U.S. patent application number 13/830668 was filed with the patent office on 2013-11-14 for methods for diagnosing or treating prostate cancer.
The applicant listed for this patent is ONCOTHERAPY SCIENCE, INC.. Invention is credited to HIDEWAKI NAKAGAWA, YUSUKE NAKAMURA, TAKUYA TSUNODA.
Application Number | 20130302799 13/830668 |
Document ID | / |
Family ID | 42198015 |
Filed Date | 2013-11-14 |
United States Patent
Application |
20130302799 |
Kind Code |
A1 |
NAKAMURA; YUSUKE ; et
al. |
November 14, 2013 |
METHODS FOR DIAGNOSING OR TREATING PROSTATE CANCER
Abstract
The present invention provides methods for detecting and/or
diagnosing cancer through the determination of the expression level
of the STC2 gene. The gene was discovered to discriminate cancer
cells from normal cells. Furthermore, the present invention
provides methods of screening for therapeutic agents useful in the
treatment of cancer, methods for treating cancer. Moreover, the
present invention provides double-stranded molecules targeting the
STC2 gene, which are suggested to be useful in the treatment of
cancer. The compositions and methods of the present invention find
particular applicability to prostate cancer, more specifically,
castration-resistant prostate cancer and aggressive prostate
cancer.
Inventors: |
NAKAMURA; YUSUKE; (TOKYO,
JP) ; NAKAGAWA; HIDEWAKI; (TOKYO, JP) ;
TSUNODA; TAKUYA; (KANAGAWA, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ONCOTHERAPY SCIENCE, INC. |
Kanagawa |
|
JP |
|
|
Family ID: |
42198015 |
Appl. No.: |
13/830668 |
Filed: |
March 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13130284 |
Aug 17, 2011 |
8420329 |
|
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PCT/JP2009/006201 |
Nov 18, 2009 |
|
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13830668 |
|
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|
61199964 |
Nov 20, 2008 |
|
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Current U.S.
Class: |
435/6.11 ;
435/6.12 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 15/1136 20130101; C12Q 1/6886 20130101; G01N 33/57434
20130101; G01N 2500/04 20130101; C12Q 2600/136 20130101; C12N
2310/14 20130101; C12Q 2600/112 20130101; A61P 13/08 20180101; C12N
2310/531 20130101 |
Class at
Publication: |
435/6.11 ;
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for diagnosing prostate cancer or determining a
predisposition for developing prostate cancer in a subject,
comprising the steps of: (a) determining the expression level of
the stanniocalcin 2 (STC2) gene in a subject-derived biological
sample wherein said expression level is determined by detecting a
transcription product of the STC2 gene; (b) comparing the
subject-derived expression level determined in step (a) with a
normal or cancerous control level; and (c) correlating an increase
of said subject-derived expression level as compared to the normal
control level to a diagnosis of prostate cancer or a predisposition
for developing prostate cancer, or correlating a similarity between
said subject-derived expression level and the cancerous control
level to a diagnosis of prostate cancer or a predisposition for
developing prostate cancer.
2. (canceled)
3. The method of claim 1, wherein said subject-derived biological
sample comprises prostate tissue, blood, or serum.
4. The method of claim 1, wherein said prostate cancer is
castration-resistant prostate cancer or aggressive prostate
cancer.
5. The method of claim 4, wherein said aggressive prostate cancer
is prostate cancer with Gleason score 8 to 10.
6.-26. (canceled)
Description
PRIORITY
[0001] The present application is a divisional of U.S. application
Ser. No. 13/130,284, filed Aug. 17, 2011, which is a U.S. National
Stage Application of PCT/JP2009/006201, filed Nov. 18, 2009, which
claims the benefit of U.S. Provisional Application No. 61/199,964,
filed on Nov. 20, 2008, the entire contents of which are
incorporated by reference herein in their entirety.
REFERENCE TO SEQUENCE LISTING
[0002] This application includes a Sequence Listing as a text file
named "87331-0018011US-869689_SEQLIST.txt" created Mar. 13, 2013,
and containing 12,339 bytes. The material contained in this text
file is incorporated by reference in its entirety for all
purposes.
TECHNICAL FIELD
[0003] The present invention relates to the field of biological
science, more specifically to the field of cancer diagnosis and
treatment. In particular, the present invention relates to methods
for detecting and diagnosing prostate cancer as well as methods for
treating and preventing prostate cancer. Moreover, the present
invention relates to methods for screening for a candidate compound
for treating and/or preventing prostate cancer. The present
invention further relates to double-stranded molecules that reduce
or inhibit the expression of STC2 and uses thereof.
BACKGROUND ART
[0004] Prostate cancer (PC) is the most common malignancy in males
and the second leading cause of cancer-related deaths in the United
States and Europe, and frequency of PC has been increasing
significantly in most developed countries probably due to prevalent
western-style life-style and the explosion of the aging population
(NPL 1:Gronberg H, Lancet 2003, 361, 859-64, and NPL 2: Hsing A W
and Devesa S S, Epidemiol Rev 2001, 23:3-13). Surgical and
radiation therapies are effective to the localized disease, but
nearly 30% of treated PC patients still suffer from the relapse of
the disease (NPL 3:Feldman B J and Feldman D, Nat Rev Cancer 2001,
1, 34-45, NPL 4:Scher H I and Sawyers C L, J Clin Oncol 2006, 23,
8253-61, and NPL 5:Han M, et al., J Urol 2001, 166, 416-9). Most of
the patients with relapsed or advanced disease respond well to
androgen-ablation therapy (medical or surgical castration) because
PCs are usually androgen-dependent at a relatively early stage.
However, they often acquire castration-resistant phenotype that
progresses aggressively and ultimately leads to the death of PC
patients. Hence, development of new therapies based on the
molecular mechanisms of prostate carcinogenesis or
castration-resistant PC (CRPC) is urgently and eagerly
required.
[0005] With that goal in mind, the present inventors previously
performed genome-wide cDNA microarray analysis of CRPC cells and
Castration-naive PC (CNPC) cells purified from clinical PC tissues
by means of microdissection and identified dozens of genes whose
expression levels were evidently increased in CRPC cells and/or
CNPC cells, as compared to normal prostatic epithelial cells (NPL
6:Tamura K, et al., Cancer Res 2007, 67, 5117-5125). However, to
date, genes useful for diagnosing and treating CRPC have not been
identified.
[0006] Both castration-naive prostate cancers (CNPCs) with high
Gleason score, and castration-resistant prostate cancers (CRPCs)
respond poorly to androgen-ablation therapy and have highly
aggressive behavior and thus are associated with poor prognosis.
True et al. demonstrated a significant association between high
levels of monoamine oxidase A (MAOA) expression and high-grade
(Gleason patterns 4 and 5) PCs (NPL 7:True L, et al. PNAS 103,
10991-6, 2006) and Heeboll et al. revealed an increased expression
of SMARCC1 protein in PC and a positive correlation with tumor
dedifferentiation, progression, metastasis and time to recurrence
(NPL 8:Heeboll S, et al., Histol Histopathol 2008, 23, 1069-76).
However, to date, candidate secreted proteins correlated with
Gleason score 8-10 have not been reported.
[0007] Accordingly, an objective of the present invention is to
provide a new biomarker and new therapeutic strategies against PCs,
in particular, CRPCs and aggressive PCs with high Gleason score. As
discussed in detail herein, analysis of genome-wide expression
profiles of PC cells suggest that the molecular target, STC2, is
such a marker, having utility in the both the treatment and
diagnosis of prostate cancer.
[0008] Stanniocalcin (STC) was first identified as a glycoprotein
hormone secreted from specific endocrine glands (corpuscle of
Stannius) in the kidney region of bony fish that is involved in
calcium and phosphate homeostasis (NPL 9:Wagner G F, et al., Mol
Cell Endocrinol 1991, 79, 129-38, and NPL 10:Wagner G F and
Jaworski E, Mol Cell Endocrinol 1994, 99, 315-22). STC is released
into the blood in response to rising serum calcium levels to
regulate the Ca.sup.2+ and phosphate uptake in different target
organs (NPL 9:Wagner G F, et al., Mol Cell Endocrinol 1991, 79,
129-38, and NPL 10:Wagner G F and Jaworski E, Mol Cell Endocrinol
1994, 99, 315-22). In mammals that lack a specific corpuscle of
stannius gland, two related mammalian genes have been identified,
STC1 and STC2, both of which are predicted to be secreted
glycosylated proteins (NPL 11:Chang A C, et al., Mol Cell
Endocrinol 1995, 112, 241-7, and NPL 12:Ishibashi K, et al. Biochem
Biophys Res Commun 1998, 250, 252-8). However, their physiological
or pathological functions in human beings and human cancers have
not been clearly elucidated, and their receptor binding partners
have not yet identified.
[0009] Recent reports suggested that HIF-1 could regulate STC2
expression (NPL 13:Law A Y, et al., Exp Cell Res 2008, 314,
1823-30). In addition, a proportion of renal cancers have been
observed to over-express STC2 and, in turn, correlated with
aggressiveness and poor prognosis of renal cancer (NPL 14:Meyer H
A, et al., Eur Urol 2008 Apr. 9). Another in-vitro study suggested
that STC2 could potentially protect the cells from various cell
stresses, especially ER stress (NPL 15:Ito D, et al., Mol Cell Biol
2004, 9456-69). However, whether STC2 is associated with PC
progression is completely unknown, and possibility of targeting
STC2 function or activity itself is yet to be known.
CITATION LIST
Non Patent Literature
[0010] [NPL 1] Gronberg H, Lancet 2003, 361, 859-64 [0011] [NPL 2]
Hsing A W and Devesa S S, Epidemiol Rev 2001, 23:3-13 [0012] [NPL
3] Feldman B J and Feldman D, Nat Rev Cancer 2001, 1, 34-45 [0013]
[NPL 4] Scher H I and Sawyers C L, J Clin Oncol 2006, 23, 8253-61
[0014] [NPL 5] Han M, et al., J Urol 2001, 166, 416-9 [0015] [NPL
6] Tamura K, et al., Cancer Res 2007, 67, 5117-5125[NPL 7] [0016]
[NPL 7] True L, et al. PNAS 103, 10991-6, 2006 [0017] [NPL 8]
Heeboll S, et al., Histol Histopathol 2008, 23, 1069-76 [0018] [NPL
9] Wagner G F, et al., Mol Cell Endocrinol 1991, 79, 129-38 [0019]
[NPL 10] Wagner G F and Jaworski E, Mol Cell Endocrinol 1994, 99,
315-22 [0020] [NPL 11] Chang A C, et al., Mol Cell Endocrinol 1995,
112, 241-7 [0021] [NPL 12] Ishibashi K, et al. Biochem Biophys Res
Commun 1998, 250, 252-8 [0022] [NPL 13] Law A Y, et al., Exp Cell
Res 2008, 314, 1823-30 [0023] [NPL 14] Meyer H A, et al., Eur Urol
2008 Apr. 9 [0024] [NPL 15] Ito D, et al., Mol Cell Biol 2004,
9456-69
SUMMARY OF INVENTION
[0025] The present invention relates to the identification of STC2
as a molecular target useful in PC treatment and diagnosis. The
STC2 protein finds particular utility as a molecular target for
development of novel treatments for PC. Preferred PCs targeted by
the prevent invention are CRPC and aggressive PC.
[0026] Accordingly, it is an object of the present invention to
provide a method for diagnosing or determining a predisposition to
PC in a subject by determining the expression level of the STC2
gene in a subject-derived biological sample, such as tissue, blood,
or serum. An increase in the level of expression of the gene as
compared to a normal control level indicates that the subject
suffers from or is at risk of developing PC. The normal control
level may be the expression level of the STC2 gene detected in a
normal healthy tissue, blood, or serum of an individual or
population known to be free from PC.
[0027] Alternatively, expression level of the STC2 gene in a sample
can be compared to a cancerous control level of the STC2 gene. A
similarity between the expression level of a sample and the
cancerous control level indicates that the subject suffers from or
is at risk of developing PC. The cancerous control level may be the
expression level of the STC2 gene detected in the cancerous tissue,
blood, or serum of an individual or population known to be
suffering from PC. It is another object of the present invention to
provide a kit for diagnosing PC, such a kit minimally containing a
reagent that measures the expression level of the STC2 gene in a
subject-derived biological sample.
[0028] It is a further object of the present invention to provide
methods for identifying agents that bind to the STC2 protein or
inhibit the activity of the STC2 protein, such methods involving
the steps of contacting the STC2 polypeptide with a test agent and
determining the binding between the polypeptide and the test agent
or a biological activity of the polypeptide. The test agent binding
the polypeptide or suppressing a biological activity of the
polypeptide may be used to reduce symptoms of PC.
[0029] The present invention also provides methods for identifying
agents that inhibit the expression or activity of the STC2 protein,
such method involving the steps of contacting a cell expressing the
STC2 protein with a test agent and determining the expression level
of the STC2 gene or the activity of the gene product, the STC2
protein. A decrease in the expression level of the gene or an
activity of its gene product as compared to a control level in the
absence of the test compound indicates that the test compound may
be used to reduce symptoms of PC.
[0030] Furthermore, the present invention provides methods for
identifying agents that inhibit the transcription of the STC2 gene,
such methods involving the steps of contacting a test compound with
the cell introduced with a vector that contains the transcriptional
regulatory region of the STC2 gene and a reporter gene expressed
under the control of the transcriptional regulatory region and
determining the expression level or activity of the reporter gene.
A decrease in the expression level of the gene or an activity of
its gene product as compared to a control level in the absence of
the test compound indicates that the test compound may be used to
reduce symptoms of PC.
[0031] It is yet a further object of the present invention to
provide double-stranded molecules and vectors encoding such
molecules, both of which find utility in the treatment of PC. The
double-stranded molecules include a sense strand and an antisense
strand complementary thereto, hybridized to each other to form the
double-stranded molecule and inhibit expression of the gene and
cell proliferation when introduced into a cell expressing the STC2
gene. Preferably, the sense strand includes the sequence
corresponding to a target sequence of SEQ ID NO:8 or 9.
[0032] Another aspect of the present invention relates to
compositions for treating PC containing at least one of those
siRNAs that inhibits cell proliferation of a cell expressing STC2
gene and methods for treating or preventing PC that includes the
step of administering to a subject those compositions. The
antisense polynucleotide or siRNA can be provided as a vector
expressing those.
[0033] One advantage of the methods and kits described herein is
that the disease may be identified prior to detection of overt
clinical symptoms of PC. Another advantage of the methods and
compositions described herein is that novel therapeutic approach
with fewer adverse effect may be provided.
[0034] Other features and advantages of the present invention will
be apparent from the following detailed description, and from the
claims. To that end, it will be understood by those skilled in the
art that one or more aspects of this invention can meet certain
objectives, while one or more other aspects can meet certain other
objectives. Each objective may not apply equally, in all its
respects, to every aspect of this invention. As such, the preceding
objects can be viewed in the alternative with respect to any one
aspect of this invention. These and other objects and features of
the invention will become more fully apparent when the following
detailed description is read in conjunction with the accompanying
figures and examples. However, it is to be understood that both the
foregoing summary of the invention and the following detailed
description are of a preferred embodiment, and not restrictive of
the invention or other alternate embodiments of the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0035] Various aspects and applications of the present invention
will become apparent to the skilled artisan upon consideration of
the brief description of the figures and the detailed description
of the present invention and its preferred embodiments that
follows:
[0036] [FIG. 1A-C]
[0037] FIG. 1 demonstrates the over-expression of STC2 in CRPC
cells and immunohistochemical analysis in PC tissues. Part (A)
depicts the results of semi-quantitative RT-PCR validating STC2
over-expression in the microdissected CRPC cells as compared to
CNPC cells and normal prostatic epithelial (NP) cells, which were
also microdissected. ACTB was used to quantify the each of cDNA
contents. Part (B) depicts the results of real-time quantitative
RT-PCR demonstrating over-expression of STC2 transcript in CRPC
cells (samples 2-8) as compared to that of CNPC cells (samples
9-15) and NP cells (sample 1). ACTB was used to quantify each of
the cDNA contents, and the relative quantity (Y-axis) was
calculated so that the expression in NP cells was one. Real-time
quantitative RT-PCR was carried out duplicated for each sample
(white and black columns). Part (C) depicts the results of Northern
blot analysis demonstrating a high level of STC2 expression in five
PC cell lines (lane 1-5), as compared to adult normal organs,
including brain, heart, kidney, liver, lung, prostate and testis
(lanes 6-12) wherein its expression is barely detectable.
[0038] [FIG. 1D-H]
[0039] Part (D) depicts the results of multiple tissue Northern
blot analysis demonstrating that STC2 was expressed only in normal
pancreas, with no or very low expression of STC2 in adult normal
organs. P. B. leukocyte means peripheral blood leukocyte. The
length of STC2 transcript was about .about.5.4 kb. Parts (E)-(H)
demonstrate the immunoreactivity with anti-STC2 antibody observed
in CRPC and CNPC with Gleason score 10 tissues examined, with
strong positive immunostaining exhibited in the cytoplasm of PC
cells. Part (E) is a representative picture of CRPC (.times.200),
part (F) is a representative picture of CNPC with Gleason score 10
(.times.200), and part (G) is a representative picture of CNPC with
Gleason score 7 (.times.200). Adjacent normal prostatic epithelium
revealed very weak or no signal for STC2 (F and G; arrowheads).
Part (H) depicts the relationship between STC2 immunohistochemical
score and Gleason score in CNPCs. (*p<0.003, Mann-Whitney's
U-test with Bonferroni method).
[0040] [FIG. 2]
[0041] FIG. 2 demonstrates the knockdown of STC2 expression by
siRNA attenuated PC cell viability and the promotion of cancer cell
growth by STC2 over-expression. Part (A) depicts the knockdown
effect of siRNA on STC2 in PC-3 cells. Semi-quantitative RT-PCR was
performed using cells transfected with each of shRNA-expressing
vectors to STC2 (si1-3) as well as a negative control vector
(siEGFP). ACTB was used to quantify RNAs. Part (B) depicts the
results of a colony formation assay of PC-3 cells transfected with
each of indicated shRNA-expressing vectors to STC2 (si1-3) and a
negative control vector (siEGFP). Cells were visualized with 0.1%
crystal violet staining after 14-day incubation with Geneticin.
Part (C) depicts the results of MTT assay of each of PC-3 cells
transfected with indicated siRNA-expressing vectors to STC2 (si1-3)
and a negative control vector (siEGFP). Each average is plotted
with error bars indicating SD (standard deviation) after 10-day
incubation with Geneticin. ABS on Y-axis means absorbance at 490
nm, and at 630 nm as reference, measured with a microplate reader.
These experiments were carried out in triplicate (*P<0.01,
Students' t-test). Part (D) depicts the results of Western blot
analysis with anti-HA-tag antibody, demonstrating that three stable
transformats (Clones 1-3) constitutively expressed an exogenous
STC2. Mock was the 22Rv1-mock clone mixture. CBB stain served as a
loading control. Part (E) depicts the in-vitro growth curve
calculated by MTT assay, showing that three stable transformants
(Clones 1-3) grew more rapidly than the 22Rv1-mock clone mixture
(*P<0.01, **P<0.05, Students' t-test). ABS on Y-axis means
absorbance at 490 nm, and at 630 nm as reference, measured with a
microplate reader.
DESCRIPTION OF EMBODIMENTS
[0042] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
embodiments of the present invention, the preferred methods,
devices, and materials are now described. However, before the
present materials and methods are described, it is to be understood
that the present invention is not limited to the particular sizes,
shapes, dimensions, materials, methodologies, protocols, etc.
described herein, as these may vary in accordance with routine
experimentation and optimization. It is also to be understood that
the terminology used in the description is for the purpose of
describing the particular versions or embodiments only, and is not
intended to limit the scope of the present invention which will be
limited only by the appended claims.
[0043] The disclosure of each publication, patent or patent
application mentioned in this specification is specifically
incorporated by reference herein in its entirety. However, nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0044] In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
DEFINITION
[0045] The words "a", "an", and "the" as used herein mean "at least
one" unless otherwise specifically indicated.
[0046] The term "biological sample" refers to a whole organism or a
subset of its tissues, cells or component parts (e.g., body fluids,
including but not limited to blood, mucus, lymphatic fluid,
synovial fluid, cerebrospinal fluid, saliva, amniotic fluid,
amniotic cord blood, urine and semen). "Biological sample" further
refers to a homogenate, lysate, extract, cell culture or tissue
culture prepared from a whole organism or a subset of its cells,
tissues or component parts, or a fraction or portion thereof.
Lastly, "biological sample" refers to a medium, such as a nutrient
broth or gel in which an organism has been propagated, which
contains cellular components, such as proteins or
polynucleotides.
[0047] The term "castration-resistant prostate cancer (CRPC)"
refers to cancers that are tolerant to androgen-ablation therapy
(castration). As used herein, the term "CRPC" includes
androgen-independent phenotype that has been termed
hormone-refractory prostate cancers (HRPCs).
[0048] The terms "isolated" and "purified" used in relation with a
substance (e.g., polypeptide, antibody, polynucleotide, etc.)
indicates that the substance is substantially free from at least
one substance that may else be included in the natural source.
Thus, an isolated or purified antibody refers to antibodies that
are substantially free of cellular material such as carbohydrate,
lipid, or other contaminating proteins from the cell or tissue
source from which the protein (antibody) is derived, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. The term "substantially free of cellular
material" includes preparations of a polypeptide in which the
polypeptide is separated from cellular components of the cells from
which it is isolated or recombinantly produced. Thus, a polypeptide
that is substantially free of cellular material includes
preparations of polypeptide having less than about 30%, 20%, 10%,
or 5% (by dry weight) of heterologous protein (also referred to
herein as a "contaminating protein"). When the polypeptide is
recombinantly produced, it is also preferably substantially free of
culture medium, which includes preparations of polypeptide with
culture medium less than about 20%, 10%, or 5% of the volume of the
protein preparation. When the polypeptide is produced by chemical
synthesis, it is preferably substantially free of chemical
precursors or other chemicals, which includes preparations of
polypeptide with chemical precursors or other chemicals involved in
the synthesis of the protein less than about 30%, 20%, 10%, 5% (by
dry weight) of the volume of the protein preparation. That a
particular protein preparation contains an isolated or purified
polypeptide can be shown, for example, by the appearance of a
single band following sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis of the protein preparation and Coomassie
Brilliant Blue staining or the like of the gel. In a preferred
embodiment, antibodies and polypeptides of the present invention
are isolated or purified. An "isolated" or "purified" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of
other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
[0049] The terms "polypeptide", "peptide", and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is a modified residue, or a non-naturally
occurring residue, such as an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0050] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that similarly functions to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those modified after translation in cells
(e.g., hydroxyproline, gamma-carboxyglutamate, and
O-phosphoserine). The phrase "amino acid analog" refers to
compounds that have the same basic chemical structure (an alpha
carbon bound to a hydrogen, a carboxy group, an amino group, and an
R group) as a naturally occurring amino acid but have a modified R
group or modified backbones (e.g., homoserine, norleucine,
methionine, sulfoxide, methionine methyl sulfonium). The phrase
"amino acid mimetic" refers to chemical compounds that have
different structures but similar functions to general amino
acids.
[0051] Amino acids may be referred to herein by their commonly
known three letter symbols or the one-letter symbols recommended by
the IUPAC-IUB Biochemical Nomenclature Commission.
[0052] The terms "gene", "polynucleotides", "oligonucleotide",
"nucleotides", "nucleic acids", and "nucleic acid molecules" are
used interchangeably unless otherwise specifically indicated and
are similarly to the amino acids referred to by their commonly
accepted single-letter codes. Similar to the amino acids, they
encompass both naturally-occurring and non-naturally occurring
nucleic acid polymers. The polynucleotide, oligonucleotide,
nucleotides, nucleic acids, or nucleic acid molecules may be
composed of DNA, RNA or a combination thereof.
[0053] Unless otherwise defined, the terms "cancer" refers to
cancers over-expressing the STC2 gene, more particularly prostate
cancers such as CRPC, more particularly castration-resistant
prostate cancer.
[0054] The STC2 gene or STC2 protein:
The present invention is based in part on the discovery of elevated
expression of the STC2 gene in cells from patients of PCs. The
expression of the gene was discovered to be particularly elevated
in CRPC. An exemplified nucleotide sequence of human STC2 gene is
shown in SEQ ID NO: 11 and is also available as GenBank Accession
No. NM.sub.--003714. Another nucleotide sequence data for the human
STC2 gene is available as GenBank Accession No. AK075406. Herein,
the STC2 gene encompasses the human STC2 gene as well as those of
other animals including non-human primate, mouse, rat, dog, cat,
horse, and cow but are not limited thereto, and includes allelic
mutants and genes found in other animals as corresponding to the
STC2 gene.
[0055] An exemplified amino acid sequence encoded the human STC2
gene is shown in SEQ ID NO: 12 and is also available as GenBank
Accession No. NP.sub.--003705. In the present invention, the
polypeptide encoded by the STC2 gene is referred to as "STC2", and
sometimes as "STC2 polypeptide" or "STC2 protein".
[0056] According to an aspect of the present invention, functional
equivalents are also included in the STC2 polypeptide. Herein, a
"functional equivalent" of a protein is a polypeptide that has a
biological activity equivalent to the protein. Namely, any
polypeptide that retains the biological ability of the STC2 protein
may be used as such a functional equivalent in the present
invention. Such functional equivalents include those wherein one or
more amino acids are substituted, deleted, added, or inserted to
the natural occurring amino acid sequence of the STC2 protein.
Alternatively, the polypeptide may be one that includes an amino
acid sequence having at least about 80% homology (also referred to
as sequence identity) to the sequence of the respective proteins,
more preferably at least about 90% to 95% homology, even more
preferably 96% to 99% homology. In other embodiments, the
polypeptide can be encoded by a polynucleotide that hybridizes
under stringent conditions to the naturally occurring nucleotide
sequence of the STC2 gene.
[0057] The phrase "stringent (hybridization) conditions" refers to
conditions under which a nucleic acid molecule will hybridize to
its target sequence, typically in a complex mixture of nucleic
acids, but not detectably to other sequences. Stringent conditions
are sequence-dependent and will vary in different circumstances.
Longer sequences hybridize specifically at higher temperatures. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, Techniques in Biochemistry and Molecular
Biology-Hybridization with Nucleic Probes, "Overview of principles
of hybridization and the strategy of nucleic acid assays" (1993).
Generally, stringent conditions are selected to be about 5-10
degrees C. lower than the thermal melting point (T.sub.m) for the
specific sequence at a defined ionic strength pH. The T.sub.m is
the temperature (under defined ionic strength, pH, and nucleic
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at T.sub.m, 50% of the
probes are occupied at equilibrium). Stringent conditions may also
be achieved with the addition of destabilizing agents such as
formamide. For selective or specific hybridization, a positive
signal is at least two times of background, preferably 10 times of
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42 degrees C., or, 5.times.SSC, 1% SDS,
incubating at 65 degrees C., with wash in 0.2.times.SSC, and 0.1%
SDS at 50 degrees C.
[0058] In the context of the present invention, a condition of
hybridization for isolating a DNA encoding a polypeptide
functionally equivalent to the human STC2 protein can be routinely
selected by a person skilled in the art. For example, hybridization
may be performed by conducting pre-hybridization at 68 degrees C.
for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE
SCIENCE), adding a labeled probe, and warming at 68 degrees C. for
1 hour or longer. The following washing step can be conducted, for
example, in a low stringent condition. An exemplary low stringent
condition may include 42 degrees C., 2.times.SSC, 0.1% SDS,
preferably 50 degrees C., 2.times.SSC, 0.1% SDS. High stringency
conditions are often preferably used. An exemplary high stringency
condition may include washing 3 times in 2.times.SSC, 0.01% SDS at
room temperature for 20 min, then washing 3 times in 1.times.SSC,
0.1% SDS at 37 degrees C. for 20 min, and washing twice in
1.times.SSC, 0.1% SDS at 50 degrees C. for 20 min. However, several
factors, such as temperature and salt concentration, can influence
the stringency of hybridization and one skilled in the art can
suitably select the factors to achieve the requisite
stringency.
[0059] In general, modification of one, two or more amino acids in
a protein will not influence the function of the protein. In fact,
mutated or modified proteins (i.e., peptides composed of an amino
acid sequence in which one, two, or several amino acid residues
have been modified through substitution, deletion, insertion and/or
addition) have been known to retain the original biological
activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984);
Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982);
Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13
(1982)). Accordingly, one of skill in the art will recognize that
individual additions, deletions, insertions, or substitutions to an
amino acid sequence that alter a single amino acid or a small
percentage of amino acids or those considered to be "conservative
modifications", wherein the alteration of a protein results in a
protein with similar functions, are acceptale in the context of the
instant invention. Thus, in one embodiment, the peptides of the
present invention may have an amino acid sequence wherein one, two
or even more amino acids are added, inserted, deleted, and/or
substituted in the STC2 sequence.
[0060] So long as the activity the protein is maintained, the
number of amino acid mutations is not particularly limited.
However, it is generally preferred to alter 5% or less of the amino
acid sequence. Accordingly, in a preferred embodiment, the number
of amino acids to be mutated in such a mutant is generally 30 amino
acids or less, preferably 20 amino acids or less, more preferably
10 amino acids or less, more preferably 5 or 6 amino acids or less,
and even more preferably 3 or 4 amino acids or less.
[0061] An amino acid residue to be mutated is preferably mutated
into a different amino acid in which the properties of the amino
acid side-chain are conserved (a process known as conservative
amino acid substitution). Examples of properties of amino acid side
chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),
hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side
chains having the following functional groups or characteristics in
common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl
group containing side-chain (S, T, Y); a sulfur atom containing
side-chain (C, M); a carboxylic acid and amide containing
side-chain (D, N, E, Q); a base containing side-chain (R, K, H);
and an aromatic containing side-chain (H, F, Y, W). Conservative
substitution tables providing functionally similar amino acids are
well known in the art. For example, the following eight groups each
contain amino acids that are conservative substitutions for one
another:
1) Alanine (A), Glycine (G);
[0062] 2) Aspartic acid (d), Glutamic acid (E);
3) Aspargine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
[0063] 8) Cystein (C), Methionine (M) (see, e.g., Creighton,
Proteins 1984).
[0064] Such conservatively modified polypeptides are included in
the present STC2 protein. However, the present invention is not
restricted thereto and the STC2 protein includes non-conservative
modifications so long as they retain at least one biological
activity of the STC2 protein. Furthermore, the modified proteins do
not exclude polymorphic variants, interspecies homologues, and
those encoded by alleles of these proteins.
[0065] Moreover, the STC2 gene of the present invention encompasses
polynucleotides that encode such functional equivalents of the STC2
protein. In addition to hybridization, a gene amplification method,
for example, the polymerase chain reaction (PCR) method, can be
utilized to isolate a polynucleotide encoding a polypeptide
functionally equivalent to the STC2 protein, using a primer
synthesized based on the sequence information of the protein
encoding DNA. Polynucleotides and polypeptides that are
functionally equivalent to the human STC2 gene and protein,
respectively, normally have a high homology to the originating
nucleotide or amino acid sequence of. "High homology" typically
refers to a homology of 40% or higher, preferably 60% or higher,
more preferably 80% or higher, even more preferably 90% to 95% or
higher, even more preferably 96% to 99% or higher. The homology of
a particular polynucleotide or polypeptide can be determined by
following the algorithm in "Wilbur and Lipman, Proc Natl Acad Sci
USA 80: 726-30 (1983)".
[0066] I. Diagnosing Cancer:
[0067] I-1. Method for Diagnosing Cancer or a Predisposition for
Developing Cancer
[0068] According to the present invention, the expression of the
STC2 gene was found to be specifically elevated in patients with
PC, more particularly, in CRPC and aggressive PC (see Example 2).
Accordingly, the STC2 genes identified herein as well as their
transcription and translation products find diagnostic utility as a
marker for PC and by measuring the expression of the STC2 gene in a
subject-derived biological sample, PC can be diagnosed. More
particularly, the present invention provides a method for
detecting, diagnosing and/or determining the presence of or a
predisposition for developing cancer, more particularly PC in a
subject by determining the expression level of the STC2 gene in the
subject.
[0069] In the context of the present invention, the term
"diagnosing" is intended to encompass predictions and likelihood
analysis. The present method is intended to be used clinically in
making decisions concerning treatment modalities, including
therapeutic intervention, diagnostic criteria such as disease
stages, and disease monitoring and surveillance for cancer.
According to the present invention, an intermediate result for
examining the condition of a subject may also be provided. Such
intermediate result may be combined with additional information to
assist a doctor, nurse, or other practitioner to diagnose that a
subject suffers from the disease. Alternatively, the present
invention may be used to detect cancerous cells in a
subject-derived tissue or biological sample and provide a doctor
with useful information to diagnose that the subject suffers from
the disease.
[0070] A subject to be diagnosed by the present method is
preferably a mammal. Exemplary mammals include, but are not limited
to, human, non-human primate, mouse, rat, dog, cat, horse, and
cow.
[0071] It is preferred to collect a biological sample from a
subject to be diagnosed to perform the diagnosis. Any biological
material can be used as the biological sample for the determination
so long as it contains the objective transcription or translation
product of the STC2 gene. The biological samples include, but are
not limited to, bodily tissues, especially some of a prostatic
tissue and bodily fluids, such as blood, sputum, semen, prostatic
fluid and urine. Preferably, the biological sample contains a cell
population including an epithelial cell, more preferably a prostate
epithelial cell derived from tissue suspected to be cancerous.
Further, if necessary, cells may be purified from an obtained
bodily tissues and fluids, and then used as a biological
sample.
[0072] According to the present invention, the expression level of
the STC2 gene is determined in a subject-derived biological sample.
The expression level can be determined at the transcription
(nucleic acid) product level, using methods known in the art. For
example, the mRNA of the STC2 gene may be quantified using a probe
by hybridization methods (e.g., Northern hybridization). The
detection may be carried out on a chip or an array. The use of an
array is preferable for detecting the expression level of a
plurality of genes (e.g., various cancer specific genes) including
the present STC2 gene. Those skilled in the art can prepare such
probes utilizing the sequence information of the STC2 gene (for
example, the sequence shown in SEQ ID NO: 11; GenBank Accession No.
NM.sub.--018423 or GenBank Accession No. AK075406). For example,
the cDNA of the STC2 gene may be used as the probes. If necessary,
the probe may be labeled with a suitable label, such as dyes and
isotopes, and the expression level of the gene may be detected as
the intensity of the hybridized labels.
[0073] Furthermore, the transcription product of the STC2 gene may
be quantified using primers by amplification-based detection
methods (e.g., RT-PCR). Such primers can also be prepared based on
an available sequence information of the gene. For example, the
primers used in Example 1 (SEQ ID NOs: 3 and 4) may be employed for
the detection by RT-PCR, but the present invention is not
restricted thereto.
[0074] Specifically, a probe or primer used for the present method
hybridizes under stringent, moderately stringent, or low stringent
conditions to the mRNA of the STC2 gene. As used herein, the phrase
"stringent (hybridization) conditions" refers to conditions under
which a probe or primer will hybridize to its target sequence, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different under different circumstances. Specific
hybridization of longer sequences is observed at higher
temperatures than shorter sequences. Generally, the temperature of
a stringent condition is selected to be about 5 degrees C. lower
than the thermal melting point (T.sub.m) for a specific sequence at
a defined ionic strength and pH. The Tm is the temperature (under
defined ionic strength, pH and nucleic acid concentration) at which
50% of the probes complementary to the target sequence hybridize to
the target sequence at equilibrium. Since the target sequences are
generally present at excess, at Tm, 50% of the probes are occupied
at equilibrium. Typically, stringent conditions will be those in
which the salt concentration is less than about 1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0
to 8.3 and the temperature is at least about 30 degrees C. for
short probes or primers (e.g., 10 to 50 nucleotides) and at least
about 60 degrees C. for longer probes or primers. Stringent
conditions may also be achieved with the addition of destabilizing
agents, such as formamide.
[0075] Alternatively, the translation product may be detected for
the diagnosis of the present invention. For example, the quantity
of the STC2 protein may be determined. A method for determining the
quantity of the protein as the translation product includes
immunoassay methods that use an antibody specifically recognizing
the protein. The antibody may be monoclonal or polyclonal.
Furthermore, any fragment or modification (e.g., chimeric antibody,
scFv, Fab, F(ab').sub.2, Fv, etc.) of the antibody may be used for
the detection, so long as the fragment retains the binding ability
to the STC2 protein. Methods to prepare these kinds of antibodies
for the detection of proteins are well known in the art, and any
method may be employed in the present invention to prepare such
antibodies and equivalents thereof.
[0076] As another method to detect the expression level of the STC2
gene based on its translation product, the intensity of staining
may be observed via immunohistochemical analysis using an antibody
against the STC2 protein. Namely, the observation of strong
staining indicates increased presence of the protein and at the
same time high expression level of the STC2 gene.
[0077] Especially, STC2 protein is secreted protein. Therefore, by
measuring the level of STC2 in subject-derived blood samples, the
occurrence of or a predisposition to develop prostate cancer
expressing STC2 in a subject can be determined. In the present
invention, any blood samples may be used for determining the level
of STC2 so long as either the gene or the protein of STC2 can be
detected in the samples. Preferably, the blood samples include
whole blood, serum, and plasma. The method of determining the level
of STC2 was known to those skilled in the art, like ELISA,
especially sandwich method.
[0078] Furthermore, the translation product may be detected based
on its biological activity. The STC2 gene have been isolated as one
of the mammalian gene related to STC gene that has been known to
regulate calcium and phosphate uptake in different target organs
(Wagner G F, et al., Mol Cell Endocrinol 1991, 79, 129-38, and
Wagner G F and Jaworski E, Mol Cell Endocrinol 1994, 99, 315-22)
and it was reported that the STC2 protein inhibited the phosphate
uptake of a kidney cell line (Ishibashi K, et al., Biochem Biophys
Res Commun 1998, Sep. 18; 250(2), 258-8). Furthermore, according to
the present invention, the translation product of STC2 gene has an
ability to promote PC cell growth. Thus, the inhibiting activity
against the phosphate uptake of a kidney cell line or the cancer
cell growth promoting activity may be used as an index of the STC2
protein existing in the biological sample.
[0079] Moreover, in addition to the expression level of the STC2
gene, the expression level of other cancer-associated genes, for
example, genes known to be differentially expressed in CRPC, may
also be determined to improve the accuracy of the diagnosis.
[0080] The expression level of the STC2 gene in a biological sample
can be considered to be increased if it increases from the control
level of the STC2 gene by, for example, 10%, 25%, or 50%; or
increases to more than 1.1 fold, more than 1.5 fold, more than 2.0
fold, more than 5.0 fold, more than 10.0 fold, or more.
[0081] The control level may be determined at the same time with
the test biological sample by using a sample(s) previously
collected and stored from a subject/subjects whose disease state
(cancerous or non-cancerous) is/are known. Alternatively, the
control level may be determined by a statistical method based on
the results obtained by analyzing previously determined expression
level(s) of the STC2 gene in samples from subjects whose disease
state are known. Furthermore, the control level can be a database
of expression patterns from previously tested cells. Moreover,
according to an aspect of the present invention, the expression
level of the STC2 gene in a biological sample may be compared to
multiple control levels, which control levels are determined from
multiple reference samples. It is preferred to use a control level
determined from a reference sample derived from a tissue type
similar to that of the patient-derived biological sample. Moreover,
it is preferred, to use the standard value of the expression levels
of the STC2 gene in a population with a known disease state. The
standard value may be obtained by any method known in the art. For
example, a range of mean plus/minus 2 S.D. or mean plus/minus 3
S.D. may be used as standard value.
[0082] In the context of the present invention, a control level
determined from a biological sample that is known to be
non-cancerous or cancer-free is called "normal control level". On
the other hand, if the control level is determined from a cancerous
biological sample, it will be called "cancerous control level".
[0083] When the expression level of the STC2 gene in a biological
sample derived from a subject is increased compared to the normal
control level or is similar to the cancerous control level, the
subject may be diagnosed to be suffering from or at a risk of
developing cancer. Furthermore, in the case where the expression
levels of multiple cancer-related genes are compared, a similarity
in the gene expression pattern between the sample and the reference
that is cancerous indicates that the subject is suffering from or
at a risk of developing cancer.
[0084] Difference between the expression levels of a test
biological sample and the control level can be normalized to the
expression level of control nucleic acids, e.g. housekeeping genes
whose expression levels are known not to differ depending on the
cancerous or non-cancerous state of the cell. Exemplary
housekeeping genes include, but are not limited to, beta-actin,
glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein
P1.
[0085] Alternatively, the present invention provides a method for
detecting or identifying prostate cancer cells in a subject-derived
prostate tissue sample, said method including the step of
determining the expression level of the STC2 gene in a
subject-derived biological sample, wherein an increase in said
expression level as compared to a normal control level of said gene
indicates the presence or suspicion of cancer cells in the
tissue.
[0086] Such result may be combined with additional information to
assist a doctor, nurse, or other healthcare practitioner in
diagnosing a subject as afflicted with the disease. In other words,
the present invention may provide a doctor with useful information
to diagnose a subject as afflicted with the disease. For example,
according to the present invention, when there is doubt regarding
the presence of prostate cancer cells in the tissue obtained from a
subject, clinical decisions can be reached by considering the
expression level of the STC2 gene, plus a different aspect of the
disease including tissue pathology, levels of known tumor marker(s)
in blood, and clinical course of the subject, etc. For example,
some well-known diagnostic prostate tumor markers in blood is PSA.
Namely, in this particular embodiment of the present invention, the
outcome of the gene expression analysis serves as an intermediate
result for further diagnosis of a subject's disease state. In
another embodiment, the present invention provides a method for
detecting a diagnostic marker of prostate cancer, said method
including the step of detecting the expression of the STC2 gene in
a subject-derived biological sample as a diagnostic marker of
prostate cancer.
[0087] According to the present invention, prostate cancer cells
can be diagnosed or detected. In preferable embodiments, the
presence of CRPC or aggressive prostate cancer cells within a
prostate tissue may be diagnosed or detected. In the present
invention, aggressive prostate cancer refers to prostate cancer
having Gleason score (Gleason tumor grade) of 8 or more. For
example, prostate cancer with Gleason score 8-10 is considered to
be aggressive prostate cancer.
[0088] The present invention reveals that the immunostaining
intensity of the STC2 polypeptide in a biological sample derived
from a prostate cancer patient is increased as compared to the
control level. Therefore, the present invention also provides a
method of correlating the immunostaining intensity of the STC2
polypeptide with the presence of prostate cancer including the
steps as follows:
(a) determining or evaluating the immunostaining intensity of the
STC2 polypeptide in a biological sample derived from a subject; and
(b) correlating the immunostaining intensity of step (a) with the
presence of prostate cancer.
[0089] In a preferred embodiment, the present invention provides a
method of correlating the immunostaining intensity of the STC2
polypeptide with the presence of prostate cancer including the
steps as follows:
(a) determining or evaluating the immunostaining intensity of the
STC2 polypeptide in a biological sample derived from a subject; (b)
comparing the immunostaining intensity of step (a) with a control
level; and (c) correlating an increase in the immunostaining
intensity determined in step (b) as compared to the control level
to the presence of prostate cancer.
[0090] Any biological samples described above are suitable for
correlating the immunostaining intensity of the STC2 polypeptide
with the presence of prostate cancer. In a preferred embodiment of
the present invention, a biological sample may be fixed chemically
or physically. For example, the formalin fixed biological sample is
preferable for evaluating the immunostaining intensity of the STC2
polypeptide Alternatively, in case where an electron microscope is
used, the biological sample can be fixed with a glutaraldehyde or
osmium teraoxide.
[0091] In the context of the present invention, a tissue derived
from a subject is preferable as the biological sample. For example,
a tissue in the present invention is a prostate tissue, more
preferably prostate epithelial tissue. In order for determining or
evaluating the immunostaining intensity, in general, sections may
be prepared from tissue samples. Methods for preparing the tissue
section are well known in the art. For example, methods for
preparing a frozen section, unfrozen section, or paraffin section
are known.
[0092] Preferably, prostate cancer of the present invention is
CRPC, CNPC, or an aggressive prostate cancer, more preferably CRPC,
CNPC, or an aggressive prostate cancer with Gleason score 8-10.
[0093] Immunostaining intensity can be determined or evaluated with
a labeled anti-STC2 antibody. For example, anti-STC2 antibody may
be labeled with enzymes. In preferable embodiment, the following
enzymes are suitable for the anti-STC2 antibody labeling;
alkaline phosphatase, horseradish peroxidase, beta-galactosidase,
or beta-glucosidase.
[0094] When the anti-STC2 antibody is labeled with an enzyme, the
antibody can be detected or measured with a substrate which reacts
with the enzyme to generate detectable signals. Substrates
generating detectable signals including color change, accumulation
of dyes, or pigments having color fluorescence, or luminescence are
well known. These detectable signals may be observed with a light
microscope or fluorescence microscope.
[0095] Alternatively, the antibody may be labeled with fluorescent
substances. In case where a fluorescent substance is labeled with
the antibody, the antibody bound to the biological sample may be
detected or measured by a fluorescence microscope. In preferable
embodiments, fluorescein isothiocyanate (FITC), or rhodamine is
suitable as the label in the present invention.
[0096] In another embodiment of the present invention, the antibody
may also be labeled with a gold colloid. When the anti-STC2
antibody is labeled with gold colloid, the bound antibody may be
detected or measured with a light microscope or electron
microscope.
[0097] In an immunohistological analysis, the number of strongly or
highly positive immunostained cells may be counted to determine or
evaluate immunostaining intensity of the biological sample. In
particular, the immunostaining intensity may determined or
evaluated by comparing numbers of cells having positive and/or
negative staining in the biological sample with control.
[0098] The immunostaining intensity of the biological sample may
also be estimated or assessed in some grades. For example, three or
more grades scoring or classifying of the immunostaining intensity
may be applied to the present invention. In particular, the
positive staining of the anti-STC2 antibody may be defined as
follows:
(1) variable weak cytoplasmic staining, and (2) segmental and
apical granular cytoplasmic staining, and (3) diffuse continuous
and intense cytoplasmic staining
[0099] According to such score, cell population of each score is
determined. Further, in order for easy comparison, the sum of the
population of cells with each score may be calculated for each
sample as described previously (Ashida S, et al., Clin Cancer Res
2006, 2767-73).
[0100] The control level may be determined at the same time with
the test biological sample by using a sample(s) previously
collected and stored from a subject/subjects whose disease state
(cancerous or non-cancerous) is/are known, as mentioned above. In a
preferable embodiment of the present invention, the control level
may be determined by using a sample(s) previously collected and
stored from a subject/subjects whose disease state is/are prostate
cancer with Gleason score 2-7 or a normal healthy subject/subjects.
The immunostaining intensity of the STC2 polypeptide in a
biological sample can be considered to be increased if it increases
from the control level of the STC2 polypeptide by, for example,
10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5
fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold,
or more.
[0101] I-2. Assessing Efficacy of Cancer Treatment
[0102] The STC2 gene differentially expressed between normal and PC
cells also allow for the course of treatment of PC to be monitored,
and the above-described method for diagnosing cancer can be applied
for assessing the efficacy of a treatment on PC. Specifically, the
efficacy of a treatment on PC can be assessed by determining the
expression level of the STC2 gene in a cell(s) derived from a
subject undergoing the treatment. If desired, test cell populations
are obtained from the subject at various time points, before,
during, and/or after the treatment. The expression level of the
STC2 gene can be, for example, determined following the method
described above under the item of `I-1. Method for diagnosing
cancer or a predisposition for developing cancer`. In the context
of the present invention, it is preferred that the control level to
which the detected expression level is compared is determined from
the STC2 gene expression in a cell(s) not exposed to the treatment
of interest.
[0103] If the expression level of the STC2 gene is compared to a
normal control level, a similarity in the expression level
indicates that the treatment of interest is efficacious and a
difference in the expression level indicates less favorable
clinical outcome or prognosis of that treatment. On the other hand,
if the comparison is conducted against a cancerous control level, a
difference in the expression level indicates efficacious treatment,
while a similarity in the expression level indicates less favorable
clinical outcome or prognosis.
[0104] Furthermore, the expression levels of the STC2 gene before
and after a treatment can be compared according to the present
method to assess the efficacy of the treatment. Specifically, the
expression level detected in a subject-derived biological sample
after a treatment (i.e., post-treatment level) is compared to the
expression level detected in a biological sample obtained prior to
treatment onset from the same subject (i.e., pre-treatment level).
A decrease in the post-treatment level as compared to the
pre-treatment level indicates that the treatment of interest is
efficacious while an increase in or similarity of the
post-treatment level to the pre-treatment level indicates less
favorable clinical outcome or prognosis.
[0105] As used herein, the term "efficacious" indicates that the
treatment leads to a reduction in the expression of a
pathologically up-regulated gene, an increase in the expression of
a pathologically down-regulated gene or a decrease in size,
prevalence, or metastatic potential of carcinoma in a subject. When
a treatment of interest is applied prophylactically, "efficacious"
means that the treatment retards or prevents the forming of tumor
or retards, prevents, or alleviates at least one clinical symptom
of cancer. Assessment of the state of tumor in a subject can be
made using standard clinical protocols.
[0106] In addition, efficaciousness of a treatment can be
determined in association with any known method for diagnosing
cancer. Cancers can be diagnosed, for example, by identifying
symptomatic anomalies, e.g., weight loss, abdominal pain, back
pain, anorexia, nausea, vomiting and generalized malaise, weakness,
and jaundice.
[0107] I-3. Assessing Prognosis of Subject with Cancer
[0108] The method for diagnosing PC described above can also be
used for assessing the prognosis of PC in a subject. Thus, the
present invention also provides a method for assessing the
prognosis of a subject with PC. The expression level of the STC2
gene can be, for example, determined following the method described
above under the item of `I-1. Method for diagnosing cancer or a
predisposition for developing cancer`. For example, the expression
level of the STC2 gene in biological samples derived from patients
over a spectrum of disease stages can be used as control levels to
be compared with the expression level of the gene detected for a
subject. By comparing the expression level of the STC2 gene in a
subject and the control level(s) the prognosis of the subject can
be assessed. Alternatively, by comparing over time the pattern of
expression levels in a subject, the prognosis of the subject can be
assessed.
[0109] For example, an increase in the expression level of STC2
gene in a subject as compared to a normal control level indicates
less favorable prognosis. Conversely, a similarity in the
expression level as compared to normal control level indicates a
more favorable prognosis for the subject.
[0110] II. Kits:
[0111] The present invention also provides kits for detecting
and/or diagnosing PC, which may also be useful in assessing the
prognosis of cancer and/or monitoring the efficacy of a cancer
therapy, i.e., kits that includes at least a reagent detecting the
transcription or translation product of the STC2 gene. Examples of
such reagents include nucleic acids that specifically bind to or
identify a transcription product of the STC2 gene. For example, the
nucleic acid that specifically bind to or identify a transcription
product of the STC2 gene include such as oligonucleotides (e.g.,
probes and primers) having a sequence that is complementary to a
portion of the STC2 gene transcription product. Alternatively,
antibodies can be exemplified as reagents for detecting the
translation product of the gene. The probes, primers, and
antibodies described above under the item of `I-1. Method for
diagnosing cancer or a predisposition for developing cancer` can be
mentioned as suitable examples of such reagents.
[0112] The translation product may also be detected based on its
biological activity. The STC2 protein was reported to inhibit the
phosphate uptake of a kidney cell line (Ishibashi K, et al.,
Biochem Biophys Res Commun 1998, Sep. 18; 250(2), 258-8). Thus, the
inhibiting activity against the phosphate uptake of a kidney cell
line may be detected for determining the expression level of the
STC2 gene. For example, labeled phosphate, or other substrate(s) of
the STC2 protein may be used as a reagent for detecting the
expression level of the gene. Furthermore, according to the present
invention, the translation product of STC2 gene has an ability to
promote PC ell growth. Thus, according to an aspect of the present
invention, a kit for detecting the expression of the STC2 gene may
include a labeled phosphate and a kidney cell line, or a PC line
and an appropriate medium.
[0113] The present kit is suited for detecting CRPC and aggressive
PC with high Gleason score, in particular, Gleason score 8-10.
[0114] These reagents may be used for the above-described diagnosis
of cancer. The assay format for using the reagents may be Northern
hybridization or sandwich ELISA, both of which are well-known in
the art.
[0115] The detection reagents may be packaged together in the form
of a kit. For example, the detection reagents may be packaged in
separate containers. Furthermore, the detection reagents may be
packaged with other reagents necessary for the detection. For
example a kit may include a nucleic acid or antibody (either bound
to a solid matrix or packaged separately with reagents for binding
them to the matrix) as the detection reagent, a control reagent
(positive and/or negative), and/or a detectable label. Instructions
(e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay
may also be included in the kit.
[0116] As an aspect of the present invention, the reagents for
detecting cancer may be immobilized on a solid matrix, such as a
porous strip, to form at least one site for detecting cancer. The
measurement or detection region of the porous strip may include a
plurality of sites, each containing a detection reagent (e.g.,
nucleic acid). A test strip may also contain sites for negative
and/or positive controls. Alternatively, control sites may be
located on a separate strip from the test strip. Optionally, the
different detection sites may contain different amounts of
immobilized detection reagents (e.g., nucleic acid), i.e., a higher
amount in the first detection site and lesser amounts in subsequent
sites. Upon the addition of test biological sample, the number of
sites displaying a detectable signal provides a quantitative
indication of the expression level of the STC2 gene in the sample.
The detection sites may be configured in any suitably detectable
shape and are typically in the shape of a bar or dot spanning the
width of a test strip.
[0117] III. Screening Methods:
[0118] Using the STC2 gene, polypeptides encoded by the gene or
fragments thereof, or transcriptional regulatory region(s) of the
gene, it is possible to screen for candidate agents or substances
that alter the expression of the gene or the biological activity of
a polypeptide encoded by the gene. Such agents or substances can be
used as pharmaceuticals for treating or preventing PC, in
particular, CRPC and aggressive PC with high Gleason score (e.g.,
Gleason score 8-10). Thus, the present invention provides methods
of screening for agents or substances for treating or preventing PC
using the STC2 gene, polypeptid encoded by the gene or fragments
thereof, or transcriptional regulatory region of the gene.
[0119] An agent or substance isolated by the screening method of
the present invention is an agent or substance that is expected to
inhibit the expression of the STC2 gene or the activity of the
translation product of the gene, and thus, is a candidate for
treating or preventing PC. The agents or substances are expected to
be particularly suited for the treatment or prevention of CRPC and
aggressive PC with high Gleason score (in particular, Gleason score
8-10). Namely, the agents or substances screened through the
present methods are deemed to have a clinical benefit and can be
further tested for its ability to prevent cancer cell growth in
animal models or test subjects.
[0120] In the context of the present invention, agents or
substances to be identified through the present screening methods
may be any compound or composition including several compounds.
Furthermore, the test agent or substance exposed to a cell or
protein according to the screening methods of the present invention
may be a single compound or a combination of compounds. When a
combination of compounds is used in the methods, the compounds may
be contacted sequentially or simultaneously.
[0121] Any test agent or substance, for example, cell extracts,
cell culture supernatant, products of fermenting microorganism,
extracts from marine organism, plant extracts, purified or crude
proteins, peptides, non-peptide compounds, synthetic micromolecular
compounds (including nucleic acid constructs, such as antisense
RNA, siRNA, Ribozymes, etc.) and natural compounds can be used in
the screening methods of the present invention. The test agent or
substances of the present invention can be also obtained using any
of the numerous approaches in combinatorial library methods known
in the art, including
(1) biological libraries, (2) spatially addressable parallel solid
phase or solution phase libraries, (3) synthetic library methods
requiring deconvolution, (4) the "one-bead one-compound" library
method and (5) synthetic library methods using affinity
chromatography selection.
[0122] The biological library methods using affinity chromatography
selection is limited to peptide libraries, while the other four
approaches are applicable to peptide, non-peptide oligomer or small
molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12:
145-67). Examples of methods for the synthesis of molecular
libraries can be found in the art (DeWitt et al., Proc Natl Acad
Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994,
91: 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho
et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed
Engl 1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994,
33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Libraries
of compounds may be presented in solution (see Houghten,
Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991,
354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (U.S.
Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484,
and 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 1992,
89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90;
Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci
USA 1990, 87: 6378-82; Felici, J Mol Biot 1991, 222: 301-10; US
Pat. Application 2002103360).
[0123] In another embodiment of the present invention, an agent or
substance that binds to STC2 polypeptide and/or reduces the
expression level of the STC2 gene may be used as a test agent or
substance for the further screening for a candidate agent or
substance that has the STC2 specific therapeutic effect.
[0124] A compound in which a part of the structure of the compound
screened by any of the present screening methods is converted by
addition, deletion and/or replacement, is included in the agents or
substances obtained by the screening methods of the present
invention.
[0125] Furthermore, when the screened test agent or substances is a
protein, for obtaining a DNA encoding the protein, either the whole
amino acid sequence of the protein may be determined to deduce the
nucleic acid sequence coding for the protein, or partial amino acid
sequence of the obtained protein may be analyzed to prepare an
oligo DNA as a probe based on the sequence, and screen cDNA
libraries with the probe to obtain a DNA encoding the protein. The
obtained DNA find use in preparing the test agent or substance
which is a candidate for treating or preventing cancer.
[0126] Test agents useful in the screenings described herein can
also be antibodies that specifically bind to a STC2 protein or
partial peptides thereof that lack the biological activity of the
original proteins in vivo.
[0127] Although the construction of test agent libraries is well
known in the art, herein below, additional guidance in identifying
test agents and construction libraries of such agents for the
present screening methods are provided.
[0128] (i) Molecular Modeling
[0129] Construction of test agent libraries is facilitated by
knowledge of the molecular structure of compounds known to have the
properties sought, and/or the molecular structure of STC2. One
approach to preliminary screening of test agents suitable for
further evaluation utilizes computer modeling of the interaction
between the test agent and its target.
[0130] Computer modeling technology allows for the visualization of
the three-dimensional atomic structure of a selected molecule and
the rational design of new compounds that will interact with the
molecule. The three-dimensional construct typically depends on data
from x-ray crystallographic analysis or NMR imaging of the selected
molecule. The molecular dynamics require force field data. The
computer graphics systems enable prediction of how a new compound
will link to the target molecule and allow experimental
manipulation of the structures of the compound and target molecule
to perfect binding specificity. Prediction of what the
molecule-compound interaction will be when small changes are made
in one or both requires molecular mechanics software and
computationally intensive computers, usually coupled with
user-friendly, menu-driven interfaces between the molecular design
program and the user.
[0131] An example of the molecular modeling system described
generally above includes the CHARMm and QUANTA programs, Polygen
Corporation, Waltham, Mass. CHARMm performs the energy minimization
and molecular dynamics functions. QUANTA performs the construction,
graphic modeling and analysis of molecular structure. QUANTA allows
interactive construction, modification, visualization, and analysis
of the behavior of molecules with each other.
[0132] A number of articles have been published on the subject of
computer modeling of drugs interactive with specific proteins,
examples of which include Rotivinen et al. Acta Pharmaceutica
Fennica 1988, 97: 159-66; Ripka, New Scientist 1988, 54-8; McKinlay
& Rossmann, Annu Rev Pharmacol Toxiciol 1989, 29: 111-22; Perry
& Davies, Prog Clin Biol Res 1989, 291: 189-93; Lewis &
Dean, Proc R Soc Lond 1989, 236: 125-40, 141-62; and, with respect
to a model receptor for nucleic acid components, Askew et al., J Am
Chem Soc 1989, 111: 1082-90. Other computer programs that screen
and graphically depict chemicals are available from companies such
as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga,
Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. See,
e.g., DesJarlais et al., J Med Chem 1988, 31: 722-9; Meng et al., J
Computer Chem 1992, 13: 505-24; Meng et al., Proteins 1993, 17:
266-78; Shoichet et al., Science 1993, 259: 1445-50.
[0133] Once a putative inhibitor has been identified, combinatorial
chemistry techniques can be employed to construct any number of
variants based on the chemical structure of the identified putative
inhibitor, as detailed below. The resulting library of putative
inhibitors, or "test agents" may be screened using the methods of
the present invention to identify test agents suited to the
treatment and/or prophylaxis of cancer and/or the prevention of
post-operative recurrence of cancer, particularly wherein, such as
prostate cancer.
[0134] (ii) Combinatorial Chemical Synthesis
[0135] Combinatorial libraries of test agents may be produced as
part of a rational drug design program involving knowledge of core
structures existing in known inhibitors. This approach allows the
library to be maintained at a reasonable size, facilitating high
throughput screening. Alternatively, simple, particularly short,
polymeric molecular libraries may be constructed by simply
synthesizing all permutations of the molecular family making up the
library. An example of this latter approach would be a library of
all peptides six amino acids in length. Such a peptide library
could include every 6 amino acid sequence permutation. This type of
library is termed a linear combinatorial chemical library.
[0136] Preparation of combinatorial chemical libraries is well
known to those of skill in the art, and may be generated by either
chemical or biological synthesis. Combinatorial chemical libraries
include, but are not limited to, peptide libraries (see, e.g., U.S.
Pat. No. 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93;
Houghten et al., Nature 1991, 354: 84-6). Other chemistries for
generating chemical diversity libraries can also be used. Such
chemistries include, but are not limited to: peptides (e.g., PCT
Publication No. WO 91/19735), encoded peptides (e.g., WO 93/20242),
random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g.,
U.S. Pat. No. 5,288,514), diversomers such as hydantoins,
benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci
USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J
Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with
glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114:
9217-8), analogous organic syntheses of small compound libraries
(Chen et al., J. Amer Chem Soc 1994, 116: 2661), oligocarbamates
(Cho et al., Science 1993, 261: 1303), and/or peptidylphosphonates
(Campbell et al., J Org Chem 1994, 59: 658), nucleic acid libraries
(see Ausubel, Current Protocols in Molecular Biology 1995
supplement; Sambrook et al., Molecular Cloning: A Laboratory
Manual, 1989, Cold Spring Harbor Laboratory, New York, USA),
peptide nucleic acid libraries (see, e.g., U.S. Pat. No.
5,539,083), antibody libraries (see, e.g., Vaughan et al., Nature
Biotechnology 1996, 14(3):309-14 and PCT/US96/10287), carbohydrate
libraries (see, e.g., Liang et al., Science 1996, 274: 1520-22;
U.S. Pat. No. 5,593,853), and small organic molecule libraries
(see, e.g., benzodiazepines, Gordon E M. Curr Opin Biotechnol. 1995
Dec. 1; 6(6):624-31.; isoprenoids, U.S. Pat. No. 5,569,588;
thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;
pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino
compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.
5,288,514, and the like).
[0137] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa.,
Martek Biosciences, Columbia, Md., etc.).
[0138] (iii) Other Candidates
[0139] Another approach uses recombinant bacteriophage to produce
libraries. Using the "phage method" (Scott & Smith, Science
1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87:
6378-82; Devlin et al., Science 1990, 249: 404-6), very large
libraries can be constructed (e.g., 10.sup.6-10.sup.8 chemical
entities). A second approach uses primarily chemical methods, of
which the Geysen method (Geysen et al., Molecular Immunology 1986,
23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74);
and the method of Fodor et al. (Science 1991, 251: 767-73) are
examples. Furka et al. (14th International Congress of Biochemistry
1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res
1991, 37: 487-93), Houghten (U.S. Pat. No. 4,631,211) and Rutter et
al. (U.S. Pat. No. 5,010,175) describe methods to produce a mixture
of peptides that can be tested as agonists or antagonists.
[0140] Aptamers are macromolecules composed of nucleic acid that
bind tightly to a specific molecular target. Tuerk and Gold
(Science. 249:505-510 (1990)) discloses SELEX (Systematic Evolution
of Ligands by Exponential Enrichment) method for selection of
aptamers. In the SELEX method, a large library of nucleic acid
molecules (e.g., 10.sup.15 different molecules) can be used for
screening.
[0141] III. Screening Methods for Anti Cancer Compound
[0142] According to the present invention, the expression of the
STC2 gene was suggested to be crucial for the growth and/or
survival of cancer cells, in particular PC cells, more particularly
CRPC cells and aggressive PC cells with high Gleason score (e.g.,
Gleason score 8-10). Therefore, it was considered that agents or
substances that suppress the function of the polypeptide encoded by
the STC2 gene would reduce or inhibit the growth and/or survival of
cancer cells, and thus find use in the treatment and prevention of
prostate cancer. Accordingly, the present invention provides
methods of screening an agent for treating or preventing cancer,
using the STC2 polypeptide.
[0143] In addition to the STC2 polypeptide, fragments of the
polypeptide may be used for the present screening so long as it
retains at least one biological activity of the natural occurring
STC2 polypeptide.
[0144] The polypeptide or fragments thereof may be further linked
to other substances so long as the polypeptide and fragments
retains at least one of its biological activity. Usable substances
include: peptides, lipids, sugar and sugar chains, acetyl groups,
natural and synthetic polymers, etc. These kinds of modifications
may be performed to confer additional functions or to stabilize the
polypeptide and fragments.
[0145] The polypeptide or fragments used for the present method may
be obtained from nature as naturally occurring proteins via
conventional purification methods or through chemical synthesis
based on the selected amino acid sequence. For example,
conventional peptide synthesis methods that can be adopted for the
synthesis include:
1) Peptide Synthesis, Interscience, New York, 1966;
2) The Proteins, Vol. 2, Academic Press, New York, 1976;
3) Peptide Synthesis (in Japanese), Maruzen Co., 1975;
4) Basics and Experiment of Peptide Synthesis (in Japanese),
Maruzen Co., 1985;
[0146] 5) Development of Pharmaceuticals (second volume) (in
Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
6) WO99/67288; and
7) Barany G. & Merrifield R. B., Peptides Vol. 2, "Solid Phase
Peptide Synthesis", Academic Press, New York, 1980, 100-118.
[0147] Alternatively, the protein may be obtained adopting any
known genetic engineering methods for producing polypeptides (e.g.,
Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss &
Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62).
For example, first, a suitable vector including a polynucleotide
encoding the objective protein in an expressible form (e.g.,
downstream of a promoter regulatory sequence) is prepared,
transformed into a suitable host cell, and then the host cell is
cultured to produce the protein. More specifically, a gene encoding
the STC2 polypeptide is expressed in host (e.g., animal) cells and
such by inserting the gene into a vector for expressing foreign
genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8. A
promoter may be used for the expression. Any commonly used
promoters may be employed including, for example, the SV40 early
promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3.
Academic Press, London, 1982, 83-141), the EF-alpha promoter (Kim
et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene
1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology
1987, 152:684-704), the SR alpha promoter (Takebe et al., Mol Cell
Biol 1988, 8:466), the CMV immediate early promoter (Seed et al.,
Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter
(Gheysen et al., Mol Appl Genet. 1982, 1:385-94), the Adenovirus
late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV
TK promoter, and such. The introduction of the vector into host
cells to express the STC2 gene can be performed according to any
methods, for example, the electroporation method (Chu et al.,
Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method
(Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran
method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman
et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method
(Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics
1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and
such.
[0148] The STC2 protein may also be produced in vitro adopting an
in vitro translation system. The STC2 polypeptide to be contacted
with a test agent or substance can be, for example, a purified
polypeptide, a soluble protein, or a fusion protein fused with
other polypeptides.
[0149] Any test agent or substance, for example, cell extracts,
cell culture supernatant, products of fermenting microorganism,
extracts from marine organism, plant extracts, purified or crude
proteins, peptides, non-peptide compounds, synthetic micromolecular
compounds (including nucleic acid constructs, such as antisense
RNA, siRNA, Ribozymes, and aptamer etc.) and natural compounds can
be used in the screening methods of the present invention. The test
agent or substance of the present invention can be also obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including (1) biological libraries, (2)
spatially addressable parallel solid phase or solution phase
libraries, (3) synthetic library methods requiring deconvolution,
(4) the "one-bead one-compound" library method and (5) synthetic
library methods using affinity chromatography selection. The
biological library methods using affinity chromatography selection
is limited to peptide libraries, while the other four approaches
are applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67).
Examples of methods for the synthesis of molecular libraries can be
found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:
6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6;
Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al.,
Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl
1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994, 33:
2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Libraries of
compounds may be presented in solution (see Houghten,
Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991,
354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (U.S.
Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484,
and 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 1992,
89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90;
Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci
USA 1990, 87: 6378-82; Felici, J Mol Biot 1991, 222: 301-10; US
Pat. Application 2002103360).
[0150] A compound in which a part of the structure of the compound
screened by any of the present screening methods is converted by
addition, deletion and/or replacement, is included in the agents or
substances obtained by the screening methods of the present
invention. Furthermore, when the screened test agent or substance
is a protein, for obtaining a DNA encoding the protein, either the
whole amino acid sequence of the protein may be determined to
deduce the nucleic acid sequence coding for the protein, or partial
amino acid sequence of the obtained protein may be analyzed to
prepare an oligo DNA as a probe based on the sequence, and screen
cDNA libraries with the probe to obtain a DNA encoding the protein.
The obtained DNA is confirmed it's usefulness in preparing the test
agent which is a candidate for treating or preventing cancer.
[0151] III-1. Identifying Agents or Substances that Bind to STC2
Polypeptide
[0152] An agent or substance that binds to a protein is likely to
alter the expression of the gene coding for the protein or the
biological activity of the protein. Thus, as an aspect, the present
invention provides a method of screening an agent for treating or
preventing cancer, that includes the steps of:
a) contacting a test agent or substance with the STC2 polypeptide
or a fragment thereof; b) detecting the binding between the
polypeptide or fragment and the test agent or substance; and c)
selecting the test agent or substance that binds to the polypeptide
as a candidate agent for treating or preventing cancer.
[0153] According to the present invention, the therapeutic effect
of the test agent or substance on inhibiting the cell growth or a
candidate agent or substance for treating or preventing STC2
associating cancer may be evaluated. Therefore, the present
invention also provides a method of screening for a candidate agent
or substance for inhibiting the cell growth or a candidate agent or
substance for treating or preventing STC2 associating cancer, using
the STC2 polypeptide or fragments thereof including the steps as
follows:
a) contacting a test agent or substance with the STC2 polypeptide
or a functional fragment thereof; and b) detecting the binding
level between the polypeptide or functional fragment and the test
agent or substance of step (a), and c) correlating the binding
level of b) with the therapeutic effect of the test agent or
substance.
[0154] In the present invention, the therapeutic effect may be
correlated with the binding level of STC2 polypeptide or a
functional fragment thereof. For example, when the test agent or
substance bind to STC2 polypeptide or a functional fragment
thereof, the test agent or substance may identified or selected as
the candidate agent or substance having the therapeutic effect.
Alternatively, when the test agent or substance does not bind to
STC2 polypeptide or a functional fragment thereof, the test agent
or substance may identified as the agent or substance having no
significant therapeutic effect.
[0155] The binding of a test agent or substance to the STC2
polypeptide may be, for example, detected by immunoprecipitation
using an antibody against the polypeptide. Therefore, for the
purpose for such detection, it is preferred that the STC2
polypeptide or fragments thereof used for the screening contains an
antibody recognition site. The antibody used for the screening may
be one that recognizes an antigenic region (e.g., epitope) of the
present STC2 polypeptide which preparation methods are well known
in the art, and any method may be employed in the present invention
to prepare such antibodies and equivalents thereof.
[0156] Alternatively, the STC2 polypeptide or a fragment thereof
may be expressed as a fusion protein having at its N- or C-terminus
a recognition site (epitope) of a monoclonal antibody, whose
specificity has been revealed, to the N- or C-terminus of the
polypeptide. A commercially available epitope-antibody system can
be used (Experimental Medicine 1995, 13:85-90). Vectors which can
express a fusion protein with, for example, beta-galactosidase,
maltose binding protein, glutathione S-transferase, green
florescence protein (GFP), and such by the use of its multiple
cloning sites are commercially available and can be used for the
present invention. Furthermore, fusion proteins containing much
smaller epitopes to be detected by immunoprecipitation with an
antibody against the epitopes are also known in the art
(Experimental Medicine 1995, 13:85-90). Such epitopes, composed of
several to a dozen amino acids so as not to change the property of
the STC2 polypeptide or fragments thereof, can also be used in the
present invention. Examples include polyhistidine (His-tag),
influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis
virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human
simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on
monoclonal phage), and such.
[0157] Glutathione S-transferase (GST) is also well-known as the
counterpart of the fusion protein to be detected by
immunoprecipitation. When GST is used as the protein to be fused
with the STC2 polypeptide or fragment thereof to form a fusion
protein, the fusion protein can be detected either with an antibody
against GST or a substance specifically binding to GST, i.e., such
as glutathione (e.g., glutathione-Sepharose 4B).
[0158] In immunoprecipitation, an immune complex is formed by
adding an antibody (recognizing the STC2 polypeptide or a fragment
thereof itself, or an epitope tagged to the polypeptide or
fragment) to the reaction mixture of the STC2 polypeptide and the
test agent or substances. If the test agent or substance has the
ability to bind the polypeptide, then the formed immune complex
will include the STC2 polypeptide, the test agent or substance, and
the antibody. On the contrary, if the test agent or substance are
devoid of such ability, then the formed immune complex only include
the STC2 polypeptide and the antibody. Therefore, the binding
ability of a test agent or substance to STC2 polypeptide can be
examined by, for example, measuring the size of the formed immune
complex. Any method for detecting the size of a substance can be
used, including chromatography, electrophoresis, and such. For
example, when mouse IgG antibody is used for the detection, Protein
A or Protein G sepharose can be used for quantitating the formed
immune complex.
[0159] Immunoprecipitation can be performed by following or
according to, for example, the methods in the literature (See, for
example, Harlow et al., Antibodies, Cold Spring Harbor Laboratory
publications, New York, 1988, 511-52).
[0160] Furthermore, the STC2 polypeptide or a fragment thereof used
for the screening of agent or substance that bind to thereto may be
bound to a carrier. Example of carriers that may be used for
binding the polypeptides include insoluble polysaccharides, such as
agarose, cellulose and dextran; and synthetic resins, such as
polyacrylamide, polystyrene and silicon; preferably commercially
available beads and plates (e.g., multi-well plates, biosensor
chip, etc.) prepared from the above materials may be used. When
using beads, they may be filled into a column. Alternatively, the
use of magnetic beads is also known in the art, and enables to
readily isolate polypeptides and agents or substances bound on the
beads via magnetism.
[0161] The binding of a polypeptide to a carrier may be conducted
according to routine methods, such as chemical bonding and physical
adsorption. Alternatively, a polypeptide may be bound to a carrier
via antibodies specifically recognizing the protein. Moreover,
binding of a polypeptide to a carrier can also be conducted by
means of interacting molecules, such as the combination of avidin
and biotin.
[0162] Screening using such carrier-bound STC2 polypeptide or
fragments thereof include, for example, contacting a test agent or
substance to the carrier-bound polypeptide, incubating the mixture,
washing the carrier, and detecting and/or measuring the agent or
substance bound to the carrier. The binding may be carried out in
buffer, for example, but are not limited to, phosphate buffer and
Tris buffer, as long as the buffer does not inhibit the
binding.
[0163] A screening method wherein such carrier-bound STC2
polypeptide or fragments thereof and a composition (e.g., cell
extracts, cell lysates, etc.) are used as the test agent or
substance, such method is generally called affinity chromatography.
For example, the STC2 polypeptide may be immobilized on a carrier
of an affinity column, and a test agent or substance, containing a
substance capable of binding to the polypeptides, is applied to the
column. After loading the test agent or substance, the column is
washed, and then the substance bound to the polypeptide is eluted
with an appropriate buffer.
[0164] A biosensor using the surface plasmon resonance phenomenon
may be used as a mean for detecting or quantifying the bound agent
or substance in the present invention. When such a biosensor is
used, the interaction between the STC2 polypeptide and a test agent
or substance can be observed real-time as a surface plasmon
resonance signal, using only a minute amount of the polypeptide and
without labeling (for example, BIAcore, Pharmacia). Therefore, it
is possible to evaluate the binding between the polypeptide and
test agent or substance using a biosensor such as BIAcore.
[0165] Methods of screening for molecules that bind to a specific
protein among synthetic chemical compounds, or molecules in natural
substance banks or a random phage peptide display library by
exposing the specific protein immobilized on a carrier to the
molecules, and methods of high-throughput screening based on
combinatorial chemistry techniques (Wrighton et al., Science 1996,
273:458-64; Verdine, Nature 1996, 384:11-3) to isolate not only
proteins but chemical compounds are also well-known to those
skilled in the art. These methods can also be used for screening
agents or substances (including agonist and antagonist) that bind
to the STC2 protein or fragments thereof.
[0166] When the test agent or substance is a protein, for example,
West-Western blotting analysis (Skolnik et al., Cell 1991,
65:83-90) can be used for the present method. Specifically, a
protein binding to the STC2 polypeptide can be obtained by
preparing first a cDNA library from cells, tissues, organs, or
cultured cells (e.g., PC cell lines) expected to express at least
one protein binding to the STC2 polypeptide using a phage vector
(e.g., ZAP), expressing the proteins encoded by the vectors of the
cDNA library on LB-agarose, fixing the expressed proteins on a
filter, reacting the purified and labeled STC2 polypeptide with the
above filter, and detecting the plaques expressing proteins to
which the STC2 polypeptide has bound according to the label of the
STC2 polypeptide.
[0167] Labeling substances such as radioisotope (e.g., .sup.3H,
.sup.14C, .sup.32P, .sup.33P, .sup.35S, .sup.125I, .sup.131I),
enzymes (e.g., alkaline phosphatase, horseradish peroxidase,
beta-galactosidase, beta-glucosidase), fluorescent substances
(e.g., fluorescein isothiocyanate (FITC), rhodamine) and
biotin/avidin, may be used for the labeling of STC2 polypeptide in
the present method. When the protein is labeled with radioisotope,
the detection or measurement can be carried out by liquid
scintillation. Alternatively, when the protein is labeled with an
enzyme, it can be detected or measured by adding a substrate of the
enzyme to detect the enzymatic change of the substrate, such as
generation of color, with absorptiometer. Further, in case where a
fluorescent substance is used as the label, the bound protein may
be detected or measured using fluorophotometer.
[0168] Moreover, the STC2 polypeptide bound to the protein can be
detected or measured by utilizing an antibody that specifically
binds to the STC2 polypeptide, or a peptide or polypeptide (for
example, GST) that is fused to the STC2 polypeptide. In case of
using an antibody in the present screening, the antibody is
preferably labeled with one of the labeling substances mentioned
above, and detected or measured based on the labeling substance.
Alternatively, the antibody against the STC2 polypeptide may be
used as a primary antibody to be detected with a secondary antibody
that is labeled with a labeling substance. Furthermore, the
antibody bound to the STC2 polypeptide in the present screening may
be detected or measured using protein G or protein A column.
[0169] Alternatively, in another embodiment of the screening method
of the present invention, two-hybrid system utilizing cells may be
used ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER
Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech);
"HybriZAP Two-Hybrid Vector System" (Stratagene); the references
"Dalton et al., Cell 1992, 68:597-612" and "Fields et al., Trends
Genet. 1994, 10:286-92"). In two-hybrid system, STC2 polypeptide or
a fragment thereof is fused to the SRF-binding region or
GAL4-binding region and expressed in yeast cells. A cDNA library is
prepared from cells expected to express at least one protein
binding to the STC2 polypeptide, such that the library, when
expressed, is fused to the VP16 or GAL4 transcriptional activation
region. The cDNA library is then introduced into the above yeast
cells and the cDNA derived from the library is isolated from the
positive clones detected (when a protein binding to the STC2
polypeptide is expressed in the yeast cells, the binding of the two
activates a reporter gene, making positive clones detectable). A
protein encoded by the cDNA can be prepared by introducing the cDNA
isolated above to E. coli and expressing the protein.
[0170] As a reporter gene, for example, Ade2 gene, lacZ gene, CAT
gene, luciferase gene and such can be used in addition to the HIS3
gene.
[0171] The agent or substance isolated by this screening is a
candidate for antagonists of the STC2 polypeptide. The term
"antagonist" refers to molecules that inhibit the function of the
polypeptide by binding thereto.
[0172] In the present invention, it is revealed that suppressing
the expression of STC2, reduces prostate cancer cell growth. Thus,
by screening for candidate agents or compounds that bind to
polypeptide, candidate compounds that find use to treat or prevent
Prostate cancers can be identified. The potential of these
candidate substance or compounds to treat or prevent prostate
cancers may be evaluated by a secondary and/or further screening to
identify therapeutic agents useful in treating or preventing
prostate cancers. For example, when a compound binding to STC2
protein inhibits, e.g., the proliferative activity or activity to
inhibit phosphate uptake of prostate cancer cells, it may be
concluded that such compound has the STC2 specific therapeutic
effect.
[0173] III-2. Identifying Agents or Substances by Detecting
Biological Activity of the STC2 Polypeptide
[0174] According to the present invention, the expression of STC2
gene was shown to be crucial for the growth and/or survival of
cancer cells, in particular, prostate cancer cells, more
particularly, CRPC cells and aggressive PC cells with high Gleason
score (e.g., Gleason score 8-10). Therefore, agents or substances
that suppress or inhibit the biological function of the
translational product of the STC2 gene is considered to serve as
candidates for treating or preventing cancer. Thus, the present
invention also provides a method for screening a compound for
treating or preventing cancer using the STC2 polypeptide or
fragments thereof including the steps as follows:
a) contacting a test agent or substance with the STC2 polypeptide
or a fragment thereof; and b) detecting the biological activity of
the polypeptide or fragment of step (a).
[0175] Any polypeptide can be used for the screening so long as it
has a biological activity equivalent to the STC2 polypeptide that
can be used as an index in the present screening method. According
to the present invention, the STC2 polypeptide has been
demonstrated to be required for the growth or viability of PC cells
(more specifically, CRPC cells and aggressive PC with high Grealon
score). Furthermore, the STC2 was reported to inhibit the phosphate
uptake of kidney cell line. Therefore, biological activities of the
STC2 polypeptide that can be used as an index for the screening
include such cell growth promoting activity and the inhibiting
activity against phosphate uptake of a kidney cell line. For
example, a human STC2 polypeptide can be used and polypeptides
functionally equivalent thereto including fragments thereof can
also be used. Such polypeptides may be expressed endogenously or
exogenously by suitable cells.
[0176] When the biological activity to be detected in the present
method is cell proliferation, detection may be accomplished, for
example, by preparing cells which express the STC2 polypeptide or a
fragment thereof (e.g., 22Rv1, LNCaP, C4-2B, DU145, PC-3, etc.),
culturing the cells in the presence of a test agent or substance,
and determining the speed of cell proliferation, measuring the cell
cycle and such, as well as by detecting wound-healing activity,
conducting Matrigel invasion assay and measuring the colony forming
activity.
[0177] According to an aspect of the present invention, the
screening further includes, after the above step (b), the steps
of:
c) comparing the biological activity of the polypeptide or fragment
with the biological activity detected in the absence of the agent
or substance; and d) selecting the agent or substance that
suppresses the biological activity of the polypeptide as a
candidate agent for treating or preventing PC.
[0178] According to the present invention, the therapeutic effect
of the test agent or substance on inhibiting the cell growth or a
candidate agent or substance for treating or preventing STC2
associating cancer may be evaluated. Therefore, the present
invention also provides a method of screening for a candidate agent
or substance for inhibiting the cell growth or a candidate agent or
substance for treating or preventing STC2 associating cancer, using
the STC2 polypeptide or fragments thereof including the steps as
follows:
a) contacting a test agent or substance with the STC2 polypeptide
or a functional fragment thereof; and b) detecting the biological
activity of the polypeptide or fragment of step (a), and c)
correlating the biological activity of b) with the therapeutic
effect of the test agent or substance.
[0179] In the present invention, the therapeutic effect may be
correlated with the biological activity STC2 polypeptide or a
functional fragment thereof. For example, when the test agent or
substance suppresses or inhibits the biological activity of STC2
polypeptide or a functional fragment thereof as compared to a level
detected in the absence of the test agent or substance, the test
agent or substance may identified or selected as the candidate
agent or substance having the therapeutic effect. Alternatively,
when the test agent or substance does not suppress or inhibit the
biological activity of STC2 polypeptide or a functional fragment
thereof as compared to a level detected in the absence of the test
agent or substance, the test agent or substance may be identified
as the agent or compound having no significant therapeutic
effect.
[0180] In the preferred embodiments, control cells which do not
express STC2 polypeptide are used. Accordingly, the present
invention also provides a method of screening for a candidate
substance for inhibiting the cell growth or a candidate substance
for treating or preventing STC2 associating disease, using the STC2
polypeptide or fragments thereof including the steps as
follows:
a) culturing cells which express a STC2 polypeptide or a functional
fragment thereof, and control cells that do not express a STC2
polypeptide or a functional fragment thereof in the presence of the
test substance; b) detecting the biological activity of the cells
which express the protein and control cells; and c) selecting the
test compound that inhibits the biological activity in the cells
which express the protein as compared to the proliferation detected
in the control cells and in the absence of said test substance.
[0181] Moreover, the present invention also provides a screening
method following the method described in III-1, including the steps
of:
a) contacting a test agent or substance with the STC2 polypeptide
or a fragment thereof; b) detecting the binding between the
polypeptide or fragment and the test agent or substance; c)
selecting the test agent or substance that binds to the
polypeptide; d) contacting the test agent or substance selected in
step c) with the STC2 polypeptide or a fragment thereof; e)
comparing the biological activity of the polypeptide or fragment
with the biological activity detected in the absence of the agent
or substance; and f) selecting the agent or substance that
suppresses the biological activity of the polypeptide as a
candidate agent for treating or preventing PC.
[0182] The agent or substance isolated by this screening is a
candidate for an antagonist of the STC2 polypeptide, and thus, is a
candidate that inhibits the in vivo interaction of the polypeptide
with molecules (including nucleic acids (RNAs and DNAs) and
proteins).
[0183] In the present invention, the STC2 gene plays the crucial
role in the cell proliferation showing in the example 4 and 5.
Therefore, a biological activity of STC2 to be detected in the
present screening method may be its cell proliferation
activity.
[0184] Moreover, STC2 polypeptide was reported to inhibit the
phosphate uptake of a kidney cell line. Accordingly, a phosphate
uptake activity in a kidney cell line may also be detected as a
biological activity of STC2. Therefore, the present invention
further provides a method for screening an agent for treating or
preventing cancer. An embodiment of this screening method includes
the steps of:
(a) contacting a cell that expresses the STC2 polypeptide or a
fragment thereof with an agent or substance; (b) detecting the
inhibiting activity of the STC2 polypeptide against the phosphate
uptake of a kidney cell line; (c) comparing the inhibiting activity
of the STC2 polypeptide against the phosphate uptake detected in
the absence of the agent or substance; and (d) selecting the agent
or substance that suppresses the inhibiting activity of the STC2
polypeptide against the phosphate uptake activity as a candidate
agent or substance for treating or preventing cancer.
[0185] According to this screening method, candidate compounds for
suppressing the growth of PC cells, especially CRPC and aggressive
PC with high Gleason score (e.g., Gleason score 8-10), can be
identified.
[0186] III-3. Identifying Agents or Substances by Detecting
Expression Level of the STC2 Gene in a Cell
[0187] As discussed in detail above, by controlling the expression
level of the STC2 gene, one can control the onset and progression
of PC. Thus, agents or substances that may be used in the treatment
or prevention of PCs, in particular, CRPC and aggressive PC with
high Gleason score (e.g., Gleason score 8-10) can be identified
through screenings that use the expression levels of STC2 gene as
indices. In the context of the present invention, such screening
may include, for example, the following steps:
a) contacting a test agent or substance with a cell expressing the
STC2 gene; b) detecting the expression level of the STC2 gene; c)
comparing the expression level with the expression level detected
in the absence of the agent or substance; and d) selecting the
agent or substance that reduces the expression level as a candidate
agent for treating or preventing cancer.
[0188] According to the present invention, the therapeutic effect
of the test agent or substance on inhibiting the cell growth or a
candidate agent or substance for treating or preventing STC2
associating cancer may be evaluated. Therefore, the present
invention also provides a method for screening a candidate agent or
substance that suppresses the proliferation of cancer cells, and a
method for screening a candidate agent or substance for treating or
preventing STC2 associating cancer.
[0189] In the context of the present invention, such screening may
include, for example, the following steps:
a) contacting a test agent or substance with a cell expressing the
STC2 gene; b) detecting the expression level of the STC2 gene; and
c) correlating the expression level of b) with the therapeutic
effect of the test agent or substance.
[0190] In the context of the present invention, the therapeutic
effect may be correlated with the expression level of the STC2
gene. For example, when the test agent or substance reduces the
expression level of the STC2 gene as compared to a level detected
in the absence of the test agent or substance, the test agent or
substance may identified or selected as the candidate agent or
substance having the therapeutic effect. Alternatively, when the
test agent or substance does not reduce the expression level of the
STC2 gene as compared to a level detected in the absence of the
test agent or substance, the test agent or substance may be
identified as the agent or substance having no significant
therapeutic effect.
[0191] An agent or substance that inhibits the expression of the
STC2 gene or the activity of its gene product can be identified by
contacting a cell expressing the STC2 gene with a test agent or
substance and then determining the expression level of the STC2
gene. Naturally, the identification may also be performed using a
population of cells that express the gene in place of a single
cell. A decreased expression level detected in the presence of an
agent or substance as compared to the expression level in the
absence of the agent or substance indicates the agent as being an
inhibitor of the STC2 gene, suggesting the possibility that the
agent is useful for inhibiting PC, thus a candidate agent to be
used for the treatment or prevention of PC, in particular, CRPC and
aggressive PC (e.g., Gleason score 8-10).
[0192] The expression level of a gene can be estimated by methods
well known to one skilled in the art. The expression level of the
STC2 gene can be, for example, determined following the method
described above under the item of `I-1. Method for diagnosing
cancer or a predisposition for developing cancer`.
[0193] The cell or the cell population used for such identification
may be any cell or any population of cells so long as it expresses
the STC2 gene. For example, the cell or population may be or
contain an immortalized cell derived from a carcinoma cell,
including PC cell. Cells expressing the STC2 gene include, for
example, cell lines established from cancers (e.g., PC cell lines
such as 22Rv1, LNCaP, C4-2B, DU145, PC-3, etc.). Furthermore, the
cell or population may be or contain a cell which has been
transfected with the STC2 gene.
[0194] The present method allows screening of various agents or
substances mentioned above and is particularly suited for screening
functional nucleic acid molecules including antisense RNA, siRNA,
and such.
[0195] III-4. Identifying Agents or Substances Using
Transcriptional Regulatory Region of STC2 Gene
[0196] According to another aspect, the present invention provides
a method which includes the following steps of:
a) contacting a test agent or substance with a cell into which a
vector, including the transcriptional regulatory region of the STC2
gene and a reporter gene that is expressed under the control of the
transcriptional regulatory region, has been introduced; b)
detecting the expression or activity of said reporter gene; c)
comparing the expression level or activity with the expression
level or activity detected in the absence of the agent or
substance; and d) selecting the agent or substance that reduces the
expression or activity of said reporter gene as a candidate agent
for treating or preventing cancer.
[0197] According to the present invention, the therapeutic effect
of the test agent or substance on inhibiting the cell growth or a
candidate agent or substance for treating or preventing STC2
associating cancer may be evaluated. Therefore, the present
invention also provides a method for screening a candidate agent or
substance that suppresses the proliferation of cancer cells, and a
method for screening a candidate agent or compound for treating or
preventing STC2 associating cancer.
[0198] According to another aspect, the present invention provides
a method which includes the following steps of:
a) contacting a test agent or substance with a cell into which a
vector, composed of the transcriptional regulatory region of the
STC2 gene and a reporter gene that is expressed under the control
of the transcriptional regulatory region, has been introduced; b)
detecting the expression level or activity of said reporter gene;
and c) correlating the expression level of b) with the therapeutic
effect of the test agent or substance.
[0199] In the context of the present invention, the therapeutic
effect may be correlated with the expression level or activity of
said reporter gene. For example, when the test agent or substance
reduces the expression level or activity of said reporter gene as
compared to a level detected in the absence of the test agent or
substance, the test agent or substance may be identified or
selected as the candidate agent or substance having the therapeutic
effect. Alternatively, when the test agent or substance does not
reduce the expression level or activity of said reporter gene as
compared to a level detected in the absence of the test agent or
substance, the test agent or substance may be identified as the
agent or substance having no significant therapeutic effect.
[0200] Suitable reporter genes and host cells are well known in the
art. For example, reporter genes are luciferase, green florescence
protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed),
Chrolamphenicol Acetyltransferase (CAT), lacZ and
beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa and
so on. The reporter construct required for the screening can be
prepared by connecting reporter gene sequence to the
transcriptional regulatory region of STC2. The transcriptional
regulatory region of STC2 herein is the region from start codon to
at least 500 bp upstream, preferably 1000 bp, more preferably 5000
or 10000 bp upstream. A nucleotide segment containing the
transcriptional regulatory region can be isolated from a genome
library or can be propagated by PCR. The reporter construct
required for the screening can be prepared by connecting reporter
gene sequence to the transcriptional regulatory region of any one
of these genes. Methods for identifying a transcriptional
regulatory region, and also assay protocol are well known
(Molecular Cloning third edition chapter 17, 2001, Cold Springs
Harbor Laboratory Press).
[0201] The vector containing the said reporter construct is
infected to host cells and the expression or activity of the
reporter gene is detected by method well known in the art (e.g.,
using luminometer, absorption spectrometer, flow cytometer and so
on). "reduces the expression or activity" as defined herein are
preferably at least 10% reduction of the expression or activity of
the reporter gene in comparison with in absence of the compound,
more preferably at least 25%, 50% or 75% reduction and most
preferably at 95% reduction.
[0202] III-5. Selecting Therapeutic Agents or Substances that are
Appropriate for a Particular Individual
[0203] Differences in the genetic makeup of individuals can result
in differences in their relative abilities to metabolize various
drugs. An agent or substance that is metabolized in a subject to
act as an anti-tumor agent or substance can manifest itself by
inducing a change in a gene expression pattern in the subject's
cells from that characteristic of a cancerous state to a gene
expression pattern characteristic of a non-cancerous state.
Accordingly, the STC2 gene differentially expressed between
non-cancerous prostate cells and cancerous prostate cells, in
particular, CRPC cells and aggressive PC cells (e.g., Gleason score
8-10), disclosed herein allow for a putative therapeutic or
prophylactic inhibitor of PC (in particular, CRPC and aggressive
PC) to be tested in a test cell population from a selected subject
in order to determine if the agent or substance are a suitable
inhibitor of PC in the subject.
[0204] To identify an inhibitor of PC that is appropriate for a
specific subject, a test cell population from the subject is
exposed to a candidate therapeutic agent or substance, and the
expression of STC2 gene is determined.
[0205] In the context of the method of the present invention, test
cell populations contain cells expressing the STC2 gene.
Preferably, the test cell is a PC cell, more preferably, a CRPC
cell or an aggressive PC cell (e.g., Gleason score 8-10).
[0206] Specifically, a test cell population may be incubated in the
presence of a candidate therapeutic agent or substance and the
expression of the STC2 gene in the test cell population may be
measured and compared to one or more reference profiles, e.g., a
cancerous reference expression profile or a non-cancerous reference
expression profile.
[0207] A decrease in the expression of the STC2 gene in a test cell
population relative to a reference cell population containing
cancerous cells indicates that the agent or substance have
therapeutic potential. Alternatively, a similarity in the
expression of the STC2 gene in a test cell population relative to a
reference cell population not containing cancerous cells indicates
that the agent or substance have therapeutic potential.
[0208] IV. Double-Stranded Molecules and Use Thereof
[0209] IV-1. Double-Stranded Molecules
[0210] Double-stranded molecules targeting the STC2 gene can
inhibit the expression of the STC2 gene and cell proliferation when
the molecule introduced into a cell expressing the STC2 gene
(Example 4). Accordingly, present invention provides for such
double-stranded molecules and methods of use the same to target the
STC2 gene.
[0211] The term "isolated double-stranded molecule" refers to a
nucleic acid molecule that inhibits expression of a target gene
including, for example, short interfering RNA (siRNA; e.g.,
double-stranded ribonucleic acid (dsRNA) or small hairpin RNA
(shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g.
double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin
chimera of DNA and RNA (shD/R-NA)).
[0212] As used herein, sense strand of a target sequence is a
nucleotide sequence within mRNA or cDNA sequence of a gene, which
will result in suppress of translation of the whole mRNA if a
double-stranded nucleic acid molecule of the present invention was
introduced within a cell expressing the gene. A nucleotide sequence
within mRNA or cDNA sequence of a gene can be determined to be a
target sequence when a double-stranded polynucleotide including a
sequence corresponding to the target sequence inhibits expression
of the gene in a cell expressing the gene. The double stranded
polynucleotide by which suppresses the gene expression may be
composed of the target sequence and 3' overhang (e.g., uu).
[0213] As used herein, the term "siRNA" refers to a double-stranded
RNA molecule which prevents translation of a target mRNA. Standard
techniques of introducing siRNA into the cell are used, including
those in which DNA is a template from which RNA is transcribed. The
siRNA includes a sense nucleic acid sequence (also referred to as
"sense strand") of the STC2 gene, a antisense nucleic acid sequence
(also referred to as "antisense strand") of the STC2 gene or both.
The siRNA may be constructed such that a single transcript has both
the sense and complementary antisense nucleic acid sequences of the
target gene, e.g., a hairpin. The siRNA may either be a dsRNA or
shRNA.
[0214] As used herein, the term "dsRNA" refers to a construct of
two RNA molecules having complementary sequences to one another and
that have annealed together via the complementary sequences to form
a double-stranded RNA molecule. The nucleotide sequence of two
strands may include not only the "sense" or "antisense" RNAs
selected from a protein coding sequence of target gene sequence,
but also RNA molecule having a nucleotide sequence selected from
non-coding rigion of the target gene.
[0215] The term "shRNA", as used herein, refers to an siRNA having
a stem-loop structure, including a first and second regions
complementary to one another, i.e., sense and antisense strands.
The degree of complementarity and orientation of the regions are
sufficient such that base pairing occurs between the regions, the
first and second regions are joined by a loop region, and the loop
results from a lack of base pairing between nucleotides (or
nucleotide analogs) within the loop region. The loop region of an
shRNA is a single-stranded region intervening between the sense and
antisense strands and may also be referred to as "intervening
single-strand".
[0216] As use herein, the term "siD/R-NA" refers to a
double-stranded polynucleotide molecule which is composed of both
RNA and DNA, and includes hybrids and chimeras of RNA and DNA and
prevents translation of a target mRNA. Herein, a hybrid indicates a
molecule wherein a polynucleotide composed of DNA and a
polynucleotide composed of RNA hybridize to each other to form the
double-stranded molecule; whereas a chimera indicates that one or
both of the strands composing the double stranded molecule may
contain RNA and DNA. Standard techniques of introducing siD/R-NA
into the cell are used. The siD/R-NA includes a sense nucleic acid
sequence (also referred to as "sense strand") of the STC2 gene, a
antisense nucleic acid sequence (also referred to as "antisense
strand") of the STC2 gene or both. The siD/R-NA may be constructed
such that a single transcript has both the sense and complementary
antisense nucleic acid sequences from the target gene, e.g., a
hairpin. The siD/R-NA may either be a dsD/R-NA or shD/R-NA.
[0217] As used herein, the term "dsD/R-NA" refers to a construct of
two molecules having complementary sequences to one another and
that have annealed together via the complementary sequences to form
a double-stranded polynucleotide molecule. The nucleotide sequence
of two strands may include not only the "sense" or "antisense"
polynucleotides sequence selected from a protein coding sequence of
target gene sequence, but also polynucleotide having a nucleotide
sequence selected from non-coding region of the target gene. One or
both of the two molecules constructing the dsD/R-NA are composed of
both RNA and DNA (chimeric molecule), or alternatively, one of the
molecules is composed of RNA and the other is composed of DNA
(hybrid double-strand).
[0218] The term "shD/R-NA", as used herein, refers to an siD/R-NA
having a stem-loop structure, having a first and second regions
complementary to one another, i.e., sense and antisense strands.
The degree of complementarity and orientation of the regions are
sufficient such that base pairing occurs between the regions, the
first and second regions are joined by a loop region, and the loop
results from a lack of base pairing between nucleotides (or
nucleotide analogs) within the loop region. The loop region of an
shD/R-NA is a single-stranded region intervening between the sense
and antisense strands and may also be referred to as "intervening
single-strand".
[0219] As used herein, an "isolated nucleic acid" is a nucleic acid
removed from its original environment (e.g., the natural
environment if naturally occurring) and thus, synthetically altered
from its natural state. In the context of the present invention,
examples of isolated nucleic acid includes DNA, RNA, and
derivatives thereof.
[0220] A double-stranded molecule targeting the STC2 gene molecule
that hybridizes to the mRNA of the STC2 gene, decreases or inhibits
production of the STC2 protein encoded by the STC2 gene by
associating with the normally single-stranded mRNA transcript of
the gene, thereby interfering with translation and thus, inhibiting
expression of the protein.
[0221] The double-stranded molecule serves as a guide for
identifying homologous sequences in mRNA for the RISC complex, when
the double-stranded molecule is introduced into cells. The
identified target RNA is cleaved and degraded by the nuclease
activity of Dicer, through which the double-stranded molecule
eventually decreases or inhibits production (expression) of the
polypeptide encoded by the RNA. Thus, a double-stranded molecule of
the present invention can be defined by its ability to generate a
single-strand that specifically hybridizes to the mRNA of the STC2
gene under stringent conditions. Herein, the portion of the mRNA
that hybridizes with the single-strand generated from the
double-stranded molecule is referred to as "target sequence" or
"target nucleic acid" or "target nucleotide". In the present
invention, nucleotide sequence of the "target sequence" can be
shown using not only the RNA sequence of the mRNA, but also the DNA
sequence of cDNA synthesized from the mRNA.
[0222] In the context of the present invention, the target sequence
of a double-stranded molecule is preferably less than 500, 200,
100, 50, or 25 base pairs in length. More preferably, the target
sequences of a double-stranded is 19-25 base pairs in length.
Accordingly, the present invention provides the double-stranded
molecules having a sense strand and an antisense strand, wherein
the sense strand includes a nucleotide sequence corresponding to a
target sequence. In preferable embodiments, the sense strand
hybridizes with antisense strand at the target sequence to form the
double-stranded molecule having between 19 and 25 nucleotide pair
in length.
[0223] Exemplary target nucleotide sequences of double-stranded
molecules targeting the STC2 gene includes the nucleotide sequences
of SEQ ID NO: 8 or 9. The nucleotide "t" in the sequence should be
replaced with "u" in RNA or derivatives thereof. Accordingly, for
example, double-stranded molecules of the present invention can
include the nucleotide sequence:
TABLE-US-00001 (SEQ ID NO: 8) 5'-GACGAACAGTCTGAGTATT-3' or (SEQ ID
NO: 9) 5'-GCAGGAGCTGGTATTGTAG-3' as a target sequence.
[0224] In order to enhance the inhibition activity of the
double-stranded molecule, nucleotide "u", "t" or other nucleotide
can be added to the 3' end of the target sequence in the antisense
strand and/or sense strand. The number of nucleotides to be added
is at least 2, generally 2 to 10, preferably 2 to 5. The added
nucleotides form a single strand at the 3' end of the antisense
strand and/or sense strand of the double-stranded molecule.
[0225] Other target sequences of suitable double-stranded molecules
for the present invention can be designed using an siRNA design
computer program available from the Ambion website (world wide
web.ambion.com/techlib/misc/siRNA_finder.html). The computer
program selects target sequences for double-stranded molecules
based on the following protocol.
[0226] Selection of Target Sites:
1. Beginning with the AUG start codon of the object transcript,
scan downstream for AA dinucleotide sequences. Record the
occurrence of each AA and the 3' adjacent 19 nucleotides as
potential siRNA target sites. Tuschl et al. Genes Cev 1999,
13(24):3191-7 don't recommend designing siRNA to the 5' and 3'
untranslated regions (UTRs) and regions near the start codon
(within 75 nucleotides) as these may be richer in regulatory
protein binding sites. UTR-binding proteins and/or translation
initiation complexes may interfere with binding of the siRNA
endonuclease complex. 2. Compare the potential target sites to the
human genome database and eliminate from consideration any target
sequences with significant homology to other coding sequences. The
homology search can be performed using BLAST (Altschul S F et al.,
Nucleic Acids Res 1997, 25:3389-402; J Mol Biot 1990, 215:403-10.),
which can be found on the NCBI server at: world wide
web.ncbi.nlm.nih.gov/BLAST/. 3. Select qualifying target sequences
for synthesis. At Ambion, preferably several target sequences can
be selected along the length of the gene to evaluate.
[0227] The double-stranded molecule of the present invention is
directed to a single target sequence or may be directed to a
plurality of target sequences.
[0228] A target sequence for the STC2 gene is meant a nucleotide
sequence that is identical to a portion of the STC2 gene (i.e, a
polynucleotide within the STC2 gene that is equal in length to and
complementary to an double-stranded molecule). The target sequence
can include the 5' untranslated (UT) region, the open reading frame
(ORF) or the 3' untranslated region of the STC2 gene.
Alternatively, the double-stranded molecule can be a nucleic acid
sequence complementary to an upstream or downstream modulator of
the STC2 gene expression. Examples of upstream and downstream
modulators include, a transcription factor that binds the STC2 gene
promoter, a kinase or phosphatase that interacts with the STC2
polypeptide, a promoter or enhancer of the STC2 gene.
[0229] A double-stranded molecule of the present invention
targeting the above-mentioned targeting sequence of the STC2 gene
include isolated polynucleotides that includes any of the nucleic
acid sequences of target sequences and/or complementary sequences
to the target sequences. Examples of polynucleotides targeting the
STC2 gene include those having the sequence of SEQ ID NO: 8 or 9
and/or complementary sequences to these nucleotides. However, the
present invention is not limited to these examples, and minor
modifications in the aforementioned nucleic acid sequences are
acceptable so long as the modified molecule retains the ability to
suppress the expression of the STC2 gene. Herein, "minor
modification" in a nucleic acid sequence indicates one, two or
several substitutions, deletions, additions or insertions of
nucleic acids to the sequence. Typically, a minor modification will
be four or fewer, sometimes three or fewer, and often two or fewer
substitutions, deletions, additions or insertions of nucleic acids
to the sequence.
[0230] According to the present invention, a double-stranded
molecule of the present invention can be tested for its ability
using the methods utilized in the Examples. In the Examples, the
double-stranded molecules having sense strands or antisense strands
complementary thereto of three portions of mRNA of the STC2 genes
were tested in vitro for their ability to decrease production of
the STC2 gene product in prostate cancer cell line according to
standard methods. Furthermore, for example, reduction in the STC2
gene product in cells contacted with the candidate double-stranded
molecule as compared to cells cultured in the absence of the
candidate molecule can be detected by, e.g. RT-PCR using primers
for STC2 mRNA mentioned under Example 1, item "Semi-quantitative
RT-PCR". Sequences that decrease the production of the STC2 gene
product in vitro cell-based assays can then be tested for their
inhibitory effects on cell growth. Sequences that inhibit cell
growth in vitro cell-based assay can then be tested for their in
vivo ability using animals with cancer, e.g. nude mouse xenograft
models, to confirm decreased production of the STC2 gene product
and decreased cancer cell growth.
[0231] When the isolated polynucleotide is RNA or derivatives
thereof, base "t" should be replaced with "u" in the nucleotide
sequences. As used herein, the term "complementary" refers to
Watson-Crick or Hoogsteen base pairing between nucleotides units of
a polynucleotide, and the term "binding" means the physical or
chemical interaction between two polynucleotides. When the
polynucleotide includes modified nucleotides and/or
non-phosphodiester linkages, these polynucleotides may also bind
each other as same manner. Generally, complementary polynucleotide
sequences hybridize under appropriate conditions to form stable
duplexes containing few or no mismatches. Furthermore, the sense
strand and antisense strand of the isolated polynucleotide of the
present invention can form double-stranded molecule or hairpin loop
structure by the hybridization. In a preferred embodiment, such
duplexes contain no more than 1 mismatch for every 10 matches. In
an especially preferred embodiment, where the strands of the duplex
are fully complementary, such duplexes contain no mismatches.
[0232] The double-stranded molecules of the present invention may
contain one or more modified nucleotides and/or non-phosphodiester
linkages. Chemical modifications well known in the art are capable
of increasing stability, availability, and/or cell uptake of the
double-stranded molecule. The skilled person will be aware of other
types of chemical modification which may be incorporated into the
present molecules (WO03/070744; WO2005/045037). In one embodiment,
modifications can be used to provide improved resistance to
degradation or improved uptake. Examples of such modifications
include phosphorothioate linkages, 2'-O-methyl ribonucleotides
(especially on the sense strand of a double-stranded molecule),
2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides,
"universal base" nucleotides, 5'-C-methyl nucleotides, and inverted
deoxyabasic residue incorporation (US20060122137). In another
embodiment, modifications can be used to enhance the stability or
to increase targeting efficiency of the double-stranded molecule.
Modifications include chemical cross linking between the two
complementary strands of a double-stranded molecule, chemical
modification of a 3' or 5' terminus of a strand of a
double-stranded molecule, sugar modifications, nucleobase
modifications and/or backbone modifications, 2-fluoro modified
ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212). In
another embodiment, modifications can be used to increased or
decreased affinity for the complementary nucleotides in the target
mRNA and/or in the complementary double-stranded molecule strand
(WO2005/044976). For example, an unmodified pyrimidine nucleotide
can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl
pyrimidine. Additionally, an unmodified purine can be substituted
with a 7-deaza, 7-alkyl, or 7-alkenyl purine. In another
embodiment, when the double-stranded molecule is a double-stranded
molecule with a 3' overhang, the 3'-terminal nucleotide overhanging
nucleotides may be replaced by deoxyribonucleotides (Elbashir S M
et al., Genes Dev 2001 Jan. 15, 15(2): 188-200). For further
details, published documents such as US20060234970 are available.
The present invention is not limited to these examples and any
known chemical modifications may be employed for the
double-stranded molecules of the present invention so long as the
resulting molecule retains the ability to inhibit the expression of
the target gene.
[0233] Furthermore, the double-stranded molecules of the present
invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA.
Specifically, a hybrid polynucleotide of a DNA strand and an RNA
strand or a DNA-RNA chimera polynucleotide shows increased
stability. Mixing of DNA and RNA, i.e., a hybrid type
double-stranded molecule composed of a DNA strand (polynucleotide)
and an RNA strand (polynucleotide), a chimera type double-stranded
molecule including both DNA and RNA on any or both of the single
strands (polynucleotides), or the like may be formed for enhancing
stability of the double-stranded molecule.
[0234] The hybrid of a DNA strand and an RNA strand may be either
where the sense strand is DNA and the antisense strand is RNA, or
vice versa, so long as it has an activity to inhibit expression of
the target gene when introduced into a cell expressing the gene.
Preferably, the sense strand polynucleotide is DNA and the
antisense strand polynucleotide is RNA. Also, the chimera type
double-stranded molecule may be either where both of the sense and
antisense strands are composed of DNA and RNA, or where any one of
the sense and antisense strands is composed of DNA and RNA so long
as it has an activity to inhibit expression of the target gene when
introduced into a cell expressing the gene. In order to enhance
stability of the double-stranded molecule, the molecule preferably
contains as much DNA as possible, whereas to induce inhibition of
the target gene expression, the molecule is required to be RNA
within a range to induce sufficient inhibition of the expression.
As a preferred example of the chimera type double-stranded
molecule, an upstream partial region (i.e., a region flanking to
the target sequence or complementary sequence thereof within the
sense or antisense strands) of the double-stranded molecule is RNA.
Preferably, the upstream partial region indicates the 5' side
(5'-end) of the sense strand and the 3' side (3'-end) of the
antisense strand. Alternatively, regions flanking to 5'-end of
sense strand and/or 3'-end of antisense strand are referred to
upstream partial region. That is, in preferable embodiments, a
region flanking to the 3'-end of the antisense strand, or both of a
region flanking to the 5'-end of sense strand and a region flanking
to the 3'-end of antisense strand composed of RNA. For instance,
the chimera or hybrid type double-stranded molecule of the present
invention include following combinations.
Sense Strand:
5'-[---DNA---]-3'
3'-(RNA)-[DNA]-5'
[0235] :antisense strand,
Sense Strand:
5'-(RNA)-[DNA]-3'
3'-(RNA)-[DNA]-5'
[0236] :antisense strand, and
Sense Strand:
5'-(RNA)-[DNA]-3'
3'-(---RNA---)-5'
[0237] :antisense strand.
[0238] The upstream partial region preferably is a domain composed
of 9 to 13 nucleotides counted from the terminus of the target
sequence or complementary sequence thereto within the sense or
antisense strands of the double-stranded molecules. Moreover,
preferred examples of such chimera type double-stranded molecules
include those having a strand length of 19 to 21 nucleotides in
which at least the upstream half region (5' side region for the
sense strand and 3' side region for the antisense strand) of the
polynucleotide is RNA and the other half is DNA. In such a chimera
type double-stranded molecule, the effect to inhibit expression of
the target gene is much higher when the entire antisense strand is
RNA (US20050004064).
[0239] In the context of the present invention, the double-stranded
molecule may form a hairpin, such as a short hairpin RNA (shRNA)
and short hairpin composed of DNA and RNA (shD/R-NA). The shRNA or
shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a
tight hairpin turn that can be used to silence gene expression via
RNA interference. The shRNA or shD/R-NA may include the sense
target sequence and the antisense target sequence on a single
strand wherein the sequences are separated by a loop sequence.
Generally, the hairpin structure is cleaved by the cellular
machinery into dsRNA or dsD/R-NA, which is then bound to the
RNA-induced silencing complex (RISC). This complex binds to and
cleaves mRNAs which match the target sequence of the dsRNA or
dsD/R-NA.
[0240] A loop sequence composed of an arbitrary nucleotide sequence
can be located between the sense and antisense strands in order to
form a hairpin loop structure. Thus, the double-stranded molecule
contained in the inventive composition may take the general formula
5'-[A]-[B]-[A']-3', wherein [A] is the sense strand having a target
sequence, [B] is an intervening single-strand and [A'] is the
antisense strand having a complementary sequence to [A]. The target
sequence may be selected from group composed of, for example,
nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO 9.
[0241] The present invention is not limited to these examples, and
the target sequence in [A] may be modified sequences from these
examples so long as the double-stranded molecule retains the
ability to suppress the expression of the targeted STC2 gene. The
region [A] hybridizes to [A'] to form a loop composed of the region
[B]. The intervening single-stranded portion [B], i.e., loop
sequence may be preferably 3 to 23 nucleotides in length. The loop
sequence, for example, can be selected from group composed of
following sequences (world wide
web.ambion.com/techlib/tb/tb.sub.--506.html). Furthermore, loop
sequence composed of 23 nucleotides also provides active siRNA
(Jacque J M et al., Nature 2002 Jul. 25, 418(6896): 435-8, Epub
2002 Jun. 26):
CCC, CCACC, or CCACACC: Jacque J M et al., Nature 2002 Jul. 25,
418(6896): 435-8, Epub 2002 Jun. 26;
UUCG: Lee N S et al., Nat Biotechnol 2002 May, 20(5): 500-5;
Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb. 18, 100(4):
1639-44, Epub 2003 Feb. 10; and
UUCAAGAGA: Dykxhoorn D M et al., Nat Rev Mol Cell Biol 2003 Jun.,
4(6): 457-67.
[0242] Exemplary, preferable double-stranded molecules having
hairpin loop structure of the present invention are shown below. In
the following structure, the loop sequence can be selected from
group composed of AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC,
and UUCAAGAGA; however, the present invention is not limited
thereto:
TABLE-US-00002 (for target sequence of SEQ ID NO: 8)
5'-GACGAACAGUCUGAGUAUU-[B]-AAUACUCAGACUGUUCGUC-3'; and (for target
sequence of SEQ ID NO: 9)
5'-GCAGGAGCUGGUAUUGUAG-[B]-CUACAAUACCAGCUCCUGC-3'.
[0243] The method of preparing the double-stranded molecule is not
particularly limited but it is preferable to use a chemical
synthetic method known in the art. According to the chemical
synthesis method, sense and antisense single-stranded
polynucleotides are separately synthesized and then annealed
together via an appropriate method to obtain a double-stranded
molecule. Specific example for the annealing includes wherein the
synthesized single-stranded polynucleotides are mixed in a molar
ratio of preferably at least about 3:7, more preferably about 4:6,
and most preferably substantially equimolar amount (i.e., a molar
ratio of about 5:5). Next, the mixture is heated to a temperature
at which double-stranded molecules dissociate and then is gradually
cooled down. The annealed double-stranded polynucleotide can be
purified by usually employed methods known in the art. Example of
purification methods include methods utilizing agarose gel
electrophoresis or wherein remaining single-stranded
polynucleotides are optionally removed by, e.g., degradation with
appropriate enzyme.
[0244] The regulatory sequences flanking STC2 sequences may be
identical or different, such that their expression can be modulated
independently, or in a temporal or spatial manner. The
double-stranded molecules can be transcribed intracellularly by
cloning STC2 gene templates into a vector containing, e.g., a RNA
pol III transcription unit from the small nuclear RNA (snRNA) U6 or
the human H1 RNA promoter.
[0245] Iv-2. Vectors Containing a Double-Stranded Molecule of the
Present Invention
[0246] Also included in the invention are vector compositions
containing one or more of the antisense nucleic acids or the
double-stranded molecules described herein, and a cell containing
the vector. A vector of the present invention preferably encodes an
antisense nucleic acid or a double-stranded molecule of the present
invention in an expressible form. Herein, the phrase "in an
expressible form" indicates that the vector, when introduced into a
cell, will express the molecule. In a preferred embodiment, the
vector includes regulatory elements necessary for expression of the
antisense nucleic acid or the double-stranded molecule. Such
vectors of the present invention may be used for producing the
present antisense nucleic acids or double-stranded molecules, or
directly as an active ingredient for treating cancer.
[0247] Alternatively, the present invention provides vectors
including each of a combination of polynucleotide having a sense
strand nucleic acid and an antisense strand nucleic acid, wherein
said sense strand nucleic acid has the nucleotide sequence of SEQ
ID NO: 8 or 9, and said antisense strand nucleic acid has a
sequence complementary to the sense strand, wherein the transcripts
of said sense strand and said antisense strand hybridize to each
other to form a double-stranded molecule, and wherein said vectors,
when introduced into a cell expressing the STC2 gene, inhibits
expression of said gene. Preferably, the polynucleotide is an
oligonucleotide of between about 19 and 25 nucleotides pair in
length (e.g., contiguous nucleotides from the nucleotide sequence
of SEQ ID NO: 11). More preferably, the combination of
polynucleotide includes a single oligonucleotide transcript
including the sense strand and the antisense strand linked via a
single-stranded nucleotide sequence. More preferably, the
combination of polynucleotide has the general formula
5'-[A]-[B]-[A']-3', wherein [A] is the nucleotide sequence of SEQ
ID NO: 8 or 9; [B] is a nucleotide sequence composed of about 3 to
about 23 nucleotide; and [A'] is a nucleotide sequence
complementary to [A].
[0248] Vectors of the present invention can be produced, for
example, by cloning STC2 gene into an expression vector so that
regulatory sequences are operatively-linked to STC2 gene sequence
in a manner to allow expression (by transcription of the DNA
molecule) of an antisense nucleic acid or both strands of a
double-stranded molecule (Lee N S et al., Nat Biotechnol 2002 May,
20(5): 500-5).
[0249] For example, in double-stranded molecules, RNA molecule that
is the antisense to mRNA is transcribed by a first promoter (e.g.,
a promoter sequence flanking to the 3' end of the cloned DNA) and
RNA molecule that is the sense strand to the mRNA is transcribed by
a second promoter (e.g., a promoter sequence flanking to the 5' end
of the cloned DNA). The sense and antisense strands hybridize in
vivo to generate a double-stranded molecule constructs for
silencing of the gene. Alternatively, two vectors constructs
respectively encoding the sense and antisense strands of the
double-stranded molecule are utilized to respectively express the
sense and anti-sense strands and then forming a double-stranded
molecule construct. Furthermore, the cloned sequence may encode a
construct having a secondary structure (e.g., hairpin); namely, a
single transcript of a vector contains both the sense and
complementary antisense sequences of the target gene.
[0250] The vectors of the present invention may also be equipped so
to achieve stable insertion into the genome of the target cell
(see, e.g., Thomas K R & Capecchi M R, Cell 1987, 51: 503-12
for a description of homologous recombination cassette vectors).
See, e.g., Wolff et al., Science 1990, 247: 1465-8; U.S. Pat. Nos.
5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647;
and WO 98/04720. Examples of DNA-based delivery technologies
include "naked DNA", facilitated (bupivicaine, polymers,
peptide-mediated) delivery, cationic lipid complexes, and
particle-mediated ("gene gun") or pressure-mediated delivery (see,
e.g., U.S. Pat. No. 5,922,687).
[0251] The vectors of the present invention may be, for example,
viral or bacterial vectors. Examples of expression vectors include
attenuated viral hosts, such as vaccinia or fowlpox (see, e.g.,
U.S. Pat. No. 4,722,848). This approach involves the use of
vaccinia virus, e.g., as a vector to express nucleotide sequences
that encode the antisense nucleic acid or the double-stranded
molecule. Upon introduction into a cell expressing the target gene,
the recombinant vaccinia virus expresses the molecule and thereby
suppresses the proliferation of the cell. Another example of
useable vector includes Bacille Calmette Guerin (BCG). BCG vectors
are described in Stover et al., Nature 1991, 351: 456-60. A wide
variety of other vectors are useful for therapeutic administration
and production of the antisense nucleic acids or the
double-stranded molecules; examples include adeno and
adeno-associated virus vectors, retroviral vectors, Salmonella
typhi vectors, detoxified anthrax toxin vectors, and the like. See,
e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al.,
J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14:
571-85.
[0252] IV-3. Methods for Treating or Preventing Cancer Using a
Double-Stranded Molecule of the Present Invention:
[0253] In present invention, two different double-stranded
molecules targeting the STC2 gene could effectively knocked down
the expression of the gene in lung cancer cell lines coincided with
suppression of cell proliferation (Example 4).
[0254] Accordingly, the present invention provides methods for
inhibiting cell growth, i.e., prostate cancer cell growth, by
inducing dysfunction of the STC2 gene via inhibiting the expression
of the STC2 gene. STC2 gene expression can be inhibited by the
aforementioned double-stranded molecules of the present invention
that specifically target of the STC2 gene or the vectors of the
present invention that can express the double-stranded
molecules.
[0255] Such ability of the present double-stranded molecules and
vectors to inhibit cell growth of cancerous cell indicates that
they can be used for methods for treating cancer. Thus, the present
invention provides methods to treat patients with prostate cancer
by administering a double-stranded molecule against STC2 gene or a
vector expressing thereof without adverse effect because that genes
were hardly detected in normal organs. In the preferred embodiment
of the present invention, the prostate cancer is CRPC or aggressive
PC (e.g., Gleason score 8-10).
[0256] The growth of cells expressing a STC2 gene may be inhibited
by contacting the cells with a double-stranded molecule against a
STC2 gene, a vector expressing the molecule or a composition
containing the same. The cell may be further contacted with a
transfection agent. Suitable transfection agents are known in the
art. The phrase "inhibition of cell growth" indicates that the cell
proliferates at a lower rate or has decreased viability as compared
to a cell not exposed to the molecule. Cell growth may be measured
by methods known in the art, e.g., using the MTT cell proliferation
assay.
[0257] Thus, patients suffering from or at risk of developing
disease related to STC2 gene may be treated with an administration
of at least one of the present double-stranded molecules, at least
one vector expressing at least one of the molecules or at least one
composition containing at least one of the molecules. For example,
patients suffering from prostate cancer may be treated according to
the present methods. The type of cancer may be identified by
standard methods according to the particular type of tumor to be
diagnosed. Prostate cancer may be diagnosed, for example, with PSA,
as prostate cancer marker. Alternatively, the diagnosing method of
the present invention described in the item "I. Diagnosing cancer"
may be used for identifying prostate cancer. In the preferred
embodiment of the present invention, patients to be treated by the
methods of the present invention are selected by detecting the
expression level of STC2 gene in a biopsy specimen from the
patient. Preferably, before the treatment of the present invention,
the biopsy specimen from the subject is confirmed for STC2 gene
over-expression by methods known in the art, for example,
immunohistochemical analysis or RT-PCR.
[0258] According to the present method to inhibit cell growth and
thereby treat cancer, through the administration of plural kinds of
the double-stranded molecules (or vectors expressing or
compositions containing the same), each of the molecules may have
different structures but act at mRNA that matches the same target
sequence of STC2 gene. Alternatively plural kinds of the
double-stranded molecules may act on at mRNA that matches different
target sequence of same gene. For example, the method may utilize
double-stranded molecules directed to STC2 gene.
[0259] For inhibiting cell growth, a double-stranded molecule of
the present invention may be directly introduced into the cells in
a form to achieve binding of the molecule with corresponding mRNA
transcripts. Alternatively, as described above, a DNA encoding the
double-stranded molecule may be introduced into cells as a vector.
For introducing the double-stranded molecules and vectors into the
cells, transfection-enhancing agent, such as FuGENE (Roche
diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine
(Invitrogen), and Nucleofector (Wako pure Chemical), may be
employed.
[0260] A treatment is deemed "efficacious" if it leads to clinical
benefit such as, reduction in expression of STC2 gene, or a
decrease in size, prevalence, or metastatic potential of the cancer
in the subject. When the treatment is applied prophylactically,
"efficacious" means that it retards or prevents cancers from
forming or prevents or alleviates a clinical symptom of cancer.
[0261] Efficaciousness is determined in association with any known
method for diagnosing or treating the particular tumor type. To the
extent that the methods and compositions of the present invention
find utility in the context of "prevention" and "prophylaxis", such
terms are interchangeably used herein to refer to any activity that
reduces the burden of mortality or morbidity from disease.
Prevention and prophylaxis can occur "at primary, secondary and
tertiary prevention levels." While primary prevention and
prophylaxis avoid the development of a disease, secondary and
tertiary levels of prevention and prophylaxis encompass activities
aimed at the prevention and prophylaxis of the progression of a
disease and the emergence of symptoms as well as reducing the
negative impact of an already established disease by restoring
function and reducing disease-related complications. Alternatively,
prevention and prophylaxis can include a wide range of prophylactic
therapies aimed at alleviating the severity of the particular
disorder, e.g. reducing the proliferation and metastasis of
tumors.
[0262] The treatment and/or prophylaxis of cancer and/or the
prevention of postoperative recurrence thereof include any of the
following steps, such as the surgical removal of cancer cells, the
inhibition of the growth of cancerous cells, the involution or
regression of a tumor, the induction of remission and suppression
of occurrence of cancer, the tumor regression, and the reduction or
inhibition of metastasis. Effectively treating and/or the
prophylaxis of cancer decreases mortality and improves the
prognosis of individuals having cancer, decreases the levels of
tumor markers in the blood, and alleviates detectable symptoms
accompanying cancer. For example, reduction or improvement of
symptoms constitutes effectively treating and/or the prophylaxis
include 10%, 20%, 30% or more reduction, or stable disease.
[0263] It is understood that a double-stranded molecule of the
present invention degrades the target mRNA in sub-stoichiometric
amounts. Without wishing to be bound by any theory, it is believed
that the double-stranded molecule of the present invention causes
degradation of the target mRNA in a catalytic manner. Thus, as
compared to standard cancer therapies, the present invention
requires the delivery of significantly less double-stranded
molecule at or near the site of cancer in order to exert
therapeutic effect.
[0264] One skilled in the art can readily determine an effective
amount of the double-stranded molecule of the present invention to
be administered to a given subject, by taking into account factors
such as body weight, age, sex, type of disease, symptoms and other
conditions of the subject; the route of administration; and whether
the administration is regional or systemic. Generally, an effective
amount of the double-stranded molecule of the present invention is
an intercellular concentration at or near the cancer site of from
about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM
to about 50 nM, more preferably from about 2.5 nM to about 10 nM.
It is contemplated that greater or smaller amounts of the
double-stranded molecule can be administered. The precise dosage
required for a particular circumstance may be readily and routinely
determined by one of skill in the art.
[0265] The present methods can be used to inhibit the growth or
metastasis of cancer expressing STC2 gene; for example prostate
cancer, especially CRPC or aggressive PC (e.g., Gleason score
8-10). In particular, a double-stranded molecule containing a
target sequence of SEQ ID NO: 8 or 9 is particularly preferred for
the treatment of prostate cancer.
[0266] For treating cancer, the double-stranded molecule of the
present invention can also be administered to a subject in
combination with a pharmaceutical agent different from the
double-stranded molecule. Alternatively, the double-stranded
molecule of the present invention can be administered to a subject
in combination with another therapeutic method designed to treat
cancer. For example, the double-stranded molecule of the present
invention can be administered in combination with therapeutic
methods currently employed for treating cancer or preventing cancer
metastasis (e.g., radiation therapy, surgery and treatment using
chemotherapeutic agents).
[0267] In the present methods, the double-stranded molecule can be
administered to the subject either as a naked double-stranded
molecule, in conjunction with a delivery reagent, or as a
recombinant plasmid or viral vector which expresses the
double-stranded molecule.
[0268] Suitable delivery reagents for administration in conjunction
with the present a double-stranded molecule include the Mims
Transit TKO lipophilic reagent; lipofectin; lipofectamine;
cellfectin; or polycations (e.g., polylysine), or liposomes. A
preferred delivery reagent is a liposome.
[0269] Liposomes can aid in the delivery of the double-stranded
molecule to a particular tissue, such as prostate tissue, and can
also increase the blood half-life of the double-stranded molecule.
Liposomes suitable for use in the context of the present invention
may be formed from standard vesicle-forming lipids, which generally
include neutral or negatively charged phospholipids and a sterol,
such as cholesterol. The selection of lipids is generally guided by
consideration of factors such as the desired liposome size and
half-life of the liposomes in the blood stream. A variety of
methods are known for preparing liposomes, for example as described
in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and U.S. Pat.
Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire
disclosures of which are herein incorporated by reference.
[0270] Preferably, the liposomes encapsulating the present
double-stranded molecule includes a ligand molecule that can
deliver the liposome to the cancer site. Ligands which bind to
receptors prevalent in tumor or vascular endothelial cells, such as
monoclonal antibodies that bind to tumor antigens or endothelial
cell surface antigens, are preferred.
[0271] Particularly preferably, the liposomes encapsulating the
present double-stranded molecule are modified so as to avoid
clearance by the mononuclear macrophage and reticuloendothelial
systems, for example, by having opsonization-inhibition moieties
bound to the surface of the structure. In one embodiment, a
liposome of the invention can include both opsonization-inhibition
moieties and a ligand.
[0272] Opsonization-inhibiting moieties for use in preparing the
liposomes of the invention are typically large hydrophilic polymers
that are bound to the liposome membrane. As used herein, an
opsonization inhibiting moiety is "bound" to a liposome membrane
when it is chemically or physically attached to the membrane, e.g.,
by the intercalation of a lipid-soluble anchor into the membrane
itself, or by binding directly to active groups of membrane lipids.
These opsonization-inhibiting hydrophilic polymers form a
protective surface layer which significantly decreases the uptake
of the liposomes by the macrophage-monocyte system ("MMS") and
reticuloendothelial system ("RES"); e.g., as described in U.S. Pat.
No. 4,920,016, the entire disclosure of which is herein
incorporated by reference. Liposomes modified with
opsonization-inhibition moieties thus remain in the circulation
much longer than unmodified liposomes. For this reason, such
liposomes are sometimes called "stealth" liposomes.
[0273] Stealth liposomes are known to accumulate in tissues fed by
porous or "leaky" microvasculature. Thus, target tissue
characterized by such microvasculature defects, for example, solid
tumors, will efficiently accumulate these liposomes; see Gabizon et
al., Proc Natl Acad Sci USA 1988, 18: 6949-53. In addition, the
reduced uptake by the RES lowers the toxicity of stealth liposomes
by preventing significant accumulation in liver and spleen. Thus,
liposomes of the invention that are modified with
opsonization-inhibition moieties can deliver the present
double-stranded molecule to prostate cancer cells.
[0274] Opsonization inhibiting moieties suitable for modifying
liposomes are preferably water-soluble polymers with a molecular
weight from about 500 to about 40,000 daltons, and more preferably
from about 2,000 to about 20,000 daltons. Such polymers include
polyethylene glycol (PEG) or polypropylene glycol (PPG)
derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate;
synthetic polymers such as polyacrylamide or poly N-vinyl
pyrrolidone; linear, branched, or dendrimeric polyamidoamines;
polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and
polyxylitol to which carboxylic or amino groups are chemically
linked, as well as gangliosides, such as ganglioside GM.sub.1.
Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives
thereof, are also suitable. In addition, the opsonization
inhibiting polymer can be a block copolymer of PEG and either a
polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine,
or polynucleotide. The opsonization inhibiting polymers can also be
natural polysaccharides containing amino acids or carboxylic acids,
e.g., galacturonic acid, glucuronic acid, mannuronic acid,
hyaluronic acid, pectic acid, neuraminic acid, alginic acid,
carrageenan; aminated polysaccharides or oligosaccharides (linear
or branched); or carboxylated polysaccharides or oligosaccharides,
e.g., reacted with derivatives of carbonic acids with resultant
linking of carboxylic groups.
[0275] Preferably, the opsonization-inhibiting moiety is a PEG,
PPG, or derivatives thereof. Liposomes modified with PEG or
PEG-derivatives are sometimes called "PEGylated liposomes".
[0276] The opsonization inhibiting moiety can be bound to the
liposome membrane by any one of numerous well-known techniques. For
example, an N-hydroxysuccinimide ester of PEG can be bound to a
phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a
membrane. Similarly, a dextran polymer can be derivatized with a
stearylamine lipid-soluble anchor via reductive amination using
Na(CN)BH. sub. 3 and a solvent mixture such as tetrahydrofuran and
water in a 30:12 ratio at 60 degrees C.
[0277] Vectors expressing a double-stranded molecule of the present
invention are discussed above. Such vectors expressing at least one
double-stranded molecule of the invention can also be administered
directly or in conjunction with a suitable delivery reagent,
including the Minis Transit LT1 lipophilic reagent; lipofectin;
lipofectamine; cellfectin; polycations (e.g., polylysine) or
liposomes. Methods for delivering recombinant viral vectors, which
express a double-stranded molecule of the invention, to an area of
cancer in a patient are within the skill of the art.
[0278] The double-stranded molecule of the present invention can be
administered to the subject by any means suitable for delivering
the double-stranded molecule into cancer sites. For example, the
double-stranded molecule can be administered by gene gun,
electroporation, or by other suitable parenteral or enteral
administration routes.
[0279] Suitable enteral administration routes include oral, rectal,
or intranasal delivery.
[0280] Suitable parenteral administration routes include
intravesical and intravascular administration (e.g., intravenous
bolus injection, intravenous infusion, intra-arterial bolus
injection, intra-arterial infusion and catheter instillation into
the vasculature); peri- and intra-tissue injection (e.g.,
peri-tumoral or intra-tumoral injection); subcutaneous injection or
deposition including subcutaneous infusion (such as by osmotic
pumps); direct application to the area at or near the site of
cancer, for example by a catheter or other placement device (e.g.,
a suppository or an implant including a porous, non-porous, or
gelatinous material); and inhalation. It is preferred that
injections or infusions of the double-stranded molecule or vector
be given at or near the site of the cancer.
[0281] The double-stranded molecule of the present invention can be
administered in a single dose or in multiple doses. Where the
administration of the double-stranded molecule of the present
invention is by infusion, the infusion can be a single sustained
dose or can be delivered by multiple infusions. Injection of the
agent directly into the tissue is at or near the site of cancer
preferred. Multiple injections of the agent into the tissue at or
near the site of cancer are particularly preferred.
[0282] One skilled in the art can also readily determine an
appropriate dosage regimen for administering the double-stranded
molecule of the invention to a given subject. For example, the
double-stranded molecule can be administered to the subject once,
for example, as a single injection or deposition at or near the
cancer site. Alternatively, the double-stranded molecule can be
administered once or twice daily to a subject for a period of from
about three to about twenty-eight days, more preferably from about
seven to about ten days. In a preferred dosage regimen, the
double-stranded molecule is injected at or near the site of cancer
once a day for seven days. Where a dosage regimen includes multiple
administrations, it is understood that the effective amount of a
double-stranded molecule administered to the subject can include
the total amount of a double-stranded molecule administered over
the entire dosage regimen.
IV-4 Compositions Containing a Double-Stranded Molecule of the
Present Invention:
[0283] In addition to the above, the present invention also
provides pharmaceutical compositions that include at least one of
the present double-stranded molecules or the vectors coding for the
molecules.
[0284] The double-stranded molecules of the present invention are
preferably formulated as pharmaceutical compositions prior to
administering to a subject, according to techniques known in the
art. Pharmaceutical compositions of the present invention are
characterized as being at least sterile and pyrogen-free. As used
herein, "pharmaceutical formulations" include formulations for
human and veterinary use. Methods for preparing pharmaceutical
compositions of the invention are within the skill in the art, for
example as described in Remington's Pharmaceutical Science, 17th
ed., Mack Publishing Company, Easton, Pa. (1985), the entire
disclosure of which is herein incorporated by reference.
[0285] The present pharmaceutical formulations contain at least one
of the double-stranded molecules or vectors encoding them of the
present invention (e.g., 0.1 to 90% by weight), or a
physiologically acceptable salt of the molecule, mixed with a
physiologically acceptable carrier medium. Preferred
physiologically acceptable carrier media are water, buffered water,
normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the
like.
[0286] According to the present invention, the composition may
contain plural kinds of double-stranded molecules, each of
molecules may be directed to the same target sequence, or different
target sequences of STC2 gene. For example, compositions may
contain double-stranded molecules directed to one, two or more
target sequences of STC2 gene.
[0287] Furthermore, the present composition may contain a vector
coding for one or plural double-stranded molecules. For example,
the vector may encode one, two or several kinds of the present
double-stranded molecules. Alternatively, the present composition
may contain plural kinds of vectors, each of the vectors coding for
a different double-stranded molecule.
[0288] Moreover, the present double-stranded molecules may be
contained as liposomes in the present composition. See under the
item of "IV-3. Methods for treating or preventing cancer using a
double-stranded molecule of the present invention" for details of
liposomes.
[0289] Pharmaceutical compositions of the present invention can
also include conventional pharmaceutical excipients and/or
additives. Suitable pharmaceutical excipients include stabilizers,
antioxidants, osmolality adjusting agents, buffers, and pH
adjusting agents. Suitable additives include physiologically
biocompatible buffers (e.g., tromethamine hydrochloride), additions
of chelants (such as, for example, DTPA or DTPA-bisamide) or
calcium chelate complexes (for example calcium DTPA,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium
salts (for example, calcium chloride, calcium ascorbate, calcium
gluconate or calcium lactate). Pharmaceutical compositions of the
invention can be packaged for use in liquid form, or can be
lyophilized.
[0290] For solid compositions, conventional nontoxic solid carriers
can be used; for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharin, talcum,
cellulose, glucose, sucrose, magnesium carbonate, and the like.
[0291] For example, a solid pharmaceutical composition for oral
administration can include any of the carriers and excipients
listed above and 10-95%, preferably 25-75%, of one or more
double-stranded molecule of the present invention. A pharmaceutical
composition for aerosol (inhalational) administration can include
0.01-20% by weight, preferably 1-10% by weight, of one or more
double-stranded molecules of the present invention encapsulated in
a liposome as described above, and propellant. A carrier can also
be included as desired; e.g., lecithin for intranasal delivery.
[0292] In addition to the above, the present composition may
contain other pharmaceutical active ingredients so long as they do
not inhibit the in vivo function of the present double-stranded
molecules of the present invention. For example, the composition
may contain chemotherapeutic agents conventionally used for
treating cancers.
[0293] In another embodiment, the present invention provides for
the use of the double-stranded nucleic acid molecules of the
present invention in manufacturing a pharmaceutical composition for
treating a prostate cancer characterized by the expression of STC2
gene. For example, the present invention relates to a use of
double-stranded nucleic acid molecule inhibiting the expression of
STC2 gene in a cell, which molecule includes a sense strand and an
antisense strand complementary thereto, hybridized to each other to
form the double-stranded nucleic acid molecule and targets to a
sequence selected from among SEQ ID NOs: 8 and 9, for manufacturing
a pharmaceutical composition for treating prostate cancer
expressing STC2 gene.
[0294] The present invention further provides a method or process
for manufacturing a pharmaceutical composition for treating a
prostate cancer characterized by the expression of STC2 gene,
wherein the method or process includes a step for formulating a
pharmaceutically or physiologically acceptable carrier with a
double-stranded nucleic acid molecule inhibiting the expression of
STC2 gene in a cell, which over-expresses the gene, which molecule
includes a sense strand and an antisense strand complementary
thereto, hybridized to each other to form the double-stranded
nucleic acid molecule and targets to a sequence selected from among
SEQ ID NOs: 8 and 9 as active ingredients.
[0295] In another embodiment, the present invention provides a
method or process for manufacturing a pharmaceutical composition
for treating a prostate cancer characterized by the expression of
STC2 gene, wherein the method or process includes a step for
admixing an active ingredient with a pharmaceutically or
physiologically acceptable carrier, wherein the active ingredient
is a double-stranded nucleic acid molecule inhibiting the
expression of STC2 gene in a cell, which over-expresses the gene,
which molecule includes a sense strand and an antisense strand
complementary thereto, hybridized to each other to form the
double-stranded nucleic acid molecule and targets to a sequence
selected from among SEQ ID NOs: 8 and 9.
[0296] Aspects of the present invention are described in the
following examples, which are not intended to limit the scope of
the invention described in the claims.
[0297] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below.
EXAMPLES
Example 1
General Methods
[0298] 1. Cell Lines
[0299] Human PC cell lines LNCaP, 22Rv1, DU-145, and PC-3 were
obtained from American Type Culture Collection (ATCC, Rockville,
Md.). LNCaP derived androgen-independent prostate cancer cell line
C4-2B was purchased from ViroMed Laboratories (Minnetonka, Minn.,
USA). All of the cell lines were cultured as monolayers in the
following medium: DMEM (Sigma-Aldrich) for 22Rv1, LNCaP, C4-2B and
DU-145; and F-12 (GIBCO) for PC-3 with 10% fetal bovine serum and
1% antibiotic/antimycotic solution (Sigma-Aldrich). Cells were
maintained in incubators containing humidified air with 5% CO.sub.2
at 37 degrees C.
[0300] 2. Semi-Quantitative RT-PCR
[0301] Purification of PC cells and normal prostatic epithelial
cells from frozen PC tissues was described previously (Tamura K, et
al., Cancer Res 2007, 67, 5117-5125). Total RNA was extracted using
RNeasy Kit (QIAGEN, Valencia, Calif.) according to manufacturer's
instruction, treated with DNase I (Roche Diagnostic, Mannheim,
Germany), and reversely transcribed to single-stranded cDNA using
random hexamer or oligo d(T) primer with Superscript reverse
transcriptase II (Invitrogen, Carlsbad, Calif.). Appropriate
dilutions of each single-strand cDNA were prepared by normalizing
cDNA content using beta-actin (ACTB) as a quantitative control,
demonstrating PCR reaction using single strand cDNA as PCR
templates. The primer sequences employed in the context of the
present invention were:
TABLE-US-00003 (SEQ ID NO: 1) ACTB forward:
5'-TTGGCTTGACTCAGGATTTA-3', (SEQ ID NO: 2) ACTB reverse:
5'-ATGCTATCACCTCCCCTGTG-3', (SEQ ID NO: 3) STC2 forward:
5'-TTACTCCATGAGCCTTCCTTTG-3', and (SEQ ID NO: 4) STC2 reverse:
5'-TCCTGTTGCCTAAATCCGTAGTA-3'.
[0302] The conditions for PCR are followed; initial denaturation at
95 degrees C. for 5 min, 23 cycles for ACTB and 30 cycles for STC2
of denaturation at 95 degrees C. for 30 sec, annealing at 55
degrees C. for 30 sec, and elongation at 72 degrees C. for 30 sec
on a GeneAmp PCR system 9700 (PE Applied Biosystems, Foster,
Calif.). Quantitative real-time PCR was also carried out using a
PRISM 7700 sequence detector with the SYBR Premix ExTaq (TaKaRa) in
accordance with the manufacturer's instructions. The primers for
real-time quantitative PCR were the same with those described
above.
[0303] 3. Northern Blot Analysis
[0304] Total RNA from five PC cell lines were extracted using
RNeasy Kit (QIAGEN, Valencia, Calif.), and their mRNAs were
purified using the mRNA Purification Kit (GE Healthcare,
Piscataway, N.J., USA), according to the manufacturer's protocols.
One-micro gram aliquot of each mRNA from the PC cell lines, as well
as those isolated from normal human brain, heart, kidney, liver,
lung, prostate and testis (BD Bioscience, Palo Alto, Calif.), were
separated on 1% denaturing agarose gels and transferred onto nylon
membranes. The membranes were hybridized for 20 hours with
.sup.32P-labeled STC2 cDNA, which was labeled by Megaprime DNA
labeling system (GE Bioscience, UK). Probe cDNA of STC2 was
prepared as a 551-bp PCR product by using the primers
followings:
TABLE-US-00004 (SEQ ID NO: 5) 5'-AACATTTACCATTAGAGAGGGGG-3' and
(SEQ ID NO: 6) 5'-CACATAGAAATGACACTCCTCCC-3'.
[0305] Pre-hybridization, hybridization, and washing were performed
according to the manufacturer's instruction. The blots were
autoradiographed at -80 degrees C. for 7 days.
[0306] 4. Generating Monoclonal Antibody to STC2 and
Immunohistochemical Analysis
[0307] Monoclonal mouse antibodies to STC2 were generated by
immunizing recombinant STC2 proteins in MBL (Nagoya, Japan).
Castration-naive PC(CNPC) tissues were obtained from the patients
who underwent prostectomy in Kochi University Medical School. CRPC
tissues were obtained in Kochi University Medical School, Iwate
Medical Collage, and Okayama University Medical School under the
appropriate informed consents. Immunohistochemical study was
carried out using the Ventana automated immunohistochemical systems
(Discovery, Ventana Medical systems, Inc., Tucson, Ariz.). Sections
were incubated with a 1:100 diluted solution of purified anti-STC2
mAb (0.75 mg/ml), for 16 minutes. The automated protocol is based
on an indirect biotin-avidin system using a biotinylated universal
secondary antibody and diaminobenzidine substrate with hematoxylin
counterstaining. The specificity of the binding was confirmed by
negative staining using mouse nonimmune serum as primary
antibody.
[0308] 5. Scoring of Immunohistochemical Staining
[0309] To evaluate both the intensity of staining and proportion of
the positive-stained cells, a scoring method previously reported
was used (Ashida S, et al., Clin Cancer Res 2006, 2767-73).
Regarding the morphology and the intensity of STC2 expression,
positive staining of anti-STC2 antibody was defined as follows:
score 1 for variable weak cytoplasmic staining, score 2 for
segmental and apical granular cytoplasmic staining, and score 3 for
diffuse continuous and intense cytoplasmic staining. For each
score, the proportion of cells with the score was estimated
visually. A combined weighed score (STC2 immunohistochemical score)
composed of the sum of the proportion of cells with each score was
calculated for each sample as described previously (Ashida S, et
al., Clin Cancer Res 2006, 2767-73). For example, a case with 70%
score 3 staining, 20% score 2 staining, and 10% score 1 staining
would be scored as follows: 70.times.3+20.times.2+10.times.1=260.
The maximum score should be 300. All sections were scored, blinded
and independently, by a clinical pathologist and a person with
considerable training in histopathological scoring. Signals were
considered positive when reaction products were localized in the
expected cellular compartment.
[0310] 6. Statistical Analysis
[0311] Comparisons of STC2 expression in 3 groups (Gleason score
2-6, 7 and 8-10) were analyzed using Kruskal-Wallis for multiple
comparisons. P<0.01 were considered significant. Additional
post-test was performed by using Mann-Whitney's U-test with
Bonferroni method to adjust for multiple pair-wise differences.
P<0.003 between 3 groups were considered significant with
Bonferroni method. All statistical calculations were done with
Statview software (ver. 5.0, SAS Institute Inc., North
Carolina).
[0312] 7. Construction of shRNA Expressing Vectors and Cell
Viability Assay
[0313] To investigate the biological function of STC2 in PC cells,
psiU6BX3.0 vector was used for expression of short hairpin RNA
(shRNA) against a target gene as described previously (Tamura K, et
al., Cancer Res 2007, 67, 5117-5125). Plasmids designed to express
shRNA were prepared by cloning of double-stranded oligonucleotides
into psiU6BX vector. The oligonucleotide sequences of target
sequences for STC2 are as followed; sense strand sequence for
si1: 5'-CAACTCTTGTGAGATTCGG-3' (SEQ ID NO: 7),
si2: 5'-GACGAACAGTCTGAGTATT-3' (SEQ ID NO: 8),
si3: 5'-GCAGGAGCTGGTATTGTAG-3' (SEQ ID NO: 9), and
[0314] siEGFP: 5'-GAAGCAGCACGACTTCTTC-3' (SEQ ID NO: 10) as a
negative control. PC-3 cells (2.times.10.sup.6) which expressed
STC2 in high level were seeded on 10-cm dishes, transfected with
psiU6-STC2 (si1-3), or psiU6-siEGFP using FuGene6 (Roche) according
to the manufacturer's instruction, and then cultured in appropriate
medium containing 800 mcg/ml of Geneticin
[0315] (Sigma-Aldrich) for 14 days. The cells were fixed with 100%
methanol, stained with 0.1% of crystal violet-H.sub.2O for colony
formation assay. In MTT assay, cell viability was measured using
Cell-counting kit-8 (DOJINDO, Kumamoto, Japan) at 10 days after
transfection. Absorbance was measured at 490 nm, and at 630 nm as
reference, with a Microplate Reader 550 (Bio-Rad). Preliminarily,
knockdown effects of these shRNA-expression vectors on endogenous
STC2 expression were validated 7 days after transfection by RT-PCR
using the primers used by semi-quantitative RT-PCR.
[0316] 8. Generation of STC2-Overexpressing Cells and In-Vitro
Growth Assay
[0317] Full-length human STC2 cDNA was amplified using primers that
were designed to contain HA-tag sequences at the COOH terminus, and
cloned into the pIRESneo3 vector (Clontech). Human
[0318] PC cell line 22Rv1 cells were seeded into 100 mm-dish
(5.times.10.sup.5 cells per a dish) and transfected with 6 micro
gram of pIRESneo3 empty vector alone or pIRESneo3-STC2-HA
expression vector using FuGENE6 reagent (Roche) according to the
manufacturer's instructions. Cells were selected with appropriate
medium containing 400 mcg/ml of Geneticin (Sigma-Aldrich) for 14
days when discrete colonies were isolated. All clones were
maintained in selective medium. Each clone was assayed for STC2
protein expression by western blot analysis using anti-HA tag
antibody (Roche). Proliferation of 22Rv1 cells that stably
expressed STC2 (22Rv1-STC2) or those transfected with pIRESneo3
empty vector (22Rv1-mock clone mixture) were examined by
Cell-counting kit-8 (DOJINDO, Kumamoto, Japan). Each of 22Rv1-STC2
and 22Rv1-mock cells was seeded at the concentration of
3.times.10.sup.3 cells per well using 48-well plates. The assay was
performed at every 48 hours for 9 days, according to the
manufacturer's instruction.
Example 2
STC2 Over Expression in CRPC Cells
[0319] The genome-wide expression profiles of CRPC cells and CNPC
cells purified from clinical PC tissues was previously reported
(Tamura K, et al., Cancer Res 2007, 67, 5117-5125). Among a number
of genes shown to be trans-activated in CRPC cells compared normal
prostate epithelial cells (NP), STC2 was focused in this invention.
Semi-quantitative RT-PCR (FIG. 1A) and real-time quantitative
RT-PCR (FIG. 1B) confirmed the elevated expression of STC2 in 6 out
of the 7 clinical CRPC cells, comparing with CNPC cells and NP
cells. Northern blot analysis using five PC cell lines and normal
adult tissues confirmed the elevated expression of STC2 in all of
PC cell lines, compared with normal prostate and adult vital organs
including brain, heart, kidney, liver, lung and testis (FIG. 1C).
Multiple tissue northern (MTN) blot analysis also revealed no or
very limited expression of STC2 in most normal adult organs, and
low expression was observed only in normal pancreas (FIG. 1D).
Example 3
Immunohistochemical Analysis in Clinical PC Tissues
[0320] To validate the over-expression of STC2 protein in CRPC
cells, immunohistochemical analysis on clinical PC tissues was
performed by using monoclonal antibody specific to human STC2. As
shown in FIG. 1E, strong immunochemical signal for STC2 was
detected predominantly in the cytoplasm of cancer cells
exceptionally in CRPC cases examined. 6 of 9 CRPCs examined showed
strong immunoreactivity to anti-STC2 antibody and one CNPC with
Gleason score 10 also showed strong immunoreactivity (FIG. 1F),
while CNPC with Gleason score 7 did not show any immunoreactivity
to anti-STC2 antibody (FIG. 1G). Adjacent normal prostatic
epithelium in the same patient revealed very weak or no signal for
STC2. Hormone ablation therapy is usually ineffective for PCs with
Gleason score 10, which progress highly aggressively, and these
findings demonstrated that STC2 was expressed specifically in CRPCs
and highly aggressively PCs.
[0321] To further investigate the clinic-pathologic significance of
STC2 expression in PC tissues, the relationship between the
calculated immunohistochemical score for STC2 and Gleason score was
analyzed. Since each PC specimen apparently showed a different
degree of staining intensity and different proportion of the
staining-positive cell, these heterogeneity were taken into
consideration, and the immunohistochemical scoring system was
applied; a combined weighed score was given by the sum of the
proportion (0-100%) of stained cells for which the score 1, 2, or 3
was given according to the signal STC2 immunohistochemical scores
and intensity as described previously (Ashida S, et al., Clin
Cancer Res 2006, 2767-73). In comparison of STC2 expression, there
were significant differences between Gleason score 2-6 group and
8-10 group, and between Gleason score 7 group and 8-10 group
(Mann-Whitney's U-test with Bonferroni method, p=0.0006 and
p=0.0004, respectively). No significant difference was observed
between Gleason score 2-6 group and 7 group (p=0.0110). It was
confirmed that STC2 immunohistochemical score revealed a strong
correlation with high-grade PCs (Gleason score 8-10) (FIG. 1H,
Table 1).
[0322] Table 1. Relationship between STC2 immunohistochemical score
and Gleason score in CNPCs.
TABLE-US-00005 TABLE 1 STC2 Gleason immunohistochemical score score
n Mean (SD) *P 2-6 12 113 (14) 7 33 123 (12) 8-10 8 194 (59)
*<0.003 Statistical significance was determined using
Mann-Whitney's U-test with Bonferroni method.
Example 4
Knockdown of STC2 Expression by siRNA Attenuated PC Cell Growth
[0323] To examine biological roles of STC2 over-expression in PC
cell, three vectors designed to express shRNA specifically to STC2
were constructed and transfected into PC cell lines PC-3, which
expressed endogenous STC2 most strongly. Among the three
shRNA-expression vectors, si2 and si3 showed the significant
knockdown effect on endogenous STC2 transcript (FIG. 2A), and this
transfection resulted in reduction of the numbers of colonies (FIG.
2B) as well as those of the viable cells measured by MTT assay for
PC-3 cells (FIG. 2C). On the other hand, the transfection of si1
and a negative control (siEGFP) showed no or little knockdown
effect on STC2 expression and did not affect cell viability of PC-3
cells. These findings indicated STC2 over-expression could play
some important roles in PC cell growth or viability.
Example 5
STC2 Over-Expression Promoted Cancer Cell Growth
[0324] To further investigate for the potential oncogenic function
of STC2, three stable transformants (Clones 1-3) were established
from 22Rv1 cells, in which exogenous STC2 expressed constitutively.
control 22Rv1 cells transfected empty vector (Mock) were also
prepared and compared their proliferation. Western blot analysis
(FIG. 2D) confirmed high level of exogenous STC2 expression in
three stable clones (Clones 1-3). MTT assay showed that the three
stable clonel, 2 and 3 grew significantly more rapidly than the
22Rv1-mock clone mixture (*P<0.01, **P<0.05,
Students't-test), indicating that STC2 over-expression promoted PC
cell growth (FIG. 2E).
INDUSTRIAL APPLICABILITY
[0325] The present invention provides a novel target molecule or
biomarker, STC2, for therapy development or diagnosis for PCs,
especially for CRPCs and aggressive PCs. PC shows relatively good
prognosis, and hormone-ablation therapy or castration is effective
in most of relapsed or advanced PC. However, once CRPC cells emerge
or it is at advanced stage with high Gleason score, there are very
limited options for PC patient care, such as doxotaxel plus
predonisone (Tannock I F, et al., N Engl J Med 2004, 351, 1502-12,
and Petrylak D P, et al., N Engl J Med 2004, 351, 1513-20) which
can still offer the minimum effect on PCs. Hence, it is most
demanded to identify molecular targets for CRPCs or aggressive PCs
and develop novel therapies for them to target those molecules.
[0326] The examples described herein indicate restrictive
expression of the STC2 gene in adult normal organs and critical
roles of STC2 in PC cells. Therefore, STC2 is a promising target
for a novel therapeutic approach with a minimal risk of adverse
effects. More specifically, the expression of the STC2 gene is
strongly related to CRPC cells viability. Thus, the detection and
the inhibition of STC2 provide a novel effective therapeutic
approach for a biomarker or a molecular treatment of CRPCs.
[0327] High-grade CNPCs, as well as CRPCs, respond poorly to
androgen-ablation therapy and have highly aggressive behavior and
poor prognosis. The immunohistochemical analysis described herein
clearly indicates STC2 is over-expressed more strongly in
high-grade CNPCs with Gleason score 8-10, as well as CRPC, than
low-grade CNPCs. It was confirmed that STC2 immunohistochemical
score revealed a strong correlation with Gleason score 8-10
(*P<0.003; FIG. 1H, Table 1). Thus, the detection and the
inhibition of STC2 is also useful for predicting Gleason score of
PC or treating aggressive PCs with high grade Gleason score (e.g.,
Gleason score 8-10).
[0328] STC2 is a secreted protein that functions to promote PC cell
viability in autocrine/paracrine manner. Accordingly, it is
possible to detect STC2 in patient serum, to thus serve as a
diagnostic biomarker diagnosing PC (more suitably CRPC), or
predicting PC aggressiveness and Gleason score, and neutlinization
of STC2 by highly specific antibody have potential as one of the
therapeutic strategies against PCs, in particular, CRPCs and
aggressive PCs.
[0329] The data provided herein add to a comprehensive
understanding of PCs, facilitate development of novel diagnostic
strategies, and provide clues for identification of molecular
targets for therapeutic drugs and preventative agents. Such
information contributes to a more profound understanding of
tumorigenesis, and provide indicators for developing novel
strategies for diagnosis, treatment, and ultimately prevention of
PCs.
[0330] All patents, patent applications, and publications cited
herein are incorporated by reference in their entirety.
[0331] Furthermore, while the invention has been described in
detail and with reference to specific embodiments thereof, it is to
be understood that the foregoing description is exemplary and
explanatory in nature and is intended to illustrate the invention
and its preferred embodiments. Through routine experimentation, one
skilled in the art will readily recognize that various changes and
modifications can be made therein without departing from the spirit
and scope of the invention. Thus, the invention is intended to be
defined not by the above description, but by the following claims
and their equivalents.
Sequence CWU 1
1
12120DNAArtificialAn artificially synthesized primer sequence for
RT-PCR 1ttggcttgac tcaggattta 20220DNAArtificialAn artificially
synthesized primer sequence for RT-PCR 2atgctatcac ctcccctgtg
20322DNAArtificialAn artificially synthesized primer sequence for
RT-PCR 3ttactccatg agccttcctt tg 22423DNAArtificialAn artificially
synthesized primer sequence for RT-PCR 4tcctgttgcc taaatccgta gta
23523DNAArtificialAn artificially synthesized primer sequence for
preparing probe 5aacatttacc attagagagg ggg 23623DNAArtificialAn
artificially synthesized primer sequence for preparing probe
6cacatagaaa tgacactcct ccc 23719DNAArtificialAn artificially
synthesized oligonucleotide for siRNA 7caactcttgt gagattcgg
19819DNAArtificialAn artificially synthesized oligonucleotide for
siRNA 8gacgaacagt ctgagtatt 19919DNAArtificialAn artificially
synthesized oligonucleotide for siRNA 9gcaggagctg gtattgtag
191019DNAArtificialAn artificially synthesized oligonucleotide for
siRNA 10gaagcagcac gacttcttc 19115361DNAHomo sapiens 11tttctccttc
cctccacggg ccgggtgaga aagtagccgg gggctatccc gacccggcgg 60ttcttgggga
gggggccgaa caagaaaagg gaggagatgg agataacttc cccggattta
120gcttttttgt ctttgttttt gttctcacca cttccatcgg atgactggag
agtaaaaggg 180aacccggagc ggggtggcga gcagcgcttt gagaaaatgc
aggagtgtgt ttggagacgc 240gtaaagttgc ctttcaagct ctggcctccg
ggcacgcgat gctccgcggc gggctgactc 300agggctgcct tgggcctccc
tgccaccctc ctggaaatga tgcaagtcct gactgtcacc 360tggatccctg
cagcccagcc tggaatgcgt ctggattagg ggaaagacga gaaacgacac
420tccaggtgtt gcacggccca ccaaagcggg aagatagggc agttgctcag
accaaatact 480gtatctagtg cttctgctcc tatcttcaat cgtggggttc
tttttaatgc aaagtgtcac 540aaggccagga attcccatgt gtgctcagtt
ggcccacagc atcattgtgc ctaggaaact 600gcttcaattt atcaagtcct
ctgggctggg aatctcactg aattccaaac ggcggaaaga 660ggaaactttc
ccaacccgat gtgggtgtga cgcgagccag gggccccagg gacactgtcc
720cagagcacac cgtccccctt taacagcaac tggagcttgg attcgctctt
atattgtaca 780gtcctttcga ccattgccct ggagcacccg cacacgcgca
cgcatctccg gccgcgctca 840cacacactca tacacacgca cgcaaacgcg
tggccgccgc caggtcggca actttgtccg 900gcgctcccag cggcgctcgg
cttcctcctg tagtagttga gcgcaggccc cgcctcccgg 960ccgtgttgtc
aaaagggccg gggtctcgga ttggtccagc cgccgggaca acacctgctc
1020gactccttca ttcaagtgac accagagctt ccagggatat ttgaggcacc
atccctgcca 1080ttgccgggca ctcgcggcgc tgctaacggc ctggtcacat
gctctccgga gagctacggg 1140agggcgctgg gtaacctcta tccgagccgc
ggccgcgagg aggagggaaa aggcgagcaa 1200aaaggaagag tgggaggagg
aggggaagcg gcgaaggagg aagaggagga ggaggaagag 1260gggagcacaa
aggatccagg tctcccgacg ggaggttaat accaagaacc atgtgtgccg
1320agcggctggg ccagttcatg accctggctt tggtgttggc cacctttgac
ccggcgcggg 1380ggaccgacgc caccaaccca cccgagggtc cccaagacag
gagctcccag cagaaaggcc 1440gcctgtccct gcagaataca gcggagatcc
agcactgttt ggtcaacgct ggcgatgtgg 1500ggtgtggcgt gtttgaatgt
ttcgagaaca actcttgtga gattcggggc ttacatggga 1560tttgcatgac
ttttctgcac aacgctggaa aatttgatgc ccagggcaag tcattcatca
1620aagacgcctt gaaatgtaag gcccacgctc tgcggcacag gttcggctgc
ataagccgga 1680agtgcccggc catcagggaa atggtgtccc agttgcagcg
ggaatgctac ctcaagcacg 1740acctgtgcgc ggctgcccag gagaacaccc
gggtgatagt ggagatgatc catttcaagg 1800acttgctgct gcacgaaccc
tacgtggacc tcgtgaactt gctgctgacc tgtggggagg 1860aggtgaagga
ggccatcacc cacagcgtgc aggttcagtg tgagcagaac tggggaagcc
1920tgtgctccat cttgagcttc tgcacctcgg ccatccagaa gcctcccacg
gcgccccccg 1980agcgccagcc ccaggtggac agaaccaagc tctccagggc
ccaccacggg gaagcaggac 2040atcacctccc agagcccagc agtagggaga
ctggccgagg tgccaagggt gagcgaggta 2100gcaagagcca cccaaacgcc
catgcccgag gcagagtcgg gggccttggg gctcagggac 2160cttccggaag
cagcgagtgg gaagacgaac agtctgagta ttctgatatc cggaggtgaa
2220atgaaaggcc tggccacgaa atctttcctc cacgccgtcc attttcttat
ctatggacat 2280tccaaaacat ttaccattag agagggggga tgtcacacgc
aggattctgt ggggactgtg 2340gacttcatcg aggtgtgtgt tcgcggaacg
gacaggtgag atggagaccc ctggggccgt 2400ggggtctcag gggtgcctgg
tgaattctgc acttacacgt actcaaggga gcgcgcccgc 2460gttatcctcg
tacctttgtc ttctttccat ctgtggagtc agtgggtgtc ggccgctctg
2520ttgtggggga ggtgaaccag ggaggggcag ggcaaggcag ggcccccaga
gctgggccac 2580acagtgggtg ctgggcctcg ccccgaagct tctggtgcag
cagcctctgg tgctgtctcc 2640gcggaagtca gggcggctgg attccaggac
aggagtgaat gtaaaaataa atatcgctta 2700gaatgcagga gaagggtgga
gaggaggcag gggccgaggg ggtgcttggt gccaaactga 2760aattcagttt
cttgtgtggg gccttgcggt tcagagctct tggcgagggt ggagggagga
2820gtgtcatttc tatgtgtaat ttctgagcca ttgtactgtc tgggctgggg
gggacactgt 2880ccaagggagt ggcccctatg agtttatatt ttaaccactg
cttcaaatct cgatttcact 2940ttttttattt atccagttat atctacatat
ctgtcatcta aataaatggc tttcaaacaa 3000agcaactggg tcattaaaac
cagctcaaag ggggtttaaa aaaaaaaaac cagcccatcc 3060tttgaggctg
atttttcttt tttttaagtt ctattttaaa agctatcaaa cagcgacata
3120gccatacatc tgactgcctg acatggactc ctgcccactt gggggaaacc
ttatacccag 3180aggaaaatac acacctgggg agtacatttg acaaatttcc
cttaggattt cgttatctca 3240ccttgaccct cagccaagat tggtaaagct
gcgtcctggc gattccagga gacccagctg 3300gaaacctggc ttctccatgt
gaggggatgg gaaaggaaag aagagaatga agactactta 3360gtaattccca
tcaggaaatg ctgacctttt acataaaatc aaggagactg ctgaaaatct
3420ctaagggaca ggattttcca gatcctaatt ggaaatttag caataaggag
aggagtccaa 3480ggggacaaat aaaggcagag agaagagaca gaactaaaaa
tacgaggaaa ggagagtgag 3540gattttcatt aaaagtctca gcagtgggtt
tcttgggtta tttaaaacat cacctaaata 3600ggccttttct tcctaattgg
ccatcaaatt aaagcctatc ctttctcaag caggagctgg 3660tattgtaggg
agtggccggg tattctgggc tgggctcttc tggagtaggg ggtcagcaaa
3720cattgtctgc aaagggccag atactgaatc cagtactttc agtttggcga
gccgtgaggt 3780ctctgtcgaa actactcaac tctgccgtcc tagcacaaaa
gcagccatag acaacacaca 3840aacgagaggg cttggctccc ttccaggaag
atttatttaa caggctccca gctgaatttc 3900actcacagga cacagtttac
tgatctctgt tctagtgagt gggtcaaaaa gcatatgcat 3960ccttatccgt
caactcatca gctcttcctc aaggcaacct gaggccagac accaagaaac
4020caagcgtatc tgctctaaaa tgacttgttc ctggggaatg ccttcaacca
aaacacagct 4080agtatttcta tgccccaaat ccaatcccag tctttcatga
tccatgccgg cggttgggtg 4140gggaggggaa tcattggttg ggggaaggga
ggaaacccca cctccagccc ccgccaccgg 4200gctccctggg cacccagcaa
gatctggggc tgcagagaac agaagagctg gtgcacttaa 4260tccagctctg
cccttggggg gaggaggacc tgtgtgtcag gctctgccat gggaacgagt
4320gtaaaccgtg gctgtctcct gcagtgagcc accgcggcag gcacgttgac
tgttttactg 4380acatcactca aaagctaaag caataacatt ctcctgcgtt
gctgagtcag ctgttcattt 4440gtccgccagc tcctggactg gatgtgtgaa
aggcatcaca tttccatttt cctccgtgta 4500aatgttttat gtgttcgcct
actgatccca ttcgttgctt ctattgtaaa tatttgtcat 4560ttgtatttat
tatctctgtg ttttccccct aaggcataaa atggtttact gtgttcattt
4620gaacccattt actgatctct gttgtatatt tttcatgcca ctgctttgtt
ttctcctcag 4680aagtcgggta gatagcattt ctatcccatc cctcacgtta
ttggaagcat gcaacagtat 4740ttattgctca gggtcttctg cttaaaactg
aggaaggtcc acattcctgc aagcattgat 4800tgagacattt gcacaatcta
aaatgtaagc aaagtagtca ttaaaaatac accctctact 4860tgggctttat
actgcataca aatttactca tgagccttcc tttgaggaag gatgtggatc
4920tccaaataaa gatttagtgt ttattttgag ctctgcatct taacaagatg
atctgaacac 4980ctctcctttg tatcaataaa tagccctgtt attctgaagt
gagaggacca agtatagtaa 5040aatgctgaca tctaaaacta aataaataga
aaacaccagg ccagaactat agtcatactc 5100acacaaaggg agaaatttaa
actcgaacca agcaaaaggc ttcacggaaa tagcatggaa 5160aaacaatgct
tccagtggcc acttcctaag gaggaacaac cccgtctgat ctcagaattg
5220gcaccacgtg agcttgctaa gtgataatat ctgtttctac tacggattta
ggcaacagga 5280cctgtacatt gtcacattgc attatttttc ttcaagcgtt
aataaaagtt ttaaataaat 5340ggcaaaaaaa aaaaaaaaaa a 536112302PRTHomo
sapiens 12Met Cys Ala Glu Arg Leu Gly Gln Phe Met Thr Leu Ala Leu
Val Leu 1 5 10 15 Ala Thr Phe Asp Pro Ala Arg Gly Thr Asp Ala Thr
Asn Pro Pro Glu 20 25 30 Gly Pro Gln Asp Arg Ser Ser Gln Gln Lys
Gly Arg Leu Ser Leu Gln 35 40 45 Asn Thr Ala Glu Ile Gln His Cys
Leu Val Asn Ala Gly Asp Val Gly 50 55 60 Cys Gly Val Phe Glu Cys
Phe Glu Asn Asn Ser Cys Glu Ile Arg Gly 65 70 75 80 Leu His Gly Ile
Cys Met Thr Phe Leu His Asn Ala Gly Lys Phe Asp 85 90 95 Ala Gln
Gly Lys Ser Phe Ile Lys Asp Ala Leu Lys Cys Lys Ala His 100 105 110
Ala Leu Arg His Arg Phe Gly Cys Ile Ser Arg Lys Cys Pro Ala Ile 115
120 125 Arg Glu Met Val Ser Gln Leu Gln Arg Glu Cys Tyr Leu Lys His
Asp 130 135 140 Leu Cys Ala Ala Ala Gln Glu Asn Thr Arg Val Ile Val
Glu Met Ile 145 150 155 160 His Phe Lys Asp Leu Leu Leu His Glu Pro
Tyr Val Asp Leu Val Asn 165 170 175 Leu Leu Leu Thr Cys Gly Glu Glu
Val Lys Glu Ala Ile Thr His Ser 180 185 190 Val Gln Val Gln Cys Glu
Gln Asn Trp Gly Ser Leu Cys Ser Ile Leu 195 200 205 Ser Phe Cys Thr
Ser Ala Ile Gln Lys Pro Pro Thr Ala Pro Pro Glu 210 215 220 Arg Gln
Pro Gln Val Asp Arg Thr Lys Leu Ser Arg Ala His His Gly 225 230 235
240 Glu Ala Gly His His Leu Pro Glu Pro Ser Ser Arg Glu Thr Gly Arg
245 250 255 Gly Ala Lys Gly Glu Arg Gly Ser Lys Ser His Pro Asn Ala
His Ala 260 265 270 Arg Gly Arg Val Gly Gly Leu Gly Ala Gln Gly Pro
Ser Gly Ser Ser 275 280 285 Glu Trp Glu Asp Glu Gln Ser Glu Tyr Ser
Asp Ile Arg Arg 290 295 300
* * * * *