Note: Descriptions are shown in the official language in which they were submitted.
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MOLECULAR PROFILING OF CANCER
This application claims priority to provisional patent application serial
number
60/732,859, filed 11/2/05, which is herein incorporated by reference in its
entirety.
This invention was made with government support under grant numbers
P50CA69568, R01AG21404 and UO1 CA111275-01 awarded by the National
Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
The present invention relates to compositions and methods for cancer
diagnostics, including but not limited to, cancer markers. In particular, the
present
invention provides cancer markers useful in the diagnosis and characterization
of
prostate and breast cancers.
BACKGROUND OF THE INVENTION
Afflicting one out of nine men over age 65, prostate cancer (PCA) is a leading
cause of male cancer-related death, second only to lung cancer (Abate-Shen and
Shen,
Genes Dev 14:2410 [2000]; Ruijter et al., Endocr Rev, 20:22 [1999]). The
American
Cancer Society estimates that about 184,500 American men will be diagnosed
with
prostate cancer and 39,200 will die in 2001.
Prostate cancer is typically diagnosed with a digital rectal exam and/or
prostate specific antigen (PSA) screening. An elevated serum PSA level can
indicate
the presence of PCA. PSA is used as a marker for prostate cancer because it is
secreted only by prostate cells. A healthy prostate will produce a stable
amount --
typically below 4 nanograms per inilliliter, or a PSA reading of "4" or less --
whereas
cancer cells produce escalating amounts that correspond with the severity of
the
cancer. A level between 4 and 10 may raise a doctor's suspicion that a patient
has
prostate cancer, while amounts above 50 may show that the tumor has spread
elsewhere in the body.
When PSA or digital tests indicate a strong likelihood that cancer is present,
a
transrectal ultrasound (TRUS) is used to map the prostate and show any
suspicious
areas. Biopsies of various sectors of the prostate are used to determine if
prostate
cancer is present. Treatment options depend on the stage of the cancer. Men
with a
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10-year life expectancy or less who have a low Gleason number and whose tumor
has
not spread beyond the prostate are often treated with watchful waiting (no
treatment).
Treatment options for more aggressive cancers include surgical treatments such
as
radical prostatectomy (RP), in which the prostate is completely removed (with
or
without nerve sparing techniques) and radiation, applied through an external
beain
that directs the dose to the prostate from outside the body or via low-dose
radioactive
seeds that are implanted within the prostate.to lcill cancer cells locally.
Anti-androgen
honnone therapy is also used, alone or in conjunction with surgery or
radiation.
Hormone therapy uses luteinizing hormone-releasing hormones (LH-RH) analogs,
which block the pituitary from producing hormones that stimulate testosterone
production. Patients must have injections of LH-RH analogs for the rest of
their lives.
While surgical and hormonal treatments are often effective for localized PCA,
advanced disease remains essentially incurable. Androgen ablation is the most
common therapy for advanced PCA, leading to massive apoptosis of androgen-
dependent malignant cells and temporary tumor regression. In most cases,
however,
the tumor reemerges with a vengeance and can proliferate independent of
androgen
signals.
The advent of prostate specific antigen (PSA) screening has led to earlier
detection of PCA and significantly reduced PCA-associated fatalities. However,
the
impact of PSA screening on cancer-specific mortality is still unknown pending
the
results of prospective randomized screening studies (Etzioni et aL, J. Natl.
Cancer
Inst., 91:1033 [1999]; Maattanen et al., Br. J. Cancer 79:1210 [1999];
Schroder et al.,
J. Natl. Cancer Inst., 90:1817 [1998]). A major limitation of the serum PSA
test is a
lack of prostate cancer sensitivity and specificity especially in the
intermediate range
of PSA detection (4-10 ng/ml). Elevated serum PSA levels are often detected in
patients with non-malignant conditions such as benign prostatic hyperplasia
(BPH)
and prostatitis, and provide little information about the aggressiveness of
the cancer
detected. Coincident with increased serum PSA testing, there has been a
dramatic
increase in the number of prostate needle biopsies performed (Jacobsen et al.,
JAMA
274:1445 [1995]). This has resulted in a surge of equivocal prostate needle
biopsies
(Epstein and Potter J. Urol., 166:402 [2001]). Thus, development of additional
seruin
and tissue biomarkers to supplement PSA screening is needed.
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SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for cancer
diagnostics, including but not liinited to, cancer marlcers. In particular,
the present
invention provides cancer markers useful in the diagnosis and characterization
of
prostate and breast cancers.
For Example, in some embodiments, the present invention provides a method
for characterizing prostate tissue in a subject, comprising: providing a
prostate tissue
sample from a subject; and detecting the level of expression of a cancer
marker (e.g.,
E2 ubiquitin ligase, UBc9, the cytosolic phosphoprotein stathmin, the death
receptor
DR3, and the Aurora A kinase (STK15), KRIP1 (KAP-1), Dynamin, CDK7, LAP2,
Myosin VI, ICBP90, ILP/XIAP, CainKK, JAM1, PICIn, or p23) in the sample,
thereby characterizing the prostate tissue sample. In some embodiments, the
detecting the level of expression of a cancer marker comprises detecting the
presence
of cancer marker mRNA (e.g., by exposing the cancer marker mRNA to a nucleic
acid probe complementary to the cancer marker mRNA). In other embodiments,
detecting the level of expression of a cancer marker comprises detecting the
presence
of a cancer marker polypeptide (e.g., by exposing the cancer marker
polypeptide to an
antibody specific to the cancer marker polypeptide and detecting the binding
of the
antibody to the cancer marker polypeptide). In some embodiments, the subject
is a
human subject. In some embodiments, the sample comprises tumor tissue. In some
embodiments, characterizing the prostate tissue comprises identifying a stage
of
prostate cancer in the prostate tissue (e.g., prostate carcinoma or metastatic
prostate
carcinoma). In some embodiments, the method further comprises the step
providing a
prognosis to the subject (e.g., the risk of developing prostate cancer).
The present invention further provides a kit for characterizing prostate
tissue
in a subject, comprising: a reagent capable of (e.g., sufficient to)
specifically detect
the level of expression of a cancer marker (e.g., E2 ubiquitin ligase, UBc9,
the
cytosolic phosphoprotein stathmin, the death receptor DR3, and the Aurora A
kinase
(STK15), KRIPI (KAP-1), Dynamin, CDK7, LAP2, Myosin VI, ICBP90, ILP/XIAP,
CamKK, JAMl, PICIn, or p23); and optionally, instructions for using the kit
for
characterizing prostate tissue in the subject. In some embodiments, the
reagent
comprises a nucleic acid probe complementary to the cancer marker mRNA. In
other
embodiments, the reagent comprises an antibody that specifically binds to the
cancer
marker polypeptide. In some embodiments, the instructions comprise
instructions
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required by the United States Food and Drug Administration for use in in vitro
diagnostic products. In some embodiments, the kit comprises software that
assists in
the collection of, analysis of, interpretation of, and/or display of data or
results
generated by or from the reagents.
In still further embodiments, the present invention provides a method for
characterizing breast tissue in a subject, coinprising: providing a breast
tissue sai.nple
from a subject; and detecting the level of expression of a cancer maker (e.g.,
CamKK,
Myosin VI, Auroara A, exportin, BM28, CDK7, TIP60, or p16 INK 4a) in the
sample,
thereby characterizing the breast tissue sample. In some embodiments, the
detecting
the level of expression of a cancer marker comprises detecting the presence of
cancer
marker inRNA (e.g., by exposing the cancer marker mRNA to a nucleic acid probe
complementary to the cancer marker mRNA). In other embodiments, detecting the
level of expression of a cancer marker comprises detecting the presence of a
cancer
marker polypeptide (e.g., by exposing the cancer marker polypeptide to an
antibody
specific to the cancer marker polypeptide and detecting the binding of the
antibody to
the cancer marker polypeptide). In some enlbodiments, the subject is a human
subject. In some embodiments, the sample comprises tumor tissue. In some
embodiments, the method further comprises the step of providing a prognosis to
the
subject (e.g., the risk of developing breast cancer).
In yet other embodiments, the present invention provides a kit for
characterizing breast tissue in a subject, comprising: a reagent capable of
(e.g.,
sufficient to) specifically detect the level of expression of a cancer marker
(e.g.,
CamKK, Myosin VI, Auroara A, exportin, BM28, CDK7, TIP60, or p16 INK 4a); and
optionally, instructions for using the kit for characterizing breast tissue in
the subject.
In some embodiments, the reagent comprises a nucleic acid probe complementary
to
the cancer marker mRNA. Iil other embodiments, the reagent comprises an
antibody
that specifically binds to the cancer marker polypeptide. In some embodiments,
the
instructions comprise instructions required by the United States Food and Drug
Administration for use in in vitro diagnostic products. In some embodiments,
the kit
comprises software that assists in the collection of, analysis of,
interpretation of,
and/or display of data or results generated by or from the reagents.
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DESCRIPTION OF THE FIGURES
Figure 1 shows high-throughput iinmunoblot analysis to define proteomic
alterations in prostate cancer progression. A, A flowchart of the general
methodology
employed to profile proteomic alterations in tissue extracts. B,
Representative high-
througliput iinmunoblots performed for pooled benign, clinically localized
prostate
cancer and metastatic prostate cancer tissues.
Figure 2 shows tissue microarray analyses of protein markers deregulated in
prostate cancer progression. A. Selected images of tissue microarray elements
representing immunohistochemical analysis of proteins altered in prostate
cancer
progression. B, Cluster analysis of twenty proteins dysregulated in prostate
cancer
progression evaluated for in situ protein levels by tissue microarrays.
Figure 3 shows integrative proteomic and transcriptomic analysis of prostate
cancer progression. A, Color map of integrative analysis relating protein
alterations
to gene expression in clinically localized prostate cancer relative to benign
prostate
tissue. B, As in A except the integrative analysis was carried out between
metastatic
prostate cancer relative to clinically localized prostate cancer. C,
Conventional
immunoblot validation of selected proteins differentially expressed between
metastatic prostate cancer and clinically localized prostate cancer.
Figure 4 shows proteomic alterations in metastatic prostate cancer nominate
gene predictors of cancer aggressiveness. A, A concordant 44-gene predictor
was
developed based on proteomic alterations that were concordant with gene
expression
(Fig. 3B) and subsequently evaluated for prognostic utility. B, The concordant
44-
gene predictor and the refined concordant 9-gene predictor were evaluated in
an
independent prostate cancer profiling dataset. C, Same as A, except the
concordant
44-gene predictor was evaluated in other solid tumors.
Figure 5 shows integrative molecular analysis of cancer to identify gene
predictors of clinical outcome.
Figure 6 shows integrative genomic and proteomic analysis of pooled and
individual prostate tissue extracts. Figure 6A shows color maps of integrative
analyses relating protein alterations observed in pooled tissues by
inununoblotting and
transcript alterations observed in the pooled and individual tissues by gene
expression
analyses. Figure 6B shows color maps depicting integrative genomic and
proteomic
analysis of individual prostate tissue samples.
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Figure 7 shows validation of proteomic alterations in prostate cancer by
conventional immunoblot analysis.
Figure 8 shows high-resolution iinages from Fig. 2. Figure 8A shows high
resolution iinages of the staining shown in Fig. 2. Figure 8B represents the
cluster
analysis of twenty proteins dysregulated in prostate cancer progression
evaluated for
in situ protein levels by tissue inicroarrays.
Figure 9 shows high-resolution matrix maps described in Fig. 3A. A, Color
map of integrative analysis relating protein alterations to gene expression in
clinically
localized prostate cancer relative to benign prostate tissue. B, As in A
except the
integrative analysis was carried out between metastatic prostate cancer
relative to
clinically localized prostate cancer.
Figure 10 shows high-resolution matrix maps for proteomic alterations in
metastatic prostate cancer nominate gene predictors of prostate cancer
aggressiveness.
A, A concordant 44-gene predictor was developed based on proteomic alterations
that
were concordant with gene expression (Fig. 3B) and subsequently evaluated for
prognostic utility. B, The concordant 44-gene predictor was evaluated in an
independent prostate cancer profiling dataset. C. Same as A, except the
refined
concordant 9-gene
predictor was evaluated in the Yu et al. study. D. Same as B, except the
refined
concordant 9-gene predictor was evaluated by using the Glinsky et al. study as
a
validation dataset.
Figure 11 shows high-resolution matrix maps described in Fig. 4C with the
addition of the Van't Veer breast cancer profiling dataset. A, A concordant 44-
gene
predictor was developed based on proteomic alterations that were concordant
with
gene expression (Fig. 3B). B, The concordant 44-gene predictor was evaluated
in an
independent prostate cancer profiling dataset. C. Same as A, except the
refined
concordant 9-gene predictor was evaluated. D. Same as B, except the refined
concordant 9-gene predictor was evaluated by using the Glinsky et al. study as
a
validation dataset.
Figure 12 shows High-resolution matrix maps described in Fig. 5C with the
addition of the Van't Veer breast cancer profiling dataset.
Figure 13 shows an immunoblot of breast cancer markers.
Figure 14 shows Table 9.
Figure 15 shows Table 10.
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GENERAL DESCRIPTION
Multiple molecular alterations occur during cancer development. To begin to
understand these processes with a systems perspective, there is a need to
characterize
and integrate these components. Experiments conducted during the course of
development of the present invention integrated such disparate inolecular data
as
RNA expression profiling and protein expression in prostate and breast cancer.
A high-throughput immunoblot approach was used to characterize proteomic
alterations in human prostate cancer progression focusing on the transition of
clinically localized disease to metastatic disease. This approach revealed
over one
hundred
proteomic alterations in prostate cancer progression. Furthermore, these
proteomic
profiles were integrated with mRNA transcript data from independent expression
profiling datasets. Proteins that were qualitatively concordant with gene
expression
could be used as a predictor of clinical outcome. In other words, this
integrative
approach revealed the presence of an "aggressive signature" in clinically
localized
prostate tumors.
Prostate cancer is a highly prevalent disease in older men of the Western
world (Chan et al., J Urol 172, S13-16, 2004; Linton and Hamdy, Cancer Treat
Rev
29, 151-160, 2003). Unlike other cancers, more men die with prostate cancer
than
from the disease (Albertsen et al., Jama 280, 975-980, 1998; Johansson et al.,
Jama
277, 467-471, 1997). Deciphering the molecular networks that distinguish
progressive disease from nonprogressive disease sheds light into the biology
of-
aggressive prostate cancer as well as leads to the identification of
biomarkers that aid
in the selection of patients that should be treated (Kumar-Sinha and
Chinnaiyan,
Urology 62 Suppl 1, 19-35, 2003). To begin to understand prostate cancer
progression with a systems perspective, it is helpful to characterize and
integrate the
molecular components involved (Grubb et al., Proteomics 3, 2142-21462003; Hood
et
al., Science 306, 640-643, 2004; Paweletz et al., Oncogene 20, 1981-1989,
2001;
Petricoin et al., J Natl Cancer Inst 94, 1576-1578, 2002). A number of groups
have
employed gene expression microarrays to profile prostate cancer tissues
(Dhanasekaran et al., Nature 412, 822-826, 2001; Lapointe et al., Proc Natl
Acad Sci
U S A 101, 811-816, 2004; LaTulippe et al., Cancer Res 62, 4499-4506, 2002;
Luo et
al., Cancer Res 61, 4683-4688, 2001; Luo et al., Mol Carcinog 33, 25-35,
2002b;
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Magee et al., Cancer Res 61, 5692-5696, 2001; Singh et al., Cancer Cell 1, 203-
209,
2002; Welsh et al., Cancer Res 61, 5974-5978, 2001; Yu et al., J Clin Oncol
22, 2790-
2799, 2004) as well as other tuinors (Alizadeh et al., Nature 403, 503-511,
2000;
Golub et al., Science 286, 531-537, 1999; Hedenfalk et al., N Engl J Med 344,
539-
548, 2001; Perou et al., Nature 406, 747-752, 2000) at the transcriptome level
but
inuch less work has been done at the protein level. Proteins, as opposed to
nucleic
acids, represent the functional effectors of cancer progression and thus serve
as
therapeutic targets as well as markers of disease.
In experiments conducted during the course of development of the present
invention, a high-throughput immunoblot approach was utilized to characterize
proteomic alterations in human prostate cancer progression focusing on the
transition
from clinically localized prostate cancer to metastatic disease. Using an
integrative
approach, proteomic profiles with mRNA transcript data from several
experiments
were analyzed. The analyses also indicated that the proteins that were
qualitatively
concordant with gene expression could be used to define a multiplex gene
predictor of
clinical outcome.
The present invention provides a general framework for the integrative
analysis of protein and transcriptomic data from human tumors (Fig. 5).
Proteomic
profiling of prostate cancer progression identified over one hundred altered
proteins in
the transition from clinically localized to metastatic disease (a significant
fraction of
which were androgen regulated). While this approach was useful to integrate
high-
throughput immunoblot data, the general paradigm can also be applied to mass
spectrometry or protein microarray based technologies. Differential proteins
were
then mapped to mRNA transcript levels to assess mRNA/protein concordance
levels
in a human disease state. Gene expression alterations that matched protein
alterations
qualitatively could be used as predictors of prostate cancer progression in
clinically
confined disease. Together, this shows that clinically aggressive prostate
cancer bears
a "signature" set of genes/proteins that is characteristic of metastatic
disease. The
observation that the concordant proteomic/genomic signature can be applied to
other
solid tumors shows commonalities in the undifferentiated state of advanced
tumors.
DEFINITIONS
To facilitate an understanding of the present invention, a number of terms and
phrases are defined below:
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The tenn "epitope" as used herein refers to that portion of an antigen that
inakes contact with a particular antibody.
When a protein or fragment of a protein is used to iinmunize a host animal,
nuinerous regions of the protein may induce the production of antibodies which
bind
specifically to a given region or three-dimensional structure on the protein;
these
regions or structures are referred to as "antigenic determinants". An
antigenic
determinant may compete with the intact antigen (i.e., the "immunogen" used to
elicit
the immune response) for binding to an antibody.
The terms "specific binding" or "specifically binding" when used in reference
to the interaction of an antibody and a protein or peptide means that the
interaction is
dependent upon the presence of a particular structure (i.e., the antigenic
determinant
or epitope) on the protein; in other words the antibody is recognizing and
binding to a
specific protein structure rather than to proteins in general. For example, if
an
antibody is specific for epitope "A," the presence of a protein containing
epitope A (or
free, unlabelled A) in a reaction containing labeled "A" and the antibody will
reduce
the amount of labeled A bound to the antibody.
As used herein, the terms "non-specific binding" and "background binding"
when used in reference to the interaction of an antibody and a protein or
peptide refer
to an interaction that is not dependent on the presence of a particular
structure (i.e.,
the antibody is binding to proteins in general rather that a particular
structure such as
an epitope).
As used herein, the tenn "siRNAs" refers to small interfering RNAs. In some
embodiments, siRNAs comprise a duplex, or double-stranded region, of about 18-
25
nucleotides long; often siRNAs contain from about two to four unpaired
nucleotides
at the 3' end of each strand. At least one strand of the duplex or double-
stranded
region of a siRNA is substantially homologous to, or substantially
complementary to,
a target RNA molecule. The strand complementary to a target RNA molecule is
the
"antisense strand;" the strand homologous to the target RNA molecule is the
"sense
strand," and is also complementary to the siRNA antisense strand. siRNAs may
also
contain additional sequences; non-limiting examples of such sequences include
linking sequences, or loops, as well as stem and other folded structures.
siRNAs
appear to function as key intermediaries in triggering RNA interference in
invertebrates and in vertebrates, and in triggering sequence-specific RNA
degradation
during posttranscriptional gene silencing in plants.
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The term "RNA interference" or "RNAi" refers to the silencing or decreasing
of gene expression by siRNAs. It is the process of sequence-specific, post-
transcriptional gene silencing in animals and plants, initiated by siRNA that
is
homologous in its duplex region to the sequence of the silenced gene. The gene
may
be endogenous or exogenous to the organism, present integrated into a
chromosome
or present in a transfection vector that is not integrated into the genome.
The
expression of the gene is either coinpletely or partially inhibited. RNAi inay
also be
considered to inhibit the function of a target RNA; the function of the target
RNA
may be complete or partial.
As used herein, the term "subject" refers to any aniinal (e.g., a mammal),
including, but not limited to, humans, non-human primates, rodents, and the
like,
which is to be the recipient of a particular treatment. Typically, the terms
"subject"
and "patient" are used interchangeably herein in reference to a human subject.
As used herein, the term "subject suspected of having cancer" refers to a
subject that presents one or more symptoms indicative of a cancer (e.g., a
noticeable
lump or mass) or is being screened for a cancer (e.g., during a routine
physical). A
subject suspected of having cancer may also have one or more risk factors. A
subject
suspected of having cancer has generally not been tested for cancer. However,
a
"subject suspected of having cancer" encoinpasses an individual who has
received an
initial diagnosis (e.g., a CT scan showing a mass or increased PSA level) but
for
whom the stage of cancer is not known. The term further includes people who
once
had cancer (e.g., an individual in remission).
As used herein, the term "subject at risk for cancer" refers to a subject with
one or more risk factors for developing a specific caricer. Risk factors
include, but are
not limited to, gender, age, genetic predisposition, environmental expose,
previous
incidents of cancer, preexisting non-cancer diseases, and lifestyle.
As used herein, the term "characterizing cancer in subject" refers to the
identification of one or more properties of a cancer sample in a subject,
including but
not limited to, the presence of benign, pre-cancerous or cancerous tissue, the
stage of
the cancer, and the subject's prognosis. Cancers may be characterized by the
identification of the expression of one or more cancer marker genes, including
but not
limited to, the cancer markers disclosed herein.
As used herein, the term "characterizing prostate tissue in a subject" refers
to
the identification of one or more properties of a prostate tissue sample
(e.g., including
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but not liinited to, the presence of cancerous tissue, the presence of pre-
cancerous '
tissue that is likely to become cancerous, and the presence of cancerous
tissue that is
likely to metastasize). In some embodiments, tissues are characterized by the
identification of the expression of one or more cancer inarlcer genes,
including but not
limited to, the cancer markers disclosed herein.
As used herein, the term "cancer marker genes" refers to a gene whose
expression level, alone or in combination with other genes, is correlated with
cancer
or prognosis of cancer. The correlation may relate to either an increased or
decreased
expression of the gene. For example, the expression of the gene may be
indicative of
cancer, or lack of expression of the gene may be correlated with poor
prognosis in a
cancer patient. Cancer marker expression may be characterized using any
suitable
method, including but not limited to, those described in the illustrative
Examples
below.
As used herein, the term "a reagent that specifically detects expression
levels"
refers to reagents used to detect the expression of one or more genes (e.g.,
including
but not limited to, the cancer markers of the present invention). Examples of
suitable
reagents include but are not limited to, nucleic acid probes capable of
specifically
hybridizing to the gene of interest, PCR primers capable of specifically
amplifying the
gene of interest, and antibodies capable of specifically binding to proteins
expressed
by the gene of interest. Other non-limiting examples can be found in the
description
and exainples below.
As used herein, the term "detecting a decreased or increased expression
relative to non-cancerous prostate control" refers to measuring the level of
expression
of a gene (e.g., the level of mRNA or protein) relative to the level in a non-
cancerous
prostate control sample. Gene expression can be measured using any suitable
method,
including but not limited to, those described herein.
As used herein, the term "detecting a change in gene expression (e.g., cancer
marker gene expression) in said prostate cell sample in the presence of said
test
compound relative to the absence of said test compound" refers to measuring an
altered level of expression (e.g., increased or decreased) in the presence of
a test
compound relative to the absence of the test compound. Gene expression can be
measured using any suitable method, including but not limited to, those
described in
the Examples below.
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As used herein, the terin "instructions for using said kit for detecting
cancer in
said subject" includes instructions for using the reagents contained in the
kit for the
detection and characterization of cancer in a sample from a subject. In some
embodiments, the instructions fizrther coinprise the stateinent of intended
use required
by the U.S. Food and Drug Administration (FDA) in labeling ifa vitro
diagnostic
products.
As used herein, the tenn "prostate cancer expression profile map" refers to a
presentation of expression levels of genes in a particular type of prostate
tissue (e.g.,
primary, metastatic, and pre-cancerous prostate tissues). The map may be
presented
as a graphical representation (e.g., on paper or on a computer screen), a
physical
representation (e.g., a gel or array) or a digital representation stored in
computer
memory. Each map corresponds to a particular type of prostate tissue (e.g.,
primary,
metastatic, and pre-cancerous) and thus provides a template for comparison to
a
patient sample. In preferred embodiments, maps are generated from pooled
samples
comprising tissue samples from a plurality of patients with the same type of
tissue.
As used herein, the terms "computer memory" and "computer memory device"
refer to any storage media readable by a computer processor. Examples of
computer
memory include, but are not limited to, RAM, ROM, computer chips, digital
video
disc (DVDs), compact discs (CDs), hard disk drives (HDD), and magnetic tape.
As used herein, the term "computer readable medium" refers to any device or
system for storing and providing information (e.g., data and instructions) to
a
computer processor. Examples of computer readable media include, but are not
limited to, DVDs, CDs, hard disk drives, magnetic tape and servers for
streaming
media over networks.
As used lierein, the terms "processor" and "central processing unit" or "CPU"
are used interchangeably and refer to a device that is able to read a program
from a
computer memory (e.g., ROM or other computer memory) and perform a set of
steps
according to the program.
As used herein, the term "stage of cancer" refers to a qualitative or
quantitative
assessment of the level of advancement of a cancer. Criteria used to determine
the
stage of a cancer include, but are not limited to, the size of the tumor,
whether the
tumor has spread to other parts of the body and where the cancer has spread
(e.g.,
within the same organ or region of the body or to another organ).
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As used herein, the term "providing a prognosis" refers to providing
infonnation regarding the iinpact of the presence of cancer (e.g., as
determined by the
diagnostic methods of the present invention) on a subject's future health
(e.g.,
expected morbidity or mortality, the likelihood of getting cancer, and the
risk of
metastasis).
As used herein, the tenn "initial diagnosis" refers to results of initial
cancer
diagnosis (e.g. the presence or absence of cancerous cells). An initial
diagnosis does
not include information about the stage of the cancer of the risk of
metastasis.
As used herein, the term "biopsy tissue" refers to a sample of tissue (e.g.,
prostate tissue) that is removed froin a subject for the purpose of
determining if the
sample contains cancerous tissue. In some embodiment, biopsy tissue is
obtained
because a subject is suspected of having cancer. The biopsy tissue is then
examined
(e.g., by microscopy) for the presence or absence of cancer.
As used herein, the term "non-human animals" refers to all non-human
animals including, but are not limited to, vertebrates such as rodents, non-
human
primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines,
canines, felines, aves, etc.
As used herein, the term "gene transfer system" refers to any means of
delivering a composition comprising a nucleic acid sequence to a cell or
tissue. For
example, gene transfer systems include, but are not limited to, vectors (e.g.,
retroviral,
adenoviral, adeno-associated viral, and other nucleic acid-based delivery
systems),
microinjection of naked nucleic acid, polymer-based delivery systems (e.g.,
liposome-
based and metallic particle-based systems), biolistic injection, and the like.
As used
herein, the term "viral gene transfer system" refers to gene transfer systems
comprising viral elements (e.g., intact viruses, modified viruses and viral
components
such as nucleic acids or proteins) to facilitate delivery of the sample to a
desired cell
or tissue. As used herein, the term "adenovirus gene transfer system" refers
to gene
transfer systems comprising intact or altered viruses belonging to the family
Adenoviridae.
As used herein, the term "site-specific recombination target sequences" refers
to nucleic acid sequences that provide recognition sequences for recombination
factors and the location where recombination takes place.
As used herein, the term "nucleic acid molecule" refers to any nucleic acid
containing molecule, including but not limited to, DNA or RNA. The term
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encoinpasses sequences that include any of the known base analogs of DNA and
RNA
including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-
inethyladenosine,
aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
fluorouracil, 5-broinouracil, 5-carboxymethylaminomethyl-2-thiouracil,
5-carboxyinethylaininoinethyluracil, dihydrouracil, inosine, N6-
isopentenyladenine,
1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-
dimethylguanine, 2-methyladenine, 2-methylguaiiine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-
methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-
thiocytosine, 5-
methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-
oxyacetic
acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-
thiocytosine, and
2,6-diaminopurine.
The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises
coding sequences necessary for the production of a polypeptide, precursor, or
RNA
(e.g., rRNA, tRNA). The polypeptide can be encoded by a full length coding
sequence or by any portion of the coding sequence so long as the desired
activity or
functional properties (e.g., enzymatic activity, ligand binding, signal
transduction,
immunogenicity, etc.) of the full-length or fragment are retained. The tenn
also
encompasses the coding region of a structural gene and the sequences located
adjacent
to the coding region on both the 5' and 3' ends for a distance of about 1 kb
or more on
either end such that the gene corresponds to the length of the full-length
mRNA.
Sequences located 5' of the coding region and present on the mRNA are referred
to as
5' non-translated sequences. Sequences located 3' or downstream of the coding
region
and present on the mRNA are referred to as 3' non-translated sequences. The
term
"gene" encompasses both cDNA and genomic forms of a gene. A genomic form or
} clone of a gene contains the coding region interrupted with non-coding
sequences
termed "introns" or "intervening regions" or "intervening sequences." Introns
are
segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may
contain regulatory elements such as enhancers. Introns are removed or "spliced
out"
from the nuclear or primary transcript; introns therefore are absent in the
messenger
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RNA (mRNA) transcript. The mRNA functions during translation to specify the
sequence or order of amino acids in a nascent polypeptide.
As used herein, the term "heterologous gene" refers to a gene that is not in
its
natural environinent. For example, a heterologous geile includes a gene from
one
species introduced into another species. A heterologous gene also includes a
gene
native to an organism that has been altered in some way (e.g., inutated, added
in
multiple copies, linked to non-native regulatory sequences, etc). Heterologous
genes
are distinguished from endogenous genes in that the heterologous gene
sequences are
typically joined to DNA sequences that are not found naturally associated with
the
gene sequences in the chromosome or are associated with portions of the
chromosome
not found in nature (e.g., genes expressed in loci where the gene is not
normally
expressed).
As used herein, the term "gene expression" refers to the process of converting
genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or
snRNA) through "transcription" of the gene (i.e., via the enzymatic action of
an RNA
polymerase), and for protein encoding genes, into protein through
"translation" of
mRNA. Gene expression can be regulated at many stages in the process. "Up-
regulation" or "activation" refers to regulation that increases the production
of gene
expression products (i.e., RNA or protein), while "down-regulation" or
"repression"
refers to regulation that decrease production. Molecules (e.g., transcription
factors)
that are involved in up-regulation or down-regulation are often called
"activators" and
"repressors," respectively.
In addition to containing introns, genomic forms of a gene may also include
sequences located on both the 5' and 3' end of the sequences that are present
on the
RNA transcript. These sequences are referred to as "flanking" sequences or
regions
(these flanking sequences are located 5' or 3' to the non-translated sequences
present
on the mRNA transcript). The 5' flanking region may contain regulatory
sequences
such as promoters and enliancers that control or influence the transcription
of the
gene. The 3' flanking region may contain sequences that direct the termination
of
transcription, post-transcriptional cleavage and polyadenylation.
The term "wild-type" refers to a gene or gene product isolated from a
naturally
occurring source. A wild-type gene is.that which is most frequently observed
in a
population and is -thus arbitrarily designed the "normal" or "wild-type" form
of the
gene. In contrast, the term "modified" or "mutant" refers to a gene or gene
product
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that displays modifications in sequence and or functional properties (i.e.,
altered
characteristics) when colnpared to the wild-type gene or gene product. It is
noted that
naturally occurring mutants can be isolated; these are identified by the fact
that they
have altered characteristics (including altered nucleic acid sequences) when
compared
to the wild-type gene or gene product.
As used herein, the terms "nucleic acid molecule encoding," "DNA sequence
encoding," and "DNA encoding" refer to the order or sequence of
deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of
these
deoxyribonucleotides determines the order of amino acids along the polypeptide
(protein) chain. The DNA sequence thus codes for the amino acid sequence.
As used herein, the terms "an oligonucleotide having a nucleotide sequence
encoding a gene" and "polynucleotide having a nucleotide sequence encoding a
gene,"
means a nucleic acid sequence comprising the coding region of a gene or in
other
words the nucleic acid sequence that encodes a gene product. The coding region
may
be present in a cDNA, genomic DNA or RNA form. When present in a DNA form,
the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense
strand) or
double-stranded. Suitable control elements such as enhancers/promoters, splice
junctions, polyadenylation signals, etc. may be placed in close proximity to
the coding
region of the gene if needed to permit proper initiation of transcription
and/or correct
processing of the primaiy RNA transcript. Alternatively, the coding region
utilized in
the expression vectors of the present invention may contain endogenous
enhancers/promoters, splice junctions, intervening sequences, polyadenylation
signals, etc. or a combination of both endogenous and exogenous control
elements.
As used herein, the term "oligonucleotide," refers to a short length of single-
stranded polynucleotide chain. Oligonucleotides are typically less than 200
residues
long (e.g., between 15 and 100), however, as used herein, the term is also
intended to
encompass longer polynucleotide chains. Oligonucleotides are often refeiTed to
by
their length. For example a 24 residue oligonucleotide is referred to as a "24-
mer".
Oligonucleotides can form secondary and tertiary structures by self-
hybridizing or by
hybridizing to other polynucleotides. Such structures can include, but are not
limited
to, duplexes, hairpins, cruciforms, bends, and triplexes.
As used herein, the terms "complementary" or "complementarity" are used in
reference to polynucleotides (i.e., a sequence of nucleotides) related by the
base-
pairing rules. For example, for the sequence "A-G-T," is complementary to the
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sequence "T-C-A." Compleinentarity may be "partial," in which only some of the
nucleic acids' bases are matched according to the base pairing rules. Or,
there may be
"coinplete" or "total" coinplementarity between the nucleic acids. The degree
of
compleinentarity between nucleic acid strands has significant effects on the
efficiency
and strength of hybridization between nucleic acid strands. This is of
particular
importance in ainplification reactions, as well as detection inethods that
depend upon
binding between nucleic acids.
The term "homology" refers to a degree of complementarity. There may be
partial hoinology or complete homology (i.e., identity). A partially
complementary
sequence is a nucleic acid molecule that at least partially inhibits a
completely
complementary nucleic acid molecule from hybridizing to a target nucleic acid
is
"substantially homologous." The inhibition of hybridization.of the completely
complementary sequence to the target sequence may be examined using a
hybridization assay (Southern or Northern blot, solution hybridization and the
like)
under conditions of low stringency. A substantially homologous sequence or
probe
will compete for and inhibit the binding (i.e., the hybridization) of a
completely
homologous nucleic acid molecule to a target under conditions of low
stringency.
This is not to say that conditions of low stringency are such that non-
specific binding
is permitted; low stringency conditions require that the binding of two
sequences to
one another be a specific (i.e., selective) interaction. The absence of non-
specific
binding may be tested by the use of a second target that is substantially non-
complementary (e.g., less than about 30% identity); in the absence of non-
specific
binding the probe will not hybridize to the second non-complementary target.
When used in reference to a double-stranded nucleic acid sequence such as a
cDNA or genomic clone, the term "substantially homologous" refers to any probe
that
can hybridize to either or both strands of the double-stranded nucleic acid
sequence
under conditions of low stringency as described above.
A gene may produce multiple RNA species that are generated by differential
splicing of the primary RNA transcript. cDNAs that are splice variants of the
same
gene will contain regions of sequence identity or complete homology
(representing
the presence of the same exon or portion of the satne exon on both cDNAs) and
regions of complete non-identity (for example, representing the presence of
exon "A"
on cDNA 1 wherein cDNA 2 contains exon "B" instead). Because the two cDNAs
contain regions of sequence identity they will both hybridize to a probe
derived from
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the entire gene or portions of the gene containing sequences found on both
cDNAs;
the two splice variants are therefore substantially homologous to such a probe
and to
each other.
When used in reference to a single-stranded nucleic acid sequence, the term
"substantially homologous" refers to any probe that can hybridize (i.e., it is
the
compleinent of) the single-stranded nucleic acid sequence under conditions of
low
stringency as described above.
As used herein, the term "hybridization" is used in reference to the pairing
of
compleinentary nucleic acids. Hybridization and the strength of hybridization
(i.e.,
the strength of the association between the nucleic acids) is impacted by such
factors
as the degree of complementary between the nucleic acids, stringency of the
conditions involved, the Tm of the formed hybrid, and the G:C ratio within the
nucleic acids. A single molecule that contains pairing of complementary
nucleic
acids within its structure is said to be "self-hybridized."
As used herein, the term "Tm" is used in reference to the "melting
temperature." The melting temperature is the temperature at which a population
of
double-stranded nucleic acid molecules becomes half dissociated into single
strands.
The equation for calculating the Tm of nucleic acids is well known in the art.
As
indicated by standard references, a simple estimate of the Tm value may be
calculated
by the equation: T. = 81.5 + 0.41(% G + C), when a nucleic acid is in aqueous
solution at 1 M NaC1(See e.g., Anderson and Young, Quantitative Filter
Hybridization, in Nucleic Acid Hybridization [1985]). Other references include
more
sophisticated computations that take structural as well as sequence
characteristics into
account for the calculation of Tm.
As used herein the term "stringency" is used in reference to the conditions of
temperature, ionic strength, and the presence of other compounds such as
organic
solvents, under which nucleic acid hybridizations are conducted. Under "low
stringency conditions" a nucleic acid sequence of interest will hybridize to
its exact
complement, sequences with single base mismatches, closely related sequences
(e.g.,
sequences with 90% or greater homology), and sequences having only partial
homology (e.g., sequences with 50-90% homology). Under 'medium stringency
conditions," a nucleic acid sequence of interest will hybridize only to its
exact
complement, sequences with single base mismatches, and closely relation
sequences
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(e.g., 90% or greater homology). Under "high stringency conditions," a nucleic
acid
sequence of interest will hybridize only to its exact complement, and
(depending on
conditions such a teinperature) sequences with single base mismatches. In
other
words, under conditions of high stringency the temperature can be raised so as
to
exclude hybridization to sequences with single base mismatches.
"High stringency conditions" when used in reference to nucleic acid
hybridization comprise conditions equivalent to binding or hybridization at 42
C in a
solution consisting of 5X SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4 H20 and 1.85
g/l
EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100
g/ml denatured salmon sperm DNA followed by washing in a solution comprising
0.1X SSPE, 1.0% SDS at 42 C when a probe of about 500 nucleotides in length is
employed.
"Medium stringency conditions" when used in reference to nucleic acid
hybridization comprise conditions equivalent to binding or hybridization at 42
C in a
solution consisting of 5X SSPE (43.8 g/l NaCl, 6.9 g/1 NaH2PO4 H20 and 1.85
g/l
EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100
g/ml denatured salmon sperm DNA followed by washing in a solution comprising
1.OX SSPE, 1.0% SDS at 42 C when a probe of about 500 nucleotides in length is
employed.
"Low stringency conditions" comprise conditions equivalent to binding or
hybridization at 42 C in a solution consisting of 5X SSPE (43.8 g/l NaCl, 6.9
g/1
NaH2PO4 H20 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1 % SDS, 5X
Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400,
Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42 C
when a probe of about 500 nucleotides in length is employed.
The art knows well that numerous equivalent conditions may be employed to
comprise low stringency conditions; factors such as the length and nature
(DNA,
RNA, base composition) of the probe and nature of the target (DNA, RNA, base
composition, present in solution or immobilized, etc.) and the concentration
of the
salts and other components (e.g., the presence or absence of formamide,
dextran
sulfate, polyethylene glycol) are considered and the hybridization solution
may be
varied to generate conditions of low stringency hybridization different from,
but
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equivalent to, the above listed conditions. In addition, the art knows
conditions that
promote hybridization under conditions of high stringency (e.g., increasing
the
teinperature of the hybridization and/or wash steps, the use of fonnamide in
the
hybridization solution, etc.) (see definition above for "stringency").
As used herein, the tenn "primer" refers to..an oligonucleotide, whether
occurring naturally as in a purified restriction digest or produced
synthetically, that is
capable of acting as a point of initiation of synthesis when placed under
conditions in
which synthesis of a primer extension product that is complementary to a
nucleic acid
strand is induced, (i.e., in the presence of nucleotides and an inducing agent
such as
DNA polynlerase and at a suitable temperature and pH). The primer is
preferably
single stranded for maximum efficiency in amplification, but may alternatively
be
double stranded. If double stranded, the primer is first treated to separate
its strands
before being used to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer nlust be sufficiently long to prime the
synthesis of extension products in the presence of the inducing agent. The
exact
lengths of the primers will depend on many factors, including temperature,
source of
primer and the use of the inethod.
As used herein, the term "probe" refers to an oligonucleotide (i. e., a
sequence
of nucleotides), whether occurring naturally as in a purified restriction
digest or
produced synthetically, recombinantly or by PCR amplification, that is capable
of
hybridizing to at least a portion of another oligonucleotide of interest. A
probe may
be single-stranded or double-stranded. Probes are useful in the detection,
identification and isolation of particular gene sequences. It is contemplated
that any
probe used in the present invention will be labeled with any "reporter
molecule," so
that is detectable in any detection system, including, but not limited to
enzyine (e.g.,
ELISA, as well as enzyme-based histochemical assays), fluorescent,
radioactive, and
luminescent systems. It is not intended that the present invention be limited
to any
particular detection system or label.
As used herein the term "portion" when in reference to a nucleotide sequence
(as in "a portion of a given nucleotide sequence") refers to fragments of that
sequence.
The fragments may range in size from four nucleotides to the entire nucleotide
sequence minus one nucleotide (10 nucleotides, 20, 30, 40, 50, 100, 200,
etc.).
As used herein, the teim "amplification reagents" refers to those reagents
(deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification
except for
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primers, nucleic acid template and the amplification enzyme. Typically,
ainplification
reagents along with other reaction components are placed and contained in a
reaction
vessel (test tube, microwell, etc.).
As used herein, the terins "restriction endonucleases" and "restriction
enzymes" refer to bacterial enzyines, each of which cut double-stranded DNA at
or
near a specific nucleotide sequence.
The terms "in operable combination," "in operable order," and "operably
linlced" as used herein refer to the linkage of nucleic acid sequences in such
a manner
that a nucleic acid inolecule capable of directing the transcription of a
given gene
and/or the synthesis of a desired protein molecule is produced. The term also
refers to
the linkage of amino acid sequences in such a manner so that a functional
protein is
produced.
The term "isolated" when used in relation to a nucleic acid, as in "an
isolated
oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid
sequence that is
identified and separated from at least one component or contaminant with which
it is
ordinarily associated in its natural source. Isolated nucleic acid is such
present in a
form or setting that is different from that in which it is found in nature. In
contrast,
non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the
state
they exist in nature. For example, a given DNA sequence (e.g., a gene) is
found on
the host cell chromosome in proximity to neighboring genes; RNA sequences,
such as
a specific mRNA sequence encoding a specific protein, are found in the cell as
a
mixture with numerous other mRNAs that encode a multitude of proteins.
However,
isolated nucleic acid encoding a given protein includes, by way of example,
such
nucleic acid in cells ordinarily expressing the given protein where the
nucleic acid is
in a chromosomal location different from that of natural cells, or is
otherwise flanked
by a different nucleic acid sequence than that found in nature. The isolated
nucleic
acid, oligonucleotide, or polynucleotide may be present in single-stranded or
double-
stranded form. When an isolated nucleic acid, oligonucleotide or
polynucleotide is to
be utilized to express a protein, the oligonucleotide or polynucleotide will
contain at a
minimum the sense or coding strand (i.e., the oligonucleotide or
polynucleotide may
be single-stranded), but may contain both the sense and anti-sense strands
(i.e., the
oligonucleotide or polynucleotide may be double-stranded).
As used herein, the term "purified" or "to purify" refers to the removal of
components (e.g., contaminants) from a sample. For example, antibodies are
purified
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by removal of contaminating non-immunoglobulin proteins; they are also
purified by
the removal of iinmunoglobulin that does not bind to the target molecule. The
removal of non-iminunoglobulin proteins and/or the reinoval of immunoglobulins
that
do not bind to the target molecule results in an increase in the percent of
target-
reactive iinmunoglobulins in the sample. In another example, recombinant
polypeptides are expressed in bacterial host cells and the polypeptides are
purified by
the reinoval of host cell proteins; the percent of recombinant polypeptides is
thereby
increased in the sample.
"Amino acid sequence" and terms such as "polypeptide" or "protein" are not
meant to limit the ainino acid sequence to the complete, native amino acid
sequence
associated with the recited protein molecule.
The tenn "native protein" as used herein-to indicate that a protein does not
contain amino acid residues encoded by vector sequences; that is, the native
protein
contains only those amino acids found in the protein as it occurs in nature. A
native
protein may be produced by recombinant means or inay be isolated from a
naturally
occurring source.
As used herein the term "portion" when in reference to a protein (as in "a
portion of a given protein") refers to fragments of that protein. The
fragments may
range in size from four amino acid residues to the entire amino acid sequence
minus
one amino acid.
The term "Southern blot," refers to the analysis of DNA on agarose or
acrylamide gels to fractionate the DNA according to size followed by transfer
of the
DNA from the gel to a solid support, such as nitrocellulose or a nylon
membrane.
The immobilized DNA is then probed with a labeled probe to detect DNA species
coinplementary to the probe used. The DNA may be cleaved with restriction
enzymes
prior to electrophoresis. Following electrophoresis, the DNA may be partially
depurinated and denatured prior to or during transfer to the solid support.
Southern
blots are a standard tool of molecular biologists (J. Sambrook et al.,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31-9.58
[1989]).
The term "Northern blot," as used herein refers to the analysis of RNA by
electrophoresis of RNA on agarose gels to fractionate the RNA according to
size
followed by transfer of the RNA from the gel to a solid support, such as
nitrocellulose
or a nylon membrane. The immobilized RNA is then probed with a labeled probe
to
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detect RNA species complementary to the probe used. Northern blots are a
standard
tool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52
[1989]).
The term "Western blot" refers to the analysis of protein(s) (or polypeptides)
immobilized onto a support such as nitrocellulose or a membrane. The proteins
are
run on acrylamide gels to separate the proteins, followed by transfer of the
protein
fiom the gel to a solid support, such as nitrocellulose or a nylon membrane.
The
immobilized proteins are then exposed to antibodies with reactivity against an
antigen
of interest. The binding of the antibodies may be detected by various methods,
including the use of radiolabeled antibodies.
The term "transgene" as used herein refers to a foreign gene that is placed
into
an organism by, for example, introducing the foreign gene into newly
fertilized eggs
or early embryos. The term "foreign gene" refers to any nucleic acid (e.g.,
gene
sequence) that is introduced into the genome of an animal by experimental
manipulations and may include gene sequences found in that animal so long as
the
introduced gene does not reside in the same location as does the naturally
occurring
gene.
As used herein, the term "vector" is used in reference to nucleic acid
molecules that transfer DNA segment(s) from one cell to another. The term
"vehicle"
is sometimes used interchangeably with "vector." Vectors are often derived
from
plasmids, bacteriophages, or plant or animal viruses.
The term "expression vector" as used herein refers to a recombinant DNA
molecule containing a desired coding sequence and appropriate nucleic acid
sequences necessary for the expression of the operably linked coding sequence
in a
particular host organism. Nucleic acid sequences necessary for expression in
prokaryotes usually include a promoter, an operator (optional), and a ribosome
binding site, often along with other sequences. Eukaryotic cells are known to
utilize
promoters, enhancers, and termination and polyadenylation signals.
The terms "overexpression" and "overexpressing" and grammatical
equivalents, are used in reference to levels of mRNA to indicate a level of
expression
approximately 3-fold higher (or greater) than that observed in a given tissue
in a
control or non-transgenic animal. Levels of mRNA are measured using any of a
number of techniques known to those skilled in the art including, but not
limited to
Northern blot analysis. Appropriate controls are included on the Northern blot
to
control for differences in the amount of RNA loaded from each tissue analyzed
(e.g.,
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the amount of 28S rRNA, an abundant RNA transcript present at essentially the
same
amount in all tissues, present in each sample can be used as a means of
nonnalizing or
standardizing the inRNA-specific signal observed on Northern blots). The
aznount of
mRNA present in the band corresponding in size to the correctly spliced
transgene
RNA is quantified; other minor species of RNA which hybridize to the transgene
probe are not considered in the quantification of the expression of the
transgenic
mRNA.
The term "transfection" as used herein refers to the introduction of foreign
DNA into eukaryotic cells. Transfection may be accomplished by a variety of
means
known to the art including calcium phosphate-DNA co-precipitation, DEAE-
dextran-
mediated transfection, polybrene-mediated transfection, electroporation,
microinjection, liposome fusion, lipofection, protoplast fusion, retroviral
infection,
and biolistics.
The term "stable transfection" or "stably transfected" refers to the
introduction
and integration of foreign DNA into the genome of the transfected cell. The
term
"stable transfectant" refers to a cell that has stably integrated foreign DNA
into the
genomic DNA.
The teml "transient transfection" or "transiently transfected" refers to the
introduction of foreign DNA into a cell where the foreign DNA fails to
integrate into
the genome of the transfected cell. The foreign DNA persists in the nucleus of
the
transfected cell for several days. During this time the foreign DNA is subject
to the
regulatory controls that govern the expression of endogenous genes in the
chromosomes. The term "transient transfectant" refers to cells that have taken
up
foreign DNA but have failed to integrate this DNA.
As used herein, the term "selectable marker" refers to the use of a gene that
encodes an enzymatic activity that confers the ability to grow in medium
lacking what
would otherwise be an essential nutrient (e.g. the HIS3 gene in yeast cells);
in
addition, a selectable marker may confer resistance to an antibiotic or drug
upon the
cell in which the selectable marker is expressed. Selectable markers may be
"doininant"; a dominant selectable marker encodes an enzymatic activity that
can be
detected in any eukaryotic cell line. Examples of dominant selectable markers
include the bacterial aminoglycoside 3' phosphotransferase gene (also referred
to as
the neo gene) that confers resistance to the drug G418 in mammalian cells, the
bacterial hygromycin G phosphotransferase (hyg) gene that confers resistance
to the
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antibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyl
transferase
gene (also referred to as the gpt gene) that confers the ability to grow in
the presence
of mycophenolic acid. Other selectable markers are not dominant in that their
use
inust be in conjunction with a ce111ine that laclcs the relevant enzyme
activity.
Examples of non-dominant selectable inarkers include the tllymidine lcinase
(tk) gene
that is used in conjunction with tlc - cell lines, the CAD gene that is used
in
conjunction with CAD-deficient cells and the inainmalian hypoxanthine-guanine
phosphoribosyl transferase (hprt) gene that is used in conjunction with hprt -
cell
lines. A review of the use of selectable markers in mammalian cell lines is
provided
in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold
Spring Harbor Laboratory Press, New York (1989) pp.16.9-16.15.
As used herein, the term "cell culture" refers to any in vitro culture of
cells.
Included within this term are continuous cell lines (e.g., with an iinmortal
phenotype),
primary cell cultures, transformed cell lines, finite cell lines (e.g., non-
transformed
cells), and any other cell population maintained in vitro.
As used, the term "eukaryote" refers to organisms distinguishable fiom
"prokaryotes." It is intended that the term encompass all organisms with cells
that
exhibit the usual characteristics of eukaryotes, such as the presence of a
true nucleus
bounded by a nuclear membrane, within which lie the chromosomes, the presence
of
membrane-bound organelles, and other characteristics commonly observed in
eukaryotic organisms. Thus, the term includes, but is not limited to such
organisms as
fungi, protozoa, and animals (e.g., humans).
As used herein, the term "ifa vitro" refers to an artificial environment and
to
processes or reactions that occur within an artificial environment. In vitro
environments can consist of, but are not limited to, test tubes and cell
culture. The
term "in vivo" refers to the natural environment (e.g., an animal or a cell)
and to
processes or reaction that occur within a natural environment.
The terms "test compound" and "candidate compound" refer to any chemical
entity, pharmaceutical, drug, and the like that is a candidate for use to
treat or prevent
a disease, illness, sickness, or disorder of bodily function (e.g., cancer).
Test
compounds comprise both known and potential therapeutic compounds. A test
compound can be determined to be therapeutic by screening using the screening
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methods of the present invention. In some embodiments of the present
invention, test
coinpounds include antisense compounds.
As used herein, the term "sample" is used in its broadest sense. In one sense,
it is meant to include a specimen or culture obtained from any source, as well
as
biological and environmental sainples. Biological samples may be obtained from
aniinals (including humans) and encompass fluids, solids, tissues, and gases.
Biological salnples include blood products, such as plasma, serum and the
like.
Enviroiunental samples include environmental material such as surface matter,
soil,
water, crystals and industrial samples. Such examples are not however to be
construed as limiting the sample types applicable to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for cancer
diagnostics, including but not limited to, cancer markers. In particular, the
present
invention provides cancer markers and cancer marker profiles associated with
prostate
and breast cancers. Accordingly, the present invention provides method of
characterizing prostate and breast tissues, kits for the detection of markers,
as well as
drug screening and therapeutic applications.
I. Cancer Markers
The present invention provides markers whose expression is specifically
altered in cancerous prostate and breast tissues. Such markers find use in the
diagnosis and characterization of prostate and breast cancer.
A. Identification of Markers
Experiments conducted during the course of development of the present
invention identified markers with altered expression levels in prostate cancer
relative
to normal prostate or in metastatic prostate cancer relative to local prostate
cancer.
Exemplary markers are described in the Figure and Tables herein. In some
preferred
embodiments, prostate cancer markers include, but are not limited to, E2
ubiquitin
ligase, UBc9, the cytosolic phosphoprotein stathmin, the death receptor DR3,
the
Aurora A kinase (STK15), KRIP1 (KAP-1), Dynamin, CDK7, LAP2, Myosin VI,
ICBP90, ILP/XIAP, CamKK, JAM1, PICIn, or p23.
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Further experiments conducted during the course of development of the
present invention identified breast cancer markers. Exemplary marlcers
include, but
are not limited to, CamIU' , Myosin VI, Auroara A, exportin, BM28, CDK7,
TIP60,
or 16 INK 4a.
B. Detection of Markers
In some embodiments, the present invention provides methods for detection of
expression of cancer markers (e.g., prostate or breast cancer marlcers). In
preferred
embodiments, expression is measured directly (e.g., at the RNA or protein
level). In
some embodiments, expression is detected in tissue sainples (e.g., biopsy
tissue). In
other embodiments, expression is detected in bodily fluids (e.g., including
but not
limited to, plasma, serum, whole blood, mucus, and urine). The present
invention
further provides panels and kits for the detection of markers. In preferred
embodiments, the presence of a cancer marker is used to provide a prognosis to
a
subject. The information provided is also used to direct the course of
treatment. For
exainple, if a subject is found to have a marker indicative of a highly
metastasizing
tumor, additional therapies (e.g., hormonal or radiation therapies) can be
started at a
earlier point when they are more likely to be effective (e.g., before
metastasis). In
addition, if a subject is found to have a tumor that is not responsive to
hormonal
therapy, the expense and inconvenience of such therapies can be avoided.
The present invention is not limited to the markers described above. Any
suitable marker that correlates with cancer or the progression of cancer may
be
utilized, including but not limited to, those described in the illustrative
examples
below. Additional markers are also contemplated to be within the scope of the
present invention. Any suitable method may be utilized to identify and
characterize
cancer markers suitable for use in the methods of the present invention,
including but
not limited to, those described in illustrative Examples below. For example,
in some
embodiments, markers identified as being up or down-regulated in PCA using the
gene expression microarray methods of the present invention are furtlier
characterized
using tissue microarray, immunohistochemistry, Northern blot analysis, siRNA
or
antisense RNA inhibition, mutation analysis, investigation of expression with
clinical
outcome, as well as other methods disclosed herein.
In some embodiments, the present invention provides a panel for the analysis
of a plurality of markers. The panel allows for the simultaneous analysis of
multiple
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marlcers correlating with carcinogenesis and/or metastasis. For example, a
panel may
include markers identified as correlating with cancerous tissue, inetastatic
cancer,
localized cancer that is likely to metastasize, pre-cancerous tissue that is
likely to
become cancerous, and pre-cancerous tissue that is not likely to become
cancerous.
Depending on the subject, panels may be analyzed alone or in combination in
order to
provide the best possible diagnosis and prognosis. Markers for inclusion on a
panel
are selected by screening for their predictive value using any suitable
method,
including but not limited to, those described in the illustrative examples
below.
In other embodiments, the present invention provides an expression profile
map
comprising expression profiles of cancers of various stages or prognoses
(e.g.,
lilcelihood of future metastasis). Such maps can be used for comparison with
patient
samples. Any suitable method may be utilized, including but not limited to, by
computer comparison of digitized data. The comparison data is used to provide
diagnoses and/or prognoses to patients.
1. Detection of RNA
In some preferred embodiments, detection of prostate or breast cancer markers
(e.g., including but not limited to, those disclosed herein) is detected by
measuring the
expression of corresponding mRNA in a tissue sample (e.g., prostate tissue).
mRNA
expression may be measured by any suitable method, including but not limited
to,
those disclosed below.
In some embodiments, RNA is detection by Northern blot analysis. Northern
blot analysis involves the separation of RNA and hybridization of a
complementary
labeled probe.
In still further embodiments, RNA (or corresponding cDNA) is detected by
hybridization to a oligonucleotide probe). A variety of hybridization assays
using a
variety of technologies for hybridization and detection are available. For
example, in
some embodiments, TaqMan assay (PE Biosystems, Foster City, CA; See e.g., U.S.
Patent Nos. 5,962,233 and 5,538,848, each of which is herein incorporated by
reference) is utilized. The assay is performed during a PCR reaction. The
TaqMan
assay exploits the 5'-3' exonuclease activity of the AMPLITAQ GOLD DNA
polymerase. A probe consisting of an oligonucleotide with a 5'-reporter dye
(e.g., a
fluorescent dye) and a 3'-quencher dye is included in the PCR reaction. During
PCR,
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if the probe is bound to its target, the 5'-3' nucleolytic activity of the
AMPLITAQ
GOLD polymerase cleaves the probe between the reporter and the quencher dye.
The
separation of the reporter dye from the quencher dye results in an increase of
fluorescence. The signal accumulates with each cycle of PCR and can be
monitored
with a fluorimeter.
In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used to
detect the expression of RNA. In RT-PCR, RNA is enzymatically converted to
complementary DNA or "cDNA" using a reverse transcriptase enzyme. The cDNA is
then used as a template for a PCR reaction. PCR products can be detected by
any
suitable method, including but not limited to, gel electrophoresis and
staining with a
DNA specific stain or hybridization to a labeled probe. In some embodiments,
the
quantitative reverse transcriptase PCR with standardized mixtures of
competitive
templates method described in U.S. Patents 5,639,606, 5,643,765, and 5,876,978
(each of which is herein incorporated by reference) is utilized.
2. Detection of Protein
In other embodiments, gene expression of cancer markers is detected by
measuring the expression of the corresponding protein or polypeptide. Protein
expression may be detected by any suitable method. In some embodiments,
proteins
are detected by immunohistochemistry. In other embodiments, proteins are
detected
by their binding to an antibody raised against the protein. The generation of
antibodies is described below.
Antibody binding is detected by techniques known in the art (e.g.,
radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich"
immunoassays, iininunoradiometric assays, gel diffusion precipitation
reactions,
immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold,
enzyrne or
radioisotope labels, for example), Western blots, precipitation reactions,
agglutination
assays (e.g., gel agglutination assays, hemagglutination assays, etc.),
complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.
In one embodiment, antibody binding is detected by detecting a label on the
primary antibody. In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary antibody.
In a
further embodiment, the secondary antibody is labeled. Many methods are known
in
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the art for detecting binding in an immunoassay and are within the scope of
the
present invention.
In some einbodiinents, an automated detection assay is utilized. Methods for
the automation of iinmunoassays include those described in U.S. Patents
5,885,530,
4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by
reference. In some embodiinents, the analysis and presentation of results is
also
automated. For example, in some embodiinents, software that generates a
prognosis
based on the presence or absence of a series of proteins corresponding to
cancer
markers is utilized.
In other embodiments, the immunoassay described in U.S. Patents 5,599,677
and
5,672,480; each of which is herein incorporated by reference.
3. Data Analysis
In some enlbodiments, a coniputer-based analysis prograin is used to translate
the raw data generated by the detection assay (e.g., the presence, absence, or
amount
of a given marker or markers) into data of predictive value for a clinician.
The
clinician can access the predictive data using any suitable means. Thus, in
some
preferred embodiments, the present invention provides the further benefit that
the
clinician, who is not likely to be trained in genetics or molecular biology,
need not
understand the raw data. The data is presented directly to the clinician in
its most
useful form. The clinician is then able to immediately utilize the information
in order
to optimize the care of the subject.
The present invention contemplates any method capable of receiving,
processing, and transmitting the information to and from laboratories
conducting the
assays, information provides, medical personal, and subjects. For example, in
soine
embodiments of the present invention, a sample (e.g., a biopsy or a serum or
urine
sample) is obtained from a subject and submitted to a profiling service (e.g.,
clinical
lab at a medical facility, genomic profiling business, etc.), located in any
part of the
world (e.g., in a country different than the country where the subject resides
or where
the information is ultimately used) to generate raw data. Where the sample
comprises
a tissue or other biological sample, the subject may visit a medical center to
have the
sample obtained and sent to the profiling center, or subjects may collect the
sample
themselves (e.g., a urine sample) and directly send it to a profiling center.
Where the
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sample comprises previously determined biological information, the information
may
be directly sent to the profiling service by the subject (e.g., an infonnation
card
containing the information may be scanned by a computer and the data
transmitted to
a computer of the profiling center using an electronic coininunication
systems). Once
received by the profiling service, the sample is processed and a profile is
produced
(i.e., expression data), specific for the diagnostic or prognostic information
desired for
the subject.
The profile data is then prepared in a format suitable for interpretation by a
treating clinician. For example, rather than providing raw expression data,
the
prepared format may represent a diagnosis or risk assessment (e.g., likelihood
of
metastasis) for the subject, along with recominendations for particular
treatment
options. The data may be displayed to the clinician by any suitable method.
For
example, in some embodiments, the profiling service generates a report that
can be
printed for the clinician (e.g., at the point of care) or displayed to the
clinician on a
computer monitor.
In some embodiments, the information is first analyzed at the point of care or
at a regional facility. The raw data is then sent to a central processing
facility for
further analysis and/or to convert the raw data to information useful for a
clinician or
patient. The central processing facility provides the advantage of privacy
(all data is
stored in a central facility with uniform security protocols), speed, and
uniformity of
data analysis. The central processing facility can then control the fate of
the data
following treatment of the subject. For example, using an electronic
communication
system, the central facility can provide data to the clinician, the subject,
or
researchers.
In some embodiments, the subject is able to directly access the data using the
electronic communication system. The subject may chose further intervention or
counseling based on the results. In some embodiments, the data is used for
research
use. For example, the data may be used to further optimize the inclusion or
elimination of markers as useful indicators of a particular condition or stage
of
disease.
4. Kits
In yet other embodiments, the present invention provides kits for the
detection
and characterization of cancer (e.g. prostate or breast cancer). In some
embodiments,
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the lcits contain antibodies specific for a cancer marker, in addition to
detection
reagents and buffers. In other embodiinents, the kits contain reagents
specific for the
detection of mRNA or cDNA (e.g., oligonucleotide probes or primers). In
preferred
embodiments, the kits contain all of the components necessary to perform a
detection
assay, including all controls, directions for perfonning assays, and any
necessary
software for analysis and presentation of results.
5. In vivo Imaging
In some einbodiments, in vivo imaging techniques are used to visualize the
expression of cancer markers in an animal (e.g., a human or non-human mammal).
For example, in some embodiments, cancer marker mRNA or protein is labeled
using
an labeled antibody specific for the cancer marker. A specifically bound and
labeled
antibody can be detected in an individual using an in vivo imaging method,
including,
but not limited to, radionuclide imaging, positron emission tomography,
computerized
axial tomography, X-ray or magnetic resonance imaging method, fluorescence
detection, and chemiluminescent detection. Methods for generating antibodies
to the
cancer markers of the present invention are described below.
The in vivo imaging methods of the present invention are useful in the
diagnosis of cancers that express the cancer markers of the present invention
(e.g.,
prostate cancer). In vivo imaging is used to visualize the presence of a
marker
indicative of the cancer. Such techniques allow for diagnosis without the use
of an
unpleasant biopsy. The in vivo imaging methods of the present invention are
also
useful for providing prognoses to cancer patients. For example, the presence
of a
marker indicative of cancers likely to metastasize can be detected. The in
vivo
imaging methods of the present invention can further be used to detect
metastatic
cancers in other parts of the body.
In some embodiments, reagents (e.g., antibodies) specific for the cancer
markers of the present invention are fluorescently labeled. The labeled
antibodies are
introduced into a subject (e.g., orally or parenterally). Fluorescently
labeled
antibodies are detected using any suitable method (e.g., using the apparatus
described
in U.S. Patent 6,198,107, herein incorporated by reference).
In other embodiments, antibodies are radioactively labeled. The use of
antibodies for in vivo diagnosis is well known in the art. Sumerdon et al.,
(Nucl.
Med. Biol 17:247-254 [1990] have described an optimized antibody-chelator for
the
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radioiirununoscintographic imaging of tumors using Indium-111 as the label.
Griffin
et al., (J Clin Onc 9:631-640 [1991]) have described the use of this agent in
detecting
tuinors in patieiits suspected of having recurrent colorectal cancer. The use
of similar
agents with parainagnetic ions as labels for magnetic resonance imaging is
known in
the art (Lauffer, Magnetic Resonance in Medicine 22:339-342 [1991]). The label
used will depend on the imaging modality chosen. Radioactive labels such as
Indium-
111, Technetium-99in, or Iodine-131 can be used for planar scans or single
photon
emission computed tomography (SPECT). Positron emitting labels such as
Fluorine-
19 can also be used for positron emission tomography (PET). For MRI,
paramagnetic
ions such as Gadoliniuni (.III) or Manganese (II) can be used.
Radioactive metals with half-lives ranging from 1 hour to 3.5 days are
available for conjugation to antibodies, such as scandium-47 (3.5 days)
gallium-67
(2.8 days), gallium-68 (68 minutes), technetiium-99m (6 hours), and indium-111
(3.2
days), of which gallium-67, technetium-99m, and indium-111 are preferable for
gamma camera imaging, gallium-68 is preferable for positron emission
tomography.
A useful method of labeling antibodies with such radiometals is by means of a
bifunctional chelating agent, such as diethylenetriaminepentaacetic acid
(DTPA), as
described, for example, by Khaw et al. (Science 209:295 [1980]) for In-111 and
Tc-
99m, and by Scheinberg et al. (Science 215:1511 [1982]). Other chelating
agents
may also be used, but the 1-(p-carboxymethoxybenzyl)EDTA and the
carboxycarbonic anhydride of DTPA are advantageous because their use permits
conjugation without affecting the antibody's immunoreactivity substantially.
Another method for coupling DPTA to proteins is by use of the cyclic
anhydride of DTPA, as described by Hnatowich et al. (Int. J. Appl. Radiat.
Isot.
33:327 [1982]) for labeling of albumin with In-111, but which can be adapted
for
labeling of antibodies. A suitable method of labeling antibodies with Tc-99m
which
does not use chelation with DPTA is the pretinning method of Crockford et al.,
(U.S.
Pat. No. 4,323,546, herein incorporated by reference).
A preferred method of labeling immunoglobulins with Tc-99m is that
described by Wong et al. (Int. J. Appl. Radiat. Isot., 29:251 [1978]) for
plasma
protein, and recently applied successfully by Wong et al. (J. Nucl. Med.,
23:229
[1981]) for labeling antibodies.
In the case of the radiometals conjugated to the specific antibody, it is
likewise
desirable to introduce as high a proportion of the radiolabel as possible into
the
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antibody molecule without destroying its immunospecificity. A further
improveinent
may be achieved by effecting radiolabeling in the presence of the specific
cancer
marlcer of the present invention, to insure that the antigen binding site on
the antibody
will be protected. The antigen is separated after labeling.
Inn still further einbodiments, in vivo biophotonic imaging (Xenogen, Almeda,
CA) is utilized for in vivo imaging. This real-time in vivo imaging utilizes
luciferase.
The luciferase gene is incorporated into cells, inicroorganisms, and animals
(e.g., as a
fusion protein with a cancer marker of the present invention). When active, it
leads to
a reaction that emits light. A CCD camera and software is used to capture the
image
and analyze it.
II. Antibodies
The present invention provides isolated antibodies. In preferred embodiments,
the present invention provides monoclonal antibodies that specifically bind to
an
isolated polypeptide comprised of at least five amino acid residues of the
cancer
markers described herein. These antibodies find use in the diagnostic methods
described herein.
An antibody against a protein of the present invention may be any
monoclonal or polyclonal antibody, as long as it can recognize the protein.
Antibodies can be produced by using a protein of the present invention as the
antigen
according to a conventional antibody or antiserum preparation process.
The present invention contemplates the use of both monoclonal and polyclonal
antibodies. Any suitable method may be used to generate the antibodies used in
the
methods and compositions of the present invention, including but not limited
to, those
disclosed herein. For example, for preparation of a monoclonal antibody,
protein, as
such, or together with a suitable carrier or diluent is administered to an
animal (e.g., a
mammal) under conditions that permit the production of antibodies. For
enhancing
the antibody production capability, complete or incomplete Freund's adjuvant
may be
administered. Normally, the protein is administered once every 2 weeks to 6
weeks,
in total, about 2 times to about 10 times. Animals suitable for use in such
methods
include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice,
rats, sheep,
goats, etc.
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For preparing monoclonal antibody-producing cells, an individual animal
whose antibody titer has been confirmed (e.g., a mouse) is selected, and 2
days to 5
days after the final iirununization, its spleen or lymph node is harvested and
antibody-producing cells contained therein are fused with inyeloina cells to
prepare
the desired monoclonal antibody producer hybridoma. Measurement of the
antibody
titer in antiserum can be carried out, for example, by reacting the labeled
protein, as
described hereinafter and antisenun and then measuring the activity of the
labeling
agent bound to the antibody. The cell fusion can be carried out according to
known
metliods, for example, the method described by Koehler and Milstein (Nature
256:495
[1975]): As a fusion promoter, for example, polyethylene glycol (PEG) or
Sendai
virus (HVJ), preferably PEG is used.
Examples of myeloina cells include NS-1, P3U1, SP2/0, AP-1 and the like.
The proportion of the number of antibody producer cells (spleen cells) and the
number of myeloma cells to be used is preferably about 1:1 to about 20:1. PEG
(preferably PEG 1000-PEG 6000) is preferably added in concentration of about
10%
to about 80%. Cell fusion can be carried out efficiently by incubating a
mixture of
both cells at about 20 C to about 40 C, preferably about 30 C to about 37 C
for about
1 minute to 10 ininutes.
Various methods may be used for screening for a hybridoma producing the
antibody (e.g., against a tumor antigen or autoantibody of the present
invention). For
example, where a supernatant of the hybridoma is added to a solid phase (e.g.,
microplate) to which antibody is adsorbed directly or together with a carrier
and then
an anti-immunoglobulin antibody (if mouse cells are used in cell fusion, anti-
mouse
immunoglobulin antibody is used) or Protein A labeled with a radioactive
substance
or an enzyme is added to detect the monoclonal antibody against the protein
bound to
the solid phase. Alternately, a supernatant of the hybridoma is added to a
solid phase
to which an anti-immunoglobulin antibody or Protein A is adsorbed and then the
protein labeled with a radioactive substance or an enzyme is added to detect
the
monoclonal antibody against the protein bound to the solid phase.
Selection of the monoclonal antibody can be carried out according to any
known method or its modification. Normally, a medium for animal cells to which
HAT (hypoxanthine, aminopterin, thymidine) are added is employed. Any
selection
and growth medium can be employed as long as the hybridoma can grow. For
example, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20% fetal
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bovine serum, GIT medium containing 1% to 10% fetal bovine serum, a serum free
medium for cultivation of a hybridoma (SFM-1 01, Nissui Seiyaku) and the like
can be
used. Normally, the cultivation is carried out at 20 C to 40 C, preferably 37
C for
about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO2 gas.
The
antibody titer of the supernatant of a hybridoma culture can be measured
according to
the same manner as described above with respect to the antibody titer of the
anti-protein in the antiserum.
Separation and purification of a monoclonal antibody (e.g., against a cancer
marker of the present invention) can be carried out according to the same
manner as
those of conveiitional polyclonal antibodies such as separation and
purification of
immunoglobulins, for exainple, salting-out, alcoholic precipitation,
isoelectric point
precipitation, electrophoresis, adsorption and desorption with ion exchangers
(e.g.,
DEAE), ultracentrifugation, gel filtration, or a specific purification method
wherein
only an antibody is collected with an active adsorbent such as an antigen-
binding
solid phase, Protein A or Protein G and dissociating the binding to obtain the
antibody.
Polyclonal antibodies may be prepared by any known method or modifications
of these methods including obtaining antibodies from patients. For example, a
complex of an immunogen (an antigen against the protein) and a carrier protein
is
prepared and an animal is immunized by the complex according to the same
manner
as that described with respect to the above monoclonal antibody preparation. A
material containing the antibody against is recovered from the immunized
animal and
the antibody is separated and purified.
As to the complex of the immunogen and the carrier protein to be used for
immunization of an animal, any carrier protein and any mixing proportion of
the
carrier and a hapten can be employed as long as an antibody against the
hapten, which
is crosslinked on the carrier and used for immunization, is produced
efficiently. For
example, bovine serum albumin, bovine cycloglobulin, keyhole limpet
hemocyanin,
etc. may be coupled to an hapten in a weight ratio of about 0.1 part to about
20 parts,
preferably, about 1 part to about 5 parts per 1 part of the hapten.
In addition, various condensing agents can be used for coupling of a hapten
and a carrier. For example, glutaraldehyde, carbodiimide, maleimide activated
ester,
activated ester reagents containing thiol group or dithiopyridyl group, and
the like
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find use with the present invention. The condensation product as such or
together
with a suitable carrier or diluent is adininistered to a site of an animal
that permits the
antibody production. For enhancing the antibody production capability,
complete or
incoinplete Freund's adjuvant may be administered. Norinally, the protein is
administered once every 2 weeks to 6 weeks, in total, about 3 times to about
10 tiines.
The polyclonal antibody is recovered from blood, ascites and the like, of an
aniinal immunized by the above method. The antibody titer in the antiserum can
be
measured according to the same manner as that described above with respect to
the
supernatant of the hybridoma culture. Separation and purification of the
antibody can
be carried out according to the same separation and purification method of
immunoglobulin as that described with respect to the above monoclonal
antibody.
The protein used herein as the immunogen is not limited to any particular type
of immunogen. For example, a cancer marker of the present invention (fu.rther
including a gene having a nucleotide sequence partly altered) can be used as
the
immunogen. Further, fiagments of the protein may be used. Fragments may be
obtained by any methods including, but not limited to expressing a fragment of
the
gene, enzymatic processing of the protein, chemical synthesis, and the like.
III. Drug Screening
In some embodiments, the present invention provides drug screening assays
(e.g., to screen for anticancer drugs). The screening methods of the present
invention
utilize cancer markers identified using the methods of the present invention.
For
example, in some embodiments, the present invention provides methods of
screening
for compound that alter (e.g., increase or decrease) the expression of cancer
marker
genes. In soine embodiments, candidate compounds are antisense agents (e.g.,
oligonucleotides) directed against cancer markers. See below for a discussion
of
antisense therapy. Ihi other embodiments, candidate compounds are antibodies
that
specifically bind to a cancer marker of the present invention.
In one screening method, candidate compounds are evaluated for their ability
to alter cancer marker expression by contacting a compound with a cell
expressing a
cancer marker and then assaying for the effect of the candidate compounds on
expression. In some embodiments, the effect of candidate compounds on
expression
of a cancer marker gene is assayed for by detecting the level of cancer marker
mRNA
expressed by the cell. mRNA expression can be detected by any suitable method.
In
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other embodiments, the effect of candidate compounds on expression of cancer
marker genes is assayed by measuring the level of polypeptide encoded by the
cancer
markers. The level of polypeptide expressed can be measured using any suitable
method, including but not limited to, those disclosed herein.
Specifically, the present invention provides, screening methods for
identifying
modulators, i.e., candidate or test compounds or agents (e.g., proteins,
peptides,
peptidomiinetics, peptoids, small molecules or other drugs) which bind to
cancer
markers of the present invention, have an inhibitory (or stimulatory) effect
on, for
exainple, cancer marlcer expression or cancer markers activity, or have a
stimulatory
or inhibitory effect on, for example, the expression or activity of a cancer
marker
substrate. Compounds thus identified can be used to modulate the activity of
target
gene products (e.g., cancer marker genes) either directly or indirectly in a
therapeutic
protocol, to elaborate the biological function of the target gene product, or
to identify
compounds that disrupt normal target gene interactions. Compounds which
inhibit
the activity or expression of cancer markers are useful in the treatment of
proliferative
disorders, e.g., cancer, particularly metastatic (e.g., androgen independent)
prostate
cancer.
In one embodiment, the invention provides assays for screening candidate or
test compounds that are substrates of a cancer markers protein or polypeptide
or a
biologically active portion thereof. In another embodiment, the invention
provides
assays for screening candidate or test compounds that bind to or modulate the
activity
of a cancer marker protein or polypeptide or a biologically active portion
thereof.
The test compounds of the present invention can be obtained using any of the
numerous approaches in combinatorial library methods known in the art,
including
biological libraries; peptoid libraries (libraries of molecules having the
functionalities
of peptides, but with a novel, non-peptide backbone, which are resistant to
enzymatic
degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et
al.,
J. Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solid phase
or
solution phase libraries; synthetic library methods requiring deconvolution;
the 'one-
bead one-compound' library method; and synthetic library methods using
affinity
chromatography selection. The biological library and peptoid library
approaches are
preferred for use with peptide libraries, while the other four approaches are
applicable
to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam
(1997) Anticancer Drug Des. 12:145).
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Examples of methods for the synthesis of molecular libraries can be found in
the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909
[1993];
Erb et al., Proc. Nad. Acad. Sci. USA 91:11422 [1994]; Zuckennann et al., J.
Med.
Chem. 37:2678 [1994]; Cho et al., Science 261:1303 [1993]; Carrell et al.,
Angew.
Chem. Int. Ed. Engl. 33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed.
Engl.
33:2061 [1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].
Libraries of compounds may be presented in solution (e.g., Houghten,
Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84 [1991]),
chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores (U.S. Patent No.
5,223,409; herein incorporated by reference), plasmids (Cull et al., Proc.
Nad. Acad.
Sci. USA 89:18651869 [1992]) or on phage (Scott and Smith, Science 249:386-390
[1990]; Devlin Science 249:404-406 [1990]; Cwirla et al., Proc. Natl. Acad.
Sci.
87:6378-6382 [1990]; Felici, J. Mol. Biol. 222:301 [1991]).
In one embodiment, an assay is a cell-based assay in which a cell that
expresses a cancer marker protein or biologically active portion thereof is
contacted
with a test compound, and the ability of the test compound to the modulate
cancer
marker's activity is determined. Determining the ability of the test compound
to
,
modulate cancer marker activity can be accomplished by monitoring, for
example,
changes in enzymatic activity. The cell, for example, can be of mammalian
origin.
The ability of the test compound to modulate cancer marker binding to a
compound, e.g., a cancer marker substrate, can also be evaluated. This can be
accomplished, for example, by coupling the compound, e.g., the substrate, with
a
radioisotope or enzymatic label such that binding of the compound, e.g., the
substrate,
to a cancer marker can be determined by detecting the labeled compound, e.g.,
substrate, in a complex.
Alternatively, the cancer marker is coupled with a radioisotope or enzymatic
label to monitor the ability of a test compound to modulate cancer marker
binding to a
cancer markers substrate in a complex. For example, compounds (e.g.,
substrates) can
be labeled with 125I, 3sS 14C or 3H, either directly or indirectly, and the
radioisotope
detected by direct counting of radioemmission or by scintillation counting.
Alternatively, compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic
label
detected by determination of conversion of an appropriate substrate to
product.
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The ability of a compound (e.g., a cancer marlcer substrate) to interact with
a
cancer marker with or without the labeling of any of the interactants can be
evaluated.
For exainple, a inicrophysiorneter can be used to detect the interaction of a
compound
with a cancer marker without the labeling of either the coinpound or the
cancer
marlcer (McConnell et al. Science 257:1906-1912 [1992]). As used herein, a
"microphysiometer" (e.g., Cytosensor) is an analytical instruinent that
measures the
rate at which a cell acidifies its environment using a light-addressable
potentiometric
sensor (LAPS). Changes in this acidification rate can be used as an indicator
of the
interaction between a compound and cancer markers.
In yet another embodiinent, a cell-free assay is provided in which a cancer
marker protein or biologically active portion thereof is contacted with a test
compound and the ability of the test compound to bind to the cancer marker
protein or
biologically active portion thereof is evaluated. Preferred biologically
active portions
of the cancer markers proteins to be used in assays of the present invention
include
fragments that participate in interactions with substrates or other proteins,
e.g.,
fragments with high surface probability scores.
Cell-free assays involve preparing a reaction mixture of the target gene
protein
and the test compound under conditions and for a time sufficient to allow the
two
components to interact and bind, thus forming a complex that can be removed
and/or
detected.
The interaction between two molecules can also be detected, e.g., using
fluorescence energy transfer (FRET) (see, for example, Lakowicz et al., U.S.
Patent
No. 5,631,169; Stavrianopoulos et al., U.S. Patent No. 4,968,103; each of
which is
herein incorporated by reference). A fluorophore label is selected such that a
first
donor molecule's emitted fluorescent energy will be absorbed by a fluorescent
label
on a second, 'acceptor' molecule, which in turn is able to fluoresce due to
the absorbed
energy.
Alternately, the'donor' protein molecule may simply utilize the natural
fluorescent energy of tryptophan residues. Labels are chosen that emit
different
wavelengths of light, such that the 'acceptor' molecule label may be
differentiated
from that of the'donor'. Since the efficiency of energy transfer between the
labels is
related to the distance separating the molecules, the spatial relationship
between the
molecules can be assessed. In a situation in which binding occurs between the
molecules, the fluorescent emission of the 'acceptor' molecule label in 15 the
assay
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should be maximal. An FRET binding event can be conveniently measured through
standard fluorometric detection means well known in the art (e.g., using a
fluorimeter).
In another einbodiment, determining the ability of the cancer marker protein
to
bind to a target molecule can be accoinplished using real-time Biomolecular
Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, Anal. Chem.
63:2338-2345 [1991] and Szabo etal. Curr. Opin. Struct. Biol. 5:699-705
[1995]).
"Surface plasmon resonance" or "BIA" detects biospecific interactions in real
time,
without labeling any of the interactants (e.g., BlAcore). Changes in the mass
at the
binding surface (indicative of a binding event) result in alterations of the
refractive
index of light near the surface (the optical phenomenon of surface plasmon
resonance
(SPR)), resulting in a detectable signal that can be used as an indication of
real-time
reactions between biological molecules.
In one embodiment, the target gene product or the test substance is anchored
onto a solid phase. The target gene product/test compound complexes anchored
on
the solid phase can be detected at the end of the reaction. Preferably, the
target gene
product can be anchored onto a solid surface, and the test compound, (which is
not
anchored), can be labeled, either directly or indirectly, with detectable
labels
discussed herein.
It may be desirable to immobilize cancer markers, an anti-cancer marker
antibody or its target molecule to facilitate separation of complexed from non-
coinplexed forms of one or both of the proteins, as well as to accommodate
automation of the assay. Binding of a test compound to a cancer marker
protein, or
interaction of a cancer marker protein with a target molecule in the presence
and
absence of a candidate compound, can be accomplished in any vessel suitable
for
containing the reactants. Examples of such vessels include microtiter plates,
test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be
provided which adds a domain that allows one or both of the proteins to be
bound to a
matrix. For example, glutathione-S-transferase-cancer marker fusion proteins
or
glutathione-S-transferase/target fusion proteins can be adsorbed onto
glutathione
Sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-derivatized
microtiter plates, which are then combined with the test compound or the test
compound and either the non-adsorbed target protein or cancer marker protein,
and
the mixture incubated under conditions conducive for complex formation (e.g.,
at
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physiological conditions for salt and pH). Following incubation, the beads or
inicrotiter plate wells are washed to reinove any unbound components, the
matrix
iininobilized in the case of beads, complex determined either directly or
indirectly, for
example, as described above.
Alternatively, the complexes can be dissociated from the matrix, and the level
of cancer marlcers binding or activity determined using standard techniques.
Other
techniques for immobilizing either cancer markers protein or a target molecule
on
matrices include using conjugation of biotin and streptavidin. Biotinylated
cancer
marker protein or target molecules can be prepared from biotin-NHS (N-hydroxy-
succinimide) using techniques known in the art (e.g., biotinylation kit,
Pierce
Chemicals, Rockford, EL), and iinmobilized in the wells of streptavidin-coated
96
well plates (Pierce Chemical).
In order to conduct the assay, the non-immobilized component is added to the
coated surface containing the anchored component. After the reaction is
complete,
unreacted components are removed (e.g., by washing) under conditions such that
any
complexes formed will remain inimobilized on the solid surface. The detection
of
complexes anchored on the solid surface can be accomplished in a number of
ways.
Where the previously non-immobilized component is pre-labeled, the detection
of
label immobilized on the surface indicates that complexes were formed. Where
the
previously non-immobilized component is not pre-labeled, an indirect label can
be
used to detect complexes anchored on the surface; e.g., using a labeled
antibody
specific for the immobilized component (the antibody, in turn, can be directly
labeled
or indirectly labeled with, e.g., a labeled anti-IgG antibody).
This assay is performed utilizing antibodies reactive with cancer marker
protein or target molecules but which do not interfere with binding of the
cancer
markers protein to its target molecule. Such antibodies can be derivatized to
the wells
of the plate, and unbound target or cancer markers protein trapped in the
wells by
antibody conjugation. Methods for detecting such complexes, in addition to
those
described above for the GST-immobilized complexes, include immunodetection of
complexes using antibodies reactive with the cancer marker protein or target
molecule, as well as enzyme-linked assays which rely on detecting an enzymatic
activity associated with the cancer marker protein or target molecule.
Alternatively, cell free assays can be conducted in a liquid phase. In such an
assay, the reaction products are separated from unreacted components, by any
of a
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number of standard techniques, including, but not limited to: differential
centrifugation (see, for exainple, Rivas and Minton, Trends Biochem Sci 18:284-
7
[1993]); chromatography (gel filtration chromatograpliy, ion-exchange
chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current
Protocols in
Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see,
for
exainple, Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J.
Wiley:
New York). Such resins and chromatographic techniques are lcnown to one
skilled in
the art (See e.g., Heegaard J. Mol. Recognit 11:141-8 [1998]; Hageand Tweed J.
Chromatogr. Biomed. Sci. App1 699:499-525 [1997]). Further, fluorescence
energy
transfer may also be conveniently utilized, as described herein, to detect
binding
without further purification of the complex from solution.
The assay can include contacting the cancer markers protein or biologically
active portion thereof with a known compound that binds the cancer marker to
form
an assay mixture, contacting the assay mixture with a test compound, and
determining
the ability of the test compound to interact with a cancer marker protein,
wherein
determining the ability of the test compound to interact with a cancer marker
protein
includes determining the ability of the test compound to preferentially bind
to cancer
markers or biologically active portion thereof, or to modulate the activity of
a target
molecule, as coinpared to the known compound.
To the extent that cancer markers can, in vivo, interact with one or more
cellular or extracellular macromolecules, such as proteins, inhibitors of such
an
interaction are useful. A hoinogeneous assay can be used can be used to
identify
inhibitors.
For example, a preformed complex of the target gene product and the
interactive cellular or extracellular binding partner product is prepared such
that either
the target gene products or their binding partners are labeled, but the signal
generated
by the label is quenched due to complex formation (see, e.g., U.S. Patent No.
4,109,496, herein incorporated by reference, that utilizes this approach for
immunoassays). The addition of a test substance that competes with and
displaces
one of the species from the preformed complex will result in the generation of
a signal
above background. In this way, test substances that disrupt target gene
product-
binding partner interaction can be identified. Alternatively, cancer markers
protein
can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay
(see, e.g.,
U.S. Patent No. 5,283,317; Zervos et al., Cell 72:223-232 [1993]; Madura et
al., J.
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Biol. Chem. 268.12046-12054 [1993]; Bartel et al., Biotechniques 14:920-924
[1993];
Iwabuchi et al., Oncogene 8:1693-1696 [1993]; and Brent WO 94/10300; each of
which is herein incorporated by reference), to identify other proteins, that
bind to or
interact with cancer markers ("cancer marker-binding proteins" or "cancer
marker-
bp") and are involved in cancer marker activity. Such cancer marlcer-bps can
be
activators or inhibitors of signals by the cancer marker proteins or targets
as, for
example, downstream eleinents of a cancer markers-mediated signaling pathway.
Modulators of cancer markers expression can also be identified. For exanple,
a cell or cell free mixture is contacted with a candidate compound and the
expression
of cancer marker mRNA or protein evaluated relative to the level of expression
of
cancer marker mRNA or protein in the absence of the candidate compound. When
expression of cancer marker mRNA or protein is greater in the presence of the
candidate compound than in its absence, the candidate compound is identified
as a
stimulator of cancer marker mRNA or protein expression. Alternatively, when
expression of cancer marker mRNA or protein is less (i.e., statistically
significantly
less) in the presence of the candidate compound than in its absence, the
candidate
compound is identified as an inhibitor of cancer marker mRNA or protein
expression.
The level of cancer markers mRNA or protein expression can be determined by
methods described herein for detecting cancer markers mRNA or protein.
26 A modulating agent can be identified using a cell-based or a cell free
assay,
and the ability of the agent to modulate the activity of a cancer markers
protein can be
confirmed in vivo, e.g., in an animal such as an animal model for a disease
(e.g., an
-animal with prostate cancer or metastatic prostate cancer; or an animal
harboring a
xenograft of a prostate cancer from an animal (e.g., human) or cells from a
cancer
resulting from metastasis of a prostate cancer (e.g., to a lymph node, bone,
or liver),
or cells from a prostate cancer cell line.
This invention further pertains to novel agents identified by the above-
described screening assays (See e.g., below description of cancer therapies).
Accordingly, it is within the scope of this invention to further use an agent
identified
as described herein (e.g., a cancer marker modulating agent, an antisense
cancer
marker nucleic acid molecule, a siRNA molecule, a cancer marker specific
antibody,
or a cancer marker-binding partner) in an appropriate animal model (such as
those
described herein) to determine the efficacy, toxicity, side effects, or
mechanism of
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action, of treatment with such an agent. Furthennore, novel agents identified
by the
above-described screening assays can be, e.g., used for treatments as
described herein.
IV. Transgenic Animals Expressing Cancer Marker Genes
The present invention contemplates the generation of transgenic animals
colnprising an exogenous cancer inarlcer gene of the present invention or
mutants and
variants thereof (e.g., truncations or single nucleotide polymorphisms). In
preferred
embodiments, the transgenic animal displays an altered phenotype (e.g.,
increased or
decreased presence of markers) as compared to wild-type animals. Methods for
analyzing the presence or absence of such phenotypes include but are not
limited to,
those disclosed herein. In some preferred embodiments, the transgenic animals
further display an increased or decreased growth of tumors or evidence of
cancer.
The transgenic animals of the present invention find use in drug (e.g., cancer
therapy) screens. In some embodiments, test compounds (e.g., a drug that is
suspected of being useful to treat cancer) and control compounds (e.g., a
placebo) are
administered to the transgenic animals and the control animals and the effects
evaluated.
The transgenic animals can be generated via a variety of methods. In some
embodiments, embryonal cells at various developmental stages are used to
introduce
transgenes for the production of transgenic animals. Different methods are
used
depending on the stage of development of the embryonal cell. The zygote is the
best
target for micro-injection. In the mouse, the inale pronucleus reaches the
size of
approximately 20 micrometers in diameter that allows reproducible injection of
1-2
picoliters (pl) of DNA solution. The use of zygotes as a target for gene
transfer has a
major advantage in that in most cases the injected DNA will be incorporated
into the
host geiiome before the first cleavage (Brinster et al., Proc. Natl. Acad.
Sci. USA
82:4438-4442 [1985]). As a consequence, all cells of the transgenic non-human
animal will carry the incorporated transgene. This will in general also be
reflected in
the efficient transmission of the transgene to offspring of the founder since
50% of the
germ cells will harbor the transgene. U.S. Patent No. 4,873,191 describes a
method
for the micro-injection of zygotes; the disclosure of this patent is
incorporated herein
in its entirety.
In other embodiments, retroviral infection is used to introduce transgenes
into
a non-human animal. In some embodiments, the retroviral vector is utilized to
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transfect oocytes by injecting the retroviral vector into the perivitelline
space of the
oocyte (U.S. Pat. No. 6,080,912, incorporated herein by reference). In other
einbodilnents, the developing non-human embryo can be cultured in vitro to the
blastocyst stage. During this time, the blastomeres caii be targets for
retroviral
infection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]). Efficient
infection
of the blastomeres is obtained by enzymatic treatment to remove the zona
pellucida
(Hogan et al., in Manipulating the Mouse Enabyyo, Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, N.Y. [1986]). The viral vector system used to
introduce
the transgene is typically a replication-defective retrovirus carrying the
transgene
(Jahner et al., Proc. Natl. Acad Sci. USA 82:6927 [1985]). Transfection is
easily and
efficiently obtained by culturing the blastomeres on a monolayer of virus-
producing
cells (Stewart, et al., EMBO J., 6:383 [1987]). Alternatively, infection can
be
performed at a later stage. Virus or virus-producing cells can be injected
into the
blastocoele (Jahner et al., Nature 298:623 [1982]). Most of the founders will
be
mosaic for the transgene since incorporation occurs only in a subset of cells
that forxn
the transgenic animal. Further, the founder may contain various retroviral
insertions
of the transgene at different positions in the genome that generally will
segregate in
the offspring. In addition, it is also possible to introduce transgenes into
the germline,
albeit with low efficiency, by intrauterine retroviral infection of the
midgestation
embryo (Jahner et al., supra [1982]). Additional means of using retroviruses
or
retroviral vectors to create transgenic animals known to the art involve the
micro-
injection of retroviral particles or mitomycin C-treated cells producing
retrovirus into
the perivitelline space of fertilized eggs or early embryos (PCT International
Application WO 90/08832 [1990], and Haskell and Bowen, Mol. Reprod. Dev.,
40:386 [1995]).
In other embodiments, the transgene is introduced into embryonic stem cells
and the transfected stem cells are utilized to form an embryo. ES cells are
obtained
by culturing pre-implantation embryos in vitro under appropriate conditions
(Evans et
al., Nature 292:154 [1981]; Bradley et al., Nature 309:255 [1984]; Gossler et
al.,
Proc. Acad. Sci. USA 83:9065 [1986]; and Robertson et al., Nature 322:445
[1986]).
Transgenes can be efficiently introduced into the ES cells by DNA transfection
by a
variety of methods known to the art including calcium phosphate co-
precipitation,
protoplast or spheroplast fusion, lipofection and DEAE-dextran-mediated
transfection. Transgenes may also be introduced into ES cells by retrovirus-
mediated
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transduction or by micro-injection. Such transfected ES cells can thereafter
colonize
an embryo following their introduction into the blastocoel of a blastocyst-
stage
embryo and contribute to the germ line of the resulting chimeric animal (for
review,
See, Jaenisch, Science 240:1468 [1988]). Prior to the introduction of
transfected ES
cells into the blastocoel, the transfected ES cells may be subjected to
various selection
protocols to enrich for ES cells which have integrated the transgene assuming
that the
transgene provides a means for such selection. Alternatively, the polymerase
chain
reaction may be used to screen for ES cells that have integrated the
transgene. This
techiiique obviates the need for growth of the transfected ES cells under
appropriate
selective conditions prior to transfer into the blastocoel.
In still other embodiments, homologous recombination is utilized to knock-out
gene function or create deletion mutants (e.g., truncation mutants). Methods
for
homologous recombination are described in U.S. Pat. No. 5,614,396,
incorporated
herein by reference.
EXPERIMENTAL
The following examples are provided in order to demonstrate and further
illustrate certain preferred embodiments and aspects of the present invention
and are
not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations
apply: N(normal); M (molar); mM (millimolar); M (micromolar); mol (moles);
mmol (millimoles); mol (micromoles); nmol (nanomoles); pmol (picomoles); g
(grams); mg (milligrams); g (micrograms); ng (nanograins);1 or L (liters); ml
(milliliters); l (microliters); cm (centimeters); nun (millimeters); m
(micrometers);
nm (nanometers); and C (degrees Centigrade).
Example 1
A. Experimental procedures
Higlz-thf oughput Imrnunoblot Analysis
Tissues utilized were from the radical prostatectomy series at the University
of
Michigan and from the Rapid Autopsy Program, which are both part of University
of
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Michigan Prostate Cancer Specialized Program of Research Excellence
(S.P.O.R.E.)
Tissue Core. Institutional Review Board approval was obtained to procure and
analyze the tissues used in this study. To develop the tissue extract pools
the
following frozen tissue blocks were identified: 5 each of benign prostate
tissues,
clinically localized prostate cancer (3 were Gleason pattern 3+3, and 1 each
of
Gleason 3+4 and 4+3), and hormone-refractory metastatic tissues (liver, lymph
node,
lung, dura and soft tissue metastasis) (Shah et al., 2004, Cancer Res 64, 9209-
9216).
Based on examination of the fiozen sections of each tissue block, specimens
were
grossly dissected maintaining at least 90% of the tissue of interest. Total
proteins
were extracted from each tissue by homogenizing samples in boiling lysis
buffer
(contains 10mM Tris-HC1 pH 7.5 containing 1% SDS and 100 molar sodium
orthovanadate). The protein concentrations were determined by using Biorad DC
(Detergent Compatible) protein assay kit (Biorad, Hercules, CA). Extracts from
each
of the 5 specimens were combined equally to establish a pool. One hundred
inicrograms of protein from each tissue extract pool was boiled in sample
buffer and
subjected to 4-15% preparative SDS-PAGE and transferred to PVDF (Amersham
Biosciences Coip, Piscataway, NJ). The membranes were incubated for 1 hour in
blocking buffer (Tris-buffered saline with 0.1 % Tween [TBS-T] and 5% nonfat
dry
milk).
Fifty-two antibodies and 4 control antibodies could be assessed in each
Miniblotter system (linmunetics, Cambridge, MA). Antibodies (n= 524) at
various
dilutions (60 L total volume in TBS-T and 5% milk were loaded in the
miniblotter
system and incubated with the membranes- for 2 hours. After washing three
times
with TBS-T buffer, the membranes were incubated with horseradish peroxidase-
linked secondary IgG antibody (mouse, rabbit or goat depending on the primary
antibody used) (Amersham Biosciences Corp, Piscataway, NJ) at 1:5000 for 2
hour at
room temperature. The signals were visualized with the ECL detection system
(Amersham Pharmacia biotech, Piscataway, NJ) and autoradiography.
To supplement the number of proteins analyzed, the same extracts were
analyzed using two commercial service providers, BD and Kinexus. Power blot
high-
throughput iinmunoblots were carried out by BD biosciences (San Diego, CA)
(Malakhov et al., 2003, J Biol Chem 278, 16608-16613). Briefly, samples were
separated on a 4-15% gradient SDS-polyacrylamide gel and transferred to
Immobilon-
P membrane (Millipore, Bedford, MA). After transfer, the membrane is dried and
re-
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wet in methanol. The membrane is then incubated for one hour with blocking
buffer
(LI-COR, Lincoln, Nebraslca USA) and is clamped with a western blotting
manifold
that provides 40 channels across the inembrane. In each channel, a complex
antibody
cocktail is added and allowed to hybridize for one hour at 37 C. The blot is
removed
froin the manifold, washed and hybridized for 30 minutes at 37 C witli
secondary goat
anti-mouse conjugated to Alexa680 fluorescent dye (Molecular Probes, Eugene,
OR).
The membrane was washed, dried and scanned using the Odyssey Infrared
Iinaging System (LI-COR, Lincoln, Nebraska USA). For phosphoptotein analyses
samples were prepared according to the instructions provided by Kinexus, Inc.
Signals from antibodies generating an iminunoreactive band at the expected
molecular
weight were evaluated visually and quantitated by densitometry or scanned
using the
Odyssey Infrared Imaging System (LI-COR). From the immunoreactive bands
assessed, visually qualified signals were selected for-further validation.
Visually
qualified proteins that were over-expressed were coded red and given a value
of 1,
under-expressed proteins were coded blue and set at a value of -1, and white
was used
for unchanged proteins.
ConventionalInzmunoblot Validation
Validation immunoblots for selected proteins in different functional classes
were carried out using 4-15% linear gradient SDS-PAGE gels. Tissue lysates
from 3
to 4 benign, 5 clinically localized and 5 metastatic prostate cancers were
separated on
a SDSPAGE and transferred to PVDF membrane. The immunoblot was carried out
using different antibodies and at specific dilutions.
Tissue Mics oarray Analysis (TMA)
A prostate cancer progression TMA composed of benign prostate tissue,
clinically localized prostate cancer, and hormone refractory metastatic
prostate cancer
was developed. These cases came from well fixed radical prostatectomy
specimens
as described previously (Rubin et al., 2002, Jasna 287, 1662-1670). Replicate
tissue
samples were placed in geographically distinct areas of the TMA in order to
evaluate
reproducibility within the same TMA based on location. Total 216 tissue
samples
were collected from 51 patients.
Pre-treatment conditions and incubation times were worked up for each
antibody optimizing signal to noise ratio. The TMA was soaked in xylene
overnight
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to remove adhesive tape used for its construction. Pre-treatments varied
depending on
the optimal conditions. Primary antibodies were incubated before washing.
Secondary antimouse or anti-rabbit antibodies avidin-conjugated were applied
before
washing. Enyzmatic reaction was completed using a strepavidin biotin detection
kit
(DalcoCytomation, Carpinteria, CA).
Protein expression was deterinined using a validated scoring method
(Dhanasekaran et al., 2001, Nature 412, 822-826; Rubin et al., 2002, supra;
Varambally et al., 2002, Nature 419, 624-629) where staining was evaluated for
intensity and the percentage of cells staining positive. Benign epithelial
glands and
prostate cancer cells were scored for staining intensity on a 4 tiered system
ranging
from negative to strong expression. An estimate of the number of cells
staining
positive over background was evaluated for each 0.6 mm core. In cases where
benign
tissue and cancer were present, only one or the other tissue type was
evaluated for
purposes of analysis.
Hierarchical clustering on samples and proteins was carried out after data
normalization. Measurements were averaged for duplicated samples in the same
patient, base 2 log-transformed, and each protein was normalized so that its
mean
across all of samples equaled zero and the variance was 1.
Integrative Molecular Afaalysis
To map the antibodies and their respective protein targets, the official gene
names were obtained from the NCBI Locuslink for antibody/protein lists. To
complement protein levels, transcriptome data was assembled from 8 publicly
available prostate cancer gene expression datasets (Dhanasekaran et al., 2001,
supra;
Lapointe et al., 2004, Proc Natl Acad Sci U S A 101, 811-816; LaTulippe et
al., 2002,
Cancer Res 62, 4499-4506; Luo et al., 2001, Cancer Res 61, 4683-4688; Luo et
al.,
2002b, Mol Carcinog 33, 25-35; Singh et al., 2002, Cancer Cell 1, 203-209;
Welsh et
al., 2001, Cancer Res
61, 5974-5978; Yu et al., 2004, J Clin Oncol 22, 2790-2799) and each probe was
mapped to Unigene Build #173 (Table S3). Expression values from multiple
clones
or probe sets mapping to the same Unigene Cluster ID were averaged. Each gene
in
each study was normalized across sainples so that the mean equaled zero and
the
standard deviation equaled to 1. Missing data was iinputed by the k-nearest
neighbors
(k=5) imputation approach (Troyanskaya et al., 2001, Bioinformatics 17, 520-
525).
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Eight prostate cancer profiling studies were included in the analysis of
clinically localized prostate cancer relative to benign prostate tissue, while
only 4
studies were included in the analysis of metastatic prostate cancer vs.
localized
prostate cancer due to the availability of metastatic samples in those
studies. Genes
that were only found in one=fourth of studies or less were excluded, leading
to 483
genes involved in the former analysis and 494 involved in the latter analysis.
A one-
sided permutation t-test was conducted per gene per study using the multtest
package
in R 2Ø A gene was considered differentially expressed if its p-value was
less than
0.05 without adjustment for multiple testing. An mRNA transcript alteration
was
considered "concordant" with a
proteomic alteration if a majority of the microarray profiling studies (at
least 50%)
showed the same qualitative differential (increased, decreased, or unchanged)
as the
highthroughput immunoblot approach. The gene/proteins were then assigned to
concordant and discordant groups based on this criterion.
Clinical Outcomes Analysis
Six different cancer profiling studies (Bhattacharjee et al., 2001, Proc Natl
Acad Sci U S A 98, 13790-13795; Freije et al., 2004, Cancer Res 64, 6503-6510;
Glinsky et al., 2004, J Clin Invest 113, 913-923; Huang et al., 2003, Lancet
361,
1590-1596; van't Veer et al., 2002, Nature 415, 530-536; Yu et al., 2004,
supra) were
used for evaluation of prognostic value of these concordant genes. Detailed
study
information is shown in Table 3. Average linkage hierarchical clustering using
an
uncentered correlation similarity metric was used to identify two main
clusters of
clinically localized prostate cancer samples based on the 44 concordant mRNA
transcripts that were qualitatively concordant with protein expression in the
Yu et al.
(Yu et al., 2004, supra) study (only 44 out of 50 of the concordant signature
were
assessed on these arrays). Kaplan-Meier suivival analysis of cluster-defined
subgroups was then conducted and the log-rank test was used to calculate the
statistical significance of difference between the two subgroups (SPSS 11.5).
High-
/low- risk labels were then assigned to each group. A permutation test was
performed
to evaluate the significance of this "lethal" concordant signature. 1000
random sets of
44 genes from the Yu et al. data set were selected and used to carry out 1000
independent clusterings of the primary prostate cancer samples. Each grouping
was
subjected each grouping to Kaplan-Meier survival analysis.
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To validate the prognostic association of the 44-gene concordant signature, an
independent (clinically localized) prostate cancer gene expression dataset
from
Glinslcy et al. (Glinsky et al., 2004, supra) was used. The Yu et al.
clustering
functioned as the "training set" to define high-/low-risk groups. Each patient
of the
Glinslcy et al. study was classified into one of the two groups based on k-
nearest
neighbor classification (k=3) using as the similarity metric the Pearson
correlation
coefficient in the space of the significant genes from the Yu et al. dataset.
Each "test"
sainple was then classified into high- / low-risk group based on which cluster
the
majority of the test patient's nearest neighbors belonged. Kaplan-Meier
survival
curves were plotted for the two groupings. This "lethal" signature was then
refined
by reducing the number of genes involved. By using Yu et al. study as a
training set,
the concordant genes were ranked by univariate cox model. Again, the
clustering
procedure was used to identify two clusters based on the top number of genes
(ranging from 5 to 44). The Glinsky et al. study was then used as a validation
set to
verify performance of the refined signature by k-nearest neighbors (k=3)
prediction
analysis.
The generality of this "lethal" signature was evaluated by using other solid
tumor datasets. The signature was applied to two breast cancer (Huang et al.,
2003,
supra)-(van't Veer et al., 2002, supra), one lung cancer (Bhattacharjee et
al., 2001,
supra) and one glioma (Freije et al., 2004, supra) gene expression study.
Clustering
was used to identify two main clusters for patients in each study and Kaplan-
Meier
survival analysis was conducted to evaluate the statistical significance of
differences
between survival curves.
Multivariable Analysis
A Cox proportional-hazards regression model was used to carry out the
multivariate analysis. The dichotomized values of the 44-gene lethal
signature,
preoperative PSA, Gleason sum score from prostatectomy specimens, preoperative
clinical stage, age, and status of surgical margins were included as
covariates. The
calculation was performed with the R 2.0 statistical package.
Pathway Analysis
To better understand the biological pathways at work in the concordant and
discordant signature, the association of these genes with gene sets defined by
Gene
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Ontology and Transfac analysis (Rhodes et al., 2005, Nat Genet 37, 579-583)
was
investigated. The overlap of the signature with each gene set was counted and
the
significance of the overlap was evaluated with Fisher's exact test.
B. Results and Discussion
In order to derive a first approximation of the prostate cancer proteome, high-
throughput immunoblot analysis was utilized. This method allowed for the
screening
of pooled tissue extracts for qualitative levels of hundreds of proteins (and
post-
translational modifications) using commercially available antibody reagents.
The
basic approach is illustrated in Figure lA. Extracts from five tissue
specimens of
benign prostate, clinically localized prostate cancer and metastatic prostate
cancer
from distinct patients were pooled. Each of the 3 pools of tissue extracts
were run on
preparative SDS-PAGE gels, transferred to PVDF, and incubated with 1484
antibodies using a miniblot apparatus. Figure 1B displays representative data
using
the high-throughput immunoblot approach. K-nown proteomics alterations in
prostate
cancer progression such as EZH2 (Varambally et al., 2002, Nature 419, 624-629)
and
AMACR (Jiang et al., 2001, Ain J Surg Pathol 25, 1397-1404; Luo et al., 2002a,
Cancer Res 62, 2220-2226; Rubin et al., 2002, supra) are highlighted in red
while
novel associations such as GSK-3beta and IRAKl are highlighted in green. To
further increase the number of proteins analyzed, an analogous high-throughput
immunoblot methodology provided by commercial services was utilized (See
Methods). Thus, in total 1484 antibodies against 1354 distinct proteins or
post-
translational modifications were assessed. Of these antibodies, 521 detected a
band of
the expected molecular weight in at least one of the pooled extracts.
Antibodies that
did not detect the correct molecular weight protein product may represent lack
of
antibody sensitivity (or poor quality antibody) or absence of protein
expression in
prostate tissues.
To validate that the proteomic alterations identified by this screen occur in
individual tissue extracts (as opposed to pooled extracts), 86 proteins were
analyzed
by conventional immunoblot analysis using 4-5 tissue extracts per class. In
order to
evaluate the proteomics alterations in situ, high-density tissue microarrays
were
utilzed.
As only a subset of the identified proteins have antibodies that are
compatible
with immunohistochemical analysis, a single tissue microarray containing 216
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specimens from 51 cases was stained using twenty of these IHC-compatible
antibodies. Representative tissue inicroarray elements are shown in Figure 2A.
Each
tissue microarray element was evaluated by a pathologist and scored for
staining
(scale of 1-4) as per cell type considered (e.g., epithelial, stromal etc...).
Using an in
situ technique such as evaluation by iininunohistochemistry allowed us to
distinguish
stromal versus epithelial expressed proteins. In general, proteins that
demonstrated a
decrease in expression in the metastatic tumors inost often were stromally
expressed
proteins. As the amount of stroma per unit area decreases with tumor
progression,
metastatic samples demonstrated a parallel decrease in protein expression of
paxillin
and ABP-280, among others. In order to visualize and cluster the tissue
microarray
data (Nielsen et al., 2003, Am J Pathol 163, 1449-1456), the qualitative
evaluations
were log transformed and normalized.
Similar to gene expression analyses (Eisen et al., 1998, Proc Natl Acad Sci U
S A 95, 14863-14868; Perou et al., 2000, Nature 406, 747-752), unsupervised
hierarchical clustering of the data revealed that the in situ protein levels
could be used
to accurately classify prostate samples as benign, clinically localized
prostate cancer,
or metastatic disease (Fig. 2B).
This high-throughput immunoblotting of prostate extracts led to the
identification of a several known and previously unknown proteomic alterations
in
prostate cancer.
The proteomic alterations identified fall into a range of functional taxonomy
including
kinases and phosphatases, cell growth and apoptosis proteins, chromatin
regulators,
proteases, and proteins involved in cell structure and motility. For example,
previous
studies have shown that the anti-apoptosis protein, XIAP (Krajewska et al.,
2003, Clin
Cancer Res 9, 4914-4925), the racemase AMACR (Jiang et al., 2001, supra; Luo
et
al., 2002a, supra; Rubin et al., 2002, supra) and the Polycomb Group protein
EZH2
(Varambally et al., 2002, supra) are dysregulated in prostate cancer
progression.
Novel associations (increases or decreases in protein expression) with
prostate cancer
progression identified by this screen include the E2 ubiquitin ligase
UBc9, the cytosolic phosphoprotein stathmin, the death receptor DR3, and the
Aurora
A kinase (STK15), among others.
Having amassed this compendium of proteomic alterations in prostate cancer
progression, the general concordance with the prostate cancer transcriptome
was
examined. An integrative model to incorporate qualitative proteomic
alterations as
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assessed by high-throughput irnmunoblotting (but applicable to other proteomic
technologies), with transcriptomic data derived from 8 prostate cancer gene
expression studies was developed (Fig. 3). As both the genomic and proteomic
approach involve analysis of grossly dissected tissues, this facilitates
molecular
comparisons to be made.
The high-throughput iinmunoblot analysis of benign prostate, clinically
localized
prostate cancer and metastatic disease yielded 521 proteins of the expected
molecular
weight.
Inununoreactive bands in each of the three tissue extracts were assessed and
comparisons were made between benign tissue and clinically localized prostate
cancer
(Fig. 3A) and between clinically localized prostate cancer and metastatic
disease (Fig.
3B). Visually qualified proteins that were over-expressed were coded red,
under-
expressed proteins were coded blue, and unchanged proteins were coded white.
Based on this analysis, 64 proteins were dysregulated in clinically localized
prostate
cancer relative to benign prostate tissue, while 156 proteins were
dysregulated
between metastatic disease relative to clinically localized prostate cancer.
The set of quantifiable proteins (n=521) was then mapped to the NCBI Locus
link database to identify each corresponding gene. Data for mRNA was extracted
for
these genes using 8 publicly available prostate cancer gene expression data
sets
(Dhanasekaran et al., 2001, supra; Lapointe et al., 2004, supra; LaTulippe et
al., 2002,
supra; Luo et al., 2001, Cancer Res 61, 4683-4688; Luo et al., 2002b, supra;
Singh et
al., 2002, supra; Welsh et al., 2001, supra; Yu et al., 2004, supra). Over 90%
of the
genes were represented in at least one microarray study allowing for
integrative
analysis to be performed. Eight of the prostate profiling studies made a
comparison
between clinically localized prostate cancer and benign tissue, while only
four of
these made a comparison between clinically localized disease and metastatic
disease.
Genes that can only be found in one-fourth of studies or less were excluded,
leading
to 483 genes involved in the former comparison and 494 involved in the latter
comparison. Since over and under-expressed genes were assessed separately, a
one-
sided t test was conducted per each gene per each profiling study (See
Methods). As
with the proteomic approach, comparisons between benign and clinically
localized
prostate cancer (Fig. 3A) and localized disease and metastatic disease (Fig.
3B) were
made. If an mRNA transcript was significantly over-expressed in a particular
study it
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was coded red, under-expressed transcripts were coded blue, and white was used
for
unchanged transcripts.
Figure 3 presents the integrative proteomic and genomic analysis of prostate
cancer progression. An mRNA transcript alteration was considered "concordant"
with a
proteomic alteration if a majority of the microarray profiling studies (at
least 50%)
showed the saine qualitative differential (increased, decreased, or unchanged)
as the
highthroughput inununoblot approach. According to these criteria, 290 (60.0%)
out
of 483 mRNA transcripts were concordant with protein levels in clinically
localized
prostate cancer relative to benign prostate tissue. Similarly, 293 (59.3%) out
of 494
mRNA transcripts were concordant with protein levels in metastatic prostate
cancer
relative to clinically localized disease. Thus, similar to studies done in
yeast (Griffin
et al., 2002, Mol Cell Proteomics 1, 323-333; Washburn et al., 2003, Proc Natl
Acad
Sci
U S A 100, 3107-3112), bacteria (Baliga et al., 2002, Proc Natl Acad Sci U S A
99,
14913-14918), and cell lines (Tian et al., 2004, Mol Cell Proteomics 3, 960-
969),
there was only weak concordance between protein and mRNA levels in prostate
cancer progression.
To further explore the poor concordance observed between protein and
metadata from transcriptomic analyses, the pooled samples were profiled as
well as
the individual samples that comprised the pools on Affymetrix HG-U133 plus 2
microarrays.
The same integrative analysis was carried out to examine the concordant
relationship
between the protein alterations observed in the pooled tissues by
immunoblotting and
transcript alterations observed in the corresponding pooled and individual
tissues.
The individual samples were included in order to calculate statistical
significance for
transcript alterations. Similar or even lower concordance was observed between
protein and transcript (61.91% concordance in clinically localized prostate
cancer
relative to benign prostate tissue, and 47.96 % for metastatic prostate cancer
relative
to clinically localized disease, Fig. 6A, Fig. l0A).
The protein and mRNA concordance in individual samples was also
investigated.
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The 86 proteins identified as outliers in the larger high-throughput screen
(see Fig 7)
were utilized. The immunoblot intensities were seini-quantitated and
correlation
coefficients were calculated for each protein (see Experiinental Procedures).
A total 55 out of 86 proteins were observed to a have a positive correlation
with
mRNA, which led to 64.0% concordance between proteins and transcripts (Fig.
6B).
On sub classification, a concordance of 54.7% and 66.3% in case of localized
prostate
cancer relative to benign prostate tissues and the metastatic disease relative
to
localized prostate cancer respectively was observed.
This proteoinic screen identified proteins that are altered from benign
prostate
to clinically localized prostate cancer and a distinct set of alterations
between
clinically localized disease to metastatic disease. The transition from
clinically
localized to metastatic disease was next investigated. As the metastatic
tissues
analyzed in this study are androgen-independent (Shah et al., 2004, Cancer Res
64,
9209-9216), and by contrast the clinically localized tumors are generally
androgen-
dependent, it was evaluated whether there was an enrichment of androgen-
regulated
proteomic alterations discovered by the screen. Androgen regulated genes
(ARGs) are
essential for the normal development of the prostate as well as the
pathogenesis of
prostate cancer (Culig et al., 1998, Prostate 35, 63-70; Koivisto et al.,
1998, Nat Med
4, 844-847; Mooradian et al., 1987, Endocr Rev 8, 1-28). Velasco et al.
developed a
meta-analysis of ARGs, which represents a cross-comparison of 4 gene
expression
(DePrimo et al., 2002, Genome Biol 3, RESEARCH0032; Nelson et al., 2002, Proc
Natl Acad Sci U S A 99, 11890-11895; Segawa et al., 2002, Oncogene 21, 8749-
8758; Velasco et al., 2004, Endocrinology 145,
3913-3924) and 2 SAGE datasets (Waghray et al., 2001, Proteomics 1, 1327-1338;
Xu et al., 2001, Int J Cancer 92, 322-328). ARGs were then defined as a union
of
these 6 datasets, all of which represented functional induction of mRNA
transcript by
androgen in vitro. 27 out of the 150 protein alterations (exclusive of post-
translational
modifications) identified as being differential between metastatic and
clinically
localized disease were designated as androgen-regulated by the Velasco et al
(Velasco
et al., 2004, supra) ARG compendium.
To demonstrate that this finding is statistically significant, random sets of
150 genes were selected from the Yu et al. (Yu et al., 2004, supra) or the
Glinsky et
al. (Glinsky et al., 2004, supra) prostate cancer profiling studies. It was
found that the
chance of selecting 27 ARGs was minimal (p< 0.0001 for the Yu et al. and
p<0.001
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for Glinslcy et al.). Thus, androgenregulated proteins are significantly
enriched in the
differential comparison between androgen-dependent and independent prostate
cancer. Out of the 156 proteomic alterations identified between metastatic and
localized prostate cancer, 50 were concordant with mRNA transcript and 90 were
discordant with mRNA transcript (Fig. 3B, left panel). Many of these proteomic
alterations were validated on individual tissue extracts to confirm the high-
throughput
immunoblot analysis (Fig. 3C). EZH2, a Polycomb group protein previously
characterized as being over-expressed in aggressive prostate and breast cancer
(Kleer
et al., 2003, Proc Natt Acad Sci U S A 100, 11606-11611; Varambally et al.,
2002,
supra) was one of the 50 proteins identified as being concordantly over-
expressed in
metastatic tissues at the mRNA and protein level (Fig. 3B and 3C). As EZH2 was
a
member of this 50 gene concordant signature, it was hypothesized that
proteomic
alterations that distinguish metastatic prostate cancer from clinically
localized disease
could serve as a multiplex "lethal" signature of prostate cancer progression
when
applied to clinically localized disease (i.e., "more aggressive" genes would
be
expressed in progressive prostate cancer). Prostate cancer gene expression
datasets
that monitored over 85% of the genes in the concordant genomic/proteomic
signature
were identified that included biochemical recurrence information (time to PSA
recurrence), as well as reported on a reasonable cohort of clinically
localized
specimens (n>50). The prostate cancer gene expression datasets that fulfilled
these
criteria were carried out by Yu et al. (Yu et al., 2004, supra) and Glinsky et
al.
(Glinsky et al., 2004, supra), both of which represent Affyrnetrix
oligonucleotide
datasets and each of which measured 44 out of the 50 genes in the concordant
signature. Prediction models were built with the Yu et al. data set and the
performance was tested on the Glinsky et al. data set. Utilizing an approach
described
earlier (Ramaswamy et al., 2003, Nat Genet 33, 49-54), unsupervised
hierarchical
clustering in the space of this 44-gene concordant signature resulted in two
main
clusters of individuals in the Yu et al. study (Fig. 4A). Kaplan-Meier (KM)
sui-vival
analysis of the clusters indicated that the two groups of individuals are
significantly
different based on time to recurrence status (P = 0.035, Fig. 4A). When the 90
discordant genes (mRNA transcripts that are not qualitatively concordant with
protein
levels) were used, it was found that these signatures did not generate a
clinical
outcome distinction (P= 0.238). By perxnutation test, it was observed that
random sets
of 44 genes did not generate such prognostic distinctions, indicating that the
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concordant signature could not be achieved by chance. To assess the validity
of this
concordant 44-gene signature, the Glinsky et al. study was used as an
independent test
set (Fig. 4B). Each of the samples in the Glinslcy dataset were classified as
high- or
low-risk based on a k-nearest neighbor (k-NN) model developed using the Yu et
al.
study as a traiiling set (k=3). Based on the class predictions derived from
the
concordant signature, F'_M survival analysis revealed a significant difference
in
survival based on the risk stratification (P = 0.001, Fig. 4B). This was not
the case
with the discordant signature when applied to the Glinsky et al. sample set
(P= 0.556).
Multivariate Cox proportional-hazards regression analysis of the risk of
recurrence
was carried out on the Glinsky et al. validation set. Table 1 shows that the
concordant
signature predicted recurrence independently of the other clinical parameters
such as
surgical margin status, Gleason sum, and pre-operative PSA. With an overall
hazard
ratio of 3.66 (95% CI: 1.36-7.02, P<0.001), it was by far the strongest
predictor of
prostate cancer recurrence in the model.
Next, the 44-gene concordant signature of prostate cancer progression was
refined by reducing the number of genes required. By using the Yu et al. study
as a
training set, the 44 concordant genes were ranked by a univariate cox model.
The
same clustering procedure was employed to identify two clusters based on the
top
number of genes ranging from a minimum of 5 to a maximum of 44. Based on this
iterative analysis, 9 genes were identified that demarcated two main clusters
that
differed most significantly by KM survival analysis (Fig. 4A). The Glinsky et
al.
study was again used as an independent validation set confirming that the 9-
gene
concordant signature identified two groups of individuals which differed
significantly
based on recurrence (Fig. 4B, Figure 8). Together, this integrative analysis
shows that
mRNA transcripts that correlate with protein levels in metastatic prostate
cancer can
be used as gene predictors of progression in clinically localized disease.
Next, the generality of the larger 44-gene concordant signature of
aggressiveness in other solid tumors was investigated. Four tumor profiling
datasets
from the Oncomine compendium (Rhodes et al., 2004, Neoplasia 6, 1-6) were
identified that fulfilled the same criteria that were used in the prostate
cancer analyses.
In 95 primaiybreast adenocarcinomas (van't Veer et al., 2002, Nature 415, 530-
536),
tumors bearing the 44-gene lethal proteomics signature were more likely to
progress
to metastasis than those lacking this signature (P =0.0025. A similar result
was
observed in 80 primary breast infiltrating ductal carcinomas (Huang et al.,
2003,
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Lancet 361, 1590-1596) (P = 0.002, Fig. 4C). This result was also observed in
a
series of 84 primary lung adenocarcinomas (Bhattacharjee et al., 2001, supra)
( P
0.03; Fig 4C) and 56 gliomas (Freije et al., 2004, Cancer Res 64, 6503-6510)
(P
=0.01; Fig. 4C). The smaller 9-gene model was only effective in discriminating
prognostic classes in the glioma study (P=0.016) but not in the other solid
tumors.
This shows that the 9-gene model is specific for prostate cancer while the 44-
gene
model has more universal applicability. It should be understood that subsets
of these
groups also find use, as well as groups that add, subtract, or substitute one
or more
martcers.
Taken together, the results of this example show that the lethal
proteomic/genomic signature identified by the integrative analysis of
metastatic
prostate cancer has utility in the prognostication of clinically localized
solid tumors in
general. While these proteomic alterations can serve as a multiplex biomarker
of
cancer aggressiveness, they may also shed light into the biology of neoplastic
progression. As proteins, rather than RNA transcripts, are the primary
effectors of the
cell, they play the central and most distal role in the functional pathways to
cancer.
EZH2, which was previously have shown to have a role in prostate cancer
progression
(Varambally et al., 2002, supra), is a member of this concordant
genomic/proteomics
signature. For example, this screen identified Aurora-A kinase (STKl5) as
being
overexpessed in metastatic prostate cancer as well as being a member of the 44-
gene
concordant signature. This serine-threonine kinase has been shown to be
amplified in
a number of human cancers (Jeng et al., 2004, Clin Cancer Res 10, 2065-2071;
Neben
et al., 2004, Cancer Res 64, 3103-3111), play a key role in G2/M cell cycle
progression (Hirota et al., 2003, Cell 114, 585-598), and inhibit p53
(Katayama et al.,
2004, Nat Genet 36, 55-62), among other functions. Another cancer regulatory
molecule in the 44-gene concordant signature was KRIP1 (KAP-1), which is known
to repress transcription via binding the methyltransferase SETDBI (Schultz et
al.,
2002, Genes Dev 16, 919-932).
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Table 1. Multivariable Proportianal.-Ha.zards Alialysi.s of the Risk of
Recurrence a.s A Fix=st ENFeut on the Glinsky et. al. Validation Set.
Variable Ilazarr~ Ratio
P ~t';jlue
(95% ~C'I)
High-Risk signatwe (irs. i.66 (1.77 - 7.59) <0.401
low-risk sigYia.tzlre )
PSA 1.04 (1.00 - 1.09) 0.043
Gleason Suin Score
Score >7 (vs. score <-7) 1.73 (0.79 - i.76) 0.17
Tuzi1or Stage
Stage T2 (vs. stage TI) 0.85 (0.42- 1.75) 0.67
Age 1.06 (1.00 --1.13) 0.06
Surgical Margins
Positive (vs. negative) 2.18 (0.92 - 5,18) 0.08
Table 2
Total Number of samples
Authoi-s Journal Array type genes
Benign Localized Metastatic
Dhanasekaran,
SM. et al. Nature,412:822 cDNA 9984 19 14 20
Cancer
Luo, J., et al. Researcla, cDNA 6500 9 16 0
61:4683
Lapointe, J., et PNAS., cDNA 19124 41 61 9
al. 101(3):811
Singh, D., et Cancer Cell, Affy HG-
1(2): 12626 50 52 0
al. 203, 2002 U95Av2
Welsh, JB., et Cancer Affy HG-
al. Researelz, U95A 12626 9 23 1
61:5974
Latulippe, E., Cancer
et al. Research, Affy HG-U95 62840 3 23 9
62:4499
Luo, JH., et a1. Mol. Carcinog., Affy HG- 12626 15 15 0
33(1):25 U95A
J. Clin. Oncol., Affy HG-
Yu, YP., et al. 22(14):2790 U95Av2, B, C 37690 23 66 25
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Table 3
Cancer Authors Journal # of Sample description
Type genes*
Yu YP. et J Clin. Oncol. 21 patients had recurrence and 39
Prostate al ' ' 22(14):2790 44 remained recurrence-free.
Glinsky, J. Clin. Invest. 37 patients with recurrent and 42
Prostate GV., 44 patients
et al. 113(6):913 with nonrecurrent disease
Glioma Freije, WA., Cancer Res. 49 38 patients were dead and 18 were alive
et al. 64(18):6503
Huang, E et Lancet. 34 patients had recurrence and 46
Breast al. 361(9369):1590 44 remained recurrence-free
45 patients advanced to inetastasis and
Van't Veer, Nature. 51 samples haven't develop distant
Breast LJ. et al. 415(6871):530 48 metastases after 5 years
Bhattacharjee PNAS. -
Lung A et al. 98(24):13 790 44 48 patients were dead and 36 were alive
*Due to different microarray platforms, some genes were missed in particular
studies.
Table 4
Androgen regulated genes among proteomic/genomic alterations between
metastatic prostate cancer
and localized prostate cancer
Segawa Velasco Deprimo Androgen-
et et et
Uiiigene ID Protein Gene Name al. al. Nelson et al. Xu et al Regulation*
Name al
Concordaiit Genes
Hs.10842 Ran RAN +
Hs.134106 Sekl MAP2K4
Hs.154103 Lim kinase LIM +
Hs.157367 Exportin XPO1 +
Hs.171280 ERAB HADH2 ~
Hs. 171952 Occludin OCLN
Hs.171995 PSA KLK3 +
Hs.234521 3PK MAPKAPK3
Hs.236030 BAF170 SMARCC2
Hs.256583 DRBP76 ILF3 4 +
Hs.298530 RAB27 RAB27A 4 +
Hs.388677 PAP ACPP
Hs.433612 KRIP-1 TRIM28
Hs.444118 MCM6 MCM6 4
Hs.446336 PAXILLIN PXN
Discordant Genes
Hs.101174 Tau-53kDMAPT Hs.15250 PECI PECI ~ +
Hs.162089 TPD52 TPD52 +
Hs.167 MAP2B MAP2
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Hs.184298 CDK7 CDK7 4 +
Hs.324473 ERK2 MAPK1
Hs.406013 Ms Cytokeratin KRTl8
Hs.408507 TFII-I GTF2I
Hs.418004 PTP I beta PTPN1 +
Hs.511397 MCAM MCAM
Hs.7557 FI{BP51 FKBP5 +
Hs.79037 HSP60 HSPDI +
*;"+" represents that the gene is androgen up-regulated; "-" represents that
the gene is androgen down-regulated.
Significance
were detennined by student t-test on our published dataset (Dhanasekaran SM et
al. FASEB J. 2005; 19(2);243-5).
Example 2
Description of Selected Proteomic Alterations Identified by this Study
This Example describes functional taxonomy of the proteomic alterations
characterized in Varambally et al, "Integrative Proteomic and Genomic
Alterations of
Prostate Cancer Progression." Results are shown in Table 5.
Table 5
Localized Metastatic
Altered Prostate Cancer Prostate Cancer Description
Proteins
RNA Protein RNA Protein
Kinases and Phosphatases
Aurora-A is a centrosome-associated oncogenic
lcinase that has been implicated in the control of
mitosis. Overexpression of Aurora-A has been
shown
to result in chromosomal aberration, genomic
instability and tumorigenesis. This kinase is
known to
STK15 be amplified in a number of human cancers and
+ U + + tumor cell lines (Jeng et al., 2004) (Neben et al.,
(Aurora-A) 2004). Aurora-A regulates the p53 pathway by
inducing increased degradation of p53, leading to
aberrant checkpoint responses and facilitating
oncogenic transformation of cells (Katayama et al.,
2004) and has been shown to play a key role in
G2/M
progression (Hirota et al., 2003).
GSK3 beta is a lcey regulator of signaling
patllways
that involves cellular responses to Wnt, receptor
tyrosine kinases, and G-protein-coupled receptors.
It
also plays a central role in a wide range of cellular
processes, such as glycogen metabolism, cell cycle
regulation and proliferation. The activity of GSK3
Glycogen beta is regulated by phosphorylation on
Synthase tyrosine/serine residues (Kim and Kimmel, 2000)
kinase + U U - (Doble and Woodgett, 2003) (Patel et al., 2004).
(GSK)
3 beta Studies have indicated that GSK-3 beta may
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function
as a repressor of AR-mediated transactivation and
cell growth (Mazor et al., 2004). It is a
downstream
substrate of PI3IQAkt and phosphorylation and
inactivation of GSK-3 beta results in the nuclear
accumulation of beta-catenin (Sharma et al.,
2002).
Beta-catenin is known to act as a coactivator of
AR.
Nuclear Pr-oteiris
Heterochrom Recruitment of mammalian HP 1 to a euchromatic
a
tin protein 1 U U U + promoter was shown to establish a silenced state
(HP1) alpha (Ayyanathan et al., 2003)
Brahma related group protein forms complexes
with
BRG1 + + + HP 1 alpha to induce a silenced state (Nielsen et
al.,
2002 .
KRAB-A interacting protein 1 (KRIP-1) represses
KRIP 1 U + + + transcription via binding to methyl transferase
SETDBI (Schultz et al., 2002)
ICBP90 ICBP90 binds to one of the inverted CCAAT
boxes of
(Inverted U U + + the topoisomerase II alpha (TopoII alpha) gene
CCAAT box promoter (Hopfiier et al., 2000) and has been
shown to
Binding be regulated by E2F-1 (Unoki et al., 2004) the
Protein of 90 functional significance of which is yet to be
kDa) understood. ICBP90 shares structural homology
with
several other proteins, including Np95, which is
known to function as core histones specific E3
ubiguitin li gase (Citterio et al., 2004)
BUB3 is a part of a large multi-protein kinetochore
BUB3 + + u + complex believed to be a lcey component of the
checkpoint regulatory pathway(Logarinho et al.,
2004).
BM28 along with other minichromosome
maintenance
proteins play an essential role in initiation and
BM28, regulation of eukaryotic DNA replication (Eward
minichromos et al., 2004). BM28 was previously reported to be
ome dysregulated in malignant prostate glands (Meng
maintenance U + + + et al., 2001). It has been proposed that BM28
2 (MCM2) expression is an independent predictor of disease-
free survival after definitive local therapy and
finds use as a
molecular marker for clinical outcome in prostate
cancer (Meng et al., 2001).
P16INK4a P16 INK4a strongly binds to cdlc4 and cdk6 to
(Cyclin- U + + + inhibit their ability to interact with cyclin D. It was
dependent shown to be upregulated in high-grade prostatic
epithelial neoplasia (Henshall et al., 2001) .
Loss of mismatch repair function leads to the
MSH2 accumulation of errors that normally occur during
mutS (E. + + + + DNA replication. Mismatch repair genes are
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coli) involved
homolog 2 in repairing these errors. MSH2 is shown to be
upregulated in prostate cancer (Velasco et al.,
2002)
Enzymes
Among various enzymes, characterized in cancer,
proteases remain the best studied group for their
role
in promoting and facilitating the spread of
MMP 19 - + - malignancy. MMP 19 has been shown to be
downregulated during transformation and
dedifferentiation of breast epithelia (Djonov et al.,
2001)
MMP23 U - - - See MMP19.
Cathepsin D, a carboxyl protease that has been
Cathepsin D U + U + implicated as an important factor in tumor cell
invasion, is known to be upregulated in prostate
cancer (Cherry et al., 1998).
SUMOylation of proteins which involves the
covalent
attachment of a SUMO (small ubiquitin-related
modifier) to substrate proteins is known to play a
key
role in protein targeting and/or stability(Lin et al.,
2003) (Hilgarth et al., 2004). The SUMO-1-
Ubc9 U + + + conjugating enzyme Ubc9 is lcnown to interact
with
androgen receptor(Poukka et al., 1999; Poukka et
al.,
2000). (AR), a ligand-activated transcription
factor,
belonging to the steroid receptor superfamily and
modulates its transcriptional regulatory
activity(Callewaert et al., 2004).
Structural proteins
Paxillin is a LIM domain containing protein. It is
Paxillin + U - - known to regulate the androgen receptor activity
by
binding and targeting the androgen receptor to the
nuclear matrix(Kasai et al., 2003).
LAP 2 beta U U + + Lamina-associated polypeptide (LAP) 2 beta is
known to function in initiation of DNA replication
Demantin is a cytoslceletal protein that bundles
actin filaments in a phosphorylation-dependent
Dematin U + manner. It also interacts with Ras-guanine
nucleotide exchange factor Ras-GRF2 and is
implicated in modulation of mitogen-activated
protein kinase pathways (Lutchman et al., 2002)
Dynamin is a cytosolic protein with a key role in
Dynamin U U U + clathrin-mediated endocytosis (Fournier et al.,
2003)
ABP280, an actin-binding cytoskeletal protein
promote orthogonal branching of actin filaments
and link actin filaments to membrane
glycoproteins (Loy et al., 2003). And It is known
to anchor various transmembrane proteins to the
actin cytoskeleton and serves as a scaffold for a
ABP280 wide range of cytoplasniic signaling proteins. Lilce
(filamin A) - - - - Paxillin, ABP280 FLNa interferesd with AR
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interdomain interactions and
competesd with the coactivator transcriptional
intermediary factor 2 to specifically down-regulate
AR function. Lilce Paxillin, ABP280 is also know
to
regulate the androgen receptor activity by
intefering with the interdomain interactions (Loy
et al., 2003).
Myosin VI plays a role in E-cadherin mediated
border cell migration(Geisbrecht and Montell,
Myosin VI + + - - 2002). It has been recently shown that Myosin VI
plays a major role in invasion in of ovarian cancer
(Yoshida et al., 2004).
Stathtnin also known as OP- 18 is a cytosolic
phosphoprotein proposed to act as a relay
integrating diverse cell signaling pathways,
Stathmin + U + + notably during the control of cell growth and
differentiation. It is one of the key regulators of
cell division known for its ability to destabilize
microtubules in a phosphorylation-dependent
manner (Curmi et al., 1999).
JAM-1 is localized at the tight junctions of
epithelial and endothelial cells and is involved in
the regulation of junctional integrity and
permeability. They are glycoproteins characterized
JAM-1 U U U + by two immunoglobulin folds (VH- and C2-type)
in the extracellular domain. This protein was
implicated in the regulation of tight junctions and
leulcocyte transmigration. This protein was
implicated in the regulation of tight junctions and
leukocyte transmigration (Mandell et al., 2004)
Apoptotic regulators
XIAP(X chromosome-linked inhibitor of apoptosis
protein) is a potent inhibitor of apoptosis. It is a
downstream target of Akt. Phosphorylation of
XIAP + + U + XIAP by Akt protects it from' degradation resulting
in increased cell survival. and undergoes
phosphorylation and stabilization.by protecting
from ubiquitination and degradation (Dan et al.,
2004)
AIF + U + Apoptosis inducing (AIF) is ubiquitously
factor
expressed protein involved in the induction of
apoptosis. The AIF precursor is synthesized in the
cytosol and is imported into mitochondria. AIF is
known to translocates through the outer
mitochondrial membrane to the cytosol and to the
nucleus upon apoptosis inducing signals(Daugas et
al., 2000).
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BID is a BH3-only member of the Bcl-2 family
that regulates cell death at the level of
BID U + U + mitochondrial membranes. It has been shown that
BID plays a role in tumor suppression (Zinkel et
al., 2003).
Bim is a pro-apoptotic protein and is known to
BIM U U U + cause degenerative disorders when overexpressed
(Bouillet et al., 2001)
BAFF, B- BAFF is critical regulator for survival of normal B
lymphocyte U U U + cells. Increased BAFF expression has been shown
stimulator in non-Hodgkin lymphoma, as tumors transform to
(BLyS) a more aggressive phenotype (Novalc et al., 2004).
*"+" or "-" denotes increased or decreased expression in localized prostate
cancer relative to benign
prostate tissue or metastatic prostate cancer relative to localized prostate
cancer. "U" denotes that the
expression level is unchanged or needs to be furtlier verified due to the
inconsistency across gene
expression profiling studies.
In order to understand the biological pathways at work in the altered
genes/proteins, pathway enrichment was analyzed using ONCOMINE analyses tools
(Rhodes et al., 2004). Such analyses of the concordant genes revealed that
there was a
disproportionate number of genes with conserved E2F 1 binding sites in their
promoters (n = 7, odds ratio [OR] = 23.8, P < 0.0001), genes localized to
chromatin (n
= 3, OR = 0.0053, P = 0.005), and genes involved in the cell cycle (n=4, OR =
9.1, P
= 0.006). The down-regulated lethal signature had a disproportionate number of
Zn-
binding proteins (n= 4, OR = 109.0, P <0.0001), genes involved in proteolysis
(n = 4,
OR = 12.1, P =0.0005), and genes involved in signal transduction (n = 6, OR =
7.6, P
= 0.001). Similarly pathway analysis of the discordant signature for
enrichment of
particular processes revealed that the discordant genes/proteins included a
disproportionate number proteins localized in the cytosol (odds ratio = 8.9, p-
value =
5.7e-5) and proteins that function in the apoptosis pathway (odds ratio = 6.9,
p-value
=1.3 e-4).
Example 3
Further Tissue Microarray Analysis
In order to further confirm the proteomic alterations as well as to
investigate
any clinical significance and diagnostic values of the novel markers, high
throughput
tissue microarray analyses was performed. Staining was done on a fraction of
the
markers identified by highthroughput screening of prostate tissue lysates that
had
IHC-compatible antibodies. The immunostaining patterns varied greatly. Results
for
a select group of proteins are presented in Fig. 2A and 8A. BM28 demonstrates
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nuclear expression in the basal cells of benign prostate glands (inset first
panel) and
both localized (inset middle) and metastatic prostate cancers. The staining
intensity is
usually, moderate to strong when positive by immunohistocheinistry. The
percentage
of epithelial cells staining positive for BM28 increases with tumor
progression. The
staining pattern is similar to that seen with Ki-67, the proliferation marker.
MSH2
protein expression is nuclear and strongest in prostate cancer samples.
However, as
previously reported, there is variable expression in localized prostate cancer
(Velasco
et al., 2002, Endocrinology 145, 3913-3924).
This is the first study that demonstrates MSH2 expression in a subset of
metastatic prostate tumors. Some of the metastatic tumors did not demonstrate
MSH2
expression. These findings are not associated with germline mutations and
therefore
the biologic significance of these alterations is unknown.
Dynamin demonstrates a cytoplasmic and membranous expression pattern.
Protein expression parallels prostate cancer progression. Expression is seen
in benign
prostate tissues but tends to be more diffuse and intense in localized tumors
and
metastatic tumors. The metastatic tumors demonstrate less membranous staining
(inset right side) as compared to the localized tumors. CDK7 protein
expression is
seen in the nucleus of benign prostate, atrophy, localized prostate cancer,
hormone
sensitive and hormone refractory prostate cancer. The staining patterns can be
generalized as follows.
CDK7 shows the strongest and most uniform expression in clinically localized
prostate cancer. The analysis does not quantify the total number of cells per
unit area.
Because tumor cells ,are more densely packed, one would expect that tissue
extracts
would demonstrate higher expression in localized prostate cancer and
metastatic
tumors. LAP2 demonstrates exclusively nuclear expression. The expression is
seen
in benign prostate tissue in the larger ductal structures and in basal cells.
The
strongest expression in the benign samples is in the ducts. Tumors demonstrate
variable levels of nuclear expression. The association with prostate cancer
progression may in part be due to the quantity of nuclei per unit area as
opposed to
significant differences in protein expression due to neoplastic
transformation. Myosin
VI demonstrates membranous and cytoplasmic protein expression. There is a
trend
towards higher expression with prostate cancer progression.
ICBP90 demonstrates intense nuclear expression that corresponds with tumor
progression. ICBP90, when expressed, is moderate to strong. The extent of
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expression or percentage of cells increased in populations of tuinor cells as
compared
to benign and atrophic prostate glands. The greatest expression was seen in
hormone
refractory ttunors. Also, in the population of clinically localized tuinors,
ICBP90
expression was most extensive in tuinors with a cribriform growth pattern
(Gleason
pattern 4), suggesting higher tumor grade. ILP/XIAP is expressed in the
cytoplasm of
neoplastic prostate epithelial cells and to a significantly lesser degree in
the benign
epithelial cells. Strong expression was seen in a few bony metastatic tumors.
In
general, the hormone naive metastatic tumors had lower expression as compared
to
the hormone refractory tumors.
CamKK demonstrates cytoplasmic and nuclear protein expression with a
slight increase going from benign prostate tissue to metastatic prostate
cancer.
However, examples of high expression in benign and low expression in
metastatic
samples could be found.
JAM1 demonstrates membranous expression, seen strongest and most
consistently in hormone refractory prostate cancer. Honnone naive inetastatic
prostate caiicer has weak protein expression. Expression can also be seen in
benign
prostate tissue and localized prostate cancer. The expression of JAMl may also
be
affected by the number of epithelial cells per unit area. PICIn demonstrates
both
nuclear and cytoplasinic protein expression. The nuclear expression is weak to
moderate in benign prostate tissue and can also be seen in neoplastic tissues.
The
cytoplasmic protein expression increases with prostate cancer progression. The
highest cytoplasmic expression is seen in metastatic PCA. A significant subset
of
metastatic tumors did not show strong expression. Co-chaperone protein p23 -
expression is predominantly cytoplasmic with some nuclear staining detected in
some
cases but always with cytoplasmic expression. Overall protein expression
appears
most consistently high in localized prostate cancer. In general, weak
expression was
seen in benign prostate tissue. Localized prostate cancer had more diffuse
moderate to
strong cytoplasmic staining. Metastatic tumors demonstrated strong protein
expression as often as having no detectable protein expression.
The changes in the staining intensity are depicted in Fig. 2A and Fig. 8.
Most of the cancer and metastatic tissues show higher staining intensity. The
tissue
microarray staining of 20 of the markers were analyzed by unsupervised
clustering.
Clustering of tissue microarrays has been reported earlier (Nielsen et al.,
2003, supra).
Similar to gene expression analyses (Eisen et al., 1998, supra; Perou et al.,
2000,
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supra), unsupervised hierarchical clustering of the data revealed that the in
situ protein
levels could be used to classify prostate sainples as benign, clinically
localized
prostate cancer, or metastatic disease (Fig. 8B). The greatest overall
increase in
expression is seen in the clinically localized tuinors. Most of the metastatic
sainples
and prostate cancer tissues cluster together as can be seen in Fig. 8B.
Additional markers validated by tissue microarray include ABP280, AMACR,
BM28, CainKK, CDK7, Dynainin, EZH2, GS28, ICBP90, JAM1, Kanadaptin, LAP2,
MSH2, Myosin VI, PAXILLIN, pICIn, RBBP, XIAP, BUB3, and GAS7.
Additional markers validated by iminunoblot include, CAMKK, CASPASE 3,
CASPASE 7, CATHEPSIN D, CDK7, C-FLIP, cIAP1, CO-chaperone protein p23,
CPKC, CRP1, DcR1, DEMATIN, DR3, DRBP76, DYNAMIN, E2F3, ECA39,
ERAB, EXPORTIN, EZH2, GAS7, GS28, GSK3-BETA, HP1 ALPHA, ICBP90,
IGFBP2, ILK, INTEGRIN 5ALPHA, IRAK, JAM1, KRIP, LAP2, LIM-KINASE,
MCAM,IVILCK, MMP-19, MMP-23, MSH2, MYOSIN VI, NEXILIIN, NTF2,
NUCLEOPORIN P62, P161NK4A, P67phox, PAXILLIN, PCNA, PICIN, P-MAPK,
p-PKR, PRO-CASPASE7, PSA, PTP1-BETA, PTP1C, RAB27, RACKl, RAL A,
RBBP, S6K, SAPK/JNK, SHC HOMOLOG, SPROUTY4, Stathmin/OP18, TGF
alpha, TROY, TRYROSINASE, UBc9, VtilB, and XIAP.
Table 6 shows genes with altered expression in benign versus prostate cancer
identified using the proteomics analysis methods of the present invention. A
(+) in
the Blot column indicates proteins that are upregulated in prostate cancer
relative to
benign prostate, while a (-) indicates proteins that are down regulated.
Table 6
l7G_CfusterlD Protein Name Gene
Symbol Blot
Benign Vs Prostate Cancer
Hs.118483 Myosin VI MY06 +
Hs.49598 AMACR AMACR +
Hs.79037 HSP60 HSPDI +
Hs.184298 CDK7 CDK7 +
Hs.162089 TPD52 TPD52 +
Hs.78202 BRGI SMARCA4 +
Hs.418533 BUB3 BUB3 +
Hs.171995 PSA KLK3 +
Hs.440394 MSH2 MSH2 +
Hs.124436 GS28 GOSR1 +
Hs.417369 plCln CLNSIA +
Hs.250822 Aurora kinase A STK6 +
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Hs.16003 RBBP RBBP4 +
Hs.318381 CK1 CSNK1A1 +
Hs.171280 ERAB HADH2 +
Hs.356076 XIAP BIRC4 +
Hs.380403 BMI-1 PCGF4 +
Hs.437508 ACT1 C6orf4 +
Hs.78996 PCNA PCNA +
Hs.250882 B2 Bradykinin receptor BDKRB2 +
Hs.349611 PKC alpha PRKCA +
Hs.76364 AIF AIF1 +
Hs.421349 p16INK4A CDKN2A +
Hs.54433 Janusin TNR +
Hs.290270 GKAP DLGAPI +
Hs.98493 XRCC XRCC1 +
Hs.348446 SAPK/JNK2 MAPK9 +
Hs.141125 Casp 3 CASP3 +
Hs.134106 Sek1 MAP2K4 +
Hs.300825 BID BID +
Hs.121575 Cathepsin D-28kD CTSD +
Hs.172865 CSTF50 CSTFI +
Hs.236030 BAF170 SMARCC2 +
Hs.154057 MMP19 MMP19 +
Hs.75360 Carboxypeptidase E CPE +
Hs.57101 BM28 MCM2 +
Hs.2007 FAS ligand TNFSF6 +
Hs.302903 Ubc9 UBE2I +
Hs.355693 co-chaperone protein p23TEBP +
Hs.433612 KRIP-1 TRIM28 +
Hs.256583 DRBP76 ILF3 +
Hs.1189 E2F3 E2F3 +
Hs.9216 Casp7 CASP7 +
Hs.82116 MYD88 MYD88 -
Hs.484782 DFF45 DFFA -
Hs.388677 PAP ACPP -
Hs.298530 RAB27 RAB27A -
Hs.324473 ERK2 MAPKI
-
Hs.241431 G alpha t GNAO1 -
Hs.211819 MMP23 MMP23B -
Hs.42806 Cab45 Cab45 -
Hs.433611 PDKI PDK1 -
Hs.226133 GAS 7 GAS7 -
Hs.437191 PTRF PTRF -
Hs.408754 E61 MAPRE1 -
Hs.149609 Integrin 5 alpha ITGA5 -
Hs.6241 P13 Kinase P{K3R1 -
Hs.390616 PAK3 PAK3 -
Hs.195464 ABP280 FLNA -
Hs.511397 MCAM MCAM -
Hs.334174 TROY TNFRSFI9 -
Hs.437191 PTRF PTRF -
Hs.408754 EB1 MAPRE1 -
Hs.149609 Integrin 5 alpha ITGA5 -
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Hs.6241 P13 Kinase PIK3R1 .
Hs.390616 PAK3 PAK3 -
Hs.195464 ABP280 FLNA .
Hs.511397 MCAM MCAM -
Hs.334174 TROY TNFRSF19 .
Table 7 shows proteins with altered expression in metastatic prostate cancer
vs. local prostate cancer identified using the proteomics analysis methods of
the
present invention. A (+) in the Blot coluinn indicates proteins that are
upregulated in
metastatic prostate cancer relative to local prostate cancer, while a(-)
indicates
proteins that are down regulated.
Table 7
UG_ClusteriDProteinName Gene Symbol Blot
Prostate Cancer Vs Metastatic tumors
Hs.250822 Aurora kinase A STK6 +
Hs.444082 EZH2 EZH2 +
Hs.528342 Nucfeoporin p62 NUP62 +
Hs.11355 LAP2 TMPO +
Hs.6906 Ral A RALA +
Hs.302903 Ubc9 UBE21 +
Hs.157367 Exportin XPOI +
Hs.421349 p161NK4A CDKN2A +
Hs.440394 MSH2 MSH2 +
Hs.419995 Vti1B VTI1B +
Hs.511739 Uba2 UBA2 +
Hs.236030 BAF170 SMARCC2 +
Hs.433612 KRIP-1 TRIM28 +
Hs.171952 Occludin OCLN +
Hs.444118 MCM6 MCM6 +
Hs.10842 Ran RAN +
Hs.348446 SAPK/JNK2 MAPK9 +
Hs.256583 DRBP76 ILF3 +
Hs.77793 Csk CSK +
Hs.171280 ERAB HADH2 +
Hs.209983 Stathmin/Metablastin STMN1 +
Hs.108106 ICBP90 UHRF1 +
Hs.159557 Karyopherin alpha 2 KPNA2 +
Hs.95577 Cdk4 CDK4 +
Hs.57101 BM28 MCM2 +
Hs.184298 CDK7 CDK7 +
Hs.89499 5-Lipoxygenase ALOX5 +
Hs.250882 B2 Bradykinin receptor BDKRB2 +
Hs.258538 Striatin-119kD STRN +
Hs.155560 Calnexin CANX +
Hs.23103 Bet1 BET1 +
Hs.254321 alpha-Catenin CTNNAI +
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Hs.166011 pp120-102kD CTNND1 +
Hs.437495 P131 PSMFI +
Hs.171626 pl9Skpl SKPIA +
Hs.380403 BMI-1 PCGF4 +
Hs.282346 TOP02 beta TOP2B +
Hs.152978 Psme3-29kD PSME3 +
Hs.300825 BID BID +
Hs.418004 PTP I beta PTPN1 +
Hs.5662 RACK1 GNB2L1 +
Hs.76930 alpha-Synuclein SNCA +
Hs.409546 p190 ARHGAP5 +
Hs.437475 Stat6 STAT6 +
Hs.356076 XIAP BIRC4 +
Hs.506845 JAM-1 F11 R +
Hs.5215 EIF-6 ITGB4BP +
Hs.121575 Cathepsin D-28kD CTSD +
Hs.305890 BCL-x BCL2L1 +
Hs.270737 BAFF TNFSF13B +
Hs.462864 Annexin II-34kD ANXA2 +
Hs.417369 plCln CLNSIA +
Hs.441202 GFRaIpha2 GFRA2 +
Hs.356630 NTF2 NUTF2 +
Hs.512638 TIP120 TIP120A +
Hs.355861 Nmt55 NONO +
Hs.271225 FACTp140 SUPT16H +
Hs.2053 Tyrosinase TYR +
Hs.141125 Casp 3 CASP3 +
Hs.389182 HP1 alpha CBX5 +
Hs.110713 DEK DEK +
Hs.418533 BUB3 BUB3 +
Hs.73722 Refl APEX1 +
Hs.78996 PCNA PCNA +
Hs.123044 NHE-3 SLC9A3 +
Hs.170009 TGFalpha TGFA +
Hs.54433 Janusin TNR +
Hs.380938 Syntaxin 8 STX8 +
Hs.156637 c-Cbl CBLC +
Hs.949 p67PHOX NCF2 +
Hs.433326 IGFBP2 IGFBP2 +
Hs.119684 DcR1 TNFRSFIOC +
Hs.436132 Dynamin DNM1 +
Hs.84063 BIM/BOD BCL2L1 1 +
Hs.274122 Dematin EPB49 +
Hs.101174 Tau-53kD MAPT +
Hs.15250 PECI PECI +
Hs.394609 Neurotensin Receptor 3-117kD SORT1 +
Hs.512640 80K-H PRKCSH +
Hs.438993 ECA39 BCAT1 +
Hs.7557 FKBP51 FKBP5
Hs.390616 PAK3 PAK3 -
Hs.406013 Ms Cytokeratin KRT18 -
Hs.75360 Carboxypeptidase E CPE -
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Hs.98493 XRCC XRCC1 -
Hs.306000 Kanadaptin SLC4AIAP -
Hs.408507 TFII-l GTF21 -
Hs.374638 FKBP12 FKBPIA -
Hs.172865 CSTF50 CSTF1
Hs.324473 ERK2 MAPK1 -
Hs.154057 MMP19 MMP19 -
Hs.82116 MYD88 MYD88 -
_
Hs.76111 Beta-Dystroglycan DAGI
Hs.226133 GAS 7 GAS7 _
Hs.162089 TPD52 TPD52 _
Hs.433795 SHC transforming protein I SHC1 _
Hs.182018 IRAK IRAK1
Hs.167 MAP2B MAP2 -
Hs.243491 Casp8 CASP8 -
Hs.1189 E2F3 E2F3 -
Hs.79037 HSP60 HSPD1
Hs.241431 G alpha t GNA01 -
Hs.511397 MCAM MCAM _
Hs.433611 PDKI PDK1 -
Hs.78202 BRG1 SMARCA4 -
Hs.1030 RINI RIN1 -
Hs.16003 RBBP RBBP4 -
Hs.349611 PKC alpha PRKCA _
Hs.124436 GS28 GOSR1 -
Hs.299558 DR3 TNFRSF25 -
Hs.282359 GSK3 beta GSK3B -
Hs.335786 TIAR TIAL1 -
Hs.79748 4F2 hc/CD98HC SLC3A2 -
Hs.86858 S6K RPS6KB1 -
Hs.268177 Phospholipase C gamma 1 PLCG1 -
Hs.298530 RAB27 RAB27A -
Hs.22370 Nexilin NEXN -
Hs.171995 PSA KLK3 -
Hs.388677 PAP ACPP -
Hs.75799 Prostasin PRSS8 -
Hs.386078 Myosin light chain kinase(MLCK) MYLK -
Hs.134106 Sek1 MAP2K4 -
Hs.437508 ACT1 C6orf4 -
Hs.377908 MYPT1 PPP1 R12A -
Hs.154103 Lim kinase LIM -
Hs.108080 CRP1 CSRP1 -
Hs.149609 Integrin 5 alpha ITGA5 -
Hs.195464 ABP280 FLNA _
Hs.9216 Casp7 CASP7
Hs.211819 MMP23 MMP23B -
Hs.1288 MSActin ACTA1 -
Hs.290270 GKAP DLGAP1 _
Hs.234521 3PK MAPKAPK3 _
Hs.289107 CIAP BIRC2
Hs.139851 Caveolin 2-20kD CAV2 _
Hs.25511 Hic-5 TGFB111 _
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Hs.446336 PAXILLIN PXN .
Hs.49598 AMACR AMACR -
Hs.408767 alphaB Crystallin CRYAB
Hs.355724 c-FLIP CFLAR
Table 8 shows cell coinparhnents and statistical analyses of 20 proteins used
for tissue microarray analysis. One-way ANOVA with post hoc tests was
conducted
for each protein. Population variances were exainined by Levene test. Tulcey's
HSD
test was used to control the Type I error rate if the population variances
were
equivalent, otherwise, the Games-Howell procedure was used.
Table 8
Multiple Comparisonsi Corcondant Analysis with the
Protein Cell transcript expression
Compartment Benign vs. PCA Benign vs. vs. BenignPCA vs. PCA vs
Met MET PCA MET
DYNAMIN Epithelium
AMACR Epithelium * * ~
CDK7 Epithelium * * ~ ~l
PICIN Epithelium * * a
XIAP Epithelium
MSH2 Epitlielium
CAMKK Epithelium * *
BUB3 Epithelium
GS28 Epithelium
RBBP Epithelium * *
GAS7 Epithelium
EZH2 Epithelium * *
ICBP90 Epithelium * *
JAM Epithelium
LAP2 Epithelium
BM28 Epithelium * *
MYQSIN6 Epithelium * *
KANADAPTIN Epithelium *
ABP-280 Stroma * *
PAXILIN Stroma * *
1. Benign: benign prostate tissue; PCA: clinically locaHzed prostate cancer;
MET: metastatic prostate cancer. One-way ANOVA with
post hoc tests was conducted for each protein. Population vat-iances were
exainined by Levene test. The Tukey's HSD test was used to
control the Type I eiror rate if the population variances were equivalent,
otheiwise, the Games-Howell procedure was used instead.
The analysis was carried out in SPSS 11.5.
*: The protein is significant at 0.051eve1; ~: The IHC result of the protein
is concordant with its transcript expressions when examined
by same procedure used in the integrative molecular analysis.
Table 9 (Figure 14) shows proteomics alterations mapped to gene expression
in clinically localized prostate cancer relative to benign prostate tissue.
*"+", "U", or
"-" denotes that the corresponding protein is increased, unchanged, or
decreased
respectively in clinically localized prostate cancer relative to benign
prostate.
Table 10 (Figure 15) shows proteomics alterations mapped to gene expression
in metastatic prostate cancer relative to clinically localized prostate
cancer.
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Table 11. Concordant proteomic/genomic signature in metastatic prostate cancer
relative to clinically
localized
disease*
UG ClusterID NAME Immunoblot Dhanasekran et al. Lapointee et al. Latulippe et
al. Yu et al.
Hs.250822 Aurora kinase A + + + + +
Hs.444082 EZH2 + + + + +
Hs.528342 Nucleoporin p62 + + + +
Hs.11355 LAP2 + u + + +
Hs.6906 RaI A + u + + +
Hs.302903 Ubc9 + + u + +
Hs.157367 Exportin + u + +
Hs.421349 p16INK4A + u + +
Hs.440394 MSH2 + + u +
Hs.419995 Vti1B + + +
Hs.511739 Uba2 + u u + +
Hs.236030 BAF170 + u + u +
Hs.433612 KRIP-1 + u + + u
Hs.171952 Occludin + u + u +
Hs.444118 MCM6 + u + + u
Hs.10842 Ran + u + +
Hs.348446 SAPIUJNK2 + + + u
Hs.256583 DRBP76 + u + + -
Hs.77793 Csk + + + u
Hs.171280 ERAB + + u
Hs.209983 StathinnvlYletablast + + u
in
Hs.108106 ICBP90 + + u
Hs.159557 ~ ~'yopherin alpha + + u
Hs.95577 Cdk4 + + u
Hs.57101 BM28 + +
Hs.298530 RAB27 - - - -
Hs.22370 Nexilin
Hs.171995 PSA - - - -
Hs.388677 PAP - - -
Hs.75799 Prostasin
Hs.386078 Myosin light chain
kinase(MLCK) - - - -
Hs.134106 Sekl - u - - -
Hs.437508 ACT1 - u - -
Hs.377908 MYPTI - u - - -
Hs.154103 Lim kinase -- - - +
Hs.108080 CRP1 - - -
Hs.149609 hitegrin 5 alpha - - -
Hs.195464 ABP280 - - -
Hs.9216 Casp7 -- -
Hs.211819 MMP23 - - -
Hs.1288 MSActin -u
Hs.290270 GKAP -+
Hs.234521 3PK -- u u -
Hs.289107 cIAP -- - u u
Hs.139851 Caveolin 2-20kD -u u
Hs.25511 Hic-5 -u u
Hs.446336 PAXILLIN u +
Hs.49598 AMACR u +
Hs.408767 alphaB Crystallin -u -
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Hs.355724 c-FLIP -- - + +
*"+", "U", or "-" denotes that the corresponding protein is increased,
unchanged, or decreased
respectively in metastatic prostate cancer relative to clinically localized
prostate cancer.
Example 4
Breast Cancer analysis
Further experiments were perforined that analyzed expression profiles in
breast cancer. Exemplary markers identified include CamKK, Myosin VI, Auroara
A,
exportin, BM28, CDI0, TIP60, and p16 INK 4a. Tissue inicroarray analysis is
shown in Figure 13.
All publications and patents mentioned in the above specification are herein
incorporated by reference. Various modifications and variations of the
described
method and system of the invention will be apparent to those skilled in the
art without
departing from the scope and spirit of the invention. Although the invention
has been
described in connection with specific preferred embodiments, it should be
understood
that the invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out
the invention which are obvious to those skilled in the relevant fields are
intended to
be within the scope of the following claims.
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