subjecting the mixture
to conditions that allow the detection of marker expression; and
classifying the tumor into a INPP5A metabolites-sensitive group
based on reduced expression of INPP5A in comparison to a control.
In the general method, when the marker comprises SEQ ID NO. 1, the
first reagent comprises a first and a second oligonucleotide
capable of binding SEQ ID NO:1; and the method for detection of the
marker is selected from the group consisting of PCR- and
hybridization-based methods. In one example, at least one of the
first and the second oligonucleotides comprises a label comprising
a fluorescent label selected from the group consisting of FAM,
dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED,
dROX, PET, BHQ+, Gold540, and LIZ. In this example, the general
method may further comprise isolating total RNA from the sample;
performing reverse transcription of total RNA isolated to obtain
cDNA; and subjecting cDNA to conditions that allow nucleic acid
amplification by the first and second oligonucleotides.
[0007] In another example, the first nucleic acid of the general
method is affixed to a substrate; and the method may comprise
performing array-based analysis of SEQ ID NO. 1 to the sample.
[0008] In the general method, when the marker comprises SEQ ID NO.
2, the first reagent comprises a first antibody capable of binding
a region of SEQ ID NO. 2. Further, the first antibody comprises a
first label selected from the group consisting of a fluorescent
compound, an enzyme, a radioisotope, and a ligand. In some
examples, the general method may further comprise adding a second
antibody to the mixture, wherein the second antibody is capable of
binding to the first antibody. Optionally, the second antibody may
comprise a second label selected from the group consisting of a
fluorescent compound, an enzyme, a radioisotope, and a ligand. The
general method of detecting the marker may also include determining
the deletion of INPP5A at chromosome 10q26.3.
[0009] The samples in the general method may be in a form selected
from the group consisting of an FFPE sample, a frozen sample, and a
fresh tumor biopsy. The control in the general method may be
selected from the group consisting of normal tissue, normal tissue
adjacent to the tumor, tissue from a less progressed tumor from the
same subject. The method may further comprise collecting a sample
from a subject selected from a group consisting of a human, a
companion animal, and a livestock animal. The tumor of the sample
in the general method may be squamous cell carcinoma (SCC),
squamous-cell carcinoma in situ (SCCIS) or actinic keratosis (AK).
Alternatively, the sample in the general method may be suspected to
be a skin squamous cell carcinoma (SCC), squamous-cell carcinoma in
situ (SCCIS) or actinic keratosis (AK).
[0010] Also provided herein is a method of assessing a risk of
progression of skin carcinoma in a subject, the method comprising:
obtaining a skin sample from the patient; adding a reagent capable
of binding a marker selected from the group consisting of SEQ ID
NO. 1 and SEQ ID NO. 2 to a mixture comprising the sample;
subjecting the mixture to conditions that allow detection of the
marker expression; and labeling the subject as having risk of skin
carcinoma progression based on the reduced expression of INPP5A in
comparison to a control. Since loss of INPP5A was shown to be
present in a significant percentage of all stages of SCC, from
precursor to metastatic disease, observing loss at AK, SCCIS, or
local SCC indicates a risk that the lesion where the loss is
observed in may continue to evolve through the stages to eventually
become metastatic SCC. In this method, if the sample comprises an
actinic keratosis, then the progression of the skin carcinoma may
comprise progression to squamous cell carcinoma or metastatic
squamous cell carcinoma. If the sample in the method comprises a
squamous cell carcinoma in situ, then the progression of the skin
carcinoma may comprise progression to squamous cell carcinoma or
metastatic squamous cell carcinoma. Alternatively, if the sample
comprises a squamous cell carcinoma, then the progression of the
disease comprises progression to metastatic squamous cell
carcinoma. The detection of the marker expression in the general
method may be selected from the group consisting of PCR-based,
hybridization-based, array-based methods and any combinations
thereof.
[0011] Further provided herein is a method of treating a subject
having skin squamous cell carcinoma at various stages, and the
method comprises: obtaining a sample from a subject; determining
tumor cell responsiveness to INPP5A metabolites of the sample;
administering a pharmaceutical composition comprising one or more
INPP5A metabolites or a pharmaceutically acceptable salt thereof to
the subject; wherein the subject has tumor cell determined to be
responsive to INPP5A metabolites. In one example, said method may
further comprise: adding to a mixture comprising the sample, a
reagent capable of binding a marker selected from the group
consisting of SEQ ID NO. 1 and SEQ ID NO. 2; subjecting the mixture
to conditions that allow detection of the marker expression;
wherein reduced expression of the marker in comparison to a control
indicates the cell responsiveness to INPP5A metabolites. The
control of the method is selected from, depending on the stage of
the sample tissue, the group consisting of normal skin tissue,
normal skin tissue adjacent to the tumor of the same subject, skin
tissue from a less progressed tumor including actinic keratosis and
squamous cell carcinoma in situ of the same subject. In one
example, the pharmaceutical composition of the method comprises IP6
or a pharmaceutically acceptable salt thereof, wherein, the
pharmaceutical composition of the method causes cessation or
reduction of cell proliferation in actinic keratosis, squamous cell
carcinoma in situ, squamous cell carcinoma or metastasized squamous
cell carcinoma, and restores cell differentiation resulting in cell
death.
[0012] In said method, determining the tumor cell responsiveness to
INPP5A metabolites of the sample is a step of detecting the
deletion of INPP5A at chromosome 10q26.3. In some examples, the
sample subjected to the method comprises a skin sample. Further,
the sample may comprise a cell selected from the group consisting
of actinic keratosis cell; squamous cell carcinoma in situ cell,
squamous cell carcinoma cell, and metastatic squamous cell
carcinoma cell.
[0013] Further provided herein is a kit for assessing skin tumor
responsiveness to INPP5A metabolites. The kit generally comprises:
a first reagent capable of specific detection of a marker selected
from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2; and an
indication signifying a result associated with INPP5A
metabolite-sensitivity of the tumor. In one example, the first
reagent comprising a first and a second oligonucleotide capable of
binding SEQ ID NO.1, and at least one of the first and the second
oligonucleotides comprises a label comprising a fluorescence moiety
or compound selected from the group consisting of FAM, dR110,
5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX,
PET, BHQ+, Gold540, and LIZ 31. In another example, the first
reagent of the kit comprises a first antibody capable of binding a
region in SEQ ID NO. 2, and the first antibody comprises a first
label selected from the group consisting of a fluorescent compound,
an enzyme, a radioisotope, and a ligand. The kit may further
comprise a second antibody capable of binding to the first antibody
comprising a second label selected from the group consisting of a
fluorescent compound, an enzyme, a radioisotope, and a ligand. The
result contained in the kit is based on the expression level of
INPP5A in comparison to a control selected from the group
consisting of normal skin tissue, normal skin tissue adjacent to
the tumor of the same subject, skin tissue from a less progressed
tumor including actinic keratosis and squamous cell carcinoma in
situ of the same subject. In other examples, the kit may further
comprise a pharmaceutical composition comprising one or more INPP5A
metabolites or a pharmaceutically acceptable salt thereof. In one
example, the pharmaceutical composition comprises IP6 or a
pharmaceutically acceptable salt thereof.
REFERENCE TO COLOR FIGURES
[0014] The application file contains at least one figure executed
in color. Copies of this patent application publication with color
figures will be provided by the Office upon request and payment of
the necessary fee.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 depicts the distribution of gene and copy number
aberrations in 40 tissues examined in this study;
[0016] FIG. 2 depicts the identification of selected INPP5A
deletions by aCGH;
[0017] FIG. 3 depicts loss of INPP5A in a squamous cell carcinoma
sample from a patient using blue fluorescence of DAPI bound to DNA
(panel labeled as "SCC") to mark nuclei and chromosomes, green
fluorescent probes to mark chromosome 10 centromeres, and red
fluorescent probes to mark the INPP5A gene near the q-terminus of
chromosome 10. A metaphase spread from normal cells is shown in
panel labeled as "INPP5A Chr10". The presence of two copies of the
chromosome 10 centromere and two copies of the INPP5A gene in
normal skin cells from a patient is shown in panel labeled as
"Normal". The presence of one copy of the chromosome 10 centromere
and one copy of the INPP5A gene in SCC skin cells from the same
patient is shown in panel labeled as "SCC";
[0018] FIG. 4 depicts the detection of INPP5A protein loss in
primary SCC tissues relative to the matched, normal epidermis.
Immunohistochemistry for INPP5A was done on FFPE tissues, and
relative intensity of INPP5A staining (dark brown) was compared
between the SCC tissue and the adjacent, histologically normal
epidermis. A, bottom, primary skin SCC tissue with low level of
INPP5A staining; top, matched, adjacent normal epidermis from the
same patient with dark staining in the dermal layer. B, a
representative case is shown to further illustrate a relative
difference in INPP5A staining between the primary SCC lesion and
the adjacent normal epidermis. Scale bar, 50 .mu.m;
[0019] FIG. 5 depicts the detection of INPP5A loss in AK.
Immunohistochemistry for INPP5A was done on FFPE tissues. Two
representative lesions are shown in the left and right panel, each
containing an area of SCCIS (evident by full epidermal thickness
neoplasia; right half of each image), arising in association with
an AK (partial epidermal thickness neoplastic change, consistent
with AK; left half of each image). Arrows highlight populations of
cells showing low level of INPP5A staining within the AK lesions.
Scale bars, 100 .mu.m;
[0020] FIG. 6 depicts increased apoptosis in a squamous cell
carcinoma cell line that carries a gene expression cassette that
expresses INPP5A (B and D) compared to the same cell line carrying
a gene expression cassette that expresses an unrelated protein,
green fluorescent protein (A and C). DNA blue fluorescence in (C)
and (D) was produced by DAPI probe, and apoptotic nuclei red
fluorescence was produced through TUNEL assay;
[0021] FIG. 7 depicts the biochemical pathway by which INPP5A
directs the production of Ins(1,3,4)P3, which is subsequently
converted to IP6;
[0022] FIG. 8 depicts increased apoptosis in a squamous cell
carcinoma cell line treated with IP6 (right panel B) relative to
the same line without treatment (left panel A), and the DNA blue
fluorescence was produced by DAPI probe. Apoptotic nuclei red
fluorescence was produced through TUNEL assay;
[0023] FIG. 9 depicts the fold change of INPP5A mRNA determined by
q-RTPCR as a function of % confluence and differentiation of normal
primary human keratinocytes;
[0024] FIG. 10 depicts the change in INPP5A protein expression
determined by Western blot as a function of the % confluence and
differentiation of normal human keratinocytes;
[0025] FIG. 11 depicts the data from FIG. 12 as a graph;
[0026] FIG. 12 depicts the effect of IP6 treatment on confluent
normal human keratinocytes. Panel A shows confluent untreated
normal human keratinocytes, while panel B shows confluent normal
human keratinocytes treated with IP6. The DNA blue fluorescence was
produced by DAPI probe. Apoptotic nuclei red fluorescence was
produced through TUNEL assay;
[0027] FIG. 13 depicts the effect of IP6 treatment on 70% confluent
keratinocytes. Panel A shows untreated 70% confluent keratinocytes
while panel B shows 70% confluent keratinocytes treated with IP6.
The DNA blue fluorescence was produced by DAPI probe. Apoptotic
nuclei red fluorescence was produced through TUNEL assay;
[0028] FIG. 14 depicts the plasmid composition of pTUNE vector used
to deliver the INPP5A gene under controlled expression. The coding
sequence for INPP5A replaces the GFP, shRNA Target region in the
construct used to deliver the INPP5A. The schematic illustrations
of the expression shut-down (A) and expression induction (B) are
provided;
[0029] FIG. 15 depicts that before and after the introduction of
controlled expression of INPP5A to cell line Cal-27, the production
of a small amount of the protein was sufficient to reduce migration
of cells in a scratch assay by 40%. A comparison of a 24 hour assay
in the absence (top panels) or presence (bottom panels) of INPP5A
in this cell line is shown;
[0030] FIG. 16 depicts SCC 15 cells after the INPP5A pTUNE
construct was delivered by transfection. The nuclei were stained
with Vybrant Violet, producing fluorescence in live cells. Normal
nuclei show as large, light gray bodies and apoptosing nuclei
appear as small, dark, pycnotic nuclei;
[0031] FIG. 17 depicts immunohistochemical staining for the
presence of INPP5A which shows that it normally appears at a higher
and higher level as keratinocytes go through the various stages of
differentiation in the transition from actively growing precursor
cells to the dead, cornified cells of the outer dermis; and
[0032] FIG. 18 depicts IP6 activity against a number of squamous
cell cancer lines SCC-4 SCC-15 SCC-9, and both a colorectal
adenocarcinoma (HT-29) and a transformed normal human embryonic
kidney cell line (HEK-293). The cells showed a dose dependent
reduction in number suggesting that effects due to INPP5A's loss
can to some extent be corrected by supplying IP6 whose synthesis
normally requires the activity of INPP5A.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Cutaneous squamous cell carcinoma (SCC) occurs commonly and
can metastasize. Identification of specific molecular aberrations
and mechanisms underlying the development and progression of
cutaneous SCC can lead to better prognostic and therapeutic
approaches and more effective chemoprevention strategies.
[0034] A genome-wide survey of gene copy number changes in skin
tissues identified frequent deletions of INPP5A gene in human SCC
tumors. As provided herein, a decrease in INPP5A protein levels is
observed in most cutaneous SCCs. This event occurs early in the
development of SCC as it can be detected even at the stage of
actinic keratosis, a common precursor to SCC. Progressive reduction
of INPP5A levels is seen in a subset of SCC patients as the tumor
progresses from primary to metastatic stage. Since loss of INPP5A
was shown to be present in a significant percentage of all stages
of SCC, from precursor to metastatic disease, observing loss at AK,
SCCIS, or local SCC indicates a risk that the lesion where the loss
is observed in may continue to evolve through the stages to
eventually become metastatic SCC.
(I) INPP5A as a Gene Marker
[0035] Provided herein is a novel gene, INPP5A, the reduced level
of which is associated with development and progression of
cutaneous SCC. INPP5A (Type I inositol-1,4,5-trisphosphate
5-phosphatase, UniProtKB/Swiss-Prot Accession Number: Q14642)
belongs to a large family of inositol polyphosphate 5-phosphatases.
This 40 kDa membrane-associated type I inositol phosphatase has
preferential substrate affinity for inositol 1,4,5-trisphosphate
(Ins(1,4,5)P3) and inositol 1,3,4,5 tetrakisphosphate
(Ins(1,3,4,5)P4), working mostly as a modulator or the metabolism
of inositol phosphates, which are widely used by cells to modulate
and regulate a variety of processes. As used in various contexts
herein, INPP5A may refer to the nucleic acid form of the gene, or
INPP5A may refer to the protein form of the gene.
[0036] Further provided herein is the discovery of supplementing
inositol phosphate metabolites downstream of the enzyme INPP5A, for
example, IP6, for reversal of cancerous cell proliferation to
cancer cell death in skin SCC. Therefore, one aspect of the
invention provides a method of identifying a tumor responsive to
INPP5A metabolites. In particular, based on the finding that
reduced INPP5A expression, including the mRNA and the protein
level, in a subset of skin SCC is associated with the disease
progression as early as AK stage, it has been found that by
supplying at least one of the downstream metabolites of INPP5A to
this subset of cancerous cells, these cells are responsive to the
administration of the metabolite, cancer cell proliferation can be
ceased, and cell differentiation can be restored, leading to cancer
cell death. In one embodiment, the identified subset of tumor cells
with INPP5A deletion is given a supplement of at least one
metabolite selected from the group including Ins(1,3,4)P.sub.3,
Ins(1,3,4,6)P.sub.4, Ins(1,3,4,5,6)P.sub.5, and
Ins(1,2,3,4,5,6)P.sub.6 (IP6), such that the supplement of INPP5A
metabolite leads to cancer cell death. In one preferred embodiment,
the identified subset of tumor cell with INPP5A deletion is given a
supplement of IP6.
[0037] Generally, the present invention provides a marker
associated with tumor cell's responsiveness to INPP5A metabolites.
A marker may be any molecular structure produced by a cell,
expressed inside the cell, accessible on the cell surface, or
secreted by the cell. A marker may be any protein, carbohydrate,
fatty acid, nucleic acid, catalytic site, or any combination of
these such as an enzyme, glycoprotein, cell membrane, virus, a
particular cell, or other uni- or multimolecular structure. A
marker may be represented by a sequence of a nucleic acid or any
other molecules derived from the nucleic acid. Examples of such
nucleic acids include miRNA, tRNA, siRNA, mRNA, cDNA, genomic DNA
sequences, or complementary sequences thereof. Alternatively, a
marker may be represented by a protein sequence. The concept of a
marker is not limited to the exact nucleic acid sequence or protein
sequence or products thereof, rather it encompasses all molecules
that may be detected by a method of assessing the expression of the
marker. Without being limited by the theory, the reduced INPP5A
expression, including the mRNA and the protein level, in a subset
of skin SCC may be caused by deletion of INPP5A gene, different
alleles of INPP5A gene, a mutation in the INPP5A gene, gene
expression regulation abnormally, increased INPP5A mRNA or protein
degradation, all of which may result in lower expression of INPP5A
individually or in any combination.
[0038] Therefore, examples of molecules encompassed by a marker
represented by a particular sequence further include alleles of the
gene used as a marker. An allele includes any form of a particular
nucleic acid that may be recognized as a form of the particular
nucleic acid on account of its location, sequence, or any other
characteristic that may identify it as being a form of the
particular gene. Alleles include but need not be limited to forms
of a gene that include point mutations, silent mutations,
deletions, frameshift mutations, single nucleotide polymorphisms
(SNPs), inversions, translocations, heterochromatic insertions, and
differentially methylated sequences relative to a reference gene,
whether alone or in combination. An allele of a gene may or may not
produce a functional protein; may produce a protein with altered
function, localization, stability, dimerization, or protein-protein
interaction; may have overexpression, underexpression or no
expression; may have altered temporal or spatial expression
specificity. An allele may also be called a mutation or a mutant.
An allele may be compared to another allele that may be termed a
wild type form of an allele. In some cases, the wild type allele is
more common than the mutant.
[0039] A mutation in INPP5A gene that causes decreased activity of
INPP5A in a test subject or a biological sample may also be called
a loss-of-function mutation. A mutation may be any detectable
change in genetic material such as DNA, or a corresponding change
in the RNA or protein product of that genetic material. A mutant
may be any biological material in which one or more mutations are
detected when compared to a control material. Examples of mutations
include gene mutations, in which the DNA sequence of a gene or any
controlling elements surrounding the gene is altered. Controlling
elements include promoter, enhancer, suppressor or silencing
elements capable of controlling a given gene. Other examples of
mutations include alterations in the products of DNA expression
such as RNA or protein that result from corresponding mutations in
the DNA. Mutants may also be interchangeably called variants. The
concept of a mutant includes any change in DNA sequence specific to
the tumor cell (not present in DNA prepared from normal,
non-neoplastic tissues).
[0040] Loss-of-function mutations display decreased total INPP5A
activity in the test subject or biological sample in comparison
with a control, e.g., a healthy subject or a sample without SCC or
SCC precursors (standard sample). Therefore, the activity of INPP5A
in a subject or a sample carrying loss-of-function mutation in
INPP5A is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%
relative to that activity in a healthy subject or a standard
sample. The decreased activity of INPP5A in a subject or a sample
carrying loss-of-function mutation may result from, for example,
decreased basal INPP5A activity, lessened activation, faster
degradation, or under-expression, e.g., due to decreased mRNA
expression level, reduced substrate binding, promiscuous or
inappropriate substrate binding, impaired recycling resulting in
abnormal signaling, increased degradation, or enzyme
activation.
[0041] A reduced expression level of INPP5A mRNA may result from,
for example, a mutation in a non-coding region of a INPP5A gene or
a mutation in a coding or non-coding gene involved in INPP5A
transcription or translation. The expression level of INPP5A can be
determined, for example, by comparing INPP5A mRNA or the level of
INPP5A protein in a test subject as compared to a control, for
example, by comparing the SCC tumor to normal skin tissue (e.g., a
normal adjacent skin tissue sample).
[0042] The INPP5A marker provided herein also included conserved
variants encompassing any mutation or other variant in which a
given amino acid residue in a protein or enzyme has been changed
without altering the overall conformation and function of the
polypeptide, including, but not limited to, replacement of an amino
acid with one having similar properties (such as, for example,
polarity, hydrogen bonding potential, acidic, basic, hydrophobic,
aromatic, and the like). Amino acids with similar properties are
well known in the art. For example, arginine, histidine and lysine
are hydrophilic-basic amino acids and may be interchangeable.
Similarly, isoleucine, a hydrophobic amino acid, may be replaced
with leucine, methionine or valine. Depending on the location of
the mutation in the overall context of the protein, some
substitutions may have little or no effect on the apparent
molecular weight or isoelectric point of the protein or
polypeptide. However some conserved variants have been found to
alter protein conformation and function, including several variants
discovered and disclosed herein.
[0043] Amino acids other than those indicated as conserved may
differ in a protein or enzyme so that the percent protein or amino
acid sequence similarity between any two proteins of similar
function may vary and may be, for example, from 70% to 99% as
determined according to an alignment scheme such as by the Cluster
Method, wherein similarity is based on the MEGALIGN algorithm. The
concept of a variant further encompasses a polypeptide or enzyme
which has at least 60%, 75%, 85%, 90%, or 95%, amino acid identity
as determined by algorithms, such as, BLAST or FASTA, and which has
the same or substantially similar properties and/or activities as
the native or parent protein or enzyme to which it is compared.
[0044] One example of such a variant is a loss-of-function variant.
Loss-of-function variants of polypeptides encompass any variant in
which a change in one or more amino acid residues in a protein or
enzyme improves the activity of the polypeptide. Examples of
activities of a polypeptide that may be impaired by a change
resulting in a gain of function variant include but are not limited
to enzymatic activity, binding affinity, phosphorylation or
dephosphorylation efficiency, activation or deactivation by a
regulatory protein, or any other activity or property of a protein
that may be quantitatively measured by some method now known or yet
to be disclosed.
[0045] Proteins that possess a common evolutionary origin may be
homologous or similar to one another. Examples of homologous or
similar proteins include proteins from superfamilies (e.g., the
immunoglobulin superfamily) and homologous proteins from different
species. Such proteins and their encoding genes have sequence
homology with one another. The homology may be expressed in terms
of percent similarity or the presence of specific residues or
motifs at conserved positions.
[0046] The presence or absence of an allele of the gene marker may
be detected through the use of any process known in the art,
including for example, using primers and probes designed
accordingly for PCR, hybridization, and for some cases, sequencing
analyses.
(II) Methods of Detecting Differential Expression of INPP5A in a
Sample
[0047] A. Method for Identifying Tumor Cells Responsive to INPP5A
Metabolites
[0048] In one embodiment of the invention, the identification of
the INPP5A metabolite responsive tumor cells generally includes
detecting tumor cells with reduced INPP5A mRNA level. In another
embodiment of the present invention, the identification of the
INPP5A metabolite responsive tumor cells generally includes
detecting tumor cells with reduced INPP5A protein level. In one
preferred embodiment, the INPP5A metabolite is IP6.
[0049] Expression of a marker may be assessed by any number of
methods used to detect material derived from a nucleic acid
template. Differential expression of a marker may be assessed or
quantified by a detector, an instrument containing a detector, or
by an aided or unaided human eye. Exemplary methods for nucleic
acid detection and/or quantification include, but are not limited
to, microarray analysis, RNA in situ hybridization, RNAse
protection assay, Northern blot, reverse transcriptase PCR,
quantitative PCR, quantitative reverse transcriptase PCR,
quantitative real-time reverse transcriptase PCR, reverse
transcriptase treatment followed by direct sequencing, or any other
method of detecting a specific nucleic acid now known or yet to be
disclosed. Exemplary methods for assessing protein expression
include, for example, flow cytometry, immunohistochemistry, ELISA,
Western blot, and immunoaffinity chromatograpy, HPLC, mass
spectrometry, protein microarray analysis, PAGE analysis,
isoelectric focusing, 2-D gel electrophoresis, or any enzymatic
assay. Methods of detecting expression may include, for example,
methods of purifying nucleic acid, protein, or some other material
depending on a nucleic acid-based or protein-based approach. Any
method of nucleic acid purification may be used, depending on the
type of marker (i.e., nucleic acid or protein), examples include
phenol alcohol extraction, ethanol extraction, guanidium
isothionate extraction, gel purification, size exclusion
chromatography, cesium chloride preparations, and silica resin
preparation. Any method of protein purification may be used,
non-limiting examples of which include size exclusion
chromatography, hydrophobic interaction chromatography, ion
exchange chromatography, affinity chromatography (including
affinity chromatography of tagged proteins), metal binding,
immunoaffinity chromatography, and HPLC.
[0050] (a) PCR Based Methods
[0051] Nucleic acids may be selectively and specifically amplified
from a template nucleic acid contained in a sample. In some nucleic
acid amplification methods, the copies are generated exponentially.
Examples of nucleic acid amplification methods known in the art
include: polymerase chain reaction (PCR), ligase chain reaction
(LCR), self-sustained sequence replication (3SR), nucleic acid
sequence based amplification (NASBA), strand displacement
amplification (SDA), amplification with Q.beta. replicase, whole
genome amplification with enzymes such as .phi.29, whole genome
PCR, in vitro transcription with T7 RNA polymerase or any other RNA
polymerase, or any other method by which copies of a desired
sequence are generated.
[0052] With PCR, it is possible to amplify a single copy of a
specific target sequence in genomic DNA or total RNA to a level
detectable by several different methodologies, for example,
hybridization with a labeled probe, incorporation of biotinylated
primers followed by avidin-enzyme conjugate detection, and,
incorporation of .sup.32P-labeled deoxynucleotide triphosphates,
such as dCTP or dATP, into the amplified segment. Generally,
nucleic acid based probes and primers are complementary to a
sequence within the target DNA sequence region. In addition to
genomic DNA, any oligonucleotide or polynucleotide sequence can be
amplified with an appropriate set of primer molecules. In
particular, the amplified segments created by the PCR process
itself are, themselves, efficient templates for subsequent PCR
amplifications.
[0053] PCR generally involves the mixing of a nucleic acid sample,
two or more primers that are designed to recognize the template
DNA, a DNA polymerase, which may be a thermostable DNA polymerase
such as Taq or Pfu, and deoxyribose nucleoside triphosphates
(dNTP's). Reverse transcription PCR, quantitative reverse
transcription PCR, and quantitative real time reverse transcription
PCR are other specific examples of PCR. In general, the reaction
mixture is subjected to temperature cycles comprising a
denaturation stage (typically 80-100.degree. C.), an annealing
stage with a temperature that is selected based on the melting
temperature (Tm) of the primers and the degeneracy of the primers,
and an extension stage (for example 40-75.degree. C.). In real-time
PCR analysis, additional reagents, methods, optical detection
systems, and devices known in the art are used that allow a
measurement of the magnitude of fluorescence in proportion to
concentration of amplified DNA. In such analyses, incorporation of
fluorescent dye into the amplified strands may be detected or
measured.
[0054] Alternatively, labeled probes that bind to a specific
sequence during the annealing phase of the PCR may be used with
primers. Labeled probes release their fluorescent tags during the
extension phase so that the fluorescence level may be detected or
measured. Generally, probes are complementary to a sequence within
the target sequence downstream from either the upstream or
downstream primer. Probes may include one or more label. A label
may be any substance capable of aiding a machine, detector, sensor,
device, or enhanced or unenhanced human eye from differentiating a
labeled composition from an unlabeled composition. Examples of
labels include but are not limited to: a radioactive isotope or
chelate thereof, dye (fluorescent or nonfluorescent) stain, enzyme,
or nonradioactive metal. Specific examples include, but are not
limited to: fluorescein, biotin, digoxigenin, alkaline phosphatese,
biotin, streptavidin, .sup.3H, .sup.14C, .sup.32P, .sup.35S, or any
other compound capable of emitting radiation, rhodamine,
4-(4'-dimethylamino-phenylazo)benzoic acid ("Dabcyl");
4-(4'-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)
("Dabsyl"); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid
("EDANS"); Psoralene derivatives, haptens, cyanines, acridines,
fluorescent rhodol derivatives, cholesterol derivatives;
ethylenediaminetetraacetic acid ("EDTA") and derivatives thereof or
any other compound that may be differentially detected. The label
may also include one or more fluorescent dyes optimized for use in
genotyping. Examples of dyes facilitating the reading of the target
amplification include, but are not limited to: CAL-Fluor Red 610,
CAL-Fluor Orange 560, dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET,
dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540, and LIZ.PCR
facilitating the reading of the target amplification.
[0055] Either primers or primers along with probes allow a
quantification of the amount of specific template DNA present in
the initial sample. In addition, RNA may be detected by PCR
analysis by first creating a DNA template from RNA through a
reverse transcriptase enzyme. The marker expression may be detected
by quantitative PCR analysis facilitating genotyping analysis of
the samples.
[0056] An illustrative example, using dual-labeled oligonucleotide
probes in PCR reactions is disclosed in U.S. Pat. No. 5,716,784 to
DiCesare. In one example of the PCR step of the multiplex Real
Time-PCR/PCR reaction of the present invention, the dual-labeled
fluorescent oligonucleotide probe binds to the target nucleic acid
between the flanking oligonucleotide primers during the annealing
step of the PCR reaction. The 5' end of the oligonucleotide probe
contains the energy transfer donor fluorophore (reporter fluor) and
the 3' end contains the energy transfer acceptor fluorophore
(quenching fluor). In the intact oligonucleotide probe, the 3'
quenching fluor quenches the fluorescence of the 5' reporter fluor.
However, when the oligonucleotide probe is bound to the target
nucleic acid, the 5' to 3' exonuclease activity of the DNA
polymerase, e.g., Taq DNA polymerase, will effectively digest the
bound labeled oligonucleotide probe during the amplification step.
Digestion of the oligonucleotide probe separates the 5' reporter
fluor from the blocking effect of the 3' quenching fluor. The
appearance of fluorescence by the reporter fluor is detected and
monitored during the reaction, and the amount of detected
fluorescence is proportional to the amount of fluorescent product
released. Examples of apparatus suitable for detection include,
e.g. Applied Biosystems.TM. 7900HT real-time PCR platform and
Roche's 480 LightCycler, the ABI Prism 7700 sequence detector using
96-well reaction plates or GENEAMP PC System 9600 or 9700 in 9600
emulation mode followed by analysis in the ABA Prism Sequence
Detector or TAQMAN LS-50B PCR Detection System. The labeled probe
facilitated multiplex Real Time-PCR/PCR can also be performed in
other real-time PCR systems with multiplexing capabilities.
[0057] "Amplification" is a special case of nucleic acid
replication involving template specificity. Amplification may be a
template-specific replication or a non-template-specific
replication (in other words, replication may be specific
template-dependent or not). Template specificity is here
distinguished from fidelity of replication (synthesis of the proper
polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)
specificity. Template specificity is frequently described in terms
of "target" specificity. Target sequences are "targets" in the
sense that they are sought to be sorted out from other nucleic
acid. Amplification techniques have been designed primarily for
this sorting out.
[0058] The term "template" refers to nucleic acid originating from
a sample that is analyzed for the presence of a molecule of
interest. In contrast, "background template" or "control" is used
in reference to nucleic acid other than sample template that may or
may not be present in a sample. Background template is most often
inadvertent. It may be the result of carryover, or it may be due to
the presence of nucleic acid contaminants sought to be purified out
of the sample. For example, nucleic acids from organisms other than
those to be detected may be present as background in a test
sample.
[0059] In addition to primers and probes, template specificity is
also achieved in some amplification techniques by the choice of
enzyme. Amplification enzymes are enzymes that, under the
conditions in which they are used, will process only specific
sequences of nucleic acid in a heterogeneous mixture of nucleic
acid. Other nucleic acid sequences will not be replicated by this
amplification enzyme. Similarly, in the case of T7 RNA polymerase,
this amplification enzyme has a stringent specificity for its own
promoters (Chamberlin et al. (1970) Nature (228):227). In the case
of T4 DNA ligase, the enzyme will not ligate the two
oligonucleotides or polynucleotides, where there is a mismatch
between the oligonucleotide or polynucleotide substrate and the
template at the ligation junction (Wu and Wallace (1989) Genomics
(4):560). Finally, Taq and Pfu polymerases, by virtue of their
ability to function at high temperature, are found to display high
specificity for the sequences bounded and thus defined by the
primers; the high temperature results in thermodynamic conditions
that favor primer hybridization with the target sequences and not
hybridization with non-target sequences (H. A. Erlich (ed.) (1989)
PCR Technology, Stockton Press).
[0060] The term "amplifiable nucleic acid" refers to nucleic acids
that may be amplified by any amplification method. It is
contemplated that "amplifiable nucleic acid" will usually comprise
"sample template." The terms "PCR product," "PCR fragment," and
"amplification product" refer to the resultant mixture of compounds
after two or more cycles of the PCR steps of denaturation,
annealing and extension are complete. These terms encompass the
case where there has been amplification of one or more segments of
one or more target sequences.
[0061] In some forms of PCR assays, quantification of a target in
an unknown sample is often required. Such quantification is often
in reference to the quantity of a control sample. The control
sample DNA may be co-amplified in the same tube in a multiplex
assay or may be amplified in a separate tube. Generally, the
control sample contains DNA at a known concentration. The control
sample DNA may be a plasmid construct comprising only one copy of
the amplification region to be used as quantification reference. To
calculate the quantity of a target in an unknown sample, various
mathematical models are established. Calculations are based on the
comparison of the distinct cycle determined by various methods,
e.g., crossing points (CP) and cycle threshold values (Ct) at a
constant level of fluorescence; or CP acquisition according to
established mathematic algorithm.
[0062] The algorithm for Ct values in Real Time-PCR calculates the
cycle at which each PCR amplification reaches a significant
threshold. The calculated Ct value is proportional to the number of
target copies present in the sample, and the Ct value is a precise
quantitative measurement of the copies of the target found in any
sample. In other words, Ct values represent the presence of
respective target that the primer sets are designed to recognize.
If the target is missing in a sample, there should be no
amplification in the Real Time-PCR reaction.
[0063] Alternatively, the Cp value may be utilized. A Cp value
represents the cycle at which the increase of fluorescence is
highest and where the logarithmic phase of a PCR begins. The
LightCycler.RTM. 480 Software calculates the second derivatives of
entire amplification curves and determines where this value is at
its maximum. By using the second-derivative algorithm, data
obtained are more reliable and reproducible, even if fluorescence
is relatively low.
[0064] The various and non-limiting embodiments of the PCR-based
method detecting marker expression level as described herein may
comprise one or more probes and/or primers. Generally, the probe or
primer contains a sequence complementary to a sequence specific to
a region of the nucleic acid of the marker gene. A sequence having
less than 60% 70%, 80%, 90%, 95%, 99% or 100% identity to the
identified gene sequence may also be used for probe or primer
design if it is capable of binding to its complementary sequence of
the desired target sequence in marker nucleic acid.
[0065] (b) Hybridization Based Methods
[0066] In addition to PCR, gene expression analysis may also be
performed using a probe that is capable of hybridizing to a nucleic
acid sequence of interest. The term "hybridization" refers to the
pairing of complementary nucleic acids. Hybridization and the
strength of hybridization (i.e. the strength of the association
between the nucleic acids) is impacted by such factors as the
degree of complementary between the nucleic acids, stringency of
the conditions involved, the Tm of the formed hybrid, and the G:C
ratio within the nucleic acids. A single molecule that contains
pairing of complementary nucleic acids within its structure is said
to be "self-hybridized."
[0067] The terms "complementary" and "complementarity" refer to
polynucleotides (i.e., a sequence of nucleotides) related by the
base-pairing rules. For example, for the sequence "A-G-T," is
complementary to the sequence "T-C-A." Complementarity may be
"partial," in which only some of the nucleic acids' bases are
matched according to the base pairing rules. Or, there may be
"complete" or "total" complementarity between the nucleic acids.
The degree of complementarity between nucleic acid strands has
significant effects on the efficiency and strength of hybridization
between nucleic acid strands. This is of particular importance in
amplification reactions, as well as detection methods that depend
upon binding between nucleic acids.
[0068] The term "homology" when used in relation to nucleic acids
refers to a degree of complementarity. There may be partial
homology, wherein some of the nucleic acids of a first sequence are
identical to the corresponding nucleic acids in a second sequence,
or complete homology, wherein the sequences are identical.
"Sequence identity" refers to a measure of relatedness between two
or more nucleic acids, and is given as a percentage with reference
to the total comparison length. The identity calculation takes into
account those nucleotide residues that are identical and in the
same relative positions in their respective larger sequences.
Calculations of identity may be performed by algorithms contained
within computer programs such as "GAP" (Genetics Computer Group,
Madison, Wis.) and "ALIGN" (DNAStar, Madison, Wis.). A partially
complementary sequence, one that at least partially inhibits (or
competes with) a completely complementary sequence from hybridizing
to a target nucleic acid is referred to using the functional term
"substantially homologous." The inhibition of hybridization of the
completely complementary sequence to the target sequence may be
examined using a hybridization assay (Southern or Northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe will
compete for and inhibit the binding, or hybridization, of a
sequence that is completely homologous to a target under conditions
of low stringency. This is not to say that conditions of low
stringency are such that non-specific binding is permitted; low
stringency conditions require that the binding of two sequences to
one another be a specific and selective interaction. The absence of
non-specific binding may be tested by the use of a second target
which lacks even a partial degree of complementarity, for example,
less than about 30% identity. In the absence of non-specific
binding the probe will not hybridize to the second
non-complementary target.
[0069] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe which can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low stringency as described infra.
[0070] Low stringency conditions when used in reference to nucleic
acid hybridization comprise conditions equivalent to binding or
hybridization at 42.degree. C. in a solution consisting of
5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4.H.sub.2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS,
5.times.Denhardt's reagent [50.times.Denhardt's contains per 500
ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)]
and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in
a solution comprising 5.times.SSPE, 0.1% SDS at 42.degree. C. when
a probe of about 500 nucleotides in length is employed.
[0071] High stringency conditions when used in reference to nucleic
acid hybridization comprise conditions equivalent to binding or
hybridization at 42.degree. C. in a solution consisting of
5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4.H.sub.2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 0.1.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0072] It is well known that numerous equivalent conditions may be
employed to comprise low stringency conditions; factors such as the
length and nature (DNA, RNA, base composition) of the probe and
nature of the target (DNA, RNA, base composition, present in
solution or immobilized, etc.) and the concentration of the salts
and other components, for example, the presence or absence of
formamide, dextran sulfate, polyethylene glycol, are considered and
the hybridization solution may be varied to generate conditions of
low stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, conditions are known in the
art that promote hybridization under conditions of high stringency,
for example, increasing the temperature of the hybridization and/or
wash steps, the use of formamide in the hybridization solution,
etc.
[0073] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe that can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low to high stringency as described above.
[0074] When used in reference to a single-stranded nucleic acid
sequence, the term "substantially homologous" refers to any probe
that can hybridize, or is the complement of, the single-stranded
nucleic acid sequence under conditions of low to high stringency as
described above.
[0075] The term "Tm" refers to the "melting temperature" of a
nucleic acid. The melting temperature is the temperature at which a
population of double-stranded nucleic acid molecules becomes half
dissociated into single strands. The equation for calculating the
Tm of nucleic acids is well known in the art. As indicated by
standard references, a simple estimate of the Tm value may be
calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic
acid is in aqueous solution at 1 M NaCl (See for example, Anderson
and Young, Quantitative Filter Hybridization (1985) in Nucleic Acid
Hybridization). Other references include more sophisticated
computations that take structural as well as sequence
characteristics into account for the calculation of Tm.
[0076] As used herein the term "stringency" refers to the
conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. With "high stringency" conditions,
nucleic acid base pairing will occur only between nucleic acid
fragments that have a high frequency of complementary base
sequences. Thus, conditions of "low" stringency are often required
with nucleic acids that are derived from organisms that are
genetically diverse, as the frequency of complementary sequences is
usually less.
[0077] Probes for hybridization may comprise nucleic acids,
oligonucleotides (DNA or RNA), proteins, protein complexes,
conjugates, natural ligands, small molecules, nanoparticles, or any
combination of molecules that includes one or more of the above, or
any other molecular entity capable of specific binding to any
allele, whether such molecular entity exists now or is yet to be
disclosed. In one aspect of the invention, the probe comprises an
oligonucleotide, as described above. Other methods used to assess
expression include the use of natural or artificial ligands capable
of specifically binding a marker in its protein or peptide form.
Such ligands include antibodies, antibody complexes, conjugates,
natural ligands, small molecules, nanoparticles, or any other
molecular entity capable of specific binding to a marker. The term
"antibody" is used herein in the broadest sense and refers
generally to a molecule that contains at least one antigen binding
site that immunospecifically binds to a particular antigen target
of interest. The term antibody thus includes, but is not limited
to, native antibodies and variants thereof, fragments of native
antibodies and variants thereof, peptibodies and variants thereof,
and antibody mimetics that mimic the structure and/or function of
an antibody or a specified fragment or portion thereof, including
single chain antibodies and fragments thereof. The term thus
includes full length antibodies and/or their variants as well as
immunologically active fragments thereof, thus encompassing
antibody fragments capable of binding to a biological molecule
(such as an antigen or receptor) or portions thereof, including but
not limited to Fab, Fab_, F(ab_)2, facb, pFc_, Fd, Fv or scFv (See,
e.g., CURRENT PROTOCOLS IN IMMUNOLOGY, (Colligan et al., eds., John
Wiley & Sons, Inc., NY, 1994-2001)).
[0078] Ligands may be associated with a label such as a radioactive
isotope or chelate thereof, dye (fluorescent or nonfluorescent)
stain, enzyme, metal, or any other substance capable of aiding a
machine or a human eye from differentiating a cell expressing a
marker from a cell not expressing a marker. Additionally,
expression may be assessed by monomeric or multimeric ligands
associated with substances capable of killing the cell. Such
substances include protein or small molecule toxins, cytokines,
pro-apoptotic substances, pore forming substances, radioactive
isotopes, or any other substance capable of killing a cell.
[0079] In some aspects of the invention, the expression level of a
marker gene may be established by binding to probes in a media or
on a microarray such as a DNA chip. Examples of DNA chips include
chips in which a number of single stranded oligonucleotide probes
are affixed to a solid substrate such as silicon glass.
Oligonucleotides with a sequence complementary to an allele are
capable of specifically binding to that allele to the exclusion of
alleles that differ from the specific allele by one or more
nucleotides. Labeled sample DNA is hybridized to the
oligonucleotides and detection of the label is correlated with
binding of the sample, and consequently, the presence of the allele
in the sample.
[0080] A nucleic acid probe may be affixed to a substrate.
Alternatively, a sample may be affixed to the substrate. A probe or
sample may be covalently bound to the substrate or it may be bound
by some non covalent interaction including electrostatic,
hydrophobic, hydrogen bonding, Van Der Waals, magnetic, or any
other interaction by which a probe such as an oligonucleotide probe
may be attached to a substrate while maintaining its ability to
recognize the allele to which it has specificity. A substrate may
be any solid or semi-solid material onto which a probe may be
affixed, either singly or in the presence of one or more additional
probes or samples as is exemplified in a microarray. Examples of
substrate materials include but are not limited to polyvinyl,
polysterene, polypropylene, polyester or any other plastic, glass,
silicon dioxide or other silanes, hydrogels, gold, platinum,
microbeads, micelles and other lipid formations, nitrocellulose, or
nylon membranes. The substrate may take any form, including a
spherical bead or flat surface. For example, the probe may be bound
to a substrate in the case of an array or an in situ PCR reaction,
and the nucleic acid probe may include a label as described
herein.
[0081] (c) Sample and Subject
[0082] Differential expression encompasses any detectable
difference between the expression of a marker in one sample
relative to the expression of the marker in another sample.
Examples include but are not limited to differential staining of
cells in an IHC assay configured to detect a marker, differential
detection of bound RNA on a microarray to which a sequence capable
of binding to the marker is bound, differential results in
measuring Real Time-PCR measured in the number of PCR cycles
necessary to reach a particular optical density at a wavelength at
which a double stranded DNA binding dye (e.g., SYBR Green)
incorporates, differential results in measuring label from a
reporter probe used in a real-time PCR reaction, differential
detection of fluorescence on cells using a flow cytometer,
differential intensities of bands in a Northern blot, differential
intensities of bands in an RNAse protection assay, differential
cell death measured by apoptotic markers, differential cell death
measured by shrinkage of a tumor, or any method that allows a
detection of a difference in signal between one sample or set of
samples and another sample or set of samples.
[0083] The expression of the marker in a sample may be compared to
a level of expression predetermined to predict the presence or
absence of a particular physiological characteristic. The level of
expression may be derived from a single control or a set of
controls. A control may be any sample with a previously determined
level of expression. A control may comprise material within the
sample or material from sources other than the sample.
Alternatively, the expression of a marker in a sample may be
compared to a control that has a level of expression predetermined
to signal or not signal a cellular or physiological characteristic.
This level of expression may be derived from a single source of
material including the sample itself or from a set of sources.
Comparison of the expression of the marker in the sample to a
particular level of expression results in a prediction that the
sample exhibits or does not exhibit the cellular or physiological
characteristic.
[0084] The sample in this general tumor identification method is
preferably a biological sample from a subject. The term "sample" or
"biological sample" is used in its broadest sense. Depending upon
the embodiment of the invention, for example, a sample may comprise
a bodily fluid including whole blood, serum, plasma, urine, saliva,
cerebral spinal fluid, semen, vaginal fluid, pulmonary fluid,
tears, perspiration, mucus and the like; an extract from a cell,
chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a
tissue; a tissue print, or any other material isolated in whole or
in part from a living subject. In one preferred embodiment, the
sample is skin tissue. Biological samples may also include sections
of tissues such as biopsy and autopsy samples, and frozen sections
taken for histologic purposes such as blood, plasma, serum, sputum,
stool, tears, mucus, hair, skin, and the like. Biological samples
also include explants and primary and/or transformed cell cultures
derived from patient tissues.
[0085] The term "subject" is used in its broadest sense. In a
preferred embodiment, the subject is a mammal. Non-limiting
examples of mammals include humans, dogs, cats, horses, cows,
sheep, goats, and pigs. Preferably, a subject includes any human or
non-human mammal, including for example: a primate, cow, horse,
pig, sheep, goat, dog, cat, or rodent, capable of developing cancer
including human patients that are suspected of having cancer, that
have been diagnosed with cancer, or that have a family history of
cancer.
[0086] Cancer cells include any cells derived from a tumor,
neoplasm, cancer, precancer, cell line, malignancy, or any other
source of cells that have the potential to expand and grow to an
unlimited degree. Cancer cells may be derived from naturally
occurring sources or may be artificially created. Cancer cells may
also be capable of invasion into other tissues and metastasis.
Cancer cells further encompass any malignant cells that have
invaded other tissues and/or metastasized. One or more cancer cells
in the context of an organism may also be called a cancer, tumor,
neoplasm, growth, malignancy, or any other term used in the art to
describe cells in a cancerous state. In one preferred embodiment,
the cancer cell is SCC cell or its precursor Actinic Keratosis
cells.
[0087] Examples of cancers that could serve as sources of cancer
cells include solid tumors such as fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal
cancer, kidney cancer, pancreatic cancer, bone cancer, breast
cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach
cancer, oral cancer, nasal cancer, throat cancer, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, uterine cancer, testicular cancer, small
cell lung carcinoma, bladder carcinoma, lung cancer, epithelial
carcinoma, glioma, glioblastoma multiforme, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
skin cancer, melanoma, neuroblastoma, and retinoblastoma. In one
preferred embodiment, the cancer is skin cancer.
[0088] Additional cancers that may serve as sources of cancer cells
include blood borne cancer, such as acute lymphoblastic leukemia
("ALL,"), acute lymphoblastic B-cell leukemia, acute lymphoblastic
T-cell leukemia, acute myeloblastic leukemia ("AML"), acute
promyelocytic leukemia ("APL"), acute monoblastic leukemia, acute
erythroleukemic leukemia, acute megakaryoblastic leukemia, acute
myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute
undifferentiated leukemia, chronic myelocytic leukemia ("CML"),
chronic lymphocytic leukemia ("CLL"), hairy cell leukemia, multiple
myeloma, lymphoblastic leukemia, myelogenous leukemia, lymphocytic
leukemia, myelocytic leukemia, Hodgkin's disease, non-Hodgkin's
Lymphoma, Waldenstrom's macroglobulinemia, Heavy chain disease, and
Polycythemia vera.
[0089] B. Method of Assessing a Risk of Progression of Skin
Carcinoma in a Subject.
[0090] Another aspect of the invention provides a prognostic method
to assess the risk of a skin SCC precursor to develop into SCC.
[0091] Prediction of a cellular or physiological characteristic
includes the prediction of any cellular or physiological state that
may be predicted by assessing the expression of a marker. Examples
include the identity of a cell as a particular cell including a
particular normal or cancer cell type, the likelihood that one or
more diseases is present or absent, the likelihood that a present
disease will progress, remain unchanged, or regress, the likelihood
that a disease will respond or not respond to a particular therapy,
or any other outcome. Further examples include the likelihood that
a cell will move, senesce, apoptose, differentiate, metastasize, or
change from any state to any other state or maintain its current
state.
[0092] Expression of a marker in a sample may be more or less than
that of a level predetermined to predict the presence or absence of
a cellular or physiological characteristic. The expression of the
marker in the sample may be more than 1,000,000.times.,
100,000.times., 10,000.times., 1000.times., 100.times., 10.times.,
5.times., 2.times., 1.times., 0.5.times., 0.1.times. 0.01.times.,
0.001.times., 0.0001.times., 0.00001.times., 0.000001.times.,
0.0000001.times. or less than that of a level predetermined to
predict the presence or absence of a cellular or physiological
characteristic.
[0093] The invention contemplates assessing the expression of the
marker in any biological sample from which the expression may be
assessed. One skilled in the art would know to select a particular
biological sample and how to collect said sample depending upon the
marker that is being assessed. Examples of sources of samples
include but are not limited to biopsy or other in vivo or ex vivo
analysis of prostate, breast, skin, muscle, fascia, brain,
endometrium, lung, head and neck, pancreas, small intestine, blood,
liver, testes, ovaries, colon, skin, stomach, esophagus, spleen,
lymph node, bone marrow, kidney, placenta, or fetus. In some
aspects of the invention, the sample comprises a fluid sample, such
as peripheral blood, lymph fluid, ascites, serous fluid, pleural
effusion, sputum, cerebrospinal fluid, amniotic fluid, lacrimal
fluid, stool, or urine. Samples include single cells, whole organs
or any fraction of a whole organ, in any condition including in
vitro, ex vivo, in vivo, post-mortem, fresh, fixed, or frozen.
[0094] One type of cellular or physiological characteristic is the
risk that a particular disease outcome will occur. Assessing this
risk includes the performing of any type of test, assay,
examination, result, readout, or interpretation that correlates
with an increased or decreased probability that an individual has
had, currently has, or will develop a particular disease, disorder,
symptom, syndrome, or any condition related to health or bodily
state. Examples of disease outcomes include, but need not be
limited to survival, death, progression of existing disease,
remission of existing disease, initiation of onset of a disease in
an otherwise disease-free subject, or the continued lack of disease
in a subject in which there has been a remission of disease.
Assessing the risk of a particular disease encompasses diagnosis in
which the type of disease afflicting a subject is determined.
Assessing the risk of a disease outcome also encompasses the
concept of prognosis. A prognosis may be any assessment of the risk
of disease outcome in an individual in which a particular disease
has been diagnosed. Assessing the risk further encompasses
prediction of therapeutic response in which a treatment regimen is
chosen based on the assessment. Assessing the risk also encompasses
a prediction of overall survival after diagnosis.
[0095] Determining the level of expression that signifies a
physiological or cellular characteristic may be assessed by any of
a number of methods. The skilled artisan will understand that
numerous methods may be used to select a level of expression for a
particular marker or a plurality of markers that signifies
particular physiological or cellular characteristics. In diagnosing
the presence of a disease, a threshold value may be obtained by
performing the assay method on samples obtained from a population
of patients having a certain type of disease, for example, cancer,
and from a second population of subjects that do not have the
disease. In assessing disease outcome or the effect of treatment, a
population of patients, all of which have a disease, such as
cancer, may be followed for a period of time. After the period of
time expires, the population may be divided into two or more
groups. For example, the population may be divided into a first
group of patients whose disease progresses to a particular endpoint
and a second group of patients whose disease does not progress to
the particular endpoint. Examples of endpoints include disease
recurrence, death, metastasis or other states to which disease may
progress. If expression of the marker in a sample is more similar
to the predetermined expression of the marker in one group relative
to the other group, the sample may be assigned a risk of having the
same outcome as the patient group to which it is more similar.
[0096] In addition, one or more levels of expression of the marker
may be selected that signify a particular physiological or cellular
characteristic. For example, Receiver Operating Characteristic
curves, or "ROC" curves, may be calculated by plotting the value of
a variable versus its relative frequency in two populations. For
any particular marker, a distribution of marker expression levels
for subjects with and without a disease may overlap. This indicates
that the test does not absolutely distinguish between the two
populations with complete accuracy. The area of overlap indicates
where the test cannot distinguish the two groups. A threshold is
selected. Expression of the marker in the sample above the
threshold indicates the sample is similar to one group and
expression of the marker below the threshold indicates the sample
is similar to the other group. The area under the ROC curve is a
measure of the probability that the expression correctly indicated
the similarity of the sample to the proper group. See, e.g., Hanley
et al., Radiology 143: 29-36 (1982) hereby incorporated by
reference.
[0097] Additionally, levels of expression for purpose of prognosis
may be established by assessing the expression of a marker in a
sample from one patient, assessing the expression of additional
samples from the same patient obtained later in time, and comparing
the expression of the marker from the later samples with the
initial sample or samples. This method may be used in the case of
markers that indicate, for example, progression or worsening of
disease or lack of efficacy of a treatment regimen or remission of
a disease or efficacy of a treatment regimen.
[0098] Other methods may be used to assess how accurately the
expression of a marker signifies a particular physiological or
cellular characteristic. Such methods include a positive likelihood
ratio, negative likelihood ratio, odds ratio, and/or hazard ratio.
In the case of a likelihood ratio, the likelihood that the
expression of the marker would be found in a sample with a
particular cellular or physiological characteristic is compared
with the likelihood that the expression of the marker would be
found in a sample lacking the particular cellular or physiological
characteristic.
[0099] An odds ratio measures effect size and describes the amount
of association or non-independence between two groups. An odds
ratio is the ratio of the odds of a marker being expressed in one
set of samples versus the odds of the marker being expressed in the
other set of samples. An odds ratio of 1 indicates that the event
or condition is equally likely to occur in both groups. An odds
ratio greater or less than 1 indicates that expression of the
marker is more likely to occur in one group or the other depending
on how the odds ratio calculation was set up.
[0100] A hazard ratio may be calculated by estimate of relative
risk. Relative risk is the chance that a particular event will take
place. It is a ratio of the probability that an event such as
development or progression of a disease will occur in samples that
exceed a threshold level of expression of a marker over the
probability that the event will occur in samples that do not exceed
a threshold level of expression of a marker. Alternatively, a
hazard ratio may be calculated by the limit of the number of events
per unit time divided by the number at risk as the time interval
decreases. In the case of a hazard ratio, a value of 1 indicates
that the relative risk is equal in both the first and second
groups. A value greater or less than 1 indicates that the risk is
greater in one group or another, depending on the inputs into the
calculation.
[0101] Additionally, multiple threshold levels of expression may be
determined. This can be the case in so-called "tertile,"
"quartile," or "quintile" analyses. In these methods, multiple
groups can be considered together as a single population, and are
divided into 3 or more bins having equal numbers of individuals.
The boundary between two of these "bins" may be considered
threshold levels of expression indicating a particular level of
risk of a disease developing or signifying a physiological or
cellular state. A risk may be assigned based on which "bin" a test
subject falls into.
[0102] C. A Method of Treating a Patient Having at Different Stage
of Skin Squamous Cell Carcinoma.
[0103] Still another aspect of the present invention provides a
method of treating a patient having skin squamous cell carcinoma or
AK cells prone to progress into SCC. The method comprises testing
one or more samples from the patient to determine the cell
sensitivity to INPP5A metabolites. If a test is positive, i.e., the
cells in the sample have reduced INPP5A expression on either mRNA
or protein level, then treating the patient with pharmaceutical
composition comprising at least one INPP5A metabolite. In one
embodiment, INPP5A metabolite is selected from the group consisting
of Ins(1,3,4)P.sub.3, Ins(1,3,4,6)P.sub.4, Ins(1,3,4,5,6)P.sub.5,
and Ins(1,2,3,4,5,6)P.sub.6 (IP6). In one preferred example, the
INPP5A metabolite is IP6 In one preferred embodiment, the
pharmaceutical composition comprises IP6.
[0104] In general, the pharmaceutical composition will comprise an
effective dosage amount of the disclosed one or more INPP5A
metabolites, i.e., an amount of INPP5A metabolites sufficient to
provide treatment to the subject being administered the
pharmaceutical composition. Determination of an effective amount of
the composition is within the capability of those skilled in the
art. The effective amount of a pharmaceutical composition used to
affect a particular purpose, as well as its toxicity, excretion,
and overall tolerance may be determined in cell cultures or
experimental animals by pharmaceutical and toxicological procedures
either known now by those skilled in the art or by any similar
method yet to be disclosed. One example is the determination of the
IC.sub.50 (half maximal inhibitory concentration) of the
pharmaceutical composition in vitro in cell lines or target
molecules. Another example is the determination of the LD.sub.50
(lethal dose causing death in 50% of the tested animals) of the
pharmaceutical composition in experimental animals. The exact
techniques used in determining an effective amount will depend on
factors such as the type, physical and/or chemical properties of
the pharmaceutical composition, the property being tested, and
whether the test is to be performed in vitro or in vivo. The
determination of an effective amount of a pharmaceutical
composition will be well known to one of skill in the art who will
use data obtained from any tests in making that determination.
Determination of an effective amount of compound for addition to a
cancer cell also includes the determination of an effective
therapeutic amount, including the formulation of an effective dose
range for use in vivo, including in humans.
[0105] In some embodiments, the pharmaceutical composition may
comprise substantially pure INPP5A metabolite. In one preferred
embodiment, the INPP5A metabolite is IP6 or a pharmaceutically
acceptable salt thereof. The amount of INPP5A IP6 or a
pharmaceutically acceptable salt thereof in such pharmaceutical
compositions, therefore, may range from about 97%, about 95%, about
90%, about 85%, about 80%, about 75%, about 70%, about 65%, about
60%, about 55%, about 50%, about 45%, about 40%, about 35%, about
30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about
3% by weight of the total amount of IP6 and its salt.
[0106] A variety of excipients commonly used in pharmaceutical
formulations may be selected on the basis of several criteria, such
as, the desired dosage form and the release profile properties of
the dosage form. Non-limiting examples of suitable excipients
include an agent selected from the group consisting of a binder, a
filler, a non-effervescent disintegrant, an effervescent
disintegrant, a preservative, a diluent, a flavoring agent, a
sweetener, a lubricant, an oral dispersing agent, a coloring agent,
a taste masking agent, a pH modifier, a stabilizer, a compaction
agent, and combinations of any of these agents.
[0107] In one embodiment, the excipient may be a binder. Suitable
binders include starches, pregelatinized starches, gelatin,
polyvinylpyrolidone, cellulose, methylcellulose, sodium
carboxymethylcellulose, ethylcellulose, polyacrylamides,
polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid
alcohol, polyethylene glycol, polyols, saccharides,
oligosaccharides, polypeptides, peptides, and combinations
thereof.
[0108] In another embodiment, the excipient may be a filler.
Suitable fillers include carbohydrates, inorganic compounds, and
polyvinylpirrolydone. By way of non-limiting example, the filler
may be calcium sulfate, both di- and tri-basic, starch, calcium
carbonate, magnesium carbonate, microcrystalline cellulose, dibasic
calcium phosphate, magnesium carbonate, magnesium oxide, calcium
silicate, talc, modified starches, lactose, sucrose, mannitol, and
sorbitol.
[0109] The excipient may be a non-effervescent disintegrant.
Suitable examples of non-effervescent disintegrants include
starches (such as corn starch, potato starch, and the like),
pregelatinized and modified starches thereof, sweeteners, clays
(such as bentonite), microcrystalline cellulose, alginates, sodium
starch glycolate, and gums (such as agar, guar, locust bean,
karaya, pecitin, and tragacanth).
[0110] In another embodiment, the excipient may be an effervescent
disintegrant. By way of non-limiting example, suitable effervescent
disintegrants include sodium bicarbonate in combination with citric
acid, and sodium bicarbonate in combination with tartaric acid.
[0111] The excipient may comprise a preservative. Suitable examples
of preservatives include antioxidants (such as alpha-tocopherol or
ascorbate) and antimicrobials (such as parabens, chlorobutanol or
phenol). In other embodiments, an antioxidant such as butylated
hydroxytoluene (BHT) or butylated hydroxyanisole (BHA) may be
utilized.
[0112] In another embodiment, the excipient may include a diluent.
Diluents suitable for use include pharmaceutically acceptable
saccharides such as sucrose, dextrose, lactose, microcrystalline
cellulose, fructose, xylitol, and sorbitol; polyhydric alcohols;
starches; pre-manufactured direct compression diluents; and
mixtures of any of the foregoing.
[0113] The excipient may include flavoring agents. Flavoring agents
may be chosen from synthetic flavor oils and flavoring aromatics
and/or natural oils, extracts from plants, leaves, flowers, fruits,
and combinations thereof. By way of example, these may include
cinnamon oils, oil of wintergreen, peppermint oils, clover oil, hay
oil, anise oil, eucalyptus, vanilla, citrus oils (such as lemon
oil, orange oil, grape and grapefruit oil), and fruit essences
(such as apple, peach, pear, strawberry, raspberry, cherry, plum,
pineapple, and apricot).
[0114] In another embodiment, the excipient may include a
sweetener. By way of non-limiting example, the sweetener may be
selected from glucose (corn syrup), dextrose, invert sugar,
fructose, and mixtures thereof (when not used as a carrier);
saccharin and its various salts such as the sodium salt; dipeptide
sweeteners such as aspartame; dihydrochalcone compounds,
glycyrrhizin; stevia-derived sweeteners; chloro derivatives of
sucrose such as sucralose; sugar alcohols such as sorbitol,
mannitol, sylitol, and the like. Also contemplated are hydrogenated
starch hydrolysates and the synthetic sweetener
3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide,
particularly the potassium salt (acesulfame-K), and sodium and
calcium salts thereof.
[0115] In another embodiment, the excipient may be a lubricant.
Suitable non-limiting examples of lubricants include magnesium
stearate, calcium stearate, zinc stearate, hydrogenated vegetable
oils, sterotex, polyoxyethylene monostearate, talc,
polyethyleneglycol, sodium benzoate, sodium lauryl sulfate,
magnesium lauryl sulfate, and light mineral oil.
[0116] The excipient may be a dispersion enhancer. Suitable
dispersants may include starch, alginic acid,
polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood
cellulose, sodium starch glycolate, isoamorphous silicate, and
microcrystalline cellulose.
[0117] Depending upon the embodiment, it may be desirable to
provide a coloring agent. Suitable color additives include food,
drug and cosmetic colors (FD&C), drug and cosmetic colors
(D&C), or external drug and cosmetic colors (Ext. D&C).
These colors or dyes, along with their corresponding lakes, and
certain natural and derived colorants may be suitable for use in
the present invention depending on the embodiment.
[0118] The excipient may include a taste-masking agent.
Taste-masking materials include cellulose hydroxypropyl ethers
(HPC); low-substituted hydroxypropyl ethers (L-HPC); cellulose
hydroxypropyl methyl ethers (HPMC); methylcellulose polymers and
mixtures thereof; polyvinyl alcohol (PVA); hydroxyethylcelluloses;
carboxymethylcelluloses and salts thereof; polyvinyl alcohol and
polyethylene glycol co-polymers; monoglycerides or triglycerides;
polyethylene glycols; acrylic polymers; mixtures of acrylic
polymers with cellulose ethers; cellulose acetate phthalate; and
combinations thereof.
[0119] In various embodiments, the excipient may include a pH
modifier. In certain embodiments, the pH modifier may include
sodium carbonate or sodium bicarbonate.
[0120] The weight fraction of the excipient or combination of
excipients in the pharmaceutical composition may be about 98% or
less, about 95% or less, about 90% or less, about 85% or less,
about 80% or less, about 75% or less, about 70% or less, about 65%
or less, about 60% or less, about 55% or less, about 50% or less,
about 45% or less, about 40% or less, about 35% or less, about 30%
or less, about 25% or less, about 20% or less, about 15% or less,
about 10% or less, about 5% or less, about 2%, or about 1% or less
of the total weight of the pharmaceutical composition.
[0121] The pharmaceutical compositions detailed herein may be
manufactured in one or several dosage forms. Suitable dosage forms
include transdermal systems or patches. The transdermal system may
be a matrix system, a reservoir system, or a system without
rate-controlling membranes. Other suitable dosage forms also
include tablets, including suspension tablets, chewable tablets,
effervescent tablets or caplets; pills; powders such as a sterile
packaged powder, a dispensable powder, and an effervescent powder;
capsules including both soft or hard gelatin capsules such as HPMC
capsules; lozenges; a sachet; a sprinkle; a reconstitutable powder
or shake; a troche; pellets such as sublingual or buccal pellets;
granules; liquids for oral or parenteral administration;
suspensions; emulsions; semisolids; or gels.
[0122] The dosage forms may be manufactured using conventional
pharmacological techniques. Conventional pharmacological techniques
include, e.g., one or a combination of methods: (1) dry mixing, (2)
direct compression, (3) milling, (4) dry or non-aqueous
granulation, (5) wet granulation, or (6) fusion. See, e.g., Lachman
et al., The Theory and Practice of Industrial Pharmacy (1986).
Other methods include, e.g., prilling, spray drying, pan coating,
melt granulation, granulation, wurster coating, tangential coating,
top spraying, extruding, coacervation and the like.
[0123] The amount of active ingredient that is administered to a
subject can and will vary depending upon a variety of factors such
as the age and overall health of the subject, and the particular
mode of administration. Those skilled in the art will appreciate
that dosages may also be determined with guidance from Goodman
& Goldman's The Pharmacological Basis of Therapeutics, Tenth
Edition (2001), Appendix II, pp. 475-493, and the Physicians' Desk
Reference. In one embodiment of the present invention, the
effective amount of the disclosed compound to results in the
slowing of expansion of the cancer cells would preferably result in
a concentration at or near the target tissue that is effective in
slowing cellular expansion in cancer cells, but have minimal
effects on non-cancer cells, including non-cancer cells exposed to
radiation or recognized chemotherapeutic chemical agents.
Concentrations that produce these effects can be determined using,
for example, apoptosis markers such as the apoptotic index and/or
caspase activities either in vitro or in vivo. In one preferred
embodiment, the effective dosage results in the resumed
differentiation of SCC cell or its precursor AK cell, reduced cell
proliferation of cancerous cell or its precursor and increased
cancerous cell death.
[0124] Methods of administration include, but are not limited to,
oral administration and parenteral administration. Parenteral
administration includes, but is not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, sublingual, intramsal, intracerebral,
iratraventricular, intrathecal, intravaginal, transdermal, rectal,
by inhalation, or topically to the ears, nose, eyes, or skin. Other
methods of administration include but are not limited to infusion
techniques including infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings such as oral mucosa,
rectal and intestinal mucosa. Compositions for parenteral
administration may be enclosed in ampoule, a disposable syringe or
a multiple-dose vial made of glass, plastic or other material.
[0125] Administration may be systemic or local. Local
administration is administration of the disclosed compound to the
area in need of treatment. Examples include local infusion during
surgery; topical application, by local injection; by a catheter; by
a suppository; or by an implant. Administration may be by direct
injection at the site (or former site) of a cancer, tumor, or
precancerous tissue or into the central nervous system by any
suitable route, including intraventricular and intrathecal
injection. Intraventricular injection can be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir. Pulmonary administration may be
achieved by any of a number of methods known in the art. Examples
include use of an inhaler or nebulizer, formulation with an
aerosolizing agent, or via perfusion in a fluorocarbon or synthetic
pulmonary surfactant. The disclosed compound may be delivered in
the context of a vesicle such as a liposome or any other natural or
synthetic vesicle.
[0126] A pharmaceutical composition formulated so as to be
administered by injection may be prepared by dissolving the
disclosed compound with water so as to form a solution. In
addition, a surfactant may be added to facilitate the formation of
a homogeneous solution or suspension. Surfactants include any
complex capable of non-covalent interaction with the disclosed
compound so as to facilitate dissolution or homogeneous suspension
of the compound.
[0127] Pharmaceutical compositions including the disclosed compound
may be prepared in a form that facilitates topical or transdermal
administration. Such preparations may be in the form of a liquid
solution, cream, paste, lotion, shake lotion, powder, emulsion,
ointment, gel base, transdermal patch or iontophoresis device.
Examples of bases used in such compositions include petrolatum,
lanolin, polyethylene glycols, beeswax, mineral oil, diluents such
as water and alcohol, and emulsifiers and stabilizers, thickening
agents, or any other suitable base now known or yet to be
disclosed.
[0128] Addition of a pharmaceutical composition to cancer cells
includes all actions by which an effect of the pharmaceutical
composition on the cancer cell is realized. The type of addition
chosen will depend upon whether the cancer cells are in vivo, ex
vivo, or in vitro, the physical or chemical properties of the
pharmaceutical composition, and the effect the composition is to
have on the cancer cell. Nonlimiting examples of addition include
addition of a solution including the pharmaceutical composition to
tissue culture media in which in vitro cancer cells are growing;
any method by which a pharmaceutical composition may be
administered to an animal including intravenous, per os,
parenteral, or any other of the methods of administration; or the
activation or inhibition of cells that in turn have effects on the
cancer cells such as immune cells (e.g. macophages and CD8+ T
cells) or endothelial cells that may differentiate into blood
vessel structures in the process of angiogenesis or
vasculogenesis.
[0129] Treatment is contemplated in living entities including but
not limited to mammals (particularly humans) as well as other
mammals of economic or social importance, including those of an
endangered status. Further examples include livestock or other
animals generally bred for human consumption and domesticated
companion animals. Treatment of a condition is the practice of any
method, process, or procedure with the intent of halting,
inhibiting, slowing or reversing the progression of a disease,
disorder or condition, substantially ameliorating clinical symptoms
of a disease disorder or condition, or substantially preventing the
appearance of clinical symptoms of a disease, disorder or
condition, up to and including returning the diseased entity to its
condition prior to the development of the disease.
[0130] The method of treating a subject having a form of cancer
includes the prevention of progression of the cancer to a
neoplastic, malignant or metastatic state. Such preventative use is
indicated in conditions known or suspected of preceding progression
to cancer, in particular, where non- or precancerous cell growth
consisting of hyperplasia, metaplasia, or most particularly,
dysplasia has occurred (for review of such abnormal growth
conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed.,
W. B. Saunders Co., Philadelphia, pp. 68-90, incorporated by
reference). Hyperplasia is a form of controlled cell proliferation
involving an increase in cell number in a tissue or organ, without
significant alteration in structure or activity. For example,
endometrial hyperplasia often precedes endometrial cancer and
precancerous colon polyps often transform into cancerous lesions.
Metaplasia is a form of controlled cell growth in which one type of
adult or fully differentiated cell substitutes for another type of
adult cell. Metaplasia can occur in epithelial or connective tissue
cells. A typical metaplasia involves a somewhat disorderly
metaplastic epithelium. Dysplasia is frequently a forerunner of
cancer, and is found mainly in the epithelia; it is the most
disorderly form of non-neoplastic cell growth, involving a loss in
individual cell uniformity and in the architectural orientation of
cells. Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism.
[0131] Alternatively or in addition to the presence of abnormal
cell growth characterized as hyperplasia, metaplasia, or dysplasia,
the presence of one or more characteristics of a transformed
phenotype or of a malignant phenotype, displayed in vivo or
displayed in vitro by a cell sample derived from a subject can
indicate the desirability of prophylactic/therapeutic
administration of the pharmaceutical composition that includes the
compound. Such characteristics of a transformed phenotype include
morphology changes, looser substratum attachment, loss of contact
inhibition, loss of anchorage dependence, protease release,
increased sugar transport, decreased serum requirement, expression
of fetal antigens, disappearance of the 250,000 dalton cell surface
protein, etc. In a preferred embodiment, the characteristics of a
transformed phenotype due to the treatment using pharmaceutical
composition comprising INPP5A metabolite(s) is the differentiation
of SCC cell or its precursor AK cell that leads to the cancerous
cell death.
[0132] Pharmaceutical compositions that include the disclosed
compound may be administered prior to, concurrently with, or after
administration of a second pharmaceutical composition that may or
may not include the compound. If the compositions are administered
concurrently, they are administered within one minute of each
other. If not administered concurrently, the second pharmaceutical
composition may be administered a period of one or more minutes,
hours, days, weeks, or months before or after the pharmaceutical
composition that includes the compound Alternatively, a combination
of pharmaceutical compositions may be cyclically administered.
Cycling therapy involves the administration of one or more
pharmaceutical compositions for a period of time, followed by the
administration of one or more different pharmaceutical compositions
for a period of time and repeating this sequential administration,
in order to reduce the development of resistance to one or more of
the compositions, to avoid or reduce the side effects of one or
more of the compositions, and/or to improve the efficacy of the
treatment.
[0133] The invention further encompasses methods of treating cancer
that comprise combination therapies that comprise the
administration of a pharmaceutical composition including the
disclosed compound and another treatment modality. Such treatment
modalities include but are not limited to, radiotherapy,
chemotherapy, surgery, immunotherapy, cancer vaccines,
radioimmunotherapy, treatment with pharmaceutical compositions
other than those which include the disclosed compound, or any other
method that effectively treats cancer in combination with the
disclosed compound now known or yet to be disclosed. Combination
therapies may act synergistically. That is, the combination of the
two therapies is more effective than either therapy administered
alone. This results in a situation in which lower dosages of both
treatment modality may be used effectively. This in turn reduces
the toxicity and side effects, if any, associated with the
administration either modality without a reduction in efficacy.
[0134] In one preferred embodiment, the cancers or precancers that
may be treated by pharmaceutical compositions including the
disclosed compound either alone or in combination with another
treatment modality is skin SCC.
(III) Kits.
[0135] The present invention further provides kits to be used in
assessing the expression of a particular RNA in a sample from a
subject to assess the risk of developing disease. Kits include any
combination of components that facilitate the performance of an
assay. A kit that facilitates assessing the expression of a RNA may
include suitable nucleic acid-based and immunological reagents as
well as suitable buffers, control reagents, and printed
protocols.
[0136] Kits that facilitate nucleic acid based methods may further
include one or more of the following: specific nucleic acids such
as oligonucleotides, labeling reagents, enzymes including PCR
amplification reagents such as Taq or Pfu; reverse transcriptase,
or one or more other polymerases, and/or reagents that facilitate
hybridization. Specific nucleic acids may include nucleic acids,
polynucleotides, oligonucleotides (DNA, or RNA), or any combination
of molecules that includes one or more of the above, or any other
molecular entity capable of specific binding to a nucleic acid
marker. In one aspect of the invention, the specific nucleic acid
comprises one or more oligonucleotides capable of hybridizing to
the marker.
[0137] A specific nucleic acid may include a label. A label may be
any substance capable of aiding a machine, detector, sensor,
device, or enhanced or unenhanced human eye from differentiating a
sample that that displays positive expression from a sample that
displays reduced expression. Examples of labels include but are not
limited to: a radioactive isotope or chelate thereof, a dye
(fluorescent or nonfluorescent) stain, enzyme, or nonradioactive
metal. Specific examples include but are not limited to:
fluorescein, biotin, digoxigenin, alkaline phosphatase, biotin,
streptavidin, .sup.3H, .sup.14C, .sup.32P, .sup.35S, or any other
compound capable of emitting radiation, rhodamine,
4-(4'-dimethylaminophenylazo)benzoic acid ("Dabcyl");
4-(4'-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)
("Dabsyl"); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid
("EDANS"); Psoralene derivatives, haptens, cyanines, acridines,
fluorescent rhodol derivatives, cholesterol derivatives;
ethylenediaminetetraaceticacid ("EDTA") and derivatives thereof or
any other compound that signals the presence of the labeled nucleic
acid. In one embodiment of the invention, the label includes one or
more dyes optimized for use in genotyping. Examples of such dyes
include but are not limited to: dR110, 5-FAM, 6FAM, dR6G, JOE, HEX,
VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, and LIZ.
[0138] An oligonucleotide is a reagent capable of binding a nucleic
acid sequence. An oligonucleotide may be any polynucleotide of at
least 2 nucleotides. Oligonucleotides may be less than 10, less
than 15, less than 20, less than 30, less than 40, less than 50,
less than 75, less than 100, less than 200, less than 500, or more
than 500 nucleotides in length. While oligonucleotides are often
linear, they may, depending on their sequence and conditions,
assume a two- or three-dimensional structure. Oligonucleotides may
be chemically synthesized by any of a number of methods including
sequential synthesis, solid phase synthesis, or any other synthesis
method now known or yet to be disclosed. Alternatively,
oligonucleotides may be produced by recombinant DNA based methods.
One skilled in the art would understand the length of
oligonucleotide necessary to perform a particular task.
Oligonucleotides may be directly labeled, used as primers in PCR or
sequencing reactions, or bound directly to a solid substrate as in
oligonucleotide arrays.
[0139] An oligonucleotide used to detect to an allele may be
affixed to a solid substrate. Alternatively, the sample may be
affixed to a solid substrate and the nucleic acid reagent placed
into a mixture. For example, the nucleic acid reagent may be bound
to a substrate in the case of an array or the sample may be bound
to a substrate as the case of a Southern Blot, Northern blot or
other method that affixes the sample to a substrate. A nucleic acid
reagent or sample may be covalently bound to the substrate or it
may be bound by some non covalent interaction including
electrostatic, hydrophobic, hydrogen bonding, Van Der Waals,
magnetic, or any other interaction by which an oligonucleotide may
be attached to a substrate while maintaining its ability to
recognize the allele to which it has specificity. A substrate may
be any solid or semi solid material onto which a probe may be
affixed, attached or printed, either singly or in the formation of
a microarray. Examples of substrate materials include but are not
limited to polyvinyl, polysterene, polypropylene, polyester or any
other plastic, glass, silicon dioxide or other silanes, hydrogels,
gold, platinum, microbeads, micelles and other lipid formations,
nitrocellulose, or nylon membranes. The substrate may take any
shape, including a spherical bead or flat surface. In some aspects
of the invention, the probe may be affixed to a solid substrate. In
other aspects of the invention, the sample may be affixed to a
solid substrate.
[0140] Kits may also contain reagents that detect proteins, often
through the use of an antibody. These kits will contain one or more
specific antibodies, buffers, and other reagents configured to
detect binding of the antibody to the specific epitope. One or more
of the antibodies may be labeled with a fluorescent, enzymatic,
magnetic, metallic, chemical, or other label that signifies and/or
locates the presence of specifically bound antibody. The kit may
also contain one or more secondary antibodies that specifically
recognize epitopes on other antibodies. These secondary antibodies
may also be labeled. The concept of a secondary antibody also
encompasses non-antibody ligands that specifically bind an epitope
or label of another antibody. For example, streptavidin or avidin
may bind to biotin conjugated to another antibody. Such a kit may
also contain enzymatic substrates that change color or some other
property in the presence of an enzyme that is conjugated to one or
more antibodies included in the kit.
[0141] A kit may also contain an indication of a result of the use
of the kit that signifies a particular physiological or cellular
characteristic. An indication includes any guide to a result that
would signal the presence or absence of any physiological or
cellular state that the kit is configured to predict. For example,
the indication may be expressed numerically, expressed as a color
or density of a color, expressed as an intensity of a band, derived
from a standard curve, or expressed in comparison to a control. The
indication may be communicated through the use of a writing. A
writing may be any communication of the result in a tangible medium
of expression. The writing may be contained physically in or on the
kit (on a piece of paper for example), posted on the Internet,
mailed to the user separately from the kit, or embedded in a
software package. The writing may be in any medium that
communicates how the result may be used to predict the cellular or
physiological characteristic that the kit is intended to predict,
such as a printed document, a photograph, sound, color, or any
combination thereof.
[0142] The invention further encompasses pharmaceutical
compositions that include the disclosed compound and/or
pharmaceutically acceptable salts of the compound.
EXAMPLES
[0143] Various embodiments of the present teachings can be
illustrated by the following non-limiting examples. The following
examples are illustrative, and are not intended to limit the scope
of the claims.
Methods and Material:
[0144] Tissues:
[0145] Tissue samples analyzed were formalin-fixed,
paraffin-embedded (FFPE), archived specimens obtained under the
Institutional Review Board-approved protocols at the Arizona Cancer
Center, University of Arizona (Tucson, Ariz.); Southern Arizona
Veterans Affairs Health Care System (Tucson, Ariz.); Loyola
University Medical Center (Chicago, Ill.); and Mayo Clinic. The
study was conducted according to the Declaration of Helsinki
Principles.
[0146] Array CGH:
[0147] To obtain genomic DNA for aCGH (array-based comparative
genomic hybridization), microscopic examination by pathologist was
used to select the areas for harvest and DNA extraction. In all
samples, only regions that showed >50% lesional content were
harvested. aCGH profiling was done using a method in which DNA was
extracted from FFPE tissue blocks using the DNeasy tissue kit
(Qiagen, Germantown, Md.). Normal pooled lymphocyte DNA (Promega,
Madison, Wis.) was used as a reference. A total of 5 .mu.g of
sample genomic DNA and 1 .mu.g of reference genomic DNA were
fragmented using the thermolabile recombinant shrimp DNase
(TS-DNase; Affymetrix, Santa Clara, Calif.) to achieve an average
DNA fragment length of 200 to 600 bp. Fragmented sample and
reference DNA were labeled with Cy5 and Cy3 fluorescent dUTP,
respectively, using the Bioprime Array CGH Genomic Labeling System
(Invitrogen, Carlsbad, Calif.). Hybridizations were done on Agilent
44K feature microarrays for aCGH (Agilent Technologies, Santa
Clara, Calif.) per the manufacturer's specifications and scanned on
an Agilent DNA Microarray scanner, followed by image analysis with
Feature Extraction software and data visualization with DNA
Analytics software using the aberration calling algorithm
ADM-1.
Fluorescence In Situ Hybridization:
[0148] Fluorescence in situ hybridization (FISH) was carried out
using a centromeric probe to chromosome 10 (Abbott Molecular, Des
Plaines, Ill.) and INPP5A-directed probes (bacterial artificial
chromosomes RP11-500B2 and RP11-288G11; BACPAC Resource Center) to
either metaphase spreads or sections prepared from FFPE blocks. The
DNA from two large plasmids carrying a large segment of human
chromosomal DNA in the INPP5A gene region (RP11-500B2 and
RP11-288G11) was isolated and labeled with Cy5 tagged nucleotides,
then hybridized to the FFPE slices from normal and SCC samples.
FFPE slices were prepared for hybridization using the Paraffin
Pretreatment Kit II (Abbott Molecular, Des Plaines, Ill.). Slides
were examined and photographed on a Zeiss Axiophot equipped with
interference filters (Chroma, Bellows Falls, Vt.) and a CoolSnap
HQ2 digital camera (Photometrics, Tucson, Ariz.). The FISH
evaluation was semiquantitative. Whenever the tissue was of
sufficient size, 100+ nuclei were examined. However, in cases where
a lesion of interest was small (e.g., AK lesions), all available
lesional nuclei (i.e., <100) were examined.
[0149] Immunohistochemistry:
[0150] FFPE tissue blocks were sectioned on glass slides at 5-.mu.m
thickness and baked for 60 minutes at 60.degree. C. Slides were
subsequently subjected to heat-induced epitope retrieval using a
proprietary citrate-based retrieval solution for 20 minutes. The
tissue sections were incubated for 30 minutes with anti-INPP5A
mouse monoclonal antibody (clone 3D8; Novus Biologicals, Littleton,
Colo.). The sections were visualized with the Bond Polymer Refine
Detection kit (Leica Microsystems, Inc., Wetzlar, Germany) using
diaminobenzidine chromogen as substrate.
[0151] IP6 Treatment of Squamous Cell Carcinoma:
[0152] The human head and neck squamous cell carcinoma lines
evaluated were: SCC-4, SCC-9 and SCC-15. The colorectal cancer cell
line HT-29 and the kidney embryonic line HEK293, purchased from the
American Type Culture Collection (ATCC, Manassas, Va.), were used
for comparison. Squamous cell lines were maintained in Dulbecco's
Modified Eagle Medium: Nutrient Mixture F12 (DMEM/F12), with 10%
Fetal bovine serum (FBS), 2 mM GlutaMAX, 25 mM Hepes buffer, 100
U/ml penicillin and 100 .mu.g/ml streptomycin. HEK293and HT-29 were
cultured in DMEM and RPMI-1640 medium respectively, with the
supplements mentioned above. All media and cell culture supplements
were purchased from Invitrogen Corporation (Carlsbad, Calif.).
Sub-confluent, rapidly growing cells were plated at a density of
600 cells/20 .mu.L/well in a clear bottom 384-well plate with
opaque walls, suitable for luminescent measurements. The next day,
IP6 (Inositol hexakisphosphate, phytic acid, dipotassium salt,
Sigma-Aldrich, St Louis, Mo.) solutions in culture medium,
corresponding to the following concentrations: 0.3125, 0.625, 1.25,
2.5 and 5 mM, were prepared, and were stored at 4.degree. C. for
4-6 h. Shortly before treatment, IP6 solutions were centrifuged to
remove any precipitates that IP6 forms with calcium chloride. Four
wells were treated for each condition. 48 h after IP6 treatments,
cells were assayed for viability using the CellTiter-Glo.RTM.
Luminescent Cell Viability Assay kit, from PromegaBioSciences, Inc.
(San Luis Obispo, Calif.), following the manufacturer's
instruction. Briefly, the assay buffer and substrate were
equilibrated at room temperature, and mixed thoroughly. Without
removing medium from wells, the assay reagent was added into each
well (1:1, volume:volume of medium per well) and the content was
mixed for 2 minutes on an orbital shaker to induce cell lysis. The
plate was incubated at room temperature for 10 minutes, and the
luminescence was read on a Perkin Elmer
Victor.sup.3microplatereader. Data from each set of four replicates
was averaged and the standard deviation was calculated.
[0153] Statistics:
[0154] The two-tailed Fisher's exact test was used to compare the
staining patterns between the cohorts of primary SCC tumors that
have subsequently metastasized with those that have not. P values
of <0.05 were considered statistically significant.
Example 1
The INPP5A Gene is Frequently Deleted in Cutaneous SCC Tumors
[0155] To identify novel genes and molecular mechanisms associated
with cutaneous SCC (squamous cell carcinoma) development and
progression, a series of archived skin tissues spanning a range
from normal skin to invasive SCC were analyzed. An optimized
high-resolution oligomer aCGH method was used on these archived
FFPE tissues. This approach enabled detecting gene copy number
changes with sensitivity and accuracy comparable with that obtained
by analysis of DNA derived from frozen tissues.
[0156] aCGH was done using the DNA from a spectrum of 40 FFPE skin
tissues, including normal skin (n=12), precancerous lesions of AK
(n=5), in situ SCC lesions (SCCIS; n=2), and invasive SCCs (SCC;
n=21). A total of 458 copy number aberrations were identified in
the examined samples, 267 (58%) of which were amplifications and
191 (42%) were deletions. It was observed that there was an
increase in the overall frequency of gene copy number aberrations
per sample in proportion to the increasing malignant
characteristics of the examined tissue, with invasive SCCs
harboring, on average, the highest number of aberrations per genome
(FIG. 1).
[0157] Examination of the genomic regions characterized by
recurrent copy number changes across samples, as detected by aCGH,
identified a prevalent copy number aberration which was deletion of
the q-ter region of chromosome 10, an area harboring the INPP5A
(inositol polyphosphate 5-phosphatases) gene (FIG. 2). aCGH
detected loss of the INPP5A gene in 1 of 2 examined SCCIS lesions,
and in 5 of 21 (24%) examined invasive SCC tumors, but in none of
the examined AK lesions or normal skin (Table 1).
TABLE-US-00001 TABLE 1 Frequency of INPP5A gene deletions as
detected by CGH Number with INPP5A Tissue Type deletion by aCGH
Normal 0/12 AK 0/5 SCCIS 1/2 SCC 5/21
[0158] To verify the accuracy of aCGH calls, FISH (fluorescent in
situ hybridization) for INPP5A was performed in two of five samples
that showed INPP5A deletions by aCGH. Both cases showed clear
INPP5A loss, whereas control tissues showed no detectable loss of
FISH signal (FIG. 3). Normal metaphase spread is provided as a
reference, and two copies of INPP5A are seen in normal
keratinocytes (FIG. 3, left and right-top panels) with INPP5A
signal in red, but only one copy in SCC (FIG. 3, right-bottom
panel).
[0159] The observed deletions of INPP5A in FIG. 3 represent a
highly selected, nonrandom genetic event in SCC. Most of 191
deletions identified among SCC samples occur only once, whereas
samples of a smaller proportion are observed as recurrent
deletions, affecting more than one sample. The region of INPP5A is
the single most frequently deleted segment in the interrogated SCC
genomes, as well as the only recurrent deletion detected in five
independent SCC samples. The core INPP5A deletion, characterized as
the smallest area of overlap among the aberrations harboring INPP5A
deletions (FIG. 2), covers a genetic segment containing 587,219 bp,
of which 91,861 bp are in the INPP5A gene itself. In addition to
INPP5A, this segment contains three other genes: GPR123, KNDC1, and
VENTX. However, INPP5A is the only gene in this cluster repeatedly
affected by the copy number transition (the edge of the
aberration), being affected in three of five samples harboring
deletion of this region. Taken together, therefore, INPP5A gene
deletions are highly selected genetic events, rather than
nonspecific bystander events in the context of the overall genomic
instability of the SCC genome.
Example 2
INPP5A Protein Level is Frequently Reduced in Primary SCC
Tissues
[0160] Genomic aberrations detected by aCGH, such as gene
deletions, can indicate a "tip of the iceberg" phenomenon, where
loss of a gene on the DNA level is seen in a subset of tumors,
whereas in remaining cases the implicated gene may be deregulated
by other mechanisms, including those affecting its mRNA and protein
products, and thus the loss of a gene on the DNA level is not the
only way by which loss of expressed protein can occur.
[0161] To evaluate whether the genetic loss of INPP5A observed with
aCGH might be similarly indicative of a more general phenomenon of
INPP5A loss in SCC, INPP5A protein levels were examined in an
independent cohort of FFPE skin tissues by immunohistochemistry
using a monoclonal antibody to INPP5A. A total of 71 archived SCC
tumors were evaluated and compared with the matched normal skin
from the same patient using the histologically normal epidermis,
immediately adjacent to the SCC tumor as control. Stained slides
were evaluated using a standard scoring system based on the
intensity of staining (0-3), with score of 0 representing no
staining and score of 3 as intense staining. If a relative
difference in signal was observed between tissues being compared,
it was recorded as a change in INPP5A protein level.
[0162] Detection of INPP5A by immunohistochemistry showed mainly
diffuse cytoplasmic signal. A comparison of INPP5A staining
intensity between SCC tissues and matched normal epidermis
identified three general staining patterns. The most prevalent
pattern of expression, observed in 51 of 71 (72%) examined tissues,
manifested as a relative reduction of INPP5A in SCC tissues when
compared with matched normal skin (FIG. 4A). Only 20 of 71 (28%)
examined tissues showed no difference in INPP5A staining between
the SCC and matched normal skin. Importantly, no single case was
observed where INPP5A staining was more intense in SCC tumor than
in matched normal skin, further highlighting the specificity of the
observed pattern (Table 2. A section; FIG. 4B). Notably, 10 SCC
lesions examined in this cohort were classified by pathology as
SCCIS (cutaneous squamous cell carcinomas in situ, a lesion with
transepidermal keratinocyte atypia, indicating that the entire
epidermis is filled with atypical keratinocytes). Six of 10 of
these SCCIS lesions showed reduced INPP5A immunohistochemical
signal, indicating no significant difference to the frequency
detected in primary SCC in general. The observed reduction of
INPP5A signal in SCC tissues is tumor specific, as the history of
sun exposure and the extent of sun damage are comparable between
the SCC lesion and the examined, adjacent normal epithelium used as
a control. Since loss of INPP5A was shown to be present in a
significant percentage of all stages of SCC, from precursor to
metastatic disease, observing loss at AK, SCCIS, or local SCC
indicates a risk that the lesion where the loss is observed in may
continue to evolve through the stages to eventually become
metastatic SCC. Taken together, a significantly higher frequency of
INPP5A loss at the protein level compared with loss at the DNA
level indicates that gene deletions may represent only one
mechanism by which loss of INPP5A protein production can occur.
There is also a sizable proportion of SCC tumors likely achieve the
same effect through deregulation of INPP5A by other mechanisms to
reduce the INPP5A protein level.
TABLE-US-00002 TABLE 2 Detection of INPP5A protein levels at
successive stages of SCC progression INPP5A Staining Intensity
Frequency A. Primary SCC compared to matched normal skin Normal
skin > Primary SCC 51/71 (72%) Normal skin = Primary SCC 20/71
(28%) Normal skin < Primary SCC 0/71 (0%) B. AK compared to
matched normal skin Normal skin > AK 9/26 (35%) Normal skin = AK
17/26 (65%) Normal skin < AK 0/26 (0%) C. Primary SCC compared
to matched metastatic tissues Primary SCC > Met 6/17 (35%)
Primary SCC = Met 11/17 (65%) Primary SCC < Met 0/17 (0%)
Example 3
INPP5A Loss on the Protein Level is an Early Event in SCC
Development
[0163] To more precisely evaluate the timing of the reduction of
INPP5A level in the development of cutaneous SCC, a series of 26
AKs (actinic keratoses, lesions in which atypical keratinocytes do
no fill the epidermis) were examined, the earliest step in SCC
development. Using immunohistochemistry, as described above, INPP5A
protein levels between the AK lesions and adjacent normal epidermis
were compared. A relative reduction of INPP5A in AK lesions was
seen in 9 of 26 (35%) examined tissues, whereas 17 of 26 (65%)
examined tissues showed no difference in INPP5A levels between the
AK and normal epidermis (Table 2. B.).
[0164] To test whether the reduction of INPP5A protein in AKs is
caused by genetic loss at the DNA level, such as seen in a subset
of SCC tumors, FISH analysis was carried out, and no INPP5A gene
deletion was detected in any of the examined cases. Although a
small lesion size and limited number of lesional nuclei available
for analysis in some of the studied AKs call for cautious
interpretation of these results, it is important to note that no
single lesion showed evidence of a clonal population with uniform
loss of INPP5A FISH signal, even in cases where such clonal loss
was suggested by immunohistochemical data. This absence of
perturbations on DNA level in AKs may explain the relative paucity
of gene copy number aberrations at early stages of disease detected
by aCGH (FIG. 1) and indicates that deregulation of INPP5A
expression in these precursor lesions occurs mainly at the mRNA or
protein level.
[0165] The less frequent reduction of INPP5A protein levels in AK
than in SCC lesions (35% versus 72%) is also informative and
reflects a selection that favors progression of AK lesions with low
INPP5A to the next stage of disease. FIG. 5 showed two
representative lesions, each containing an area of SCCIS (evident
by full epidermal thickness neoplasia; right half of each image),
arising in association with an AK (partial epidermal thickness
neoplastic change, consistent with AK; left half of each image). A
pattern of INPP5A protein reduction in a subset of AKs, occurring
in the form of strikingly demarcated regions of low INPP5A signal
is an evidence for clonally expanding populations of affected cells
within epidermis (FIGS. 5A and 5B). As SCCIS often arise within the
preexisting lesions of AKs, this focal loss of INPP5A in AKs
represents an early step toward a full oncogenic transformation
along the spectrum of evolving epidermal neoplasia.
Example 4
Loss of INPP5A in Association with Progression to Metastatic
Disease
[0166] The above data showed that deregulation of INPP5A levels as
an early event in the development of keratinocyte neoplasia,
provides a selective advantage in progression from AK to SCC. To
assess a potential role of INPP5A loss in the process of tumor
maintenance and progressions, the potential association of
reduction of INPP5A level with the subsequent biological step in
SCC progression and development of metastatic disease was tested.
INPP5A protein levels were evaluated in a selected cohort of 17
patients with cutaneous SCC tumors that have subsequently
metastasized, and INPP5A protein levels were also evaluated where
both primary tumor tissue and matched regional metastatic tissue
were available for examination. Immunohistochemical analysis of
these paired tissues detected further reduction of INPP5A levels in
the transition from primary to metastatic SCC in 6 of 17 (35%)
examined tissue pairs (Table 2. C).
[0167] Although the remaining 11 of 17 (65%) studied pairs show no
further loss of INPP5A levels in transition from primary to
metastatic disease, there is no single case was identified where
INPP5A staining was stronger in the metastatic tissue than in the
primary SCC tumor. This observation further highlighted the
specificity of the observed INPP5A loss in SCC progression. These
data demonstrated that reduction of INPP5A levels, although an
early event in development of SCC, also plays a role in progression
of SCC from primary to metastatic disease in a significant subset
of aggressive primary SCC tumors.
[0168] It was further noted during the INPP5A staining in the
cohort of 17 patients with metastatic SCCs, there were normal
epidermis present immediately adjacent to the primary SCC tissue in
13 of 17 examined primary SCCs. Twelve of 13 (92%) of these
aggressive primary SCC tumors, which is a strikingly high
frequency, showed loss of INPP5A staining when compared with the
adjacent, normal epidermis. In comparison, in randomly selected
primary SCC tumors, 51 of 71 (72%) SCC tumors showed reduction of
INPP5A protein levels by immunohistochemistry (FIG. 4; Table 2A).
Thus, higher frequency of INPP5A loss in primary SCC tumors that
have shown an aggressive clinical course (i.e., subsequent
development of metastases) indicates more aggressive primary
disease. INPP5A level is, therefore, of prognostic value in
assessing the risk of progression in primary SCC tumors.
Example 5
IP6 Reinitiates Terminal Differentiation of Cells with Reduced
INPP5A Expression
[0169] In normal skin, a gradient of increasing levels of INPP5A
expression is seen as keratinocytes move from the basal
proliferative layer and pass through the various non-proliferative
differentiation stages that end at the cornified layer. In both AKs
(actinic keratosis) and tumors, loss of INPP5A expression may
represent an escape mechanism that inappropriately allows these
cells to retain high proliferative capacity. Ectopic expression of
INPP5A in SCC cell lines leads to apoptosis. INPP5A is known to
play a role in the synthesis of inositol hexaphosphate (IP6), and
this compound exhibits effects on growth. Loss of INPP5A activity
allows squamous cells to avoid cessation of growth and terminal
differentiation. Exposure of cells that have lost INPP5A expression
to IP6 reinitiates the terminal differentiation process.
Specifically, exposure to IP6 induces apoptosis in SCC lines and
primary, undifferentiated, proliferating keratinocytes in vitro.
Therefore, the level of INPP5A expression identifies patients that
would benefit from IP6 therapy.
[0170] SCC4 cells were infected with lentiviral vectors that
delivered either CMV-GFP (control) or CMV-INPP5A. The cells were
evaluated two weeks later. A 5-fold induction of INPP5A expression
over baseline was confirmed by Quantitative Real-Time PCR. The q
Real-Time PCR was done on an ABI PRISM.RTM. 7000 device using an
ABI kit per the manufacturer's instructions (Applied Biosystems
Carlsbad, Calif.). The primer pair used for detecting INPP5A m-RNA
expression level were 5'-TTGCAGACTGTCCTTTGAC-3' (SEQ ID NO: 3) and
5'-AAACCCTTCTCGAATCGCTGA-3' (SEQ ID NO: 4). FIG. 6 depicts
increased apoptosis in a squamous cell carcinoma cell line that
carries a gene expression cassette that expresses INPP5A (B and D)
compared to the same cell line carrying a gene expression cassette
that expresses an unrelated protein, green fluorescent protein (A
and C). The top panels of FIG. 6 depict CMV-GFP infected cells and
CMV-INPP5A transfected cells in culture 2 weeks after infection
(20.times.). The bottom panels show CMV-GFP and CMV-INPP5A infected
SCC-4 cells after performance of TUNEL staining for cells
undergoing apoptosis. Nuclei were stained blue using DAPI probe and
apoptosis-positive cells are stained in red (lighter, more
punctuate). Overexpression of INPP5A results in significant cell
death by apoptosis.
[0171] IP6 is an inositol metabolite that is dependant upon INPP5A
activity for its synthesis (See FIG. 7). Addition of exogenous IP6
inhibits the cell proliferation brought about by INPP5A loss and
drives cells towards a differentiated phenotype. In FIG. 8, the
left panel (A) shows untreated SCC-4 cells and the right panel (B)
shows SCC-4 cells treated with IP6. Increased TUNEL staining (red)
is seen in the IP6 treated cells. Therefore, treatment of SCC-4
cells with IP6 results in significant cell death by apoptosis,
similar to the result seen in FIG. 8 with the overexpression of
INPP5A.
[0172] IP6 is selective for cells with reduced INPP5A expression.
Normal human keratinocytes were harvested at various stages of
confluency. Cell cultures were assessed by morphological evaluation
of the cell and segregated into distinct culture confluence
intervals at or near the plateau phase of growth, generally; lower
confluence (L), 70-80%; medium confluence (M), 80-90%, and higher
confluence (H), >95%, INPP5A mRNA expression at selected culture
confluence levels was evaluated by qPCR. INPP5A mRNA expression by
q-RTPCR as a function of % of confluence of normal human
keratinocytes are provided in Table 3 and FIG. 9.
TABLE-US-00003 TABLE 3 INPP5A mRNA expression at selected
confluence level Confluence INPP5A mRNA Fold Change 70% 1 90% 2.8
100% 3.4 4-d post 4.1
[0173] INPP5A protein level in cells at different confluence levels
was measured using Western Blot (FIG. 10 and FIG. 11).
Cytokeratin-10 (CK10) and integrin .beta.4 are two known markers
associated with keratinocyte differentiation. Keratinocyte
differentiation level as a function of increased confluence was
demonstrated by increased expression of Cytokeratin-10 (CK10) mRNA
and decreased expression of integrin .beta.4 mRNA as determined by
qPCR using ABI TaqMan kits for detecting human KRT10 and human
ITGB4 mRNA and provided in Table 4. These results are consistent
with the expression patterns in keratinocyte differentiation.
TABLE-US-00004 TABLE 4 Expression of known markers of keratinocyte
differentiation at selected confluence level Fold Induction mRNA
NHK Confluence KRT10 ITGB4 70% 1.00 1.00 90% 6.34 1.51 100% 124.34
0.38 4-day post 5294.22 0.66
[0174] FIG. 12 and FIG. 13 depict reduced apoptosis in more highly
confluent and differentiated keratinocytes. FIG. 12, left panel (A)
shows 100% confluent keratinocytes that were untreated. and the
right panel (B) shows 100% confluent keratinocytes treated with 2
mM IP6 for 24 hours. FIG. 13 left panel (A) shows 70% confluent
keratinocytes that were untreated; and the right panel (D) shows
70% confluent keratinocytes that were treated with 2 mM IP-6 for 24
hours. Greater apoptosis relative to background was seen in the 70%
confluent keratinocytes in comparison to the 100% confluent
keratinocytes.
Example 6
INPP5A Cellular Characterization
[0175] INPP5A was delivered to cells in a controlled expression
vector pTUNE, a derivative developed from a commercial vector (FIG.
14). Introduction of INPP5A with controlled expression allows
production of a small amount of the protein. However, in the head
and neck cancer cell line Cal27, the small amount of the INPP5A
protein expression was sufficient to reduce migration in a scratch
assay by 40%. A comparison of a 24-hour assay in the absence or
presence of INPP5A in the Cal27 line is shown by the scratch assay
shown in FIG. 15. While CAL27 did not show a growth reduction after
INPP5A transfection, it did show a marked loss in motility (FIG.
15). A scratch assay shows fill-in of an approximately 1 mm scratch
in 8 hours for the unmodified CAL27 line, but no fill in over 24
hours in the Cal-27+INPP5A transfectants. The growth rates of head
and neck squamous cell carcinoma cell lines with and without the
INPP5A expression plasmid were tested. Cell lines (Table 5) were
plated in 12 well plates. Cal-27 and SCC-15 were grown in DMEM
media, and SCC-9, -4, and -25 were grown in DMEM F12 media. All
cell lines were grown to 70% confluence. 1.6 micrograms of AhdI
linearized pTUNE INPP5A (Origene, Rockville, Md.) were transfected
into the cell lines using lipofectamine and Optimem media
(Invitrogen, Carlsbad, Calif.). After transfection, cells carrying
the INPP5A bearing vector were selected with G418 at 1 microgram/ml
for CAL27 and at 0.5 microgram/ml for the remaining cell lines.
Cells were examined microscopically for outgrowth of transformants
and those cell lines not growing were examined with the vital
fluorescent stain Vybrant Violet (Invitrogen, Carlsbad, Calif.) to
observe apoptotic nuclear decay. The results are provided in Table
5 below.
TABLE-US-00005 TABLE 5 Effects of INPP5A expression on growth of
selected cell lines: Cell Doubling time Doubling Line Control Time
+ INPP5A Comments Cal-27 1-2 days 1-2 days no change SCC-9 4-5 Days
4-5 days no change SCC-15 2-3 days NA Slowly dying over 16 days
post transfection (FIG. 18) SCC-25 5-6 days NA Slowly dying over 12
days post transfection SCC-4 4-5 days NA Cells all died within 4
days of transfection
[0176] Note that in SCC15+INPP5A and SCC-25+INPP5A, a vital nuclear
dye shows pycnotic nuclei and apoptosis. FIG. 16 depicts SCC
15+INPP5A stained with Vybrant Violet, and the small, dark pycnotic
nuclei were shown in apopotosis. Similar results were seen in
SCC-25. Table 6 showed the q-Real-Time PCR results of INPP5A mRNA
level in selected cell lines normalized to ACTB, a control gene.
The primer used for INPP5A q-Real-Time PCR were
5'-TTGCAGACTGTCCTTTGAC-3' (SEQ ID NO: 3) and
5'-AAACCCTTCTCGAATCGCTGA-3' (SEQ ID NO: 4). The primers used for
ACTB q-Real-Time PCR was 5'-TCATGAAGTGTGACGTGGACATC-3' (SEQ ID NO:
5) and 5'-CAGGAGGAGCAATGATCTTGATCT-3' (SEQ ID NO: 6).
TABLE-US-00006 TABLE 6 q-Real-Time PCR of INPP5A mRNA level in
selected cell lines normalized to ACTB Cell Line Critical Cycle
INPP5A-ACTB % of ACTB CAL27 3.46 9.1% CAL27 + INPP5A 2.99 12.6%
SCC25 3.44 9.2% SCC15 3.32 10.0% SCC9 3.80 7.2% SCC4 3.66 7.9%
Example 7
Dose-Dependent IP6 Treatment of Squamous Cell Carcinoma
[0177] The high frequency of loss of INPP5A in skin squamous cell
carcinoma (SCC) and as well as its loss in sun damaged skin, an
early precursor of SCC, was demonstrated in above examples. As
shown in FIG. 5 and FIG. 17, immunohistochemical staining for the
presence of INPP5A demonstrated that INPP5A normally appears at a
higher and higher level as keratinocytes go through the various
stages of differentiation in the transition from actively growing
precursor cells to the dead, cornified cells of the outer dermis.
Without being bound by theory, one way that INPP5A could be
involved in this differentiation process is that it acts as a
requisite enzyme in the formation of a small molecule involved in
cell signaling. As INPP5A mediates the dephosphorylation of the 5
phospho group on either Ins(1,4,5)P3 or Ins(1,3,4,5)P4, the test in
this example was carried out to determine whether restoring the
phospho-inositol metabolites that are downstream of the
INPP5A-mediated step to SCC cancer cell lines could lead to
differentiation and death.
[0178] The diagram in FIG. 7 shows two inositol phosphate metabolic
branches resulting directly from the action of INPP5A. In this
invention, IP6 was tested for activity against a number of squamous
cell cancer lines derived from head and neck tumors (SCC-4 SCC-15
SCC-9), and both a colorectal adenocarcinoma (HT-29) and a
transformed normal human embryonic kidney cell line (HEK-293).
Cultures of 600 cells for each of these lines in growth media were
established in wells of a 384 well plate, and then sets of four
replicates per dose were treated by the addition of no drug, 0.31
mM, 0.63 mM, 1.25 mM, 2.5 mM and 5 mM IP6. These treated and
untreated cultures were allowed to grow for 72 hours in a tissue
culture incubator and then were treated with and equal volume of
Cell Titer Glow reagent, which lyses the cells and produces light
through a reaction utilizing the ATP available from the cells.
[0179] The proportion of cells present after treatment relative to
the number of cells in the untreated cells was shown in FIG. 18. As
demonstrated, the cells showed a dose-dependent reduction in
number, which is much greater for the SCC cells than for the
colorectal adenocarcinoma or the transformed human embryonic kidney
cells. This data presented that INPP5A's loss leads to a failure of
cells produced by less differentiated basal cells to cease
proliferating and differentiate to from a liner layer and that this
loss of regulation can to some extent be corrected by supplying an
inositol phosphate metabolite whose synthesis normally requires the
activity of INPP5A. Therefore the detection of loss of INPP5A
activity in tumors that are dependent on this loss for their
survival will prove useful in directing therapy for these tumors.
Sequence CWU 1
1
612938DNAHomo sapiens 1gcgcgggccg ctgtgaggcg cggcggcgag cgacgggcgc
ggggccgcgg agcagcgagc 60gagcgagcga gcgcgaggcc ggagccccgg ccaggcccgg
ccgacccgcc gagcccgcga 120tgcgccccgg ggccgccccc cggcgcagct
gacgccccgc ggccccgcga agaccccggc 180cggccggtcc cggaggaagc
ggccgccgcc gccgccgccc agcccagcgc ccgcgccgcc 240cgggcaccat
ggcggggaag gcggccgccc cgggcaccgc ggtgctgctg gtcacggcca
300acgtgggctc gctcttcgac gacccagaaa acctgcagaa gaactggctt
cgggaatttt 360accaggtcgt gcacacacac aagccgcact tcatggcctt
gcactgtcag gagtttggag 420ggaagaacta cgaggcctcc atgtcccacg
tggacaagtt cgtcaaagaa ctattgtcga 480gtgatgcgat gaaagaatat
aacagggctc gagtctacct ggatgaaaac tacaaatccc 540aggagcactt
cacggcacta ggaagctttt attttcttca tgagtcctta aaaaacatct
600accagtttga ctttaaagct aagaagtata gaaaggtcgc tggcaaagag
atctactcgg 660ataccttaga gagcacgccc atgctggaga aggagaagtt
tccgcaggac tacttccccg 720agtgcaaatg gtcaagaaaa ggcttcatcc
ggacgaggtg gtgcattgca gactgtgcct 780ttgacttggt gaatatccat
cttttccatg atgcttccaa tctggtcgcc tgggaaacaa 840gcccttccgt
gtactcggga atccggcaca aggcactggg ctacgtgctg gacagaatca
900ttgatcagcg attcgagaag gtttcctact ttgtatttgg tgatttcaac
ttccggctgg 960attccaagtc cgtcgtggag acgctctgca caaaagccac
catgcagacg gtccgggccg 1020ccgacaccaa tgaagtggtg aagctcatat
ttcgtgagtc ggacaacgac cggaaggtta 1080tgctccagtt agaaaagaaa
ctcttcgact acttcaacca ggaggttttc cgagacaaca 1140acggcaccgc
gctcttggag tttgacaagg agttgtctgt ctttaaggac agactgtatg
1200aactggacat ctcgttccct cccagctacc cgtacagtga ggacgcccgc
cagggtgagc 1260agtacatgaa cacccggtgc ccagcctggt gtgaccgcat
cctcatgtcc ccgtctgcca 1320aggagctggt gctgcggtcg gagagcgagg
agaaggttgt cacctatgac cacattgggc 1380ccaacgtctg catgggagac
cacaagcccg tgttcctggc cttccgaatc atgcccgggg 1440caggtaaacc
tcatgcccat gtgcacaagt gttgtgtcgt gcagtgacgt ggtgggaaga
1500gatgccagcg ccacgagagg acacttcgtg agcctccctg tagccgtgga
ccgaatacgc 1560actcttgaaa gctgcatcga gaacccgccc aagcgccacc
tgctagacgg ccagccccac 1620acttcgcttc agcctccgga ccattccgga
gcagcctcac atacctcact gtctcgtctg 1680tctatgtgac attaagtaga
aatattggtt tttttttttt ttttttaaat aagtcacagt 1740cctgttgtca
aaactctaat agacagcaaa gagggtctgt accgtagact tcacagtttt
1800cagtttttaa tgattgccag tggaggggct tcttcagcac agagaccccc
cactgtgtcc 1860agggaccccc tctgccaggt ggaggtgtgt ccaggggctg
gggaagccga gacgggcact 1920ccctctgccg gccggcagcg tggccctgag
catggcaagg gggtctgtct ctgccgatgc 1980tccttccgcg gcactgactc
tgcgccgtgt cacatggttt ttgaatcaca ctgcagctgc 2040tttccatttt
tatatatata taaatatata taaatatata ctttttaaaa ataatttata
2100aatcttacca aaacttatgc taaatatact ttccagtatg aacgcacagg
agagtcccat 2160cagcaggcgg cattggagtc taggagctca gctgtgtgtc
catcaacaca caaattcgta 2220aaaaacacac atggcctcgc catcgtgggt
aaaatcggcc ccacagcacg tctgcaccag 2280cgggccgtta ctcccatgcc
gttcttctgt gtaatattaa gaactgaatg tgaagtttat 2340agctagcctg
ggtgtacctt ttaagaattt tgtaaaccgt ttgtctgtct tttgttactg
2400ttttatggtg ccaagtatcc tacgttacaa caataatatc atgggagaaa
tagaaatagc 2460ctagtttgct tccaatagaa actgctttta acatgggctg
tatataaaaa tattaaagag 2520aaacaaaact gtacatttcc tcattgctcc
gctacagaca acccatgtca taaccttgtt 2580gcaaatattt ttctcctata
gcagtaagta cagcattaga aggtgattag agagtctgtt 2640gatgaaacac
aaatgtatgt tttattgatt ttactttaga acactacaga gttcctggac
2700cgggtgaagg cattagctgg gtgtttgtgt gggataaata ctaccactgc
aagtgactgc 2760tgtccgctgc ggaatctgtt cttggtggaa gcacaggtcc
gtgtcgctgc tgtggttgcc 2820gctgtccgcg gttcaacacg gagtccgccc
cgcgggtttc agctgttggt cgttctgagg 2880ggcctttgga agtgaccggt
ctggttccta agcaataaaa ttgaccgtgg tgaaaata 29382412PRTHomo sapiens
2Met Ala Gly Lys Ala Ala Ala Pro Gly Thr Ala Val Leu Leu Val Thr 1
5 10 15 Ala Asn Val Gly Ser Leu Phe Asp Asp Pro Glu Asn Leu Gln Lys
Asn 20 25 30 Trp Leu Arg Glu Phe Tyr Gln Val Val His Thr His Lys
Pro His Phe 35 40 45 Met Ala Leu His Cys Gln Glu Phe Gly Gly Lys
Asn Tyr Glu Ala Ser 50 55 60 Met Ser His Val Asp Lys Phe Val Lys
Glu Leu Leu Ser Ser Asp Ala 65 70 75 80 Met Lys Glu Tyr Asn Arg Ala
Arg Val Tyr Leu Asp Glu Asn Tyr Lys 85 90 95 Ser Gln Glu His Phe
Thr Ala Leu Gly Ser Phe Tyr Phe Leu His Glu 100 105 110 Ser Leu Lys
Asn Ile Tyr Gln Phe Asp Phe Lys Ala Lys Lys Tyr Arg 115 120 125 Lys
Val Ala Gly Lys Glu Ile Tyr Ser Asp Thr Leu Glu Ser Thr Pro 130 135
140 Met Leu Glu Lys Glu Lys Phe Pro Gln Asp Tyr Phe Pro Glu Cys Lys
145 150 155 160 Trp Ser Arg Lys Gly Phe Ile Arg Thr Arg Trp Cys Ile
Ala Asp Cys 165 170 175 Ala Phe Asp Leu Val Asn Ile His Leu Phe His
Asp Ala Ser Asn Leu 180 185 190 Val Ala Trp Glu Thr Ser Pro Ser Val
Tyr Ser Gly Ile Arg His Lys 195 200 205 Ala Leu Gly Tyr Val Leu Asp
Arg Ile Ile Asp Gln Arg Phe Glu Lys 210 215 220 Val Ser Tyr Phe Val
Phe Gly Asp Phe Asn Phe Arg Leu Asp Ser Lys 225 230 235 240 Ser Val
Val Glu Thr Leu Cys Thr Lys Ala Thr Met Gln Thr Val Arg 245 250 255
Ala Ala Asp Thr Asn Glu Val Val Lys Leu Ile Phe Arg Glu Ser Asp 260
265 270 Asn Asp Arg Lys Val Met Leu Gln Leu Glu Lys Lys Leu Phe Asp
Tyr 275 280 285 Phe Asn Gln Glu Val Phe Arg Asp Asn Asn Gly Thr Ala
Leu Leu Glu 290 295 300 Phe Asp Lys Glu Leu Ser Val Phe Lys Asp Arg
Leu Tyr Glu Leu Asp 305 310 315 320 Ile Ser Phe Pro Pro Ser Tyr Pro
Tyr Ser Glu Asp Ala Arg Gln Gly 325 330 335 Glu Gln Tyr Met Asn Thr
Arg Cys Pro Ala Trp Cys Asp Arg Ile Leu 340 345 350 Met Ser Pro Ser
Ala Lys Glu Leu Val Leu Arg Ser Glu Ser Glu Glu 355 360 365 Lys Val
Val Thr Tyr Asp His Ile Gly Pro Asn Val Cys Met Gly Asp 370 375 380
His Lys Pro Val Phe Leu Ala Phe Arg Ile Met Pro Gly Ala Gly Lys 385
390 395 400 Pro His Ala His Val His Lys Cys Cys Val Val Gln 405 410
320DNAArtificial SequenceOligonucleotide Primer 3ttgcagactg
tgcctttgac 20420DNAArtificial SequenceOligonucleotide Primer
4aaaccttctc gaatcgctga 20523DNAArtificial SequenceOligonucleotide
Primer 5tcatgaagtg tgacgtggac atc 23624DNAArtificial
SequenceOligonucleotide Primer 6caggaggagc aatgatcttg atct 24
* * * * *