U.S. patent application number 10/408133 was filed with the patent office on 2004-01-29 for composition and method for the treatment and prevention of metastatic disorders.
This patent application is currently assigned to Baylor College of Medicine. Invention is credited to Ren, Chengzhen, Thompson, Timothy C..
Application Number | 20040018173 10/408133 |
Document ID | / |
Family ID | 22140884 |
Filed Date | 2004-01-29 |
United States Patent
Application |
20040018173 |
Kind Code |
A1 |
Thompson, Timothy C. ; et
al. |
January 29, 2004 |
Composition and method for the treatment and prevention of
metastatic disorders
Abstract
The invention relates to methods for the isolation of metastatic
sequences and the isolated sequences. Cells from a cell line or an
animal tissue are treated to form a cell line predisposed to
cancer. Treated cells are implanted in an animal and incubated for
a period of time sufficient for the cells to proliferate and
develop malignant transplants. RNA from the malignant transplant
and the primary tumor are analyzed by differential display
polymerase chain reaction. Differentially expressed genes are
cloned, reanalyzed, and sequenced. These genes and sequences can be
used as probes in the diagnosis of neoplastic disorders, as probes
to isolate metastatic sequences and as a therapeutic agent in the
treatment of neoplastic disorders. The metastatic sequence may be a
dominant metastatic sequence or a recessive metastatic
sequence.
Inventors: |
Thompson, Timothy C.;
(Houston, TX) ; Ren, Chengzhen; (Pearland,
TX) |
Correspondence
Address: |
Tracey B. Davies
VINSON & ELKINS L.L.P.
2300 First City Tower
1001 Fannin Street
Houston
TX
77002-6760
US
|
Assignee: |
Baylor College of Medicine
Houston
TX
77030
|
Family ID: |
22140884 |
Appl. No.: |
10/408133 |
Filed: |
April 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10408133 |
Apr 7, 2003 |
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09266764 |
Mar 12, 1999 |
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6545139 |
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60077934 |
Mar 13, 1998 |
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Current U.S.
Class: |
424/93.2 ;
424/94.4; 435/320.1; 435/325; 435/456; 536/23.2 |
Current CPC
Class: |
A61K 38/45 20130101;
C12Y 104/03013 20130101; C12N 15/113 20130101; A61K 38/208
20130101; C07K 14/47 20130101; A61K 38/2013 20130101; A61K 38/00
20130101; A61K 38/44 20130101; A61K 48/00 20130101; C07K 16/18
20130101; G01N 33/57434 20130101; C12Y 207/01021 20130101; C07K
16/28 20130101; G01N 33/50 20130101 |
Class at
Publication: |
424/93.2 ;
424/94.4; 435/456; 435/320.1; 435/325; 536/23.2 |
International
Class: |
A61K 048/00; C07H
021/04; A61K 038/44; C12N 015/86 |
Goverment Interests
[0002] The invention was made with support from the United States
government under grant numbers RO1-CA50588, RO1-CA68814 and
P50-CA58204, awarded by the National Institutes of Health, and the
United States government has certain rights in this invention.
Claims
We claim:
1. A composition for the treatment or prevention of a metastatic
disorder comprising a recombinant vector containing a
metastatic-specific promoter functionally linked to a gene whose
expression will result in death of a host cell.
2. The composition of claim 1 wherein the recombinant vector is an
adenovirus vector, an adenovirus-associated vector, a vaccinia
virus vector, a lentivirus vector, a herpes virus vector or a
combination thereof.
3. The composition of claim 1 wherein the metastatic-specific
promoter is a caveolin gene promoter, a p99 gene promoter or a
lysyl oxidase gene promoter.
4. The composition of claim 1 wherein the gene encodes IL-2, IL-12,
a thymidine kinase gene or a toxin.
5. The composition of claim 1 wherein the recombinant vector is
transformed in the host cell.
6. The composition of claim 1 wherein the host cells are mammalian
cells.
7. Cells containing a recombinant vector that comprises a
metastatic-specific promoter functionally linked to a gene whose
expression will result in death of a host cell.
8. A recombinant vector comprising a metastatic-specific promoter
functionally linked to a gene whose expression will result in death
of a host cell.
9. An isolated nucleic acid that encodes the sequence of p99.
10. The nucleic acid of claim 9 wherein the sequence comprises the
p99 promoter.
11. A pharmaceutical composition comprising an effective amount of
a lysyl oxidase protein, or an active fragment thereof, and a
pharmaceutically acceptable carrier.
12. The composition of claim 11 wherein the active fragment
comprises the complete amino acid sequence of the p99 protein.
13. The composition of claim 11 wherein the pharmaceutically
acceptable carrier is selected from the group consisting of water,
alcohol, oil, saccharide, fatty acid or a combination thereof.
14. A method for treating or preventing a metastatic disorder
comprised of administering a recombinant vector containing a
metastatic-specific promoter functionally linked to a gene whose
expression will result in death of a host cell to a patient.
15. The method of claim 14 wherein the metastatic disorder is
metastatic prostate cancer.
16. The method of claim 14 wherein administration is by parenteral
administration.
17. The method of claim 14 wherein the recombinant vector is an
adenovirus vector, an adenovirus-associated vector, a vaccinia
virus vector, a lentivirus vector, a herpes virus vector or a
combination thereof.
18. The method of claim 14 wherein the metastatic-specific promoter
is a promoter from the caveolin gene, the p99 gene or the lysyl
oxidase gene.
19. The method of claim 14 wherein the gene encodes IL-2, IL-12, a
thymidine kinase or a toxin.
20. The method of claim 14 wherein the metastatic-specific promoter
is induced in metastatic cells.
21. The method of claim 14 wherein the metastatic-specific promoter
is suppressed in metastatic cells.
22. The method of claim 14 wherein the host cell is a mammalian
cell.
23. The method of claim 14 wherein the patient is a human.
24. A method for treating or preventing a metastatic disorder
comprising administering a therapeutically effective amount of an
antisense nucleic acid that alters expression of a
metastatic-specific gene to a patient.
25. The method of claim 24 wherein the antisense nucleic acid is
DNA, RNA or PNA.
26. The method of claim 24 wherein the antisense nucleic acid
contains the antisense of a portion of the gene the encodes
caveolin, lysyl oxidase or p99.
27. The method of claim 24 wherein expression is decreased.
28. The method of claim 24 wherein the antisense nucleic acid is
expressed from a viral vector.
29. The method of claim 28 wherein the viral vector is a vaccinia
viral vector, a retroviral vector, an adenoviral vector, an
adeno-associated viral vector, a lentiviral vector, a herpes viral
vector or a combination thereof.
30. A method for treating a metastatic disorder comprising
administering an effective amount of a metastatic-specific protein
to a patient.
31. The method of claim 30 wherein the metastatic disorder is
prostate or breast cancer.
32. The method of claim 30 wherein the metastatic-specific protein
is lysyl oxidase or a functional equivalent of lysyl oxidase.
33. The method of claim 30 wherein the metastatic-specific protein
is an antibody with specific affinity to caveolin or p99.
34. A method for evaluating metastatic potential of a primary
prostate tumor comprising: contacting a sample of the tumor with a
metastatic-specific marker; determining the amount of marker bound
to the sample; and determining the metastatic potential of the
tumor.
35. The method of claim 34 wherein the metastatic-specific marker
is coupled to a detectable label.
36. The method of claim 35 wherein the marker is a monoclonal or a
polyclonal antibody.
37. The.about.method of claim 34 wherein the marker is a nucleic
acid sequence
38. The method of claim 37 wherein the nucleic acid sequence is a
sequence of the gene for caveolin, lysyl oxidase or p99.
39. A method for treating a patient having a prostate tumor
comprising: suppressing the expression of at least one metastatic
sequence in prostrate tumor cells; and reducing the level of
androgen in the patient.
40. The method of claim 39 wherein expression of the metastatic
sequence is suppressed by administering an anti-sense nucleic
acid.
41. The method of claim 39 wherein the level of androgen is reduced
by administering anti-androgen therapy.
42. A method for increasing the sensitivity of a malignant tumor to
anti-neoplastic therapy comprising: suppressing expression of a
multidrug resistant gene in tumor cells; administering a
therapeutically effective dose of an anti-cancer drug.
43. The method of claim 42 wherein the multidrug resistant gene is
caveolin.
44. A method of treating or preventing a metastatic disorder
comprising administering a therapeutically effective amount of an
anti-metastatic sequence to metastatic cells of the patient.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S.
Provisional Patent Application, entitled Metastatic Sequences,
serial No. 60/077,934, filed Mar. 13, 1998.
BACKGROUND OF THE INVENTION 1. Field of the Invention
[0003] The present invention relates to methods for the
identification, isolation and use of metastatic genes and their
sequences, to sequences identified by these methods, and to the use
of diagnostic and therapeutic agents based on these sequences for
the treatment of metastatic and other neoplastic disorders. 2.
Description of the Background
[0004] The development of higher organisms is characterized by an
exquisite pattern of temporal and spatially regulated cell
division. Disruptions in the normal physiology of cell division are
almost invariably detrimental. One such type of disruption is
cancer, a disease that can arise from a series of genetic
events.
[0005] Cancer cells are defined by two heritable properties,
uncontrolled growth and uncontrolled invasion of normal tissue. A
cancerous cell can divide in defiance of the normal growth
constraints in a cell leading to a localized growth or tumor. In
addition, some cancer cells also gain the ability to migrate away
from their initial site and invade other healthy tissues in a
patient. It is the combination of these two features that make a
cancer cell especially dangerous.
[0006] An isolated abnormal cell population that grows
uncontrollably will give rise to a tumor or neoplasm. As long as
the neoplasm remains noninvasively in a single location, it is said
to be benign, and a complete cure may be expected by removing the
mass surgically. A tumor or neoplasm is counted as a cancer if it
is malignant, that is, if its cells have the ability to invade
surrounding tissue. True malignancy begins when the cells cross the
basal lamina and begin to invade the underlying connective tissue.
Malignancy also occurs when the cells gain the ability to detach
from the main tumor mass, enter the bloodstream or lymphatic
vessels, and form secondary tumors or metastases at other sites in
the body. The more widely a tumor metastasizes, the harder it is to
eradicate and treat.
[0007] As determined from epidemiological and clinical studies,
most cancers develop in slow stages from mildly benign into
malignant neoplasms. Malignant cancer usually begins as a benign
localized cell population with abnormal growth characteristics
called dysplasia. The abnormal cells acquire abnormal growth
characteristics resulting in a neoplasia characterized as a cell
population of localized growth and swelling. If untreated, the
neoplasia in situ may progress into a malignant neoplasia. Several
years, or tens of years may elapse from the first sign of dysplasia
to the onset of full blown malignant cancer. This characteristic
process is observed in a number of cancers. Prostate cancer
provides one of the more clear examples of the progression of
normal tissue to benign neoplasm to malignant neoplasm.
[0008] Prostate cancer is the most common malignancy in men in the
USA, resulting in an estimated 41,800 deaths in 1997. (Parker S L,
et al., CA Cancer J Clin 47: 5-27, 1997). The widespread use of
prostate-specific antigen (PSA) has dramatically increased the
number of patients diagnosed with prostate cancer and generally
lowered the stage of disease at diagnosis. (Scardino P T, Urol.
Clin. N. Am. 16:635-655, 1989; Epstein J L, et al., JAMA 271:
368-374, 1994). Nevertheless, 5%-10% of cancers detected by PSA
screening are clinically advanced and not candidates for radical
prostatectomy. Despite surgical removal of the prostate, 30%-60% of
men treated will have recurrence of cancer within 5 years,
suggesting that the clinical stage of the patients undergoing
surgery was highly inaccurate. 20%-57% of patients undergoing
definitive surgery with presumed localized disease will have rising
PSA following treatment, also indicative of local or distant
residual disease. (Ohori M, et al., J. Urol. 154: 1818-1824, 1995;
Zeitman A L, et al., Urology 43: 828-833, 1994). Neither of these
conditions is amenable to curative therapy.
[0009] The walnut-sized prostate is an encapsulated organ of the
mammalian male urogenital system. Located at the base of the
bladder, the prostate is partitioned into zones referred to as the
central, peripheral and transitional zones, all of which surround
the urethra. Histologically, the prostate is a highly
microvascularized gland comprising fairly large glandular spaces
lined with epithelium which, along with the seminal vesicles,
supply the majority of fluid to the male ejaculate. As an
endocrine-dependent organ, the prostate responds to both the major
male hormone, testosterone, and the major female hormones, estrogen
and progesterone. Testicular androgen is considered important for
prostate growth and development because, in both humans and other
animals, castration leads to prostate atrophy and, in most cases,
an absence of any incidence of prostatic carcinoma.
[0010] The major neoplastic disorders of the prostate are benign
enlargement of the prostate, also called benign prostatic
hyperplasia (BPH), and prostatic carcinoma, a type of neoplasia.
BPH is very common in men over the age of 50. It is characterized
by the presence of a number of large distinct nodules in the
periurethral area of the prostate. Although benign and not
malignant, these nodules can produce obstruction of the urethra
causing nocturia, hesitancy to void, and difficulty in starting and
stopping a urine stream upon voiding the bladder. Left untreated, a
percentage of these prostate hyperplasias and neoplasias may
develop into malignant prostatic carcinoma.
[0011] In its more aggressive form, malignant transformed prostatic
tissues escape from the prostate capsule and metastasize invading
locally and throughout the bloodstream and lymphatic system.
Metastasis, defined as tumor implants which are discontinuous with
the primary tumor, can occur through direct seeding, lymphatic
spread and hematogenous spread. All three routes have been found to
occur with prostatic carcinoma. Local invasion typically involves
the seminal vesicles, the base of the urinary bladder, and the
urethra. Direct seeding occurs when a malignant neoplasm penetrates
a natural open field such as the peritoneal, pleural or pericardial
cavities. Cells seed along the surfaces of various organs and
tissues within the cavity or can simply fill the cavity spaces.
Hematogenous spread is typical of sarcomas and carcinomas.
Hematogenous spread of prostatic carcinoma occurs primarily to the
bones, but can include massive visceral invasion as well. It has
been estimated that about 60% of newly diagnosed prostate cancer
patients will have metastases at the time of initial diagnosis.
[0012] Surgery or radiotherapy is the treatment of choice for early
prostatic neoplasia. Surgery involves complete removal of the
entire prostate (radical prostatectomy), and often removal of the
surrounding lymph nodes, or lymphadenectomy. Radiotherapy,
occasionally used as adjuvant therapy, may be either external or
interstitial using .sup.125I. Endocrine therapy is the treatment of
choice for more advanced forms. The aim of this therapy is to
deprive the prostate cells, and presumably the transformed prostate
cells as well, of testosterone. This is accomplished by orchiectomy
(castration) or administration of estrogens or synthetic hormones
which are agonists of luteinizing hormone-releasing hormone. These
cellular messengers directly inhibit testicular and organ synthesis
and suppress luteinizing hormone secretion which in turn leads to
reduced testosterone secretion by the testes. In normal prostate,
removal of androgenic hormones results in regression of the gland
involving apoptosis of more than 60% of the luminal epithelial
cells. Although often initially sensitive to removal of androgens,
prostate cancer cells eventually lose this response and continue to
grow and spread even in the absence of androgenic steroids. Despite
the advances made in achieving a pharmacologic orchiectomy, the
survival rates for those with late stage carcinomas are rather
bleak
[0013] Current therapeutic regimens for metastatic disease
typically involve both chemical and surgical androgen ablation,
which although has been demonstrated to extend life when compared
to untreated patients, almost invariably results in the development
of hormone-refractory disease and the demise of the patient. The
fundamental concepts upon which current androgen ablation therapy
was developed were reported more than 50 years ago by Huggins and
Hodges. (Huggins C, et al., Cancer Res. 1:293-297, 1941). These
experiments reported the phenomenon in which removal of androgenic
steroids by castration resulted in reduced growth and biochemical
activities in prostate cancer.
[0014] With the advent of molecular biology, various investigators
in laboratories have attempted to understand the molecular biology
of castration-induced regression of the prostate at a more
mechanistic level. The model systems selected almost invariably
compared mRNAs produced prior to castration and during
castration-induced regression using rat prostate model systems in
vivo. These model systems yield gene activities that may be
involved in castration-induced regression but could also be
involved in activities that are not directly relevant or related to
castration-induced regression but were stimulated by removal of
androgenic steroids. It is anticipated that only a small fraction
of gene activities modulated by steroid withdrawal would indeed be
involved in castration-induced regression and, therefore,
significant confounding background activity would be seen in these
existing model systems. There is therefore a need for a model
system in which the androgenic-stimulated gene activities not
associated with castration-induced regression, or "background" gene
activities, would be normalized. Moreover, a better understanding
of the molecular basis of metastasis, in prostate cancer, as well
as other forms of cancer, would allow rational efforts toward the
development of novel effective anti-metastatic therapy to
proceed.
SUMMARY OF THE INVENTION
[0015] The present invention overcomes the problems and
disadvantages associated with current strategies and designs and
provides new compositions and methods for the for the evaluation,
diagnosis and treatment of metastatic and other neoplastic
disorders.
[0016] One embodiment of the invention is directed to compositions
and methods for treating a patient having a metastatic tumor.
Compositions may contain agents that selectively target metastatic
cells for destruction. Such agents include nucleic acid sequences
that selectively suppress metastasis or protein sequences that
selectively destroy metastatic cells. Methods involve administering
a therapeutically effective amount of, for example, an anti-sense
nucleic acid that selectively suppresses expression of a gene
encoding a metastatic-specific protein or metastatic-specific
protein that selectively inhibits the proliferation of or destroys
the metastatic cell, to the patient. The nucleic acid may comprise,
for example, RNA, DNA or PNA, and be expressed using any suitable
means, such as a viral vector which is a vector containing one or
more virally-derived sequences. Useful viral vectors include
vaccinia virus vectors, herpes virus vectors, retrovirus vectors,
adenovirus vectors, adeno-associated virus vectors, lenti viral
vectors and combinations thereof. The anti-sense sequence may
encode the entirety of or, alternately, an effective portion of the
gene encoding the metastatic sequence, such as a functional domain
like a scaffolding domain or a dimerization domain. Alternately,
the effective portion may comprise the transcription promoter
region of the gene.
[0017] Another embodiment of the invention is directed to methods
for treating a metastatic disorder, such as metastatic prostate or
breast cancer, by administering to a patient having the disorder an
effective amount of an anti-sense sequence, a metastatic-specific
product or an antibody to such products. The antibody may be
reactive against all or an effective portion of the sequence or its
product, such as the scaffolding domain or the dimerization domain
of a caveolin protein.
[0018] Another embodiment of the invention is directed to methods
for evaluating the metastatic potential of a primary prostate tumor
by contacting a sample of the tumor with an antibody to the product
of the metastatic sequence coupled to a detectable marker and then
determining the amount of antibody bound to the sample. The
antibody may be a monoclonal or polyclonal antibody, and may be
optionally coupled to a detectable label.
[0019] Another embodiment of the invention is directed to methods
for treating a metastatic disorder, such as a metastatic prostate
cancer, by administering to a patient having the disorder an
effective amount of the metastatic sequence, said metastatic
sequence functioning as a metastatic suppressor, such as lysyl
oxidase. Transcription of the metastatic sequence may be driven by
a promoter that is up-regulated by metastasis-specific factors in
metastatic cells, such as the caveolin promoter.
[0020] Another embodiment of the invention is directed to methods
for treating a neoplastic disorder, preferably a metastatic
disorder, comprising administering a pharmaceutically effective
amount of a metastatic nucleic acid to a patient. The nucleic acid
may be single stranded in the sense or the anti-sense direction.
Alternatively, the nucleic acid may be packaged in a viral vector
such as, for example, herpes viral vectors, retroviral vectors,
adenoviral vectors, adeno-associated viral vectors, lenti viral
vectors, vaccinia viral vectors and combinations thereof.
Administration may be performed by injection, pulmonary absorption,
topical application or delayed release of the nucleic acid along
with a pharmaceutically acceptable carrier such as water, alcohols,
salts, oils, fatty acids, saccharides, polysaccharides and
combinations thereof.
[0021] Another embodiment of the invention is directed to isolated
promoters that are specific for expression in metastatic cells. The
promoter may further be functionally coupled to a gene which
encodes an anti-metastatic therapeutic agent. The therapeutic agent
may be a toxin, an apoptotic inducer, a cytokine such as IL-2 or
IL-12, or another suitable agent or combination of agents.
[0022] Another embodiment of the invention is directed to methods
for the isolation of a metastatic sequence. One or more oncogenic
sequences are transfected into a cell to form a transfected cell.
The transfected cell is introduced into a primary site of a host
animal to establish a colony which is incubated in the animal for a
period of time sufficient to develop both a primary tumor and a
malignant tumor. RNA is harvested from the primary tumor and from
the malignant tumor and the two groups of RNA sequences are
compared to each other. The harvested tumor RNAs are also compared
to normal nonmalignant tissues to identify sequences specific for
nonmetastatic tumors as well as those specific for metastatic
tumors. Dominant metastatic genes are genes whose expression leads
to metastasis. Such genes are typically expressed at high levels in
metastatic tumors and not significantly expressed in normal or
nonmetastatic cells. Recessive metasutic genes, genes whose
expression prevents metastasis, may be selectively expressed in
normal and nonmetastatic cells and absent in metastatic cells.
Dominant and recessive metastatic genes may act directly or act
pleiotropically by enhancing or inhibiting the expression or
function of other dominant and recessive metastatic genes.
[0023] Another embodiment of the invention is directed to sequences
isolated by the methods of the invention. Sequences may be in the
form of DNA, RNA or PNA. The nucleic acid may be single stranded or
double stranded. Single stranded nucleic acid may be in the form of
a sense strand or an anti-sense strand. In addition, the sequence
may be part of a homologous recombination vector designed to
recombine with another metastatic sequence.
[0024] Another embodiment of the invention is directed to methods
for the identification of metastatic sequences. One or more
oncogenic sequences are transfected into a mammalian urogenital
cell to form a transfected cell. The transfected cell is introduced
into a site of a host animal and incubated for a period of time
sufficient for cells to proliferate and to develop malignancies at
secondary sites. RNA is isolated from the primary and secondary
sites and reverse transcribed into cDNA. cDNA sequences from the
primary tumor and the secondary metastasis are compared by
differential display polymerase chain reaction to detect and
subsequently isolate metastatic sequences. The host mammal may be
an allogenic, a xenogenic, a transgenic or an immunocompromised
host.
[0025] Another embodiment of the invention is directed to kits that
contain one or more metastatic sequences that can be used for
staging a tumor. DNA or RNA may be isolated from a tumor and
detected using a probe comprising a metastatic sequence. The
presence or absence of metastatic DNA or RNA sequences in the
tumor, will indicate the oncogenic and metastatic potential of the
tumor.
[0026] Other objects and advantages of the invention are set forth
in part in the description which follows, and in part, will be
obvious from this description, or may be learned from the practice
of the invention.
DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 Nucleotide and amino acid sequences of p99.
[0028] FIG. 2 A compilation of the sequences obtained using methods
of the invention for identifying dominant and recessive metastatic
sequences.
[0029] FIG. 3 Data obtained through the initial characterization of
some of the sequences resulting from the Met screen.
[0030] FIG. 4 Data obtained through the initial characterization of
some of the sequences (also) resulting from the Met screen.
[0031] FIG. 5 Data obtained through the initial characterization of
some of the sequences (also) resulting from the Met screen
[0032] FIG. 6 Data obtained through the initial characterization of
some of the sequences (also) resulting from the p53 screen.
[0033] FIG. 7 Data obtained through the initial characterization of
some of the sequences (also) resulting from the TGF-.beta.
screen.
[0034] FIG. 8 Data obtained through the initial characterization of
some of the sequences resulting from screening a phage library.
[0035] FIG. 9 Summaries of study results on the expression of the
metastatic sequences in primary cells and in metastatic cells using
RNA blots.
DESCRIPTION OF THE INVENTION
[0036] Identification of Metastatic Sequences
[0037] As embodied and broadly described herein, the present
invention is directed to compositions and methods for detection,
diagnosis, treatment and prevention of metastatic disease and
disorders.
[0038] The ability of cancers to metastasize makes tumors difficult
to eradicate by any means. Malignant cancer involves a multistage
progression from, for example, normal tissue through hyperplasia,
early adenoma, early carcinoma and finally to a metastatic tumor.
Cells of a typical tumor loosen their adhesion to their original
cellular neighbors and cross the basal lamina and endothelial
lining to enter the body's circulation. Once in circulation, the
metastatic cell exits from the circulation to disseminate
throughout the body and proliferate in a new environment.
[0039] Like the initial oncogenic event, the ability of a cell to
metastasize requires additional mutations or epigenetic changes. An
understanding of the molecular mechanisms of metastasis allows for
the design of treatments to inhibit metastasis. Knowledge of stage
specific gene expression for neoplastic disorders allows for early
detection and typing of tumors. With early detection and typing,
proper treatment may be administered to a patient with the
neoplastic disorder earlier, which will lead fo a higher
probability of a complete cure.
[0040] For human prostate tumors, the study of stage specific
tumors is difficult, if not impossible, as cell lines are extremely
difficult to grow and it is rare that tissue becomes available from
the primary tumor as well as metastatic disease from the same
patient. This problem is exacerbated because of the infrequent
biopsy of metastatic deposits in concordance of isolation of
material from the primary tumor. Furthermore, the growth of cell
lines from malignant prostates has proved to be problematic over
the last few decades. This is evidenced by the lack of cell lines
from prostate cancer obtained under any conditions.
[0041] Expression metastatic sequences are either increased or
decreased in metastatic cancer cells as compared to primary tumors
depending upon the function of the sequence. Recessive metastatic
genes functioning as tumor suppressors are downregulated in
metastatic cells while dominant metastatic genes are up-regulated.
Using the methods of the present invention, certain cancers may be
treated, for example, by either suppressing or inducing the
expression, as the situation requires, of these genes in metastatic
cells or cells predisposed to metastasis. As expression correlates
with metastasis, application of biological technologies designed to
either block activity or up-regulate the activity of these genes or
their products and may be used for the treatment and prevention of
metastatic and other neoplastic disorders. Variations of
conventional techniques that take advantage of this observation can
also be used for the treatment and prevention of metastatic
disease.
[0042] One embodiment of the invention is directed to a method for
identifying a metastatic sequence. A mammalian cell is transformed
into a pre-neoplastic or neoplastic state or phenotype by
transfection with one or more oncogenic sequences (e.g. DNA, RNA,
PNA). Alternatively, or in addition to transfection, the mammalian
cell may be treated with an agent or subjected to a condition that
potentiates the metastatic character of the cell or predisposes the
cell to metastasis. The transfected or treated cell is implanted
into a host animal at a primary site and grown for a period of time
sufficient to develop a metastasis at a secondary site. Expressed
sequences from cells of the primary site and cells at the secondary
site are amplified by differential display polymerase chain
reactions. PCR products from these reactions are compared and the
metastatic sequence identified by alteration in the levels or
patterns of the resulting products.
[0043] Mammalian cells from a wide variety of tissue types and
species are suitable for transfection or treatment including
surgically obtained or primary or immortalized cells and cell
lines. Cells may be from humans or primates, mice, rats, sheep,
cows, rabbits, horses, pigs or guinea pigs or from transgenic or
xenogencic host mammals. Cells may be obtained from adult, juvenile
or fetal tissue, and used directly from the mammal, from
cryogenically preserved samples, or after culturing in vitro or in
vivo for a period of time. In vitro culturing typically involves
tissue culture conditions (e.g. 37.degree. C., 5% CO.sub.2) while
in vivo culturing may involve successive passage of cells through
host animals such as, for example, mice or rabbits. Cells passed in
vivo may be obtained from sites proximal or distal to the site of
implantation. The tissue type from which the cells are derived or
obtained may be any tissue which is susceptible to transfection, or
other treatment including, for example, urogenital tissues,
epithelial cells, hepatic cells, fibroblasts lymphatic tissues,
hematopoietic cells, cells of the immune system, cells of the
gastrointestinal system and cells of the nervous system.
[0044] Starting cells may also be treated prior to use to enhance
detection sensitivity. Treatment may comprise contact with reagents
which affects the neoplastic, metastatic, differentiation,
activation or growth of the cell. Treatment may be in vitro or in
vivo and may include, for example, direct or indirect induction or
suppression of well known oncogenic sequences and genes isolated by
the invention such as, for example, TGF-.beta.1, cyclin D1, p53,
lysyl oxidase, caveolin, actin binding protein, ubiquitin
activating enzyme E1, nmb, .alpha. actinin 3, and p34. Gene
expression induction includes transfecting expression vectors
encompassing the coding region of the gene. Gene repression
comprises introducing a gene ablation sequence or a repressor of
the gene to the cell.
[0045] Cells which have one or more genes ablated may also be used.
For example a metastatic suppression gene may be ablated to prevent
inhibition to metastasis. A useful gene for ablation is a gene
capable of affecting the phenotype and behavior of a cell or tumor.
For example, with prostate tumors, suitable genes include both well
known genes and genes isolated by the methods of the invention such
as for example, TGF-.beta.1, cyclin D1, p21, p34, p53, lysyl
oxidase, caveolin, actin binding protein, ubiquitin activating
enzyme E1, nmb, and .alpha. actinin 3. Genetic ablation (gene
knockout) refers to a process of silencing the expression of a
particular gene in a cell. The silencing process may include for
example, gene targeting or anti-sense blocking. Gene targeting
refers to a process of introducing a nucleic acid construct into a
cell to specifically recombine with a target gene. The nucleic acid
construct inactivates the targeted gene. Inactivation may be by
introduction of termination codons into a coding region or
introduction of a repression site into a regulatory sequence.
Anti-sense blocking refers to the incorporation into a cell of
expression sequences which directs the synthesis of anti-sense RNA
to block expression of a target gene. Anti-sense RNA hybridizes to
the mRNA of the target gene to inhibit expression.
[0046] Another embodiment of the invention is directed to analysis
of a cell line before it is used as starting material to isolate
metastatic genes in a particular pathway. Analysis is useful in
identifying cells, and consequently sequences specific to these
cells, which are particularly susceptible or resistant to
metastatic transformation. For example, a cell highly predisposed
to metastasis may be especially sensitive for detecting metastatic
genes. Conversely, a cell showing high resistance to metastasis can
be used to isolate especially potent metastatic sequences. One
method to analyze susceptibility to metastasis is to determine the
cellular response to growth factors or growth inhibitors. Briefly,
a control population and a test population of cells are exposed to
a growth factor or a growth inhibitor and the cellular response
(e.g. proliferation, metabolism) recorded. Cells showing abnormal
responses to the growth factor or growth inhibitor may be used as
the starting material for metastatic gene isolation. Cellular
responses include changes in the rate of cellular division (e.g.
thymidine uptake), changes in the expression of RNA and/or
proteins, changes in cellular localization or modification of
patterns of RNA and/or proteins, and changes in the rate of uptake,
release or metabolism of nutrients.
[0047] Especially potent or weak metastatic genes may be detected
by treating and analyzing the metastatic potential of different
cells and selecting a suitable cell type as the starting material.
For example, cells may be treated with myc, ras and p53 or
combinations thereof, and analyzed for cyclin D1 expression which
is shown to correlate with metastasis. The gene expression pattern
of cyclin D1 in MPR correlates with that of human prostate tumors
analyzed with stains specific for cyclin D1 expression. Normal
human tissue shows no cyclin D1 expression or staining. Moderately
differentiated prostate cancers with dispersed or focal positive
staining show moderate staining. Advanced, poorly differentiated
prostate cancer shows strong nuclear as well as cytoplasmic
staining implying strong expression of cyclin D1. After treatment
with myc, ras or p53, cyclin D1 expression shows correlation with
the metastatic potential of the cell. Thus, cyclin D1 expressing
cells are a source of cells with high metastatic potential.
Conversely, cells with low cyclin D1 expression are a source of
potentially metastatically resistant cells.
[0048] This method may be adjusted for the isolation of metastatic
sequences expressed along a particular developmental or
differentiation pathway by combining the various treatment and
analytical techniques. For example, a mammalian cell may be
genetically ablated for TGF-.beta.1, Cyclin D1, p53, lysyl oxidase,
caveolin, actin binding protein, ubiquitin activating enzyme E1,
nmb, .alpha. actinin 3, or p34. The genetically altered cell is
used in an in vivo mouse prostate reconstitution (MPR) model.
Metastatic and nonmetastatic cells isolated from the MPR may be
analyzed directly or after induction with an agent such as the
TGF-.beta. gene or its product. Analysis involves the use of
differential display polymerase chain reaction to identify
differentially expressed bands. Sequences identified may be used
for subsequent gene ablation, transformation or differential
analysis.
[0049] Cell types useful for the identification of metastatic
sequences related to prostate cancer include cells and cell lines
of the fetal prostate lineage from normal or transgenic animals.
Prostate cells may be derived from normal prostates or from
reconstituted prostate tissue. One method of generating
reconstituted prostate cells is to isolate fetal prostate tissue
and micro dissect the fetal prostate epithelium away from fetal
mesenchyme. Fetal prostate epithelium may be genetically
manipulated before reassociation with fetal mesenchyme. Genetic
manipulation involves treatment or transfection with a metastatic
agent or nucleic acid sequence that affects neoplastic or
metastatic potential of the cell. Reassociation of fetal epithelium
and mesenchyme is performed by implanting epithelial tissue within
a pocket of mesenchymal tissue. After manipulation, cells are
reimplanted into a mammalian host in a similar manner as other
cells, such as reimplantation into or under the renal capsule.
[0050] The metastatic potential of a cell may be altered, for
example, by gene ablation with a sequence specific for a recessive
oncogene. Recessive oncogenes are those genes which encode products
which can suppress oncogenesis and metastasis. A gene ablation
sequence can be designed to specifically suppress a recessive
oncogene. Ablation may include pre-transcriptional inhibition such
as homologous recombination with endogenous recessive oncogenes and
post transcriptional inhibition such as the expression of
anti-sense oncogenes to suppress translation. Gene ablation
sequences may be targeted towards well known recessive oncogenes
such as, for example, the retinoblastoma gene (Rb) or Bc1. Other
candidates for ablation include metastatic genes previously
isolated by the invention such as, for example, TGF-.beta.1, cyclin
D1, p21, P34, p53, lysyl oxidase, caveolin, actin binding protein,
ubiquitin activating enzyme E1, nmb and .alpha. actinin 3. The
effects of ablating a recessive oncogene may include oncogenesis
and metastases.
[0051] Genetic ablation (gene knockout) may be performed after a
cell is selected for use or a cell already comprising a genotype
with the proper genetic ablation may be selected. Cells already
comprising gene ablation may be acquired from a cell depository,
from other laboratories or from a transgenic animal. A transgenic
animal which comprises a genetically ablated gene will carry the
genotype in every cell in its body. Thus, any tissue from a
transgenic animal may be used as the starting material.
[0052] After selection, the cell is transformed with an oncogenic
sequence. An oncogene is a sequence which can predispose, or induce
the cell into a pre-neoplastic or neoplastic condition or otherwise
enhance the metastatic potential of the cell. Oncogenes can be
classified into two types, dominant oncogenes and recessive
oncogenes. One or more dominant oncogenes can confer a neoplastic
or preneoplastic phenotype to a cell when transfected. One or more
recessive oncogenes, when silenced, may also confer a neoplastic or
preneoplastic phenotype. Gene silencing is performed by
transfecting cells with nucleic acids which cause genetic ablation
or by anti-sense suppression. While any oncogene may be used, the
preferred oncogenes are those that are normally associated with
prostate tumors. Thus, TGF-.beta.1, Cyclin D1, p53, lysyl oxidase,
caveolin, actin binding protein, ubiquitin activating enzyme E1,
nmb, .alpha. actinin 3, or p34 are among the preferred genes. Other
oncogenes which may also be used include abl, ahi, akt, bcl, crk,
dsi, erb, ets, evi, fes/fps, fim, fis, fgr, flv, fms, fos, gin,
gli, int, jun, kit, mas, lck, met, mil/raf, mis, mlv, mos, myb,
myc, neu, onc, pim, raf, ras, rel, ros, seq, sis, ski, spi, src,
tcl, thy, trk, and yes. Metastatic-specific genes may be used
individually or in combination with other oncogenes.
[0053] Many oncogenes represent members of multigene families or
homolog families. Members of a multigene family or a homolog of an
oncogene may also be used. Two genes are homologs when they encode
for proteins which have very similar primary, secondary or tertiary
structures. Thus two genes may differ in nucleic acid sequence or
encoded peptide sequence and still be homologs to each other
because the confirmation of the encoded polypeptides have similar
spatial folding. Homologs genes may have arisen from divergent
evolution from a common ancestor or from convergent evolution from
different ancestral genes. Convergent evolution refer to the effect
of two genes evolving under similar evolutionary pressure to encode
proteins which are similar to each other.
[0054] Some oncogenes, such as ras, are oncogenic when mutated.
Other oncogenes, such as myc, are oncogenic when overexpressed. In
using oncogenes for transfection, mutated or overexpress forms of
the oncogenes may also be used. Another method to predispose a cell
to metastasis and neoplasia is to ablate recessive oncogenes.
Ablation may include pre-transcriptional inhibition such as
homologous recombination with endogenous recessive oncogenes and
post transcriptional inhibition such as the expression of
anti-sense oncogenes to suppress translation.
[0055] The effects of oncogenes are at least additive and often
synergistic. Thus, dominant oncogenes may be transfected together
or multiple recessive oncogenes ablated together for a stronger
effect. Furthermore, both methods may be combined and dominant
oncogene transfection may be accompanied by recessive oncogene
ablation.
[0056] Genetic manipulation of the starting material may be
accomplished by well-established laboratory procedures. Numerous
methods, either direct or indirect, have been developed for
cellular transfection. Mammalian cells may be transfected by a
variety of techniques, all of which are well-known to those of
ordinary skill. Direct methods involve the introduction of genetic
material into the nucleus of a cell by injection. These techniques
include high velocity projectile injection, microinjection, and
electroporation. Indirect methods involve the active or passive
uptake of the genetic information by the cell. Indirect techniques
include transduction with recombinant vectors, and chemical or
physical treatments such as calcium phosphate uptake, lipofection
or dextran sulfate transfection. Chemical techniques rely on
chemical carriers to introduce nucleic acids into a cell. These
methods, for example, utilize unilamellar phospholipid vesicles
(e.g. liposomes) loaded with DNA (or RNA). The approach relies on
the fusion of the DNA containing vesicles with the plasma membrane
of the recipient cells. After entry, DNA traverses the cytoplasm
and enters the nucleus. Another lipofection technique uses a
synthetic cationic lipid such as
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA). DOTMA spontaneously associates with nucleic acids and
forms unilamellar vesicles upon sonication. Genetic material is
incorporated into these vesicles and subsequently transfected into
the cell. Calcium phosphate co-precipitation involves mixing of
purified nucleic acid with buffers containing phosphate and calcium
chloride which results in the formation of a fine precipitate.
Presentation of this precipitate to cells results in incorporation
of the nucleic acid into cellular genome. Other chemicals, such as
DEAE dextran or polybrene, when present in media with nucleic
acids, can also cause the transfection of mammalian cells.
[0057] Physical methods of transfection rely on electric fields,
needles and particles to enable nucleic acids to traverse the
cellular membrane. Electric field mediated DNA transfection,
commonly called electroporation, is based on the principle that
membranes, when subjected to an electric field, undergo a
reversible breakdown resulting in pores large enough to permit the
passage of nucleic acids. In micro-projectile mediated gene
transfer, micro-projectiles of subcellular dimensions are coated
with nucleic acid and propelled at high velocity into a cell using
a particle gun. The nucleic acid is introduced into the nucleus
directly when the particles impinge upon the nucleus. In
microinjection, nucleic acid is injected directly into the nucleus
of a cell with a needle. Lasers have also been used to introduce
minute holes in cellular membranes to allow introduction of nucleic
acids. All these methods may be used for transfection and the
selection of the method will depend on the cell type, the desired
transfection efficiency and the equipment available.
[0058] The efficiency of transfection, the fraction of live cells
transfected with the oncogene, may be monitored and enhanced by the
co-transfection of a selectable marker. If a marker is
co-transfected with a genetic construct, positively transformed
cells may be separated from nontransformed cells by chemical
selection. The efficiency of transfection will be increased in most
cases because the chemicals will selectively kill nontransfected
cells. The number of transfected cells may also be monitored by
analyzing the degree of chemical resistance of the transfected
cells. Markers commonly used for selection purposes include, for
example, nucleic acids encoding dihydrofolate reductase,
metallothionein, CAD, adenosine deaminase, adenylate deaminase, UMP
synthetase, IMP 5'-dehydrogenase, xanthine-guanine
phosphoribosyltransferase, mutant thymidine kinase, mutant
HGPRTase, thymidylate synthetase, P-glycoprotein 170,
ribonucleotide reductase, glutamine synthetase, asparagine
synthetase, arginosuccinate synthetase, ornithine decarboxylase,
HMG-CoA reductase, N-acetylglucosaminyl transferase, theronyl-tRNA
synthetase, sodium or potassium dependent ATPase or derivatives or
mutants of these nucleic acids. Markers may be used individually or
in combination. Chemicals useful for selection include
methotrexate, cadmium, PALA, Xyl-A, adenosine, 2'-deoxycoformycin,
adenine, azaserine, coformycin, 6-azauridine, pyrazofuran,
mycophenolic acid, limiting xanthine, hypoxanthine, aminopterin,
thymidine, 5-fluorodeoxyuridine, adriamycin, vincristine,
colchicine, actinomycin D, puromycin, cytocholasin B, emetine,
maytansine, Bakers' antifolate, aphidicolin, methionine
sulfoximine, .beta.-aspartyl hydroxamate, albizziin, canavanine,
.alpha.-difluoromethylornithine, compactin, tunicamycin,
borrelidin, ouabain, and derivatives and analogs and combinations
of these chemicals. Some chemicals, such as methotrexate, may be
used individually while other chemicals, such as HAT (hypoxanthine,
aminopterin and thymidine), need to be used in combination to be
effective.
[0059] Alternatively, or in addition to transfecting the mammalian
cell may be treated with an agent, either before or after
transfection, that alters the expression of the cell's nucleic
acids. Treatment may comprise contacting the cells with one or more
agents which affect the neoplastic character (e.g. neoplastic
agents; phorbol esters), metabolization (e.g. metabolic agents),
metastatic character (e.g. metastatic agents), differentiation
(e.g. differentiation agents; retinoic acid), activation or
proliferation (e.g. growth factors) of the cell. Agents which can
alter gene expression include chemicals such as benzanthracene
(BA), dimethyl benzanthracene (DMBA) or 5-azacytidine.
Alternatively, treatment may also comprise altered conditions such
as hypoxia which involves subjecting a cell to a reduced oxygen
content, exposable to radiation or other stresses to the cell.
[0060] The host animal is preferably the same species as the cell
to be implanted. In cases of xenogeneic transplants, the host may
be immunocompromised by genetically or by treatment with drugs such
as immunosuppressants. A host may be immunocompromised genetically
by breeding such as with nude mice or severe combined
immunodeficient (SCID) mice. A host may also be immunocompromised
by chemical or irradiation methods. An additional route to
immunocompromise a host is to use transgenic technology to
introduce an immunosuppressing gene or to introduce a foreign
antigen gene. An immunosuppressing gene is a gene that affects the
efficiency of the immune system such as a gene which inhibits the
formation of cells of the B cell or T cell lineage. A foreign
antigen gene, when expressed may cause the host to tolerate the
antigens in a xenogeneic transplant and not mount an immune
response.
[0061] The preferred site for reimplantation, the primary site, may
be any site receptive to implantation. Some sites are preferred for
implantation purposes such as the renal capsule, the testes, the
prostate and the ovaries. A number of reasons may exist for
choosing a site that they may include ease of implant, similar
tissue type, immunoprivileged position and ease of inspection.
Metastasises may migrate from the primary tumor to any secondary
site on an animal including the usual sites consisting of lung,
kidney, liver, lymph nodes, testis, spleen, ovaries and mammary. To
avoid histocompatibility problems, the implant may be placed into a
histocompatible host animal. Such problems are generally avoided if
the host animal is syngenic. Alternatively, a non-histocompatible
host may be used if the host can be made immunotolerant.
Immunotolerant hosts may be a transgenic animal, or an
immunocompromised animal. Immunocompromised animals may be derived
from established mouse lines such as nude mice of severe combined
immune deficiency (SCID) mice, or by laboratory treatments such as
radiation, chemical, transgenic treatment, pharmaceutical or
genetic targeting. Hosts may also be transgenic or
immunocompromised animals or genetically matched to the mammalian
cells to be introduced. Sufficiently immunosuppressed animals can
be made tolerant to xenogeneic transplants.
[0062] After implantation the host animal is maintained under
normal conditions to develop metastases. Alternatively, the host
animal may be subjected to an altered treatment or environmental
condition to stimulate or repress metastasis or induce other
cellular functions. In metastasis, a sub-population of cells of the
implantation site invade and establish one or more secondary
colonies in the host animal. The behavior of the implanted cell
will depend on the cell type, the transfected sequence and the
implantation location. Typical secondary sites for metastatic
colonies include lung, kidney, liver, lymph nodes, brain, testis,
spleen, bone, ovary, skin and mammary tissue. Metastatic
development times vary from days to weeks even months. Cells with a
high metastatic potential tend to progress to metastasis quickly
while cells with a low metastatic potential may require very long
periods of time that span significant portions of the life span of
the animal.
[0063] The host animal may be analyzed for metastatic development
weekly, from one week to 20 weeks to six months, nine months or one
year after implantation. For animals with longer life spans such as
sheep, the animal may be inspected yearly from one year on up to
ten years for metastatic tumors. Metastases can be detected by
examinations such as palpation, biopsy, imaging, exploratory
surgery, CAT scans, autopsy, X-ray and direct observation. In
addition, tissue samples may be taken surgically from the host
mammal and subjected to histological or other examination for the
detection of metastases.
[0064] Expressed sequences include mRNA, rRNA, hnRNA, DNA, cDNA and
any nucleic acid sequence that is expressed in the cell. These
sequences may be amplified by in situ techniques or by purification
of nucleic acid from collected cells. Expressed sequences may be
obtained by extracting nucleic acids from cells before
implantation, at the primary site or at the secondary site. Cells
collected at these sites may optionally be cultured for a time
before nucleic acid extraction. The effects of treatment with gene
expression modifying agents or environmental conditions can be
ascertained by collecting cells before and after treatment.
Treatment may be applied to the cells while the cells are in the
host mammal or after the cells are excised and in culture. Nucleic
acids are collected from cells using techniques that are well known
to those of ordinary skill in the art.
[0065] Expressed sequences may be used directly for polymerase
chain reaction (PCR) analysis using, for example, the technique of
reverse transcriptase polymerase chain reaction (RT-PCR).
Alternatively, RNA may be enriched for mRNA using a poly-A RNA
enrichment method. Numerous poly-A RNA enrichment methods exist and
are commercially available. Techniques used for poly-A RNA
enrichment include oligo-dT columns, oligo-dT magnetic beads, and
oligo-dT cellulose. RNA may be further processed into cDNA before
analysis by reverse transcription using reverse transcriptase. The
cells or the extracted nucleic acid may be preserved, such as by
freezing, and analyzed at a later time.
[0066] Differential display polymerase chain reactions (DD-PCR) are
performed on the expressed sequences using two variable primers
which may contain the same or entirely different sequences or an
anchor primer and a variable primer. If an anchor primer is used,
one anchor primer and one variable primer create a single or a
single set of reaction products for each reaction. A complete
profile may include 25 or more different PCR reactions per sample
wherein each PCR reaction is performed with the same anchor primer
and a different variable primer. DD-PCR may also be performed using
anchor and variable primers which contain the same sequence.
Whether a particular reaction is used depends on whether a
difference exists between the products of two PCR reactions using
the same primers. When a significant difference exists between the
expression sequences amplified, one pair of PCR reactions may be
sufficient and informative.
[0067] Anchor primers are preferably oligonucleotides with a poly-T
sequence at the 5' terminals and a dinucleotide selected from the
group consisting of AA, AG, AC, AT, GA, GG, GC, GT, CA, CG, CC and
CT at the 3' terminals. The length of the poly-T sequence is
typically between about five to about 30 bases in length and
preferably between about ten to about twenty nucleotides in length.
The total length of the anchor primer can vary greatly for each
experiment but is preferably between about seven to about 32 and
more preferably between about twelve and about 22. Differential
diagnostic polymerase chain reaction may also be performed using an
anchor primer of any sequence and a length between about five to
about 30, preferably between about five to about 20 and more
preferably between about seven to about 12 bases. The variable
primer may comprise a random sequence, or a specific sequence.
Variable primers preferably are oligonucleotides with a length
between about five to about 30, preferably between about five to
about 20, and more preferably between about seven to about twelve
bases in length.
[0068] To enhance detection of the PCR product, the anchor primer
or the variable primer or both may comprise a detectable moiety.
Examples of detectable moieties include radioactive moieties,
phosphorescent moieties, magnetic moieties, luminescent moieties,
conjugatable moieties or other detectable moiety. A plurality of
detectable moieties may be used to enhance detection or to simplify
data analysis. Other detectable moieties include conjugatable
moieties and molecules which can bind specifically to other
molecules which are themselves detectable. Examples of conjugatable
moieties include avidin, streptavidin, biotin, antibody, antigen,
cell adhesion molecules and other molecules with similar
activities.
[0069] Detectable moieties are preferably labeled nucleotides. A
nucleotide may be any natural or synthetic nucleotide or nucleotide
analog capable of incorporation into an elongation reaction in a
polymerase chain reaction. Labeled nucleotides include nucleotide
triphosphates labeled with one or more radioactive atoms such as
.sup.32P, .sup.33P, .sup.3H, .sup.14C, .sup.125I and .sup.35S.
[0070] Products of the DD-PCR reactions are compared to detect the
metastatic sequences. A dominate metastatic gene product is
expected to be present in a metastatic tumor while a recessive
metastatic gene product is present in a non-metastatic tumor.
Comparisons can be performed between expressed sequences from cells
at secondary sites with cells at any stage in the method, including
untreated, transfected or treated mammalian cells, implanted cells,
or cells from the primary site in the host animal. DD-PCR products
may be analyzed by any method which reliably compares the products
of two polymerase chain reactions. Typical analytical methods used
for this purpose include polyacrylamide gel electrophoresis,
capillary electrophoresis and high pressure liquid chromatography
(HPLC). Product produced from DD-PCR may be analyzed in
double-stranded form or single-stranded form. When the products of
the DD-PCR reaction are labeled the sizes and distribution of the
products may be monitored and analyzed by following the labels
using a radiation monitor or by autoradiography. For example,
DD-PCR performed in the presence of radioactive primers or
nucleotide triphosphates can be analyzed by gel electrophoresis, by
capillary electrophoresis, or by HPLC. Products are easily
monitored by the presence of radioactivity.
[0071] Another method for analyzing and isolating metastatic
sequences is to sequence the amplified nucleic acid sequences.
Sequencing may be performed using standard methods well known to
those of ordinary skill in the art. The resulting sequence may be
compared to a sequence database created or well-known, such as
Genbank, for identification or for locating homologs. The
sequencing information may be used to calculate the physical
characteristics of the nucleic acids such as melting temperature
and secondary structure. The primary sequence and the physical
characteristic may be used to synthesize optimal nucleic acid
probes for the detection or staging of metastasis or conditions
that are predictive of the presence or absence of the metastatic
condition.
[0072] Another embodiment of the invention is directed to a method
for identifying a metastatic sequence. A mammalian cell is
pretreated with a metastatic agent to form a population of cells
predisposed to metastasize. The treated cells are introduced into a
host mammal at a primary site. The host animal is maintained for a
period of time sufficient to develop a metastasis at a secondary
site. Expressed sequences of cells at the primary site and cells at
the secondary site are treated with a genotoxic agent or subjected
to genotoxic conditions. Expressed sequences of the treated cells
are amplified by differential display polymerase chain reaction and
compared with untreated cells from any previous step to identify
the metastasis sequence.
[0073] The metastatic agent may be a chemical compound, a nucleic
acid or a protein that alters the metastatic potential of a cell or
relates to or is associated with the metastatic process. Chemical
compounds include retinoids such as 4-hydroxyphenyl (4HP). Other
agents include the proteins TGF-.beta.1, Cyclin D1, p21, p34, p53,
lysyl oxidase, caveolin, actin binding protein, ubiquitin
activating enzyme E1, nmb or .alpha.-actinin 3, or their respective
genes. The metastatic agent may be a metastatic stimulant or a
metastatic suppressant. Metastatic stimulants may be used to
enhance the sensitivity of the metastasis sequence detection
method. Conversely metastatic suppressants may be used to decrease
the sensitivity of the method enabling the selective identification
of potent metastatic sequences or sequences specific to a
particular tissue type or metastatic disorder. Treatment may
comprise direct contact with the metastatic agent or incubation for
a period of time. Metastatic agents enhance the metastatic
potential of the implanted cells and increase the sensitivity and
the speed of the overall method.
[0074] The cells at the primary site and the metastatic cells at
the secondary site may be treated with a genotoxic agent in vivo or
in vitro. In vivo treatment may comprise injecting genotoxic agents
directly into the host mammal or specifically applying the agent
with, for example, topical formulations. The cells at the primary
site and the secondary site may also be isolated from the host
animal and treated with the genotoxic agent in culture. Genotoxic
agents are chemical compounds, nucleic acids or proteins that alter
gene expression by affecting the nucleic acid genome directly by,
for example, chemical modification, or indirectly by, for example,
altering components associated with gene expression. Such agents
include, for example, benzanthracene (BA), dimethyl benzanthracene
(DMBA) and 5-azacytidine, and may include metastatic agents as
well. In addition to or in place of genotoxic agents, the cells may
be treated to hypoxic conditions or radiation to alter gene
expression. Metastatic sequences identified in these methods may be
specific for particular genotoxic agents or conditions.
[0075] Another embodiment of the invention is directed to the use
of a host animal with an altered genotypic or phenotypic
predisposition for metastases. A host animal may be screened for
endogenous expression of a metastasis gene. Particularly useful
metastatic sequences include TGF-.beta.. A host animal with reduced
levels of a metastatic gene product may be used to isolate novel
metastatic genes. Host animals may be screened for reduced levels
of metastatic gene expression. In addition, transgenic technology
may be used to ablate a metastatic gene in the germline of a host
animal.
[0076] The function of the metastatic sequences identified by the
methods of the invention may be ascertained through differential
expression pattern. For example, a dominant metastatic gene will be
present in a metastatic cell while a recessive metastatic gene is
present in a non-metastatic cell. Metastatic sequences may be
detected as bands which are present in the DD-PCR of metastases
isolated in secondary sites and absent from DD-PCR products of
primary cells. These sequences may be dominant metastatic genes
whose expression is directly responsible for metastasis, or they
may be metastasis-associated genes whose expression correlates with
metastasis. Either are useful for therapy and/or diagnosis.
Conversely, DD-PCR bands which are present in primary site tumors,
but absent in secondary metastatic sites, may be dominant
metastasis suppression genes. Dominant metastasis suppression genes
comprise genes whose expression suppresses metastasis while
nonmetastatic genes comprise genes whose expression correlates with
non-metastatic tissue. Genes which are highly correlative with
either the metastatic phenotype or the non-metastatic phenotype may
be isolated. Isolation can be performed by cutting the appropriate
nucleic acid in the band of a polyacrylamide gel or by collecting
the appropriate fraction in an HPLC or capillary electrophoresis.
The nucleic acid may be cloned into a plasmid vector, and
sequenced, or synthetically prepared.
[0077] Another embodiment of the invention is directed to a method
for identifying sequences in a metastatic pathway which are
responsive or unresponsive to extracellular signals. Such sequences
may be used in therapy and diagnosis of metastatic disorders.
Implanted cells or cells from a primary site and cells from a
secondary site are treated with extracellular signals. RNA
sequences from the treated cells are compared with RNA sequences of
the untreated cells. Treated cells and untreated cells may be
derived from a short term or long term in vitro culture of primary
tumor and malignant tumors. Alternatively, a part of a primary
tumor and a part of a malignant tumor may be collected before the
animal is treated with an extracellular cytokine or other factor.
Long term cultures, or cell lines of primary and malignant cells
may also be used as recipients of extracellular growth signal
treatment. Suitable signals for each experiment will depend on the
cell type. Generally, growth factors, lymphokines, inhibitory
factors, migratory factors or hormones may be used. Factors
previously isolated by commercial or methods of the invention and
factors associated with or causative or suppressive of metastasis
are preferred.
[0078] Thus, transforming growth factor .beta.1 (TGF-.beta.1) may
be used to treat cells before DD-PCR analysis. Proteins encoded by
the genes isolated by this method are especially useful for the
treatment of cells for the isolation of additional sequences. The
identification of one sequence responsive to the extracellular
signal pathway allows for the identification of additional genes
upstream and downstream from that sequence.
[0079] Another embodiment of the invention is directed to
metastatic sequences identified by the methods of the invention.
Metastatic sequences are sequences associated with the presence or
absence of a metastasis or related to the metastatic process.
Metastatic sequences can be used in the therapeutic treatment of
metastases. Metastatic-related sequences include dominant
metastatic sequences, recessive metastatic sequences,
metastasis-associated sequences, dominant oncogenes, recessive
oncogenes and cell cycle genes. These genes encode, for example,
proteins involved in cell cycle, signal processing, DNA
replication, growth regulation, inter and intra cellular signaling
transcription control and translation control.
[0080] Metastatic sequences identified by the methods of the
invention were obtained by the following screens for metastatic
sequences: (1) Met; (2) p53 and (3) T TGF-.beta.1. The screen
designated Met was performed by comparing RNA extracted from a cell
line established from a primary tumor and a cell line derived from
an associated metastatic tumor-derived cell line. The screen
designated p53 was a screen for dominant or recessive metastatic
sequences in which cells of a murine prostate cancer cell line were
infected with either an adenoviral vector expressing wild-type p53
or mock-stimulated with E1 deleted adenoviral vector prior to
starting the screen. The TGF-.beta. designation indicates that the
cells were pre-treated with or without TGF-.beta. prior to the step
in the screen in which cellular RNA is extracted. Sequences
isolated by the methods described herein are useful in the
treatment and detection of metastasis and other disorders.
Disorders whih may be treated comprise both malignant or
nonmalignant disorders. Examples of nonmalignant disorders include
benign enlargement of the prostate, hyperplasia, such as nodular
hyperplasia, hypertrophy, such as benign prostatic hypertrophy and
dysplasia. Malignant disorders include prostate cancer, breast
cancer and other non-benign cancers.
[0081] Another embodiment of the invention is directed to nucleic
acids which comprise a sequence identified by the method of the
invention such as, for example, caveolin, ABP280 (actin binding
protein 280), lysyl oxidase, the gene encoding p99 (clone 99) and
the nmb gene (clone 29). Nucleic acids comprising a sequence
corresponding to any of these genes may be used in treatment of
neoplastic disorders and malignant tumors, or in diagnostic kits
for screening biological samples for the presence or absence of
metastasis or metastatic potential. The nucleic acid may be DNA,
RNA or PNA and may comprise additional sequences such as a promoter
sequence for expression of a sense or antisense message,
recombination sequences for gene targeting, selectable markers for
transfections, or replication origins for passage in a prokaryotic
or eukaryotic host such as animal cells, bacteria or yeast.
Treatment may involve using the sequences in gene therapy,
including gene ablation, gene expression and antisense suppression.
Diagnosis may involve genotypic analysis of samples to determine
the existence and expression levels of the expressed sequence.
[0082] Characterization of three metastatic sequences identified by
the methods described herein reveal different expression patterns
in metastatic cells. Also, two of these sequences were encoded for
by known genes, while the third is the product of a novel gene.
These expression patterns suggest that the genes encoding the
metastatic sequences represent different classes of metastatic
genes. One such gene, the lysysl oxidase gene, was found to be
repressed in metastatic tissues as compared to expression levels in
primary tumor or normal tissues, suggesting lysyl oxidase may
function in a tumor suppressing pathway. Contrasting results were
obtained for a second gene, the caveolin gene. Unlike Iysyi
oxidase, caveolin levels were found to be increased in metastatic
tissues suggesting a role for caveolin in metastasis-associated
processes.
[0083] A third metastatic sequence was identified and designated
p99 (FIG. 1). This sequence contains the predicted peptide sequence
of the p99 protein. A Northern blot of p99 expression in mouse
normal prostate-derived, mouse prostate primary tumor-derived and
mouse metastasis-derived cell lines showed that expression of p99
was found to be induced by the tumor suppressor p53 and, similar to
caveolin levels, p99 levels were found to be up-regulated in
metastatic prostate cells as compared to primary tumor-derived
cells and cells from normal prostate tissue.
[0084] Although p99 has not previously been associated with
metastasis, the deduced amino acid sequence of p99 shares up to 44%
homology with several members of the epithelial membrane protein
family having four transmembrane domains. Northern blot analysis
show up-regulation of p99 in response to gamma radiation and DNA
damaging agents in a p53-dependent manner with relatively high
expression in mouse urogential tissues including prostate, bladder,
kidney and seminal versicles. In addition, high p99 p53-dependent
expression levels were detected in liver, colon and heart.
Expression of p99 as an N-terminal fusion protein with EGFP
demonstrates perinuclear localization of the fusion protein, with
accumulation in the endoplasmic reticulum and Golgi apparatus.
FIGS. 2-8 summarize the isolation and characterization of
metastatic sequences identified using the herein described
methods.
[0085] Specifically, FIG. 2 contains a compilation of the sequences
obtained using the described methods for identifying dominant and
recessive metastatic sequences. The following information is
provided in FIG. 2 for each clone: the name/number of the clone,
the length of the clone, the degenerate oligonucleotide primer used
in the differential display PCR, and the type of screen--Met, p53
or TGF-.beta.- from which the clone was derived.
[0086] The screen designated Met was performed by comparing RNA
extracted from a cell line established from a primary tumor and a
cell line derived from metastatic cells. The screen designated p53
was a screen for dominant or recessive metastatic sequences in
which cells of a murine prostate cancer cell line were infected
with either an adenoviral vector expressing wild-type p53 or
mock-stimulated with E1 deleted adenoviral vector prior to starting
the screen. 148-1 cells are p53 null and carry integrated
retrovirally expressed ras and myc oncogenes in their genome. The
TGF-.beta. designation indicates that the cells were pre-treated
with or without TGF-.beta. prior to the step in the screen in which
cellular RNA is extracted.
[0087] FIG. 3 contains data obtained through the initial
characterization of some of the sequences resulting from the Met
screen. The following data, as available, is shown in FIG. 3: the
name/number of the clone, the cell line from which the clone was
derived; the degenerate oligonucleotide primer used in the
differential display (DD) polymerase chain reaction (PCR), size of
the DD-PCR product, the most homologous murine gene or sequence,
the most homologous human gene or sequence, whether the clone is
transcriptionally up- or down-regulated in the indicated cell line
as determined by Northern Blot analysis, and whether the clone is
transcriptionally up- or down-regulated.
[0088] FIG. 4 contains data obtained through the initial
characterization of some of the sequences (also) resulting from the
Met screen. The following data, as avalilable, is provided in FIG.
4: the primer used in the DD-PCR and the size of the resulting
product, the most homologous murine gene or sequence, the most
homologous human gene or sequence, whether the clone is
transcriptionally up- or down-regulated in the indicated cell line
as determined by Northern Blot analysis.
[0089] FIG. 5 contains data obtained through the initial
characterization of some of the sequences (also) resulting from the
Met screen. The following data, as available, is provided in FIG.
5: the primer used in the DD-PCR and the size of the resulting
product, the most homologous murine gene or sequence, the most
homologous human gene or sequence, whether or not the clone is
transcriptionally up- or down-regulated in the indicated cell line
and performed in duplicate (screen 1 and screen 2).
[0090] FIG. 6 contains data obtained through the initial
characterization of some of the sequences (also) resulting from the
p53 screen. The following data, as available, is provided in FIG.
6: the primer used in the DD-PCR and the size of the resulting
product, the most homologous murine gene or sequence, the most
homologous human gene or sequence, whether or not the clone is
transcriptionally up- or down-regulated in the indicated cell line
as a result of a p53 expression after 12 (+12) or 24 (+24) hours
and performed in duplicate (screen 1 and screen 2).
[0091] FIG. 7 contains data obtained through the initial
characterization of some of the sequences (also) resulting from the
TGF-.beta. screen. The following data, as available, is provided in
FIG. 7: the primer used in the DD-PCR and the size of the resulting
product, the most homologous murine gene or sequence, the most
homologous human gene or sequence, whether or not the clone is
transcriptionally up- or down-regulated in the indicated cell line
as a result of a 12 hour treatment/exposure to TGF-.beta. and
performed in duplicate (screen 1 and screen 2).
[0092] FIG. 8 contains data obtained through the initial
characterization of some of the sequences resulting from screening
a phage library. The following data, as available, is provided in
FIG. 8: the primer used in the DD-PCR and the size of the resulting
product, the most homologous murine gene or sequence, the most
homologous human gene or sequence, whether or not the clone is
transcriptionally up- or down-regulated in the indicated cell line
and performed in triplicate (screen 1, screen 2 and screen 3), and
the number of the positive plaques obtained in the phage
screen.
[0093] The sequences identified by the methods of the invention
have numerous potential applications. For example, one embodiment
of the invention is directed to methods for treating a neoplastic
disorder comprising administering a pharmaceutically effective
amount of composition containing a nucleic acid having a sequence
identified according to the methods of this invention, its
expression product or fragments of either. The nucleic acid may be
in the form of a sense or anti-sense single-stranded or
doublestranded nucleic acid. The nucleic acid may be administered
by injection, pulmonary absorption, or topical application and
delayed release. The nucleic acid may be combined with a
pharmaceutically acceptable carrier such as water, alcohols, salts,
oils, fatty acids, saccharides, polysaccharides administered by
injection, pulmonary absorption, topical application or delayed
release. More than one carrier may be used together to create a
pharmaceutical with desirable properties.
[0094] Treatment may involve gene replacement, gene targeting,
anti-sense inhibition, gene expression or gene suppression. Gene
replacement involves replacing a copy of a defective gene with
another copy by homologous recombination. Gene targeting involves
the disruption of a cellular copy of a gene by homologous
recombination. Anti-sense inhibition exploits the specificity of
hybridization reactions between two complementary nucleic acid
chains to suppress gene expression. Cloned genes can be engineered
to express RNA from only one or the other DNA strands. The
resultant nucleic acid, which may be DNA, RNA or PNA, or another
synthetic sequence, hybridizes to the sense RNA and inhibits gene
expression. Gene expression and gene suppression involve the
introduction of genes whose expression actively inhibits neoplastic
transformation and metastasis.
[0095] Another embodiment of the invention is directed to a kit or
diagnostic aid useful for screening biological samples for
detection of metastasis, neoplasia or for the staging of a tumor
comprising sequences isolated according to the methods of the
invention. Kits contain nucleic acid such as DNA or PNA of a
sequence that hybridizes (or fail to hybridize for example with
competition assays) to metastatic-specific sequences in a
biological sample. Hybridization may be detected using conventional
detection reagents and methods well known to those of ordinary
skill in the art. The kit further comprises reagents and materials
useful in such kits, such as, for example, buffers, salts,
preservatives, and carriers, all of which are well known to those
of ordinary skill in the art. Such kits are useful for the analysis
of biological samples containing fluids and/or tissues to screen
for the determination of normal nonmalignant, neoplastic or
malignant cells. Kits may comprise additional reagents useful for
the extraction of nucleic acids from a tissue sample. Reagents for
analyzing the nucleic acid extracted from a tissue sample such as
polymerase chain reaction reagents and Southern blot reagents may
also be included.
[0096] Correlation of Caveolin Expression with Neoplastic
Disorders
[0097] One of the gene products found to be associated with
metastasis in the mouse model as well as in human prostate cancer
is caveolin, a major structural component of an organelle termed
caveolae. Caveolin is an integral membrane protein and a principal
component of caveolae. Caveolae are small invaginations at or near
the plasma membrane of most smooth muscle cells. Caveolin was
initially identified as a major v-src substrate for phosphorylation
in Rous sarcoma virus transformed chicken embryo fibroblasts
(Glenney, J. R., J. Biol. Chem. 264, 10163-20166, 1989). Recent
studies suggest that caveolae may function in some capacity as a
component of specific signal transduction pathways. In the only
published report which documents caveolin expression in
non-transformed and transformed cells, it was demonstrated that
both caveolin expression as well as caveolae are lost during
transformation of NIH-3T3 cells by v-abl, bcr-abl, middle T antigen
and activated ras (Koleske, A. J., Baltimore, D. and Lisanti, M. P.
Proc. Natl. Acad. Sci., USA 92, 1381-1385, 1995).
[0098] The association of caveolin with metastasis has been
confirmed in animal models and in human prostate and breast cancer
that increased levels of the caveolin protein are associated with
metastasis (Yang, G. et al., Clin. Can. R. 4:1873-1880, 1998).
Thus, the identification of caveoli as a metastatic sequence
provides another example validating the importance of the selected
animal model since many discoveries made using this model have also
been found to be relevant to human prostate cancer (Truong L D, et
al., Hum. Pathol. 24:4-9, 1993; Thompson T C, et al., J. Cell.
Biochem. 16(S):54-61, 1992; Eastham J A, et al., Lab Invest.
73:628-635, 1995; Williams R H, et al., Clin. Cancer Res.
2:635-640, 1996; Eastham J, et al., Clin. Cancer Res. 1:1111-1118,
1995; Yang G, et al., Clin. Cancer Res. 2:635-640, 1996; Stapleton
A M F, et al., Clin. Cancer Res. 3:1389-1397, 1997; Aihara M, et
al., Hum. Pathol. 25:797-801, 1994; Aihara M, et al., Cancer
75:522-529, 1995; Yang G, et al., Cancer 78:1267-1271, 1996).
[0099] The subsequent production of stably selected clones with
antisense caveolin resulted in a significant reduction in
metastatic activities relative to vector-control clones and parent
cell lines. Surprisingly, it has been discovered that tumors
produced by the antisense caveolin clones significantly regressed
in response to surgical castration in vivo. Eleven days following
androgen ablation, tumors derived from three independent antisense
clones regressed by approximately 30% relative to the wet weights
produced in either vector-control clones or parental clones which
did not respond to castration therapy under the same conditions.
The antisense caveolin tumors that responded to castration therapy
also demonstrated significantly increased levels of apoptosis
relative to either vector-control clones or parental cell lines.
The data indicates that reduction of caveolin levels not only
suppresses metastatic activity, but also restores androgen
sensitivity. Thus, caveolin-1 is a metastasis-related gene as well
as a candidate androgen resistance gene for prostate cancer in man.
Caveolin-2 forms a heterodimer with a caveolin-1 and is also
expected to be useful in the invention. Caveolin-3 is specifically
expressed in muscle cells and may be useful for certain types of
metastatic disorders and not others. These results are believed to
establish a new paradigm for understanding androgen refractory
disease and open the door for new innovations in prostate cancer
therapy.
[0100] Surprisingly, caveolin expression increases in metastatic
human prostate cells as compared to human primary prostate tumors.
The general observation that caveolin mRNA and protein levels are
consistently elevated in metastatic prostate cancer compared to
non-metastatic prostate cancer in both mouse and human prostate
cancer models is different from the caveolin gene transfer results
in NIH-3T3 cells. However the difference may be attributed to the
difference in the underlying mechanism of transformation to
tumorigenicity (soft agar assay) in NIH-3T3 cells compared with
those involved in carcinogenesis/metastasis in vivo using the mouse
prostate reconstitution model system.
[0101] As caveolin expression correlates with metastasis,
application of biological technologies designed to block the
activity of caveolin or the function of caveolae may have
therapeutic benefits for the treatment of neoplastic disorders such
as human prostate tumors. Specific treatment approaches using
caveolin may include the delivery of antisense or dominant negative
caveolin sequences using expression or viral vectors, as well as
the use of specific anti-caveolin antibodies. As the caveolin gene
is expressed specifically in metastatic cells, the gene promoter
(as well as the promoter of other metastatic-specific genes such as
p99 {which is selectively expressed in metastatic cells} and lysyl
oxidase {which is selectively suppressed in metastatic cells}) may
also be specifically induced in metastatic cells. Thus, by
functionally incorporating into normal cells a gene encoding a
toxic product downstream of the caveolin promoter, metastatic
disease should not develop because when and if it does, the toxic
product is expressed and the cells expressing it are destroyed.
This has been shown for the caveolin-1 promoter and is believed to
be true for the caveolin-2 and caveolin-3 promoters as well as
other metastatic-specific genes. It is well known to those of
ordinary skill in the art how to create genetic constructs that
take advantage of selective expression, or the lack thereof, in
metastatic cells for the treatment and prevention of metastatic
disease.
[0102] Additional approaches could also target the caveolae, but
are not specifically based on caveolin function. Additional protein
and non-protein components of caveolae could also be targeted for
abrogation or the local or systemic administration of a nutritional
or biological agent may also be used. For example, caveolae are
extremely rich in cholesterol and disruption or depletion of this
molecule may alter the function of caveolae.
[0103] Multiple genetic activities are involved in androgen
ablation-induced prostate regression, yet very little is known
regarding the rate limiting steps in the molecular cascade that
leads to regression, or the molecular basis of hormone resistance
in prostate cancer. A mouse model has been developed to identify
metastasis-related genes in prostate cancer. This model includes a
series of clonal cell lines derived from prostate cancer metastases
that developed in vivo using the mouse prostate reconstitution
(MPR) model system.
[0104] Specifically, panels of clonal cell lines were derived from
primary prostate tumors as well as metastases from the same animal
using the MPR mouse model system for prostate cancer metastasis.
Within this panel of cell lines, there are sets that are both
genetically and biologically matched such that the primary genetic
difference between these cell lines should be related to metastatic
activities. This is made possible, in part, by unique retroviral
integration sites that serve as markers for clonality. Using a
modified differential display-polymerase chain reaction (DD-PCR)
approach, numerous genes were identified that are related to
metastasis in human prostate cancer. For example, the caveolin gene
was found to be up-regulated in metastasis-derived cells relative
to their primary tumor-derived counterparts. (Yang, G. et al.,
Clin. Can. R. 4:1873-1880, 1998). The caveolin gene, first
identified as the major phospho-protein in src transformed cells
(Glenney J R, J. Biol. Chem. 264:20163-20166, 1989), was shown to
be the major structural component of caveolae. (Lisanti M P, et
al., Mol. Memb. Biol. 12:121-124, 1995). Caveolae are membrane
domains which may compartmentalize some single transduction
pathways, and recent identification of an integrin/urokinase
plasminogen activator receptor (uPAR)/caveolin complex (Wei Y, et
al., Science 273:1551-1555, 1996) provides a mechanistic framework
for linking alterations of caveolin expression with two potentially
important properties of malignant progression of
metastasis-integrin mediated cell-cell adhesion and uPAR mediated
proteolytic activity. With commercially available antiserum to
caveolin, increased protein levels in both mouse and human prostate
metastases have been confirmed. (Yang, G. et al., Clin. Can. R.
4:1873-1880, 1998). Further, the expression of caveolin in three
mouse metastatic cell lines has been experimentally suppressed by
expression of an antisense cDNA construct. Suppression of caveolin
does not reduce the growth potential of mouse prostate cancer
cells, but does reduce both the incidence of metastatic spread and
the actual tumor volume of lymph node metastases. (Yang, G. et al.,
Clin. Can. R. 4:1873-1880, 1998).
[0105] Surprisingly, it has been discovered that the antisense
caveolin clones have also acquired hormone sensitivity. Orthotopic
tumors that form from antisense caveolin clones, but not
vector-control clones or parental cells, regress by approximately
30% in wet weight following surgical castration. Further studies
confirm increased levels of apoptosis in antisense caveolin tumors
relative to non-regressing control tumors. Continuous massaging of
three antisense caveolin tumors in castrated male hosts resulted in
increased caveolin protein levels. This data indicates that
caveolin alone is responsible, in part, for the development of
hormone-refractory prostate cancer in the present model system.
These novel results should have a significant impact on prostate
cancer by: 1) spawning additional investigations that will reveal
the molecular pathway leading from the androgen ablation stimulus
to regression of prostate cancer in vivo; 2) leading to a more
complete understanding of the molecular basis of hormone-refractory
prostate cancer; and 3) ultimately leading to the development of
anti-metastasis therapy based on small molecule, immunological or
gene therapy approaches. Future studies will likely result in
tremendous therapeutic impact on men in that suppression of
caveolin or other molecules in the caveolin-androgen resistance
pathway will lead to effective anti-metastasis therapy.
[0106] As noted, an animal model has been developed for
experimental prostate cancer research, the mouse prostate
reconstitution (MPR) model. (Thompson, T C, et al., Cell
56:917-930, 1989). A unique and significant feature of this
"transgenic gland" model is that by manipulating the number and
types of initiating genetic events, it can be used to produce and
study every step of carcinogenesis, from premalignant changes
through the metastatic cascade. (Thompson T C, et al., Mol.
Carcinog. 7:165-179, 1993; Thompson T C, et al., Oncogene
10:869-879, 1995). Experiments using the MPR model have also
provided numerous cell lines that have been utilized extensively
for both in vitro and in vivo studies. Significant advances in
understanding prostate cancer metastasis have come from analyzing
cell lines that were derived from either a primary tumor or a tumor
at a metastatic site. Since the tumors are initiated as a result of
retroviral infection, primary tumor- and metastasis-derived cells
from the same animals are clonally related and may be compared,
based on unique retrovirus integration sites. Differential display
PCR has been adapted and refined to compare mRNA from clonally
matched cell lines and identify numerous genes whose expression
appears to be metastasis specific.
[0107] Surprisingly, it has been discovered that when tumor-bearing
animals are castrated following orthotopic injection of metastatic
cell lines with antisense caveolin (ABAC3, ABAC5, and BACS4) tumor
volume is reduced relative to sham surgery-treated animals or
castrated animals that received a testosterone implant. For
example, antisense caveolin has been shown to restore androgen
sensitivity in 3 independent cell lines, Orthotopic tumors were
initiated by injecting 5000 cells into the dorsal prostate of 129
male mice. Three days later the animals were castrated (cast) or
sham operated. Some animals also received implants of silastic
tubing containing testosterone proprionate (cast+T) or an empty
pellet (cast EP). Tumor volume was determined after 2 weeks. All
values for ABAC3, ABAC5, and BACS4 in the cast and cast EP groups
are significantly different from cast+T pellet and sham controls
(p<0.05).
[0108] In contrast to the antisense tumors, two parental cell lines
(148-1LMD and 151-2LMC) as well as control vector only clones
(ABH11, ABH14 and BHS3) did not respond to androgen withdrawal.
Significantly increased apoptosis is believed responsible, in part,
for the regression, and that in addition to growth suppression, a
reduction in metastasis also occurs following castration only in
the antisense clones.
[0109] The model system has been generated based on the finding
that high caveolin levels block castration-induced prostatic
regression, and reduction in caveolin levels appear to release this
block. This model is believed to normalize the nonrelevant
androgenic-stimulated gene activities. This model system involves
clonal cell lines in which caveolin levels have been selectively
reduced by stable antisense caveolin transfection as well as the
production of clonal vector control cell lines. When these cell
lines are injected orthotopically in vivo, allowed to produce
tumors (that are of equivalent size at 3 days post inoculation),
and subjected to hormone manipulation, only the antisense caveolin
stable clones undergo castration-induced regression, whereas the
vector control clones and their parental cells do not. Therefore,
in this model system, the gene activities that are not directly
involved with castration-induced regression, but are induced or
repressed following the castration stimulus, should be present in
both vector control clones as well as antisense caveolin clones.
The only differences in the gene activities between the two groups
of cell clones in response to the castration stimulus should be
related to castration-induced regression which occurs only in the
antisense caveolin clones.
[0110] During normal development, the mouse prostate undergoes
extensive growth and morphogenesis in response to androgenic
steroids (Cunha G R, et al., J. Androl 13:465-475, 1992).
Preliminary data indicates that overexpression of caveolin can
block castration-induced regression of mouse prostate cancer.
Therefore, it seems likely that overexpression of caveolin would
block the normal testosterone-stimulated growth and development of
mouse prostate. Growth and development that occurs in prostatic
tissue in the mouse is not only dependent on increasing
testosterone concentrations that occur with reproductive maturity,
but also fluctuations in testosterone that occur shortly after
birth (Cunha G R, et al., J. Androl 13:465-475, 1992). It is
believed that under the influence of caveolin overexpression, a
normal mouse prostate would be insensitive to such changes and an
aberrant phenotype would be produced.
[0111] One embodiment of the invention is directed to the caveolin
gene and caveolin protein, or portions thereof, in the treatment of
neoplastic disorders and preferably metastatic disease such as, for
example, metastatic prostate cancer. Treatment involves
administration of the sequence of the gene, which may comprise only
the promoter region and a toxic gene to a patient to destroy
metastatic cells.
[0112] Therapies for prostate cancer metastasis become apparent to
those of ordinary skill in the art from an increased understanding
of the molecular mechanisms of cancer progression. Preliminary data
indicates that overexpression of caveolin blocks the
castration-induced pathway that leads to apoptosis-mediated
regression of mouse prostate cancer in vivo. One possible
explanation is that the caveolin protein is binding inducible
nitric oxide synthase and inhibiting activity following an initial
castration-induced stimulus (Chamness S L, et al., Fertil. Steril.
63:1101-1107, 1995). Therefore, reduction of caveolin levels in
human prostate cancer prior to androgen ablation therapy would
likely convert it from androgen-insensitive to androgen-sensitive
and result in increased tumor regression. The present invention
relates to methods for producing significant reductions in caveolin
protein prior to androgen ablation therapy. The molecular tools for
applying anti-caveolin therapy include recombinant viral vector
systems (such as recombinant vectors derived from retro viruses,
herpes viruses, lentiviruses, adenoviruses, adenoassociated viruses
and T cell leukemia viruses such as HTLV), antisense
oligonucleotides, and small molecule and antibody interference.
[0113] Another embodiment of the invention is directed to the
production of a transgenic mouse over expressing caveolin. In a
preferred embodiment, caveolin expression is under the
transcriptional control of the MMTV or the probasin promoter and is
targeted for expression in the prostate gland. Transgenic mouse
model systems have thus far proved to be invaluable tools for
understanding gene functions within a complex biological milieu in
vivo. Over the past 10 years hundreds if not thousands of
transgenic mice have been generated and used to unravel the
phenotypic effects of tissue-specific gene expression within the
context of normal gene activities, i.e. normal mouse development in
vivo. A transgenic mouse with targeted expression of caveolin to
the prostate gland may result in overgrowth and abnormal
differentiation of the mouse prostate in vivo. During development,
the mouse prostate is exquisitely sensitive to changes in
circulating androgen levels. Aberrant expression of caveolin will
block the response to androgenic stimuli to a great extent, and
prostatic epithelium in male caveolin-transgene mice will be
deregulated for growth and differentiation. Analyses of these mice
can be used to determine the extent of developmental abnormalities
and pathological changes that occur. Hormone manipulation of these
mice can provide insights into prostatic growth, morphogenesis and
therapeutic efficacy in vivo. The caveolin transgenic mouse model
is expected to provide significant and important information
regarding the impact of overexpression of caveolin on normal mouse
prostatic development as well as the possible pathological
consequences of this gene activity.
[0114] Another embodiment of the invention is directed to
significantly reducing caveolin levels (which may be caveolin-1,
-2, -3 or combinations thereof) using specific gene-based
technologies to result in reacquisition of sensitivity to
castration in hormone-refractory mouse and human prostate cancer.
In a preferred embodiment, recombinant adenoviral vectors may be
used to over express antisense caveolin directly in mouse prostate
cancer cells, thereby reducing caveolin levels. In a preferred
embodiment, treatment with antisense caveolin adenovirus is
combined with subsequent castration therapy to result in enhanced
sensitivity of prostatic tumors to the stimulus and, therefore,
more widespread apoptosis in cancer cells. Such therapy has direct
application to human disease.
[0115] Another embodiment of the invention is directed to
potentiation of androgen ablation therapy by reduction of caveolin
protein using adenoviral vector systems, antisense
oligonucleotides, antisense retroviral vectors, small molecules
that interact with caveolin function and antibodies. For example,
caveolin is involved in molecular transport and cell-cell
interactions. Interference with these functional properties may
interfere with metastatic development. Accordingly, caveolin-mimics
(i.e. agents that mimic caveolin function) as well as mimics of
other metastatic-specific functions can be used to treat and/or
prevent metastatic disease. Optimal methodologies may be selected
for the coupling of these therapies with surgical castration in an
attempt to produce more widespread cell death in mouse prostate
cancer.
[0116] Another embodiment of the invention is directed to methods
to determine the molecular pathways of castration-induced
regression in mouse prostate cancer vis-a-vis the caveolin
overexpression model system by, inter alia, assessment of specific
apoptotic activities and gene activities previously shown to
accompany castration-induced regression in both rodent and human
model systems. In addition, differential display (DD)-PCR may be
used to identify specific gene activities that are directly related
to castration-induced regression using the unique model system of
the present invention and ultimately, specific signal transduction
pathways may be tested.
[0117] Reduced Lysyl Oxidase mRNA Levels in Metastatic Prostate
Cancer
[0118] Previous studies indicate that acquisition of differential
responses to transforming growth factor .beta.1 (TGF-.beta.1) by
specific cell clones within a primary tumor can result in
phenotypic traits that facilitate cancer progression or tumor
metastasis in those cells. Cells of lung metastases-derived cell
lines have been shown to secrete relatively large amounts of total
TGF-.beta.s, and to lack most or all TGF-.beta.1-induced growth
inhibition, yet retain the ability to respond to TGF-.beta.1 as
indicated by TGF-.beta.1-induced expression of type IV collagenase
matrix metalloproteinase-9. In contrast, cell lines derived from
primary lung tumors were found to secrete relatively smaller
amounts of total TGF-.beta.s and retain TGF-.beta.1-induced
collagenase activity (Sehgal L, et al., Cancer Research
56:3359-3365, 1996). TGF-.beta.1 has also been identified in
association with prostate cancer (Truong L D, et al., Human Path.
24:4-9, 1993; Eastham J A, et al., Laboratory Invest. 5:628-635,
1995). Thus, a correlation can be drawn between increased
TGF-.beta.1 expression and increased cancer progression.
[0119] Another embodiment of the invention is directed to the
identification of genes that serve as downstream targets of
TGF-.beta.1 and may thus underlie the selective advantage of
metastatic clones. To identify genes that are differentially
induced or repressed by TGF-.beta.1 treatment in association with
prostate cancer progression, a strategy was utilized involving the
use of a modified DD PCR technique (Liang P, et al., Cancer Res.,
52:6966-6968, 1991; Liang P, et al., Science, 257:967-971, 1992;
Ralph D, et al., Proc. Natl. Acad. Sci. USA, 90:10710-10714, 1993),
and a panel of mouse prostate cancer cell lines derived from
genetically matched primary tumor and metastasis. In the
modification of the DD-PCR technique, the oligo (dT) primer was
substituted with a semiarbitrary 10-mer for the initial reverse
transcription and for subsequent amplification steps. Each primer
amplifies a large number of primer-specific bands that are
similarly expressed in either the presence or absence of
TGF-.beta.1, but in some cases, bands are induced or repressed in
the 148-1 PA cells by TGF-.beta.1 stimulation.
[0120] The nucleotide sequence of the lysyl oxidase gene, a
metastatic sequence, was also identified by the methods of the
invention. A homology comparison between lysyl oxidase clones
isolated by the invention and lysyl oxidase sequences entered in
the GenBank database show a very close homology.
[0121] The approach used to identify TGF-.beta.1-regulated genes
involved establishing mouse prostate cancer cell lines, which were
derived from primary tumors or metastatic deposits in the same host
animal, implanted with a ras+myc-initiated p53-nullizygous MPR
(Thompson, T C, et al., Oncogene, 10:869-879, 1995). Multiple sets
of early-passage clonal cell lines were established from both
primary and metastatic tumor foci recovered from the same
experimental animal. As the strains of mice used in these
experiments are inbred strains and because conditions for outgrowth
of both primary and metastasis-derived cell lines are closely
controlled, resulting cell-line systems are both genetically and
biologically matched. Therefore, the predominant genetic
differences between the primary tumor-derived and
metastasis-derived cell lines should be related to the metastatic
process.
[0122] Cells of the primary mouse prostate cancer cell line,
148-1PA (Sehgal, L, et al., Cancer Res., 56:3359-3365, 1996), were
either treated in vitro with 2 ng/ml TGF-.beta.1 for 12 hours to
induce TGF-.beta.1-dependent gene expression, or left untreated for
12 hours prior to use in the modified DD-PCR protocol. DD-PCR was
performed on RNA extracted from the TGF-.beta.1 treated and
untreated cells of primary tumor- versus metastases-derived cell
lines. Cloned cDNA fragments were used to probe cellular RNA
extracted from primary versus metastatic mouse prostate cancer
cells for sequences that are differentially regulated by
TGF-.beta.1, using Northern blot analysis. Sequences that were
found to be differentially regulated by TGF-.beta.1 were used as
probes to analyze constitutive gene expression by Northern blot
analysis using a panel of primary and metastatic mouse prostate
cancer cell lines. Numerous differentially regulated DD-PCR
fragments were cloned, sequenced, and compared to mouse and human
gene sequence databases. In approximately 90% of the cases, a
portion of a known mouse gene or a mouse homologue for a human gene
had been cloned. One of the cDNA fragments isolated by this
approach was found to encode a portion of the mouse lysyl oxidase
(LO) gene.
[0123] The detection of LO mRNA in the mouse prostate cancer cell
line 148-1PA was demonstrated in a DD-PCR gel. 148-1PA cells were
either treated with TGF-.beta.1 (Lanes +) or left untreated (Lanes
-) in serum free media (SFM), both for 12 hours, then DD-PCR was
carried out using one of three different primers, P10, P11, or P12.
as both the 5' and 3' primer. A TGF-.beta.1-induced band was
isolated, cloned and sequenced. Upon performing database (GenBank
Version 86.0) searches with this sequence, the 307-bp fragment was
found to be 100% identical (excluding three mismatched primer
sequences) with the mouse LO gene and 93% identical with the human
LO gene. The cloned LO fragment was then used in experiments with
both the primary mouse prostate cancer cell line 148-1PA and its
metastatic tumor-derived counterpart, 148-1 LMD, to analyze and
compare the extent of LO induction by TGF-.beta.1.
[0124] A Northern blot analysis was performed on total RNA from the
primary tumor-derived cell line 148-1PA and the lung
metastasis-derived cell line 148-1LMD. Cells were either
TGF-.beta.1 treated, or left untreated, for both 12 and 24 hours in
SPM. Induction of LO gene expression in 148-1PA cells was detected
at both 12 and 24 hours after TGF-.beta.1 treatment. In 148-1LMD
cells, the constitutive level of LO was barely detectable, and no
significant increase was observed as a result of
TGF-.beta.1-treatment. The blot was stripped and reprobed for PAI-1
gene expression. The PAI-1 gene is a TGF-.beta.1-induced gene and
encodes an extracellular matrix protein. PAI-1 gene expression has
been reported to undergo stimulation to a similar extent in both
primary tumor- and metastases-derived cell lines (Sehgal, L, et
al., Cancer Research 56:3359-3365, 1996). Accordingly, PAI-1
expression was found to be induced by TGF-.beta.1 to similar levels
at both 12 and 24 hours in both 148-IPA and 148-1LMD cell lines.
Equivalent RNA loading was verified by striping the blot and
reprobing it with a rat glyceraldehyde 3-phosphate dehydrogenase
(GAPDI-1) probe.
[0125] A Northern blot analysis was performed of total RNA from
cell lines derived from primary tumors (148-1PA, 148-1PC 151-1PA,
151-1PB, 151-1PF and 151-2PA) and their genetically matched lung
metastases (148-1LMA, 148-1LMC, 148-1LMD, 151-1LM 1, 151-1LM2,
151-2LMA and 151-2LMC). These cell lines were derived from three
independent animals (148-1, 151-1 and 151-2) (Thompson, T C, et
al., Oncogene, 10:869-879, 1995; Sehgal, L, et al., Cancer Res.,
56:3359-3365, 1996) LO expression was readily detected in five of
the six primary tumor-derived cell lines, and in only two of the
seven lung metastasis-derived cell lines. Also, the expression of
LO was significantly reduced in ras+myc-transformed NIH3T3 (3T3R/M)
cells compared to parental NIH3T3 cells (3T3). The blot was
stripped and reprobed with glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) as a control for RNA loading and transfer, and Endo B
(cytokeratin 18), a marker for prostatic luminal epithelial cells,
as a control for the epithelial origin of the cell lines (Thompson,
T C, et al., Cell, 56:917-930, 1989). Endo B was present in all of
the mouse prostate cancer cell lines documenting the epithelial
origin of the cell lines. The GAPDH blot verified that RNA had been
loaded equally in all lanes. Together, the results indicate that LO
represents a TGF-.beta.1-stimulated gene in nonmetastatic prostate
cancer cells, but that during cancer progression, reduced LO
expression becomes an acquired feature synonymous the metastatic
phenotype.
[0126] In situ hybridization analyses were performed on LO
expressed in mouse prostate tissues. To analyze mRNA levels in
normal and malignant mouse prostate, the 307-bp LO fragment that
was originally cloned by DD-PCR from 148-1PA cells was used for in
situ hybridization analysis of normal, primary tumor, and
metastatic mouse prostate cancer tissues. LO mRNA was detected
predominantly in the epithelium and to a lesser extent in the
stroma in normal mouse prostate. LO mRNA was detected predominantly
in the epithelium and to a lesser extent in the stroma in primary
mouse prostate cancer. LO mRNA was progressively less abundant in
the lymph node metastasis associated with the primary tumor. The
minimal background associated with this technique in which normal
mouse prostate tissue was probed with an LO sense probe. The
results indicate that significant mRNA levels are present in normal
mouse prostate epithelium, and also in the stroma of normal mouse
prostate albeit at a significantly reduced level. In the matched
set of primary and metastatic mouse prostate cancer tissues, in
situ hybridization analysis reveal significantly reduced levels of
LO mRNA in primary mouse prostate cancer relative to normal mouse
prostate epithelium and even further reduced levels of LO
expression in the metastatic lesions.
[0127] In situ hybridization analysis was performed of LO
expression in normal and malignant human prostate tissues. The
307-bp mouse LO DD-PCR fragment was utilized as a probe. This probe
fragment is 93% homologous with the human LO gene and was,
therefore, suitable for such studies. LO mRNA was abundant in
benign glandular epithelium in human prostate. LO expression was
markedly reduced in human prostate cancer. A further loss of LO
mRNA signal was shown in lymph node metastatic disease. A control
for background hybridization in which normal human prostate was
probed with an LO sense probe to demonstrated the specificity of
the analysis. The results of the in situ analysis of multiple
normal and malignant human tissues revealed a pattern similar to
that seen in the mouse. Significant levels of LO mRNA were detected
in benign prostate epithelium. LO was also detected in normal
prostatic stroma, but at a significantly reduced level. In primary
human prostate cancer, LO mRNA levels were reduced relative to that
seen in benign prostatic epithelium, and analysis of metastatic
specimens revealed a further diminution of LO mRNA (Table 1).
1TABLE 1 LO mRNA expression in normal prostate and prostate cancer
relative LO mRNA levels in human prostate tissues were scored as
negative (-) to strongly positive (+ + +), depending on the
fluorescence intensity on tissue sections. n.sup.a - + ++ +++
Normal stroma 7 0 7 0 0 Normal epithelium 7 0 0 0 7 Primary cancer
7 0 2 4 1 Lymph node metastases 4 0 3 1 0 n.sup.a number of
specimens examined.
[0128] The identification of the LO gene as one that is
differentially expressed in metastatic prostate tissue as compared
to primary tumor and normal tissue reveals the usefulness of LO in
diagnostic and treatment protocols according to the present
invention provides a novel cancer progression/metastasis-related
candidate gene for further studies. Because low LO expression
levels correlate with metastasis, LO gene expression is useful as a
prognostic marker for staging the metastatic state of a tumor and
for monitoring a patient, such as a prostate cancer patient, after
surgical treatment. The LO gene is also useful as a therapeutic
agent. The LO gene, or a portion thereof, may be cloned into an
expression system, such as, for example, an adenoviral vector, an
adeno-associated viral vector, a lentiviral vector or a retroviral
vector, and targeted for expression in a metastatic tumor, such as
a metastatic prostate tumor, to suppress the metastatic potential
of the tumor.
[0129] The LO gene product has been previously described as a
copper-dependent enzyme expressed predominantly in bone, blood
vessels, and connective tissue, and leads to the stabilization of
matrix structure by cross-linking collagen and elastin in the
extracellular matrix (Kagan, H. M., In: Regulation of Matrix
Accumulation, R. P. Mechiam (ed.), Vol. 1, pp. 321-398. New York:
Academic Press. 1986; Kagan, H. M., et al., Am. J. Respir. Cell
Mol. Biol., 5:206-210, 1991). Expression of LO has been reported to
be stimulated by TGF-.beta.1 in osteoblasts, vascular smooth muscle
cells and lung fibroblasts (Feres-Filhot, E J, et al., JBC
270:30797-30803, 1995; Shanley, C J, et al., J. Vase. Surg.,
25:446-452, 1997; Boak, A. M., et al., Am. J. Respir. Cell Mol.
Biol. 11:751-755, 1994). Increased LO activity has also been
reported in fibrotic disorders, whereas decreased LO activity is
associated with inherited disorders of collagen (Kivirikko, K I, et
al., In J. Uitte and A. J. Pere (eds.), Connective Tissue Disease,
pp. 263-292. New York: Marcel Dekker, Inc., 1987; Danks, D M, In:
Connective Tissue and Its Heritable Diseases: Molecular, Genetic
and Medical Aspects, P. M. Royce and B. Steinmann (eds.), pp.
487-506. New York: Wiley-Liss, Inc. 1992; Kivirikko, K I, Ann.
Med., 25:113-126, 1993).
[0130] Increased expression of the LO gene has also been reported
for ras-transformed mouse fibroblasts which revert from a
ras-transformed phenotype to a normal phenotype (Contente, S, et
al., Science 249:796-798, 1990; Hajnal, A, et al., Cancer Res.,
53:4670-4675, 1993). LO is also regulated by the anti-oncogenic
transcription factor IRF-1 (Tan, R S, et al., Cancer Res.,
56:2417-2421, 1996) indicating that LO may function as a tumor
suppressor. Low levels of LO mRNA have also been detected by RT-PCR
in several human tumor cell lines (Kuivaniemi, H, et al., FEBS
Lett., 195:261-264, 1986; Hmlinen, E, et al., J. Biol. Chem.,
270:21590-21593, 1995). Additionally, LO expression has been
detected in the extracellular matrix of breast cancer cells
(Peyrol, S, et al., Am. J. Pathol., 150:497-507, 1997).
[0131] As demonstrated herein, the LO gene has been identified for
the first time to be a TGF-.beta.1-induced gene in primary
tumor-derived cell lines, but not in cells derived from a
genetically matched metastatic counterpart tumors. Northern
blotting analysis of a panel of primary tumor cell lines revealed
increased LO expression, but significantly reduced or nondetectable
LO expression in their genetically matched metastatic counterparts.
Further analysis using in situ hybridization revealed expression of
the LO gene in normal mouse prostate epithelium but, in most cases,
progressive loss of expression in primary prostate cancer and
associated metastatic lesions. The progressive loss of LO
expression during prostate cancer progression provides information
that may increase understanding of the mechanisms that underlie
this disease. Furthermore, LO gene expression may now serve as a
useful molecular marker and/or a novel therapeutic target for
prostate cancer. Accordingly, administration of LO or a functional
equivalent of LO may be used as a therapeutic in the treatment of
metastatic disease and other neoplastic disorders.
[0132] The invention is the first documentation of LO expression in
normal prostate cells vs. prostate cancer cells. LO mRNA is present
predominantly in epithelium and, to a lesser extent, in the stromal
cells of normal mouse and human prostatic tissues. This information
adds to the limited understanding of the localization of LO and
glandular tissues in general. Previous studies of the expression
and regulation of LO have been carried out predominantly using
stromal-derived cell lines (Feres-Filhot, E J, et al., JBC 270:
30797-30803, 1995; Shanley, C J, et al., J. Vasc. Surg., 25:
446-452, 1997; Boak, A M, et al., Am. J. Respir. Cell Mol. Biol.
11: 751-755, 1994; Contente, S, et al., Science 249: 796-798, 1990;
Hajnal, A, et al., Cancer Research 53: 4670-4675, 1993; Tan, R S,
et al., Cancer Research 56: 2417-2421, 1996; Kuivaniemi, H, et al.,
FEBS Lett., 195: 261-264, 1986; Hmlinen, ER, et al, JBC 270:
21590-21593, 1995).
[0133] Studies of LO expression in malignant human breast tissues
have been reported (Peyrol, S, et al., Am. J. Pathol., 150:497-507,
1997). In the Peyrol study, LO was undetectable in normal breast
tissue using immunostaining for LO protein. However, in situ
hybridization analysis for LO demonstrated that the presence of
mRNA in stromal cells surrounding glandular epithelium and reduced
LO expression was associated with loose or scirrhous stroma that
accompanied invading tumors (Peyrol, 5, et al., Am. J. Pathol.,
150:497-507, 1997.)
[0134] The present studies on prostate cancer progression also
associate with loss of LO expression with cancer progression. In
the case of prostate cancer, the malignant cells per se demonstrate
a progressive reduction in LO mRNA during primary tumor development
and metastasis. Studies are currently ongoing to further define the
compartmentalization of LO in normal and malignant prostate and to
better understand its biological and clinical significance.
Although results indicate loss of LO mRNA during mouse and human
prostate cancer progression, the molecular basis for this loss is
less clear. Alterations may occur not only in the expression of the
LO gene, but also in the anti-oncogenic transcription factor IRF-1
which regulates expression (Tan, R S, et al., Cancer Research
56:2417-2421, 1996).
[0135] These results have implications for the biological
underpinnings of prostate cancer progression. It has been
previously suggested that the protective effect of a collagenized
matrix might be determined by collagen cross-linking which could
dictate fibrillar and basement membrane stability against
metalloproteinase activities (Vater, C A, Biochem. J., 181:639-635,
1979). This concept was recently supported by studies involving
invasive ductal breast carcinoma (Peyrol, S, et al., Am. J.
Pathol., 150:497-507, 1997). This protective effect may underlie,
in part, the phenomenon associated with the reversion of
ras-transformed 3T3 cells by LO (Contente, S, et al., Science
249:796-798, 1990; Hajnal, A, et al., Cancer Res., 53:4670-4675,
1993). In addition to promoting a physical extracellular matrix
cross-linking barrier against malignant progression, it is also
conceivable that maintaining the integrity of the extracellular
matrix prevents the release of mitogenic growth factors (Thompson,
K, et al., J. Cell. Physiol. 166:495-505, 1996). These concepts are
now relevant for prostate cancer progression. Progressive loss
and/or unusual patterns of LO expression in prostate cancer biopsy
material may be of prognostic significance. The LO gene can now be
considered a potential tumor suppressor gene or component of a
tumor suppressor pathway for prostate cancer and, therefore, a
target for molecular therapeutic strategies.
[0136] In addition, as discussed in below in the examples, LO
expression is an independent prognostic factor for prostate cancer
recurrence, indicating that it plays a role in prostate cancer
progression. Thus, this novel marker will likely have clinical
significance for predicting prostate cancer recurrence in various
clinical settings.
[0137] Additional Operations
[0138] Selected potential therapeutic and diagnostic uses of the
invention include:
[0139] 1. Antisense Therapy: An antisense sequence, such as an
antisense caveolin sequence, may be used for a number of
applications, including antisense blocking or antisense inhibition.
Antisense blocking refers to the incorporation into a cell of
expression sequences which direct the synthesis of antisense
nucleic acid to block expression of a target gene. Antisense
hybridizes to the mRNA of the target gene to inhibit
expression.
[0140] Antisense inhibition also exploits the specificity of
hybridization reactions between two complementary nucleic acid
chains to suppress gene expression. If a cloned gene is engineered
so that only the opposite DNA strand is transcribed, the resultant
RNA may hybridize to the sense RNA and inhibit gene expression.
[0141] 2. Domain-specific uses: Some of the identified metastatic
sequences may encode polypeptides with domains useful in specific
applications. For example, there are three genes in the caveolin
family, caveolin-1, caveolin-2 and caveolin-3. Two domains have
been identified on caveolin-1 that have important biological
functions. One of these domains mediates dimerization between
caveolin-1 and caveolin-2. This dimerized form of caveolin
spontaneously leads to the formation of caveolae. The other domain,
the scaffolding domain, mediates the binding of some, but certainly
not all, of the specific proteins that can initiate signal
transduction. These sites on caveolin can serve as potential
targets for drugs or peptides that interfere with or modify these
biological activities, For example, these sequence sites could be
the subject of gene targeting or other diagnostic and therapeutic
strategies.
[0142] 3. Interference peptides: In addition, small interference
peptides may be chemically linked to steroids to allow for both
specificity of cell target as well as the specificity of
intercellular pathway.
[0143] 4. Viral vectors and non-viral approaches: Specific
treatment approaches using metastatic sequences may include the
delivery of sense, antisense or dominant negative forms of
metastatic sequences such as caveolin, lysyl oxidase or p99, using
expression or viral vectors or non-viral approaches.
[0144] 5. Gene promoter approaches: The gene promoter, for example,
the caveolin gene promotor, which has been cloned, can be used for
cell targeting of the expression of therapeutic gene sequences. The
prostate cancer cells expressing high levels of caveolin, and
possibly the breast cancer cells expressing high caveolin levels,
are the cells that are more likely to metastasize, because they can
survive in the vasculature and lymphatic system where
concentrations of growth factors and testosterone are very low
compared to the prostate per se. In addition, the normal blood
vessel endothelium expresses high levels of caveolin; thus, the
killing of these cells would promote overall tumor death. Because
the caveolin promoter appears to be highly up regulated in
metastatic cells, it is an advantageous promoter for use in driving
the expression of therapeutic gene sequences. The therapeutic genes
sequences could be lysyl oxidase, antisense caveoli, antisense p99,
or other sequences with anti-metastatic properties.
[0145] 6. Metastatic sequences as biomarkers: Metastatic sequences,
such as lysyl oxidase and caveolin may have applications as
biomarkers. For example, caveolin is up-regulated by cholesterol,
insulin-like growth factor 1 and testosterone. As these factors may
be risk-factors for the development and/or progression of prostate
and breast cancer, sequences caveolin may serve as an intermediate
biomarker for the adverse effects of these dietary/hormonal
elements.
[0146] Caveolin levels in African-American prostate cancer are four
times higher than that in Caucasian prostate cancer, which has been
controlled for stage and grade of cancer. African-Americans have a
much higher rate of progression and mortality from prostate cancer
than Caucasians.
[0147] The caveolin promoter (in the mouse gene) has an unusual
region of di-nucleotide and tri-nucleotide repeats that could be
highly mutable. This may explain the "genetic difference" in
caveolin expression in African Americans versus Caucasians in
response to, perhaps, dietary cholesterol.
[0148] Lysysl oxidase is down-regulated in metastatic cells and may
thus be useful as a marker for metastasis. The LO biomarker would
have application in monitoring patients, for example, a prostate
cancer patient, after surgical treatment methods, or for staging
cancer progression of a tumor, such as a prostate primary
tumor,
[0149] 7. Gene transfer-initiated "in vivo priming" of tumor
infiltrating lymphocytes: Solid tumors attract lymphocytes that
migrate into the tumor and initiate various levels of antitumor
activities. These tumor infiltrating lymphocytes, or TILs, reflect,
as well as alter, the biological milieu of the tumor and
potentially have the capacity to destroy tumor cells if sufficient
activation levels are achieved. Previously, methodologies have been
developed for the isolation of TILs from solid tumors, their
activation with specific cytokines in vitro and their
administration to patients for potential therapeutic benefits. The
results of multiple clinical trials using TIL therapies indicate
that in a relatively low but consistent percentage of the patients,
TIL therapy can result in measurable therapeutic benefits and in
some cases outright cure of the disease. The reasons for this
limited therapeutic activity of ex vivo activated TIL appear to be
multifaceted and likely include: 1) the lack of substantial TIL
recovery from the tumor that could reflect limited infiltration, 2)
the limited activation state of the TIL recovered from tumors, and
3) the inability to fully ex vivo activate the TIL to the cytotoxic
in vivo state.
[0150] Another embodiment of the invention is directed to
compositions and methods that involve administration of viral
vector-delivered gene therapies having immunomodulatory activities
for cancer and, preferably, metastatic disease. These therapies
include the delivery of the Herpes Simplex Virus thymidine kinase
(HSV-tk) gene followed by the administration of systemic
ganciclovir. This gene therapy approach results in hemorrhagic
necrosis within the tumor that leads to high levels of lymphocytic
infiltration and increased activity of specific lymphocytes
including T cells. Based on these and other preliminary data,
specific in vivo gene therapy approaches, in combination with TIL
transfer, are expected to produce significantly enhanced
therapeutic activities compared to current TIL methods. The
enhanced therapeutic activities will result from the "in vivo
priming" that occurs in response to the activities of the
transferred genes. This "in vivo priming" will produce: 1) enhanced
lymphocytic infiltration and 2) a higher activated state of the
infiltrating lymphocytes. A preferred embodiment of the present
invention comprises pretreating a patient with adenoviral vector
delivered HSV-tk+ganciclovir therapy, and possibly other cytokine
genes, in combination with traditional TIL therapy to produce a
distinct therapeutic advantage by increasing the amount of
lymphocytic infiltration and activation state within the context of
high levels of tumor antigen in vivo. This combination "in vivo
priming"/TIL therapy approach may be used directly following
surgery or biopsy, or the activated cells could be frozen for
future use. Diagnostic and/or prognostic information may be
recovered from the more fully expanded and activated cells.
[0151] 8. Suppression of genes associated with the drug resistant
phenotype: It is possible to increase the susceptibility of a tumor
to drug treatment. There are genes, such as mdrl and caveolin, that
encode for proteins that confer a drug-resistant phenotype to
cells. This drug-resisitant phenotype presents an obstacle in
treatment due to the cells' insensitivity to cancer drugs.
Therefore an approach to increase the susceptibility of these cells
to cancer drugs is to decrease or suppress the expression of the
multidrug resistance gene(s) combined with apoptosis-inducing drug
therapy. This suppression of the drug resisitant genes could be via
antibodies or antisense sequences.
[0152] 9. Recombinant adenoviral vectors for gene therapy:
overexpression of cyclin-dependent kinase inhibitors p18 and p19
using Adcp18 and Adcp19: Many human malignancies present
therapeutic dilemmas for the clinician. Prostate cancer therapy is
remarkable in this regard. The incidence of pathological prostate
cancer is exceedingly high and with current methods of detection
(primarily PSA screening) available hundreds of thousands of men
are being diagnosed with prostate cancer. However, only a small
percentage of these cancers would normally progress to clinical
significance. Yet the dominant therapy for these cancers is radical
prostatectomy, a serious surgical procedure with significant
associated risks. In addition the current modalities used to stage
prostate cancer are not sufficiently accurate to be confident that
the cancer has not metastasized from the primary tumor site.
Currently there are no satisfactory treatments for metastatic
prostate cancer. Therefore it is imperative to develop novel and
effective therapies for localized prostate cancer that are less
dangerous than radical surgery as well as novel and effective
therapies for metastatic disease. In vivo gene therapy for prostate
cancer has the potential to provide effective and relatively
harmless therapy for both local and metastatic prostate cancer.
[0153] One gene therapy approach of the invention for the control
of localized prostate cancer is to replace or increase the dose of
genes that suppress the growth of malignant prostate cells.
Previous studies have examined the effects of vector mediated gene
transfer of the wild type p53 gene, a known G1->S checkpoint
control gene. p53 has been shown to produce growth suppression
through the transcriptional activation of p21, a cyclin dependent
kinase inhibitor (CKI). More recently we and other laboratories
have tested the therapeutic effects of p21 gene transfer (Eastham
et at., Cancer Res. 55:5151-5155, 1995). The p21 protein is
transcriptionally regulated by p53 and functions as a primary
effector for arresting cells at the G1->S interface. Recently
the concept of CKI-mediated gene therapy has been extended by
constructing and testing two recombinant adenoviral vectors which
transduce the p18 and p19, unique CKIs that may also have activity
in malignant prostatic epithelium. Transcriptional regulation of
p18 and p19 is under the transcriptional control of the
cytomegalovirus promoter and the recombinant virus was generated by
co-transfection of the expression cassette with Ad5 virus that had
been previously digested with restriction endonucleases cleave the
virus in the E1A region, allowing recombination to occur. These
recombinant viruses have been tested for activity in mouse and
human prostate cancer cells in vitro. The results indicate that
both recombinant adenoviruses are capable of suppressing the growth
of these cells. Further studies will test the activities of these
recombinant viruses in prostate cancer in vivo to further define a
role for these reagents in human gene therapy protocols.
[0154] The following experiments are offered to illustrate
embodiments of the invention and should not be viewed as limiting
the scope of the invention.
EXAMPLES
Example 1
[0155] Production of Mouse Prostate Reconstitution Tumors and
Metastasis
[0156] Mouse Urogenital Sinus (UGS) tissue was isolated from 17 day
old mice embryos. Each isolated UGS was digested with 1% trypsin
for three hours at 4.degree. C. The trypsin was inactivated by the
addition of fetal calf serum, UGS cells were digested with 0.125%
collagenase for 1.5 hours, counted and mixed at the appropriate
cell rations prior to infection with retrovirus in the presence of
polybrene. Retro viruses used include Zipras/myc-9. Control
experiments were performed using BAGa virus. After a two-hour
infection, the infected cells were centrifuged and individual
reconstitutions containing 1.5.times.10.sup.6 cells produced by
resuspending the cells in rat tail collagen at a density of
6.0.times.10.sup.7 cells per ml. Aliquots of the infected UGS cells
were placed in (DME) with 10% fetal calf serum overnight at
37.degree. C., 5% CO.sub.2. The next morning each cell/collagen
reconstitution was implanted under the renal capsule of an adult
male +/+ animal. Reconstitutions were harvested from the mice five
weeks later when they showed signs of obvious distress from the
tumor burden. Metastasized tumors were isolated from the same mice
at sites outside the renal capsule. Isolated tumors and
metastasises were either stored in liquid nitrogen or in
preservatives such as 10% buffered formalin. Cell lines were
derived from fresh tumors by mincing a small portion of the primary
metastatic or nonmetastatic tumor and placing each in explant
culture in Dulbecco's Modified Eagle Medium (DMEM) supplemented
with 10% fetal calf serum. Cells which grow from each explant were
propagated in DMEM and 10% fetal calf serum. For histological
analysis, a portion of a fresh tumor was fixed in 10% buffered
formalin and embedded in paraffin for sectioning and staining with
hematoxylin and eosin (H&E) or immunohistochemical staining.
Immunohistochemical localization of cytokeratin was detected using
polyclonal cytokeratin antiserum A575 (Dake Co., Carpinteria,
Calif.) and Vectastain ABC kit (Vector Laboratories, Burlingame,
Calif.).
Example 2
[0157] Isolation of C-DNA for DD-PCR
[0158] Total cellular RNA was isolated by ultracentrifugation
through cesium chloride. Briefly, up to one gram of cells from
culture, tumors or organs was placed into 4 ml of ice-cold GIT
buffer (4M guanidine isothiocyanate, 0.025 M sodium acetate, 0.1
M.beta.-mercaptoethanol) and homogenized in a tissue homogenizer
(Polytron or equivalent). The homogenate was carefully layered over
4 ml of 5.7 M CsC1, 0.024 M sodium acetate (1.8 g CsCl per ml) in a
centrifuge tube. The layers were centrifuged at 35,000 RPM for 18
hours in a SW50. 1 rotor. DNA was collected from the interface
between the cushion and the supernatant, diluted two folds with
water, added to 2.5 volumes of ethanol and spooled out on a glass
rod. RNA that formed a pellet on the bottom of the CsCl layer was
resuspended, and once extracted with an equal volume of
phenol:chloroform (1:1), twice with chloroform and precipitated
with ethanol and resuspended in diethylpyrocarbonate treated water.
The concentration of DNA and RNA were be determined by absorption
at 260 nanometers.
Example 3
[0159] Differential Display Polymerase Chain Reaction
[0160] mRNA isolated from primary tumors or metastasis was reverse
transcribed with one of the primers and subjected to DD-PCR using
the same primer as both the forward and reverse primer. A set of 24
primers comprising short oligonucleotides were used for both the
reverse transcription of mRNA into c-DNA and for differential
display polymerase chain reaction.
[0161] PCR was performed using standard conditions with 40 cycles
of denaturation at 94.degree. C. for 40 seconds, annealing at
40.degree. C. for 2 minutes, and elongation at 72.degree. C. for 35
seconds. After PCR, the products were analyzed with non-denaturing
polyacrylamide gel electrophoresis (PAGE) at 12 watts for 15 hours.
Bands which differed between test and control samples were eluted
from the gel, subjected to reamplification by PCR and cloned.
Northern blot analysis was performed showing that the sequence
isolated by DD-PCR is differentially expressed.
Example 4
[0162] p53 Allelotype Determination
[0163] The p53 allelotype of a cell sample was determined by PCR.
Briefly, nucleic acid is extracted from a tissue sample or a cell
culture sample. An aliquot of nucleic acids in placed in 45 .mu.l
aliquot of a master mix which contained a final concentration of
0.2 mM of each dATP, dTTP, dGTP, dCTP, 1.5 mM MgCl.sub.2, 0.5 unit
Taq polymerase, 0.05 .mu.M of each of two primers set specific for
the normal wildtype allele of p53. A control set of primers
specific for the fibroblast growth factor-7 gene was used to
monitor the polymerase chain reaction experiment. One .mu.l of the
reaction from the first round of PCR was used as the starting
material for a second round of PCR using a second set of wildtype
p53 specific primer. This second round of PCR was also monitored
using a control set of primers specific for the fibroblast growth
factor-7. After PCR the products were analyzed with non-denaturing
polyacrylamide gel electrophoresis (PAGE) at 12 watts for 15 hours.
Bands which differed between test and control were eluted from the
gel, subjected to reamplification by PCR and cloned.
Example 5
[0164] Induction of Cell Lines with TGF-.beta.1 Influence Cellular
Gene Expression
[0165] 1481PA cells were grown overnight in DME supplemented with
10% fetal calf serum overnight at 37.degree. C., and 5% CO.sub.2.
Induction was performed by treatment with TGF-.beta.1 at a
concentration of 2 ng/ml. The treated cells were returned to the
incubator and cultured for 12 hours. After induction, cells were
washed in phosphate buffered saline and harvested and concentrated
by centrifugation. RNA was extracted from treated and untreated
cells and subjected to DD-PCR. Differentially expressed bands
detected by DD-PCR were cloned and differential expressions were
confirmed using RNA blots. Subsequent cloning and sequencing
identified the bands as ABP28O or filamin. One gene isolated showed
differential expression in cells induced by TGF-.beta. (clone 29),
while a control probe on the same cell line showed no difference in
expression levels (GAPDH). RNA blot analysis of total RNA (20
.mu.g) from primary tumor and metastasis derived cell lines in
three independent mouse prostate reconstitutions (148-1, 151-1
& 151-2) showed differential expression. Control experiments
using the same cell lines, but using a probe to GAPDH, showed no
differential expression.
Example 6
[0166] Metastatic Sequences Isolated
[0167] Using the methods of Examples 1, 2, 3, 4, and 5, a plurality
of metastatic sequences were isolated and sequenced. The expression
of the metastatic sequences in primary cells and in metastatic
cells were determined using RNA blots. The results of these studies
are summarized in FIG. 9.
Example 7
[0168] Caveolin Immunoassay in Human Prostate Cancers
[0169] Primary site human prostate tumors and metastases were
isolated and analyzed for caveolin expression by immunoassay. The
results of the assay is shown in Table 2 Metastases shows higher
levels of caveolin proteins in metastases than in primary tumors.
Immunohistology of tissue sections reveals both elevated levels and
distinct distribution of caveolin protein in metastatic human
prostate when compared to a primary human prostate tumor.
2TABLE 2 Metastases in Patients Primary-site lymph node 1 + ++ 2 ++
+++ 3 ++ +++ 4 ++ ++ 5 + + 6 ++ ++ 7 ++ +++ 8 + + 9 - - 10 + + 11 +
+ 12 ++ ++ 13 + + 14 ++ +++
Example 8
[0170] Production of Primary and Metastatic Mouse Prostate Cancer
Lines
[0171] Mouse prostate cancer cell lines were derived from primary
tumors or metastatic deposits in the same host animal implanted
with a ras+myc-initiated p53-nullizygous MPR (Thompson, T C, et
al., Oncogene, 10:869-879, 1995). The cell lines were analyzed for
retroviral integration patterns by Southern blotting (Thompson, T
C, et al., Oncogene, 10:869-879, 1995) and cultured as described
previously (Sehgal, L, et al., Cancer Res., 56:3359-3365, 1996).
All murine cell lines were used at early passages, i.e., passages
7-10. The primary tumor-derived cell line 148-IPA was seeded into
15-cm diameter plates at 3.times.10.sup.6 cells per plate, and the
next day, the medium changed to SFM (serum-free medium, which was
DMEM with 10 mM HEPES, penicillin, streptomycin, and 0.1% BSA),
without or with 2 ng/ml TGF-.beta.1, and the cells were incubated
at 37.degree. C. for 12 or 24 hours.
Example 9
[0172] Isolation and Analysis of RNA
[0173] RNA was isolated from cell lines as described previously
(Thompson, T C, et al., Oncogene, 10:869-879, 1995), or with
commercially available RNA isolation reagents (Biotex). mRNA was
purified from total RNA with PolyATtract mRNA Isolation System
(Promega). For Northern blot analysis, 20 .mu.g of total RNA were
fractionated under denaturing conditions on a 1% agarose-6.7%
formaldehyde gel and transferred onto Hybond-N nylon membrane
(Amersham). The membrane was baked at 80.degree. C. for 2 hours
prior to prehybridization at 65.degree. C. for 2 hours in 7.5% SDS,
0.5 M sodium phosphate buffer (pH 7.2), 1 mM EDTA,
4.times.Denhardt's solution (50.times.Denhardt's solution=1%
Ficoll, 1% polyvinylpyrrolidone, and 1% BSA), and 50 .mu.g/ml
salmon testis DNA. Hybridization was carried out by addition of a
.sup.32P-labeled probe that had been purified with a QIAquick spin
column (Qiagen). The hybridization was incubated overnight at
65.degree. C. Blots were washed at 65.degree. C. for 20 min with
wash solution (40 mM sodium phosphate (pH 7.2), 5% SDS) two
times.
Example 10
[0174] Differential Display Polymerase Chain Reaction
[0175] One of a set of unique 10-mer deoxyoligonucleotide primers,
primer 11, with an arbitrarily chosen sequence (primer
11=CTGCTTGATG), was used for reverse transcription. Primer 11 was
also used as both a 5' and 3' primer for amplification by PCR. The
primers were selected based on having approximately the same
G+C:A+T ratio, with no uninterrupted self-complementary sequence of
more than 2 nucleotides (Ralph, D, et al., PNAS USA,
90:10710-10714, 1993.). Reverse transcription (RT) of mRNA was with
the Perkin-Elmer/Cetus GeneAmp RNA PCR kit. The RT reaction volume
was 10 .mu.l and was contained in 5 mM MgCl.sub.2, 1.times.PCR
buffer II, 1 mM each dNTP (dATP, dCTP, dGTP, and dTTP), 1
unit/.mu.l RNase inhibitor, 2.5 units/.mu.l reverse transcriptase,
250 nanograms of primer, and 60 nanograms of mRNA. The reaction
mixture was covered with 50 .mu.l of mineral oil and incubated at
22.degree. C. for 10 min, 42.degree. C. or 15 minutes, and
99.degree. C. for 5 minutes (termination). The reaction was
immediately diluted to 50 .mu.l and adjusted such that it contained
2 mM MgCl.sub.2, 1.times.PCR buffer II, 1.25 units of AmpliTaq DNA
polymerase, and 20 .mu.Ci of [.sup.33P]dATP (3000 Ci/mmol). No
additional dNTPs or primer were added, so that the final
concentrations were 0.2 mM each dNTP and 250 negotiating of primer.
The PCR consisted of 40 cycles at 94.degree. C. for 40 seconds,
40.degree. C. for 2 minutes, and 72.degree. C. for 35 seconds, with
a final extension period of 72.degree. C. for 4 minutes.
[0176] Samples from the PCR were separated on a nondenaturing 5%
polyacrylamide gel (29:1) with 5% glycerol at 9 W for 18 hours. The
gel was transferred to Whatman 3 MM paper, dried, and exposed to
X-ray film overnight. The differentially displayed bands were
excised from the dried polyacrylamide gel and soaked in 500 .mu.l
of H.sub.2O for 15 min at room temperature to remove the filter
paper, and the gel slice was transferred to 20 .mu.l of 10 mM Tris
(pH-8)-1 mM EDTA buffer, smashed, and incubated at room temperature
from 2 h to overnight. A 5-.mu.l aliquot was reamplified in a
50-.mu.l PCR mixture containing 1.times.PCR buffer II, 2 mM
MgCl.sub.2, 0.25 mM dNTP, 1.25 units of AmpliTaq DNA polymerase,
and 1 .mu.g of primer. The PCR was 45 cycles, with the same
parameters as above. The reamplified cDNA fragments were purified
on 2% NuSieve agarose (FMC Bioproducts) by gel electrophoresis. The
bands were excised and used to make a .sup.32P-labeled probe for
Northern blot analysis as described above or cloned into TA cloning
vector (PCR 2.0 vector; Invitrogen). The cloned DD-PCR fragments
were sequenced with Sequenase Version 2.0 (United States
Biochemical).
Example 11
[0177] Histological Analysis of RNA Expression
[0178] Frozen sections 6 .mu.m thick from normal mouse prostate and
mouse prostate cancer and lymph nodes with metastatic deposits
produced in the MPR system were used for in situ hybridization In
addition, frozen sections 6 .mu.m thick from seven histologically
normal human prostates obtained from cystoprostatectomy specimens
and seven primary cancers from radical prostatectomies, together
with their lymph node metastases (four of seven cases), were also
analyzed. Sections were air-dried and fixed in a fixative
containing 2% paraformaldehyde, 75% ethanol, and 23% acetic acid
for 30 min. After treatment with 0.2 N HCl for 10 min, the slides
were digested for 20 min in 5 .mu.g/ml proteinase K, rinsed in 0.2
mM glycine/PBS, and fixed for 20 min in 4% paraformaldehyde,
Antisense (cRNA of LO) or sense RNA probes were obtained by run-off
transcription of the vector pCR 2.0 (Invitrogen), into which the
307 bp fragment of the mouse LO cDNA had been subcloned. The
recombinant DNAs were linearized with HindIII or XbaI. The sense
and antisense probes were prepared with T7 or SP6 polymerase,
respectively, using the DIG (digoxigenin) RNA labeling kit from
Boehringer Mannheim by SP6/T7 transcription, according to the
procedure recommended by the manufacturer. The sections were
prehybridized in 50% formamide, 5.times.SSC, 5.times.Denhardt's
solution, 250 .mu.g/ml yeast t-RNA, 4 mM EDTA, and 1 mg/ml salmon
sperm DNA for 60 minutes at 37.degree. C. and hybridized in the
prehybridization buffer (without the salmon sperm DNA) containing
20 negotiating/.mu.l DIG-labeled cRNA probes at 48.degree. C. for
16 hours. Following washing in 4.times.SSC for 20 minutes, the
sections were digested with 20 .mu.g/ml RNase A at 37.degree. C.
for 20 minutes, followed by further washing in 2.times.SSC (10
minutes), 1.times.SSC (20 minutes), and 0.1.times.SSC (30 minutes)
at 48.degree. C. DIG-labeled hybrids on sections were
immunocytochemically detected by using an anti-DIG IgG conjugated
with fluorescein (Boehringer Mannheim). Sections from primary and
metastatic cancers were always processed in parallel on the same
slides using the same batches of probes and reagents.
Example 12
[0179] Reduction of LO Expression as an Independent Predictor of
Recurrence Following Radical Prostatectomy
[0180] As noted, lysyl oxidase has enzymatic activity that can
stabilize extracellular matrix and may have tumor suppressor
activities. LO mRNA is predominantly expressed in normal mouse and
human prostatic epithelial cells and to a lesser extent in
prostatic stroma and that LO mRNA levels were significantly reduced
in primary prostate cancer and further reduced in metastatic
lesions. In this example, immunohistochemical (IHC) staining was
used to analyze LO in human prostate cancer specimens.
Specifically, sixty-one specimens obtained from radical
prostatectomies were analyzed by IHC staining using the
avidin-biotin complex technique in conjunction with a polyclonal LO
antibody. LO-positive cancer cells were counted and expressed as a
percent of cancer cell population. LO positive cancer cells were
found to have heterogeneous distribution and LO levels in cancer
were remarkably lower when compared with their adjacent,
histologically normal glandular epithelia. LO labeling rates in
cancers tended to decrease with increasing Gleason sum, although
the differences did not reach statistical significance (p=0.088).
No significant differences in LO levels were found in regard to
seminal vesicle involvement, surgical margin status and lymph node
metastases. However, a highly significant association was found
between reduced LO positive cells and recurrence of cancer
following radical prostatectomy (p=0.0041). Multivariate logistic
regression analyses were performed to assess the effects of LO
labeling rate, Gleason sum, lymph node metastases, surgical margins
and seminal vesicle involvement as possible prognostic markers for
recurrence. It was found that LO positive cells (p<0.042) as
well as Gleason sum (p<0.016) added independent prognostic
information. This demonstrates that LO expression is an independent
prognostic factor for prostate cancer recurrence, suggesting that
it plays a role in prostate cancer progression. This novel marker
may be used to predict prostate cancer recurrence in various
clinical settings.
[0181] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. All documents cited
herein including U.S. Pat. No. 5,783,182, entitled "Method for
Identifying Metastatic Sequences," which issued Jul. 21, 1998, and
U.S. Provisional Patent Application, entitled Metastatic Sequences,
serial No. 60/077,934, filed Mar. 13, 1998, are specifically
incorporated by reference. The specification and examples should be
considered exemplary only with the true scope and spirit of the
invention indicated by the following claims.
Sequence CWU 1
1
28 1 1870 DNA homo sapiens sapiens CDS (1)..(669) 1 ttt agt cgc ggt
gtc agc gct cgc agg acc act ctt ggc cgc tgc tcc 48 Phe Ser Arg Gly
Val Ser Ala Arg Arg Thr Thr Leu Gly Arg Cys Ser 1 5 10 15 tgc ccg
gcg ttc ctc cgc tcc gcg ccc gcc gcc acc gac gac atg ctg 96 Cys Pro
Ala Phe Leu Arg Ser Ala Pro Ala Ala Thr Asp Asp Met Leu 20 25 30
cgc tgc ggc ctg gcc tgc gag cgc tgc agg tgg atc ctg ccc ctg ctg 144
Arg Cys Gly Leu Ala Cys Glu Arg Cys Arg Trp Ile Leu Pro Leu Leu 35
40 45 ctg ctc agc gcc atc gcc ttc gac atc atc gcg ctg gcc ggc cgc
ggc 192 Leu Leu Ser Ala Ile Ala Phe Asp Ile Ile Ala Leu Ala Gly Arg
Gly 50 55 60 tgg ctg cag tct agc aac cac atc cag aca tcg tcg ctt
tgg tgg agg 240 Trp Leu Gln Ser Ser Asn His Ile Gln Thr Ser Ser Leu
Trp Trp Arg 65 70 75 80 tgt ttc gac gag ggc ggc ggc agc ggc tcc tac
gac gat ggc tgc cag 288 Cys Phe Asp Glu Gly Gly Gly Ser Gly Ser Tyr
Asp Asp Gly Cys Gln 85 90 95 agc ctc atg gag tac gca tgg gga cga
gca gct gca gcc acg ctt ttc 336 Ser Leu Met Glu Tyr Ala Trp Gly Arg
Ala Ala Ala Ala Thr Leu Phe 100 105 110 tgt ggc ttt atc atc ctg tgc
atc tgc ttc att ctc tcg ttc ttc gcc 384 Cys Gly Phe Ile Ile Leu Cys
Ile Cys Phe Ile Leu Ser Phe Phe Ala 115 120 125 ctg tgt gga ccc cag
atg ctt gtt ttc ctg aga gtc att gga ggc ctc 432 Leu Cys Gly Pro Gln
Met Leu Val Phe Leu Arg Val Ile Gly Gly Leu 130 135 140 ctc gca ctg
gct gcc ata ttc cag atc atc tcc ctg gta atc tac ccc 480 Leu Ala Leu
Ala Ala Ile Phe Gln Ile Ile Ser Leu Val Ile Tyr Pro 145 150 155 160
gtg aag tac aca cag acc ttc agg ctt cac gat aac cct gct gtt aat 528
Val Lys Tyr Thr Gln Thr Phe Arg Leu His Asp Asn Pro Ala Val Asn 165
170 175 tac atc tat aac tgg gcc tat ggc ttc gga tgg gcg gcc acc atc
atc 576 Tyr Ile Tyr Asn Trp Ala Tyr Gly Phe Gly Trp Ala Ala Thr Ile
Ile 180 185 190 ttg att ggt tgt tcc ttc ttc ttc tgc tgc ctc ccc aac
tac gag gat 624 Leu Ile Gly Cys Ser Phe Phe Phe Cys Cys Leu Pro Asn
Tyr Glu Asp 195 200 205 gac ctt ttg ggg gcc gcc aag ccc agg tac ttc
tat ccc cca gcc taa 672 Asp Leu Leu Gly Ala Ala Lys Pro Arg Tyr Phe
Tyr Pro Pro Ala 210 215 220 tgt ggg agg aag agc ctg aga aaa gcc tgc
tgc aag atg gat ctg agg 720 Cys Gly Arg Lys Ser Leu Arg Lys Ala Cys
Cys Lys Met Asp Leu Arg 225 230 235 agg aaa ctg ttc tcc aag gca caa
gga acc tac gtt tgg gca atg ttc 768 Arg Lys Leu Phe Ser Lys Ala Gln
Gly Thr Tyr Val Trp Ala Met Phe 240 245 250 255 ata tga tca gaa atg
cta gaa taa atg cta aag aaa att ctt cat aat 816 Ile Ser Glu Met Leu
Glu Met Leu Lys Lys Ile Leu His Asn 260 265 tagtgttaa gtt tca tgt
atg tcg tgt gga gtt aaa aag act tga att ctg 867 Val Ser Cys Met Ser
Cys Gly Val Lys Lys Thr Ile Leu 270 275 280 ttt gct aag tat atg cta
att ttt cct tat gtc aat tct ata cca ttt 915 Phe Ala Lys Tyr Met Leu
Ile Phe Pro Tyr Val Asn Ser Ile Pro Phe 285 290 295 aag ctt cat ttg
tta aag aat atg cct gtg aaa ctt gat aag gta gaa 963 Lys Leu His Leu
Leu Lys Asn Met Pro Val Lys Leu Asp Lys Val Glu 300 305 310 atg tag
cag cct ctc att taataa tct gat ggg gct tct gtt ttt cca 1011 Met Gln
Pro Leu Ile Ser Asp Gly Ala Ser Val Phe Pro 315 320 325 cat aga atg
ggt tgt ttc tgc taa ggg cta cag agg agg aaa gtc act 1059 His Arg
Met Gly Cys Phe Cys Gly Leu Gln Arg Arg Lys Val Thr 330 335 340 ggc
aaa act tcc gtg acc aaa tat cct gaa att agt att ttt tta aaa 1107
Gly Lys Thr Ser Val Thr Lys Tyr Pro Glu Ile Ser Ile Phe Leu Lys 345
350 355 aga cct tat ttt gag ttt tca gtt aca taa aaa agc aga agc aga
ttg 1155 Arg Pro Tyr Phe Glu Phe Ser Val Thr Lys Ser Arg Ser Arg
Leu 360 365 370 gtt tcc taa gtg agc atc gtt tgt gag aat ttt
tagtcagtgt tttga aca 1206 Val Ser Val Ser Ile Val Cys Glu Asn Phe
Thr 375 380 att att gtt ttt cta agc ttc gtg ttg act ttc tct gat gcg
tag aaa 1254 Ile Ile Val Phe Leu Ser Phe Val Leu Thr Phe Ser Asp
Ala Lys 385 390 395 agt gtt cta acg tag cca agg tta agc cgc tgt cac
tac tga aat gct 1302 Ser Val Leu Thr Pro Arg Leu Ser Arg Cys His
Tyr Asn Ala 400 405 410 aag aat ttt cct ctt ttc ccg tag tgt aga ggg
gta ggg tgt ggg aag 1350 Lys Asn Phe Pro Leu Phe Pro Cys Arg Gly
Val Gly Cys Gly Lys 415 420 425 aag ccg tgt tag cac atc tgt agt att
ctg tgt gta tgc tta gaa cca 1398 Lys Pro Cys His Ile Cys Ser Ile
Leu Cys Val Cys Leu Glu Pro 430 435 440 gcg tag acc gga tgg gag gat
gga cta ggc cta atc cct ccc aac tgg 1446 Ala Thr Gly Trp Glu Asp
Gly Leu Gly Leu Ile Pro Pro Asn Trp 445 450 455 tgg atg tga aga ggt
cag gta gga agg cac agg agg gtc acc act gtc 1494 Trp Met Arg Gly
Gln Val Gly Arg His Arg Arg Val Thr Thr Val 460 465 470 aca gca gtg
cca tgc aga cat cct agg aga aga cat ggc agt gtt tct 1542 Thr Ala
Val Pro Cys Arg His Pro Arg Arg Arg His Gly Ser Val Ser 475 480 485
tct cag tgc ttc ttc cct taa ctg agc tct gct cac aga cag cta gaa
1590 Ser Gln Cys Phe Phe Pro Leu Ser Ser Ala His Arg Gln Leu Glu
490 495 500 tag att tta act gta aca gaa acc taaatgtaa tta aaa cct
ggt ctt cct 1641 Ile Leu Thr Val Thr Glu Thr Leu Lys Pro Gly Leu
Pro 505 510 515 tgg taa gca gac tta aaa tat ctg tat agt aca tgc aag
tgg aaa att 1689 Trp Ala Asp Leu Lys Tyr Leu Tyr Ser Thr Cys Lys
Trp Lys Ile 520 525 530 tgg gaa tgc gtg tct ctg aat aca tac cgg aag
ggc tac tat tac ctt 1737 Trp Glu Cys Val Ser Leu Asn Thr Tyr Arg
Lys Gly Tyr Tyr Tyr Leu 535 540 545 ttc ctt acc att tat act tac cta
atg gaa acg agc ttg ttt taa cta 1785 Phe Leu Thr Ile Tyr Thr Tyr
Leu Met Glu Thr Ser Leu Phe Leu 550 555 560 tca gaa cac tat ttt gta
agg tgc tgc aaa gac agt tga agt ttt cat 1833 Ser Glu His Tyr Phe
Val Arg Cys Cys Lys Asp Ser Ser Phe His 565 570 575 tac caa ctt ccc
caa taa acc agg tgt tca aaa aaa a 1870 Tyr Gln Leu Pro Gln Thr Arg
Cys Ser Lys Lys 580 585 2 223 PRT homo sapiens sapiens 2 Phe Ser
Arg Gly Val Ser Ala Arg Arg Thr Thr Leu Gly Arg Cys Ser 1 5 10 15
Cys Pro Ala Phe Leu Arg Ser Ala Pro Ala Ala Thr Asp Asp Met Leu 20
25 30 Arg Cys Gly Leu Ala Cys Glu Arg Cys Arg Trp Ile Leu Pro Leu
Leu 35 40 45 Leu Leu Ser Ala Ile Ala Phe Asp Ile Ile Ala Leu Ala
Gly Arg Gly 50 55 60 Trp Leu Gln Ser Ser Asn His Ile Gln Thr Ser
Ser Leu Trp Trp Arg 65 70 75 80 Cys Phe Asp Glu Gly Gly Gly Ser Gly
Ser Tyr Asp Asp Gly Cys Gln 85 90 95 Ser Leu Met Glu Tyr Ala Trp
Gly Arg Ala Ala Ala Ala Thr Leu Phe 100 105 110 Cys Gly Phe Ile Ile
Leu Cys Ile Cys Phe Ile Leu Ser Phe Phe Ala 115 120 125 Leu Cys Gly
Pro Gln Met Leu Val Phe Leu Arg Val Ile Gly Gly Leu 130 135 140 Leu
Ala Leu Ala Ala Ile Phe Gln Ile Ile Ser Leu Val Ile Tyr Pro 145 150
155 160 Val Lys Tyr Thr Gln Thr Phe Arg Leu His Asp Asn Pro Ala Val
Asn 165 170 175 Tyr Ile Tyr Asn Trp Ala Tyr Gly Phe Gly Trp Ala Ala
Thr Ile Ile 180 185 190 Leu Ile Gly Cys Ser Phe Phe Phe Cys Cys Leu
Pro Asn Tyr Glu Asp 195 200 205 Asp Leu Leu Gly Ala Ala Lys Pro Arg
Tyr Phe Tyr Pro Pro Ala 210 215 220 3 33 PRT homo sapiens sapiens 3
Cys Gly Arg Lys Ser Leu Arg Lys Ala Cys Cys Lys Met Asp Leu Arg 1 5
10 15 Arg Lys Leu Phe Ser Lys Ala Gln Gly Thr Tyr Val Trp Ala Met
Phe 20 25 30 Ile 4 5 PRT homo sapiens sapiens 4 Ser Glu Met Leu Glu
1 5 5 8 PRT homo sapiens sapiens 5 Met Leu Lys Lys Ile Leu His Asn
1 5 6 11 PRT homo sapiens sapiens 6 Val Ser Cys Met Ser Cys Gly Val
Lys Lys Thr 1 5 10 7 35 PRT homo sapiens sapiens 7 Ile Leu Phe Ala
Lys Tyr Met Leu Ile Phe Pro Tyr Val Asn Ser Ile 1 5 10 15 Pro Phe
Lys Leu His Leu Leu Lys Asn Met Pro Val Lys Leu Asp Lys 20 25 30
Val Glu Met 35 8 4 PRT homo sapiens sapiens 8 Gln Pro Leu Ile 1 9
15 PRT homo sapiens sapiens 9 Ser Asp Gly Ala Ser Val Phe Pro His
Arg Met Gly Cys Phe Cys 1 5 10 15 10 33 PRT homo sapiens sapiens 10
Gly Leu Gln Arg Arg Lys Val Thr Gly Lys Thr Ser Val Thr Lys Tyr 1 5
10 15 Pro Glu Ile Ser Ile Phe Leu Lys Arg Pro Tyr Phe Glu Phe Ser
Val 20 25 30 Thr 11 8 PRT homo sapiens sapiens 11 Lys Ser Arg Ser
Arg Leu Val Ser 1 5 12 8 PRT homo sapiens sapiens 12 Val Ser Ile
Val Cys Glu Asn Phe 1 5 13 15 PRT homo sapiens sapiens 13 Thr Ile
Ile Val Phe Leu Ser Phe Val Leu Thr Phe Ser Asp Ala 1 5 10 15 14 5
PRT homo sapiens sapiens 14 Lys Ser Val Leu Thr 1 5 15 8 PRT homo
sapiens sapiens 15 Pro Arg Leu Ser Arg Cys His Tyr 1 5 16 9 PRT
homo sapiens sapiens 16 Asn Ala Lys Asn Phe Pro Leu Phe Pro 1 5 17
11 PRT homo sapiens sapiens 17 Cys Arg Gly Val Gly Cys Gly Lys Lys
Pro Cys 1 5 10 18 13 PRT homo sapiens sapiens 18 His Ile Cys Ser
Ile Leu Cys Val Cys Leu Glu Pro Ala 1 5 10 19 16 PRT homo sapiens
sapiens 19 Thr Gly Trp Glu Asp Gly Leu Gly Leu Ile Pro Pro Asn Trp
Trp Met 1 5 10 15 20 35 PRT homo sapiens sapiens 20 Arg Gly Gln Val
Gly Arg His Arg Arg Val Thr Thr Val Thr Ala Val 1 5 10 15 Pro Cys
Arg His Pro Arg Arg Arg His Gly Ser Val Ser Ser Gln Cys 20 25 30
Phe Phe Pro 35 21 9 PRT homo sapiens sapiens 21 Leu Ser Ser Ala His
Arg Gln Leu Glu 1 5 22 7 PRT homo sapiens sapiens 22 Ile Leu Thr
Val Thr Glu Thr 1 5 23 7 PRT homo sapiens sapiens 23 Leu Lys Pro
Gly Leu Pro Trp 1 5 24 44 PRT homo sapiens sapiens 24 Ala Asp Leu
Lys Tyr Leu Tyr Ser Thr Cys Lys Trp Lys Ile Trp Glu 1 5 10 15 Cys
Val Ser Leu Asn Thr Tyr Arg Lys Gly Tyr Tyr Tyr Leu Phe Leu 20 25
30 Thr Ile Tyr Thr Tyr Leu Met Glu Thr Ser Leu Phe 35 40 25 13 PRT
homo sapiens sapiens 25 Leu Ser Glu His Tyr Phe Val Arg Cys Cys Lys
Asp Ser 1 5 10 26 8 PRT homo sapiens sapiens 26 Ser Phe His Tyr Gln
Leu Pro Gln 1 5 27 6 PRT homo sapiens sapiens 27 Thr Arg Cys Ser
Lys Lys 1 5 28 10 DNA Artificial pcr primer 28 ctgcttgatg 10
* * * * *