A polynucleotide sequence as shown in SEQ ID NO:1 is associated
with metastatic potential of cancer cells, especially breast cancer
cells. Methods are provided for determining the risk of metastasis
of a tumor, by determining whether a tissue sample from a tumor
expresses a polypeptide or mRNA encoded by a polynucleotide as shown
in SEQ ID NO:1. Also provided are therapeutic methods and compositions.
1. An isolated nucleic acid molecule comprising a polynucleotide
selected from the group consisting of: (a) a polynucleotide encoding
amino acids from about 1 to about 273 of SEQ ID NO:2; (b) a polynucleotide
encoding amino acids from about 2 to about 273 of SEQ ID NO:2; (c)
a polynucleotide encoding amino acids from about 26 to about 273
of SEQ ID NO:2; (d) the polynucleotide complement of the polynucleotide
of (a), (b), or (c); and (e) a polynucleotide at least 90% identical
to the polynucleotide of (a), (b), (c), or (d).
2. An isolated nucleic acid molecule comprising 24-738 contiguous
nucleotides from the coding region of SEQ ID NO:1.
3. The isolated nucleic acid molecule of claim 2, which comprises
50-500 contiguous nucleotides from the coding region of SEQ ID NO:1.
4. The isolated nucleic acid molecule of claim 3, which comprises
75-250 contiguous nucleotides from the coding region of SEQ ID NO:1.
5. An isolated nucleic acid molecule comprising a polynucleotide
encoding a polypeptide wherein, except for at least one conservative
amino acid substitution, said polypeptide has an amino acid sequence
selected from the group consisting of: (a) amino acids about 1 to
about 273 of SEQ ID NO:2; (b) amino acids about 2 to about 273 of
SEQ ID NO:2; and (c) amino acids 26 to 273 of SEQ ID NO:2.
6. The isolated nucleic acid molecule of claim 1, which is DNA.
7. A method of making a recombinant vector comprising inserting
a nucleic acid molecule of claim 1 into a vector in operable linkage
to a promoter.
8. A recombinant vector produced by the method of claim 7.
9. A method of making a recombinant host cell comprising introducing
the recombinant vector of claim 8 into a host cell.
10. A recombinant host cell produced by the method of claim 9.
11. A recombinant method of producing a polypeptide, comprising
culturing the recombinant host cell of claim 10 under conditions
such that said polypeptide is expressed and recovering said polypeptide.
12. An isolated polypeptide comprising amino acids at least 95%
identical to amino acids selected from the group consisting of:
(a) amino acids about 1 to about 273 of SEQ ID NO:2; (b) amino acids
about 2 to about 273 of SEQ ID NO:2; and (c) amino acids 26 to 273
of SEQ ID NO:2.
13. An isolated polypeptide wherein, except for at least one conservative
amino acid substitution, said polypeptide has an amino acid sequence
selected from the group consisting of: (a) amino acids about 1 to
about 273 of SEQ ID NO:2; (b) amino acids about 2 to about 273 of
SEQ ID NO:2; and (c) amino acids 26 to 273 of SEQ ID NO:2.
14. An isolated polypeptide comprising amino acids selected from
the group consisting of: (a) amino acids about 1 to about 273 of
SEQ ID NO:2; (b) amino acids about 2 to about 273 of SEQ ID NO:2;
and (c) amino acids 26 to 273 of SEQ ID NO:2.
15. An epitope-bearing portion of the polypeptide of SEQ ID NO:2.
16. The epitope-bearing portion of claim 15, which comprises 8-25
contiguous amino acids of SEQ ID NO:2.
17. The epitope-bearing portion of claim 15, which comprises 10
contiguous amino acids of SEQ ID NO:2.
18. An isolated antibody that binds specifically to the polypeptide
of claim 12.
19. An isolated antibody that binds specifically to a polypeptide
of claim 13.
20. An isolated antibody that binds specifically to the polypeptide
of claim 14.
21. A method for detecting a human gene encoding SEQ ID NO:2 said
method comprising obtaining in computer-readable format SEQ ID NO:1,
comparing said sequence with polynucleotide sequences of a human
genome, and identifying one or more human genome sequences having
at least 95% sequence identity to SEQ ID NO:1 as determined by the
Smith-Waterman algorithm using an affine gap search with a gap open
penalty of 12 and a gap extension penalty of 1 as parameters.
22. A non-naturally occurring fusion protein comprising a first
protein segment and a second protein segment fused to each other
by means of a peptide bond, wherein the first protein segment comprises
at least six contiguous amino acids selected from an amino acid
sequence encoded by the nucleotide sequence of SEQ ID NO:1 or the
23. The fusion protein of claim 22 wherein said first protein segment
comprises at least six contiguous amino acids of SEQ ID NO:2.
24. The fusion protein of claim 23 wherein said first protein segment
comprises at least twelve contiguous amino acids of SEQ ID NO:2.
25. The fusion protein of claim 22 wherein said first protein segment
comprises amino acids 20-30 of SEQ ID NO:2.
26. The fusion protein of claim 24 wherein said first protein segment
comprises at least 50 contiguous amino acids of SEQ ID NO:2.
27. The fusion protein of claim 26 wherein said first protein segment
comprises at least 100 contiguous amino acids of SEQ ID NO:2.
28. The fusion protein of claim 22 wherein said first protein segment
comprises amino acids 26-287 of SEQ ID NO:2.
29. A method for comparing metastatic potential of tumor cells
in a first and second tissue sample, comprising: measuring in said
tissue samples an expression product of a gene which comprises a
polypeptide coding region of SEQ ID NO:1, wherein at least a two-fold
greater expression of the product in the first tissue sample indicates
a greater metastatic potential compared to the second tissue sample.
30. The method of claim 29 wherein the expression product is protein.
31. The method of claim 30 wherein the protein is measured using
an antibody which specifically binds to the protein.
32. The method of claim 29 wherein the expression product is mRNA.
33. The method of claim 32 wherein said mRNA is measured using
a polynucleotide probe comprising at least 20 contiguous nucleotides
of nucleotides 365-1173 of SEQ ID NO:1.
34. A composition for inhibiting expression of protein by a mammary
carcinoma cell, said composition comprising the polynucleotide of
SEQ ID NO:4.
35. A method of inhibiting expression of a protein by a mammary
carcinoma cell, said method comprising contacting said cell with
a composition comprising the polynucleotide of SEQ ID NO:4.
CROSS-REFERENCE TO RELATED APPLICATIONS
 This application claims priority from U.S. Patent Application
No. 60/175,462 filed Jan. 10, 2000, which is incorporated by reference
herein in its entirety.
 This invention relates to methods for predicting the behavior
of tumors. More particularly, the invention relates to methods in
which a tumor sample is examined for expression of a specified gene
sequence thereby to indicate propensity for metastatic spread.
BACKGROUND OF THE INVENTION
 Breast cancer is one of the most common malignant diseases
with about 1,000,000 new cases per year worldwide. Despite use of
a number of histochemical, genetic, and immunological markers, clinicians
still have a difficult time predicting which tumors will metastasize
to other organs. Some patients are in need of adjuvant therapy to
prevent recurrence and metastasis and others are not. However, distinguishing
between these subpopulations of patients is not straightforward,
and course of treatment is not easily charted. There is a need in
the art for new markers for distinguishing between tumors which
will or have metastasized and those which are less likely to metastasize
SUMMARY OF THE INVENTION
 It is an object of the present invention to provide markers
for distinguishing between tumors which will or have metastasized
and those which are less likely to metastasize. These and other
objects of the invention are provided by one or more of the embodiments
 One embodiment of the invention provides an isolated and
purified human protein having an amino acid sequence which is at
least 85% identical to an amino acid sequence encoded by the nucleotide
sequence of SEQ ID NO:1 or the complement thereof.
 Another embodiment of the invention provides a fusion protein
which comprises a first protein segment and a second protein segment
fused to each other by means of a peptide bond. The first protein
segment consists of at least six contiguous amino acids selected
from an amino acid sequence encoded by a nucleotide sequence SEQ
ID NO:1 or the complement thereof, and the second protein segment
comprises an amino acids sequence not found adjacent to the first
protein segment in the native protein encoded by SEQ ID NO:1.
 Yet another embodiment of the invention provides an isolated
and purified polypeptide consisting of at least six contiguous amino
acids of a human protein having an amino acid sequence encoded by
a nucleotide sequence of SEQ ID NO:1 or the complement thereof.
 Still another embodiment of the invention provides a preparation
of antibodies which specifically bind to a human protein which comprises
an amino acid sequence encoded by a nucleotide sequence of SEQ ID
NO:1 or the complement thereof.
 Even another embodiment of the invention provides an isolated
and purified subgenomic polynucleotide comprising at least 11 contiguous
nucleotides of a nucleotide sequence which is at least 95% identical
to a nucleotide sequence of SEQ ID NO:1 or the complement thereof.
 Another embodiment of the invention provides an isolated
and purified polynucleotide which comprises a coding sequence comprising
a nucleotide sequence of SEQ ID NO:1 or the complement thereof.
 Yet another embodiment of the invention provides a method
for determining metastasis in a tissue sample. An expression product
of a gene which comprises a coding sequence of SEQ ID NO:1 is measured
in a tissue sample. A tissue sample which expresses the product
at a higher level than in a control sample is categorized as having
greater metastatic potential.
 Yet a further embodiment of the invention provides a method
for detecting a human gene encoding SEQ ID NO:2, the method comprising
obtaining in computer-readable format SEQ ID NO:1, comparing the
sequence with polynucleotide sequences of a human genome, and identifying
one or more human genome sequences having at least 95% sequence
identity to SEQ ID NO:1 as determined by the Smith-Waterman algorithm
using an affine gap search with a gap open penalty of 12 and a gap
extension penalty of 1 as parameters.
 The invention thus provides the art with a number of polynucleotides
and polypeptides, which can be used as markers of metastasis. These
are useful for more rationally prescribing the course of therapy
for breast cancer patients.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 illustrates the polynucleotide sequence of human
Out at First (SEQ ID NO:1).
 FIG. 2 illustrates the amino acid sequence encoded by SEQ
ID NO:1 (SEQ ID NO:2).
 FIG. 3 illustrates the putative signal peptide (SEQ ID NO:3).
 FIG. 4 illustrates the translation of SEQ ID NO:1 (SEQ ID
NO:1, polynucleotide; SEQ ID NO:2, amino acid sequence).
 FIG. 5 illustrates the expression of hsOAF relative to .beta.-Actin
in tumor cell lines and tumor tissues from SCID mice developed from
the cell lines. "PT" refers to primary tumor.
 FIG. 6 illustrates the growth of colonies by MDA-MB-435
cells in soft agar following treatment with antisense oligo SEQ
ID NO:4 (66-2as) or reverse control SEQ ID NO:5 (66-2rc), relative
to untreated cells (WT).
 FIG. 7 is an alignment of the human OAF amino acid sequence
with the Drosophila OAF amino acid sequence.
 FIG. 8. FIG. 8A illustrates the expression of hsOAF protein
in COS-7 and MCF-7 cell lines. FIG. 8B illustrates the expression
of hsOAF protein in mammory carcinoma cell lines.
 FIG. 9 illustrates the expression of hsOAF in normal human
 FIG. 10. FIG. 10A illustrates the morphological changes
seen in MDA-MB-435 cells following treatment with antisense oligo
(SEQ ID NO:4). AS=antisense; RC=reverse control (SEQ ID NO:5); M=conditioned
medium. FIG. 10B illustrates cell invasion following treatment of
MDA-MB-435 cells with AS, RC and RC+M.
 FIG. 11 illustrates the predicted signal sequence of human
OAF (double underline).
 FIG. 12. FIG. 12A and 12B illustrate the secretion of hsOAF
by MDA-MB-435 cells treated with antisense oligo (SEQ ID NO:4) or
reverse control oligo (SEQ ID NO:5).
DETAILED DESCRIPTION OF THE INVENTION
 The most lethal cause in patients with breast cancer is
the metastasis of breast carcinomas and their proliferation at distant
loci (lung and bone, mainly). Metastasis is a multistep process
by which tumor cells emigrate from the primary tumor, disseminate
through blood and lymph vessels, and then are deposited in specific
target organs where they re-colonize. Schirrmacher, V., Adv. Cancer
Res. 43:1-73, 1985 and Liotta, L. A. et al., Cell 64(2):327-36 (1991).
During this process the invasiveness of tumor cells is crucial since
they must encounter and pass through numerous basement membranes.
Liotta, L. A., Am. J. Pathol. 117(3):339-48 (1984) and Fidler, I.
J., Cancer Res. 38(9):2651-60 (1978). Therefore the elucidation
of the molecular causes of tumor cell invasion and metastasis is
essential for the development of efficient treatment procedures
for breast cancer patients. Genes differentially expressed in breast
tumor metastasis are potential targets that play critical roles
during metastasis. Identification of such genes and their biological
function will significantly contribute to the development of therapy
and diagnosis for breast cancer.
 Some important genes involved in breast tumor metastasis
have been discovered. Loss of estrogen receptor and presence of
vimentin have been associated with human breast tumor invasiveness
and poor prognosis, and also correlate with the invasiveness and
metastatic potential of human breast cancer cell lines. Aamdal S.,
et al., Cancer 53(11):2525-9 (1984); Clark, G. M., et al., Semin
Oncol., 2 Suppl 1:20-5 (1988); Raymond, W. A. et al., J. Pathol.
157(4):299-306 (1989); Raymond, W. A., et al., J. Pathol. 158(2):107-14
(1989); and Thompson, E. W. et al., J. Cell Physiol. 150(3):534-44
(1992). E-cadherin underexpression has been implicated in mammary
tumor invasiveness. Vleminckx, K., et al., Cell 66(1):107-19 (1991)
and Oka, H., et al., Cancer Res. 53(7):1696-701 (1993). Maspin,
a protease inhibitor expressed in normal mammary epithelial cells'
but not in most breast carcinoma cell lines, was able to suppress
MDA-MB-435 cells ability to induce tumors and metastasize in mice
and to invade basement membrane in vitro. Loss of maspin expression
occurred most frequently in advanced cancers. Zou, Z., et al., Science
263(5146):526-9 (1994) and Seftor, R. E., et al., Cancer Res. 58(24):5681-5
 Overexpression of TIMP-4 (tissue inhibitor of metalloproteinases-4)
or CLCA2 (Ca.sup.2+-activated chloride channel-2) in MDA-MB-435
cells by transfection inhibited the tumorigenicity, invasiveness
and metastasis ability of the cells. Wang, M., et al., Oncogene
14(23):2767-74 (1997) and Gruber, A. D., et al., Cancer Res. 59(21):
5488-91 (1999). Overexpression of the growth factor receptors IGF-IR
and p185.sup.ErbB-2 has been found to be involved in breast cancer
metastasis. Surmacz, E., et al., Breast Cancer Res. Treat 47(3):255-67
(1998); Dunn, S. E., et al., Cancer Res. 58(15):3353-61 (1998);
Tan, M., et al., Cancer Res. 57(6):1199-205 (1997); Dhingra, K.,
et al., Semin Oncol. 23(4):436-45 (1996); and Revillion, F., et
al., Eur. J. Cancer 34(6):791-808 (1998).
 The aspartyl protease cathepsin D has been reported to be
a marker of poor prognosis for breast cancer patients and there
is a significant correlation between high cathepsin D concentration
in the cytosol of primary breast cancer and development of metastasis,
though no correlation was found between cathepsin D secretion and
invasion ability of breast cancer cell lines. Rochefort, H., Breast
Cancer Res Treat 16(1):3-13 (1990); Johnson, M. D., et al., Cancer
Res. 53(4):873-7 (1993); and Rochefort, H., et al., Clin Chim Acta.
29(2):157-70 (2000). Osteopontin, a secreted integrin-binding glycoprotein
that is thought to be involved in bone resorption and bone formation,
can induce migration and invasion of mammary carcinoma cells. Osteopontin
levels (tumor cell or plasma levels) have been associated with enhanced
malignancy of breast cancer. Denhardt, D. T., et al., FASEB J. 7(15):1475-82
(1993); Denhardt, D. T., et al., J. Cell Biochem Suppl., 30-31:92-102
(1998); Tuck, A. B., et al., J. Cell Biochem. 78(3):465-75 (2000);
Tuck, A. B., et al., Oncogene 18(29):4237-46 (1999); and Singhal,
H., et al., Clin Cancer Res. 3(4):605-11 (1997).
 The invention relates to the cloning of a novel gene overexpressed
in highly metastatic human breast cancer cell lines. It encodes
a secreted protein and its protein secretion has been confirmed
to be much greater in highly metastatic human breast cancer cell
lines than in low metastatic/nonmetastatic cell lines. Knockout
of the secretion of this protein of the aggressive MDA-MB-435 cell
line by antisense oligo technology resulted in significant morphological
alteration along with reduced invasiveness and proliferation rate
of the cells. The gene is named hsOAF based on its homology with
the Drosophila gene OAF (out at first). Bergstrom, D. E., et al.,
Genetics 139(3):1331-46 (1995) and Merli, C., et al., Genes Dev.
 It is a discovery of the present invention that a polynucleotide
is differentially expressed between high metastatic breast cancer
cells and non-metastatic or low metastatic cancer cells. This polynucleotide
therefore relates to a metastatic marker gene. This information
can be utilized to make diagnostic reagents specific for the expression
products of the differentially expressed gene. It can also be used
in diagnostic and prognostic methods which will help clinicians
in planning appropriate treatment regimes for cancers, especially
of the breast.
 The polynucleotide is shown in FIG. 1 (SEQ ID NO:1), and
the predicted open reading frame (ORF) encodes a polypeptide shown
in FIG. 2 (SEQ ID NO:2). The first 30 amino acid residues (SEQ ID
NO:3) comprise a putative signal peptide, with a predicted protease
cleavage site indicated by "*"; APLLG*TGAPA (between amino
acids at positions 25 and 26 of SEQ ID NO:3).
 The polynucleotide sequence of the invention shares some
homology with a Drosophila gene known as "Out at First"(oaf).
Transcription of oaf results in three classes of alternatively polyadenylated
RNAs, the expression of which is developmentally regulated. All
oaf transcripts contain two adjacent ORFs separated by a single
UGA stop codon. Suppression of the UGA codon during translation
could lead to the production of different proteins from the same
RNA molecule. During oogenesis, oaf RNA is expressed in nurse cells
of all ages, and is maternally contributed to the egg.
 During embryonic development, zygotic transcription of the
oaf gene occurs in small clusters of cells in most or all segments
at the time of germband extension and later in a segmentally repeated
pattern in the developing central nervous system. The oaf gene is
also expressed in the embryonic, larval and adult gonads of both
sexes. (Bergstrom, D. E. et al., Genetics 139:1331-1346, 1995.)
 The polynucleotide of the invention was differentially expressed
in seven pairs of high metastatic versus non-metastatic or low metastatic
breast cancer cell lines. The cell lines used are MDA-MB-361 (derived
from human breast adenocarcinoma), MDA-MB-231 (human breast cancer
cells metastatic to bone and/or lung); MDA-MB-468 (derived from
human estrogen receptor-negative breast cancer cells); MCF-7 (non-metastatic
human breast cancer cells); ZR-75-1 (derived from estrogen receptor-positive
human breast carcinomas, Engle et al., Cancer Res. 38:3352-64 (1978));
and MDA-MB-435 (derived from estrogen receptor-negative human breast
carcinoma cells, Rishi et al., Cancer Res. 56:5246-5252 (1996)).
 The expression profile is as follows:
1 TABLE 1 Cell Line Pair Ratio of Expression MDA-MB-361/MDA-MB-231
0.11 MDA-MB-468/MDA-MB-231 0.44 MCF-7/MDA-MB-231 0.17 ZR-75-1/MDA-MB-231
0.12 MDA-MB-361/MDA-MB-435 0.06 MDA-MB-468/MDA-MB-435 0.36 MCF-7/MDA-MB-435
 The upregulation of the mRNA expression was confirmed by
Northern blot analysis using total RNA from the cell lines (FIG.
 The cell lines in which expression of the polynucleotide
of the invention was compared represent human breast cancers of
varying metastatic potential. Cell line ZR-75-1 cultures were derived
from malignant ascitic effusion of a breast cancer patient. The
cell lines grown in vitro closely resembled the morphology seen
in biopsies or cell preparations from the donors of the original
cells. ZR-75-1 cells are specifically stimulated by estrogen, and
have been used as a model system for studying estrogen responsiveness.
Engel, L. W. et al., Cancer Res. 38:3352-3364, 1978.
 Cell line MDA-MB-435 is an estrogen receptor-negative cell
line that has been studied as a model for human breast cancer, for
example, for studying the mechanism of action of growth inhibition
in the presence of retinoic acid. Rishi, A. K. et al., Cancer Res.
56:5246-5252, 1996. Growth inhibition by retinoids has also been
studied in MCF-7 cells and MDA MB 468 cells. Tin-U, C. K. et al.,
Am. Soc. Clin. Onc. Proceedings, Vol. 17,2125, 1998.
 Cell line MDA-MB-361 was derived from a human breast adenocarcinoma,
specifically from a malignant site. ATCC Number HTB-27. Differential
expression of human Wnt genes has been studied in this cell line.
Huguet, E. L. et al., Cancer Res. 54:2615-2621, 1994.
 Once metastasis occurs, mammary primary tumor cells invade
basement membranes and spread to other organs of the body and the
survival chance of patients with breast cancer becomes quite slim.
It is critical to identify genes participating in breast cancer
invasion and metastasis on behalf of clinical diagnosis and therapy.
Such genes are potential markers for diagnosis or candidate targets
for therapeutic drug development. For instance, presence of vimentin
in human breast tumor has been associated with lack of estrogen
receptor and tumor invasiveness as a marker of poor prognosis. Raymond,
W. A. et al., J. Pathol. 157(4):299-306 (1989); Raymond, W. A.,
et al., J. Pathol. 158(2):107-14 (1989); and Thompson, E. W. et
al., J. Cell Physiol.150(3):534-44 (1992). Increased activities
of matrix metalloproteinases are related with the metastatic phenotype
of carcinomas, especially breast cancer. Basset, P., et al., Nature
348(6303):699-704 (1990) and Basset, P., et al., Cancer 74(3 Suppl):1045-9
(1994). Osteopontin, a secreted integrin-binding glycoprotein, is
able to induce increased invasiveness of human mammary epithelial
cells and has been associated with enhanced malignancy in breast
cancer. Tuck, A. B., et al., J. Cell Biochem. 78(3):465-75 (2000);
Tuck, A. B., et al., Oncogene 18(29):4237-46 (1999); and Singhal,
H., et al., Clin Cancer Res. 3(4): 605-11 (1997).
 The invention relates to identification of a novel secreted
protein (hsOAF) involved in breast cancer metastasis. The human
breast cancer cell lines used in elucidating the role of the protein
are divided into three groups according to their metastatic abilities:
highly metastatic, low metastatic, and nonmetastatic. Taking advantage
of different metastatic potentials among these cell line groups
and utilizing the advanced microarray technology, genes were identified
which are differentially expressed between highly metastatic human
breast cancer cell lines and low metastatic/nonmetastatic ones.
These genes are good candidates for expression validation and function
studies that could lead to better understanding of the molecular
mechanism of breast cancer metastasis. hsOAF gene is the focus of
this invention as it encodes a novel secreted protein. hsOAF mRNA
expression is quite common in various human normal tissues. However,
hsOAF protein secretion is much stronger by highly metastatic human
breast cancer cell lines than by low metastatic/nonmetastatic ones,
and MDA-MB-435 has the greatest hsOAF secretion. hsOAF gene was
named based on its homology with the Drosophila OAF (out at first)
gene. However, the Drosophila OAF protein may not be a secreted
protein since it does not possess a typical signal peptide sequence
at N-terminus (FIG. 7).
 To address the importance of secreted hsOAF protein in breast
cancer metastasis, antisense oligo technology was used to specifically
knock out hsOAF expression. Antisense oligo technology is an efficient,
fast way to dramatically reduce gene expression for gene functional
studies. Stein, C. A., et al., Science 261(5124):1004-12 (1993);
Defacque, II. et al., J. Cell Physiol. 178(1):109-19 (1999). Knockout
of hsOAF protein secretion of highly metastatic MDA-MB-435 cells
resulted in cell shape change, reduced cell invasiveness and slower
cell proliferation. Treatment of cells with the conditioned medium
(culture medium of normal MDA-MB-435 cells) was able to recover
all those phenotypic alterations caused by the knockout of hsOAF
protein secretion to some degree. Although the inventors are not
bound by a specific mechanism, the secreted hsOAF protein is believed
to be essential for the invasiveness and proliferation of MDA-MB-435
cells. However, knockout of hsOAF protein secretion of another highly
metastatic cell line, MDA-MB-231, by antisense oligo technology
did not cause any significant cellular changes. MDA-MB-435 and MDA-MB-231
are quite different metastatic cell lines and MDA-MB-435 shows much
stronger hsOAF protein secretion than does MDA-MB-213.
 hsOAF gene is located at chromosome 11q23 region where loss
of heterozygosity occurs frequently in human breast tumors. Negrini,
M., et al., Cancer Res 55(14):3003-7 (1995) and Tomlinson, I. P.,
et al., J. Clin. Pathol. 48(5):424-8 (1995). Loss of heterozygosity
at 11q23 in primary human breast tumors has been reported to be
associated with poor survival after metastasis. Winqvist, R., et
al., Cancer Res. 55(12):2660-4 (1995). 11q23 also contains loci
such as ATM (Ataxia-telangiectasia, mutated), and MLL (which is
frequently disrupted by chromosomal rearrangement in acute leukemia).
Rasio, D., et al., Cancer Res. 55(24):6053-7 (1995) and Rubnitz,
J. E., et al., Leukemia 10(1):74-82 (1996). The relationship between
mutation at chromosome 11q23 and hsOAF gene expression in breast
cancer metastasis remains unclear.
 Secreted hsOAF protein may be a suitable target for drug
development against breast cancer and a good diagnostic marker for
the malignancy of breast tumor. SEQ ID NO:1 and polynucleotides
comprising this sequence are therefore useful as metastatic markers.
Reference to metastatic marker nucleotide or amino acid sequences
includes variants which have similar expression patterns in high
metastatic relative to non-metastatic or low metastatic cells. Metastatic
marker polypeptides can differ in length from full-length metastatic
marker proteins and contain at least 6, 8, 10, 12, 15, 18, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120,
140, 160, 180, 200, 220, 240, 260, 265, 270 or 271 or more contiguous
amino acids of a metastatic marker protein. Exemplary polynucleotides
include those encoding amino acids from about 1 to about 273; from
1 to 273; from about 2 to about 273; from 2 to 273; from about 26
to about 273; and from 26 to 273 of SEQ ID NO:2.
 Variants of marker proteins and polypeptides can also occur.
Metastatic marker protein or polypeptide variants can be naturally
or non-naturally occurring. Naturally occurring metastatic marker
protein or polypeptide variants are found in humans or other species
and comprise amino acid sequences which are substantially identical
to a protein encoded by a gene corresponding to the nucleotide sequence
shown in SEQ ID NO:1 or its complement. Non-naturally occurring
metastatic marker protein or polypeptide variants which retain substantially
the same differential expression patterns in high metastatic relative
to low-metastatic or non-metastatic breast cancer cells as naturally
occurring metastatic marker protein or polypeptide variants are
also included here. Preferably, naturally or non-naturally occurring
metastatic marker protein or polypeptide variants have amino acid
sequences which are at least 85%, 90%, 91%, 92%, 93%, 94%, or 95%
identical to amino acid sequences encoded by the nucleotide sequence
shown in SEQ ID NO:1. More preferably, the molecules are at least
96%, 97%, 98% or 99% identical. Percent sequence identity between
a wild-type protein or polypeptide and a variant is determined by
aligning the wild-type protein or polypeptide with the variant to
obtain the greatest number of amino acid matches, as is known in
the art, counting the number of amino acid matches between the wild-type
and the variant, and dividing the total number of matches by the
total number of amino acid residues of the wild-type sequence.
 Preferably, amino acid changes in metastatic marker protein
or polypeptide variants are conservative amino acid changes, i.e.,
substitutions of similarly charged or uncharged amino acids. A conservative
amino acid change involves substitution of one of a family of amino
acids which are related in their side chains. Naturally occurring
amino acids are generally divided into four families: acidic (aspartate,
glutamate), basic (lysine, arginine, histidine), non-polar (alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), and uncharged polar (glycine, asparagine, glutamine,
cystine, serine, threonine, tyrosine) amino acids. Phenylalanine,
tryptophan, and tyrosine are sometimes classified jointly as aromatic
 It is reasonable to expect that an isolated replacement
of a leucine with an isoleucine or valine, an aspartate with a glutamate,
a threonine with a serine, or a similar replacement of an amino
acid with a structurally related amino acid will not have a major
effect on the biological properties of the resulting metastatic
marker protein or polypeptide variant. Properties and functions
of metastatic marker protein or polypeptide variants are of the
same type as a metastatic marker protein or polypeptide comprising
amino acid sequences encoded by the nucleotide sequence shown in
SEQ ID NO:1, although the properties and functions of variants can
differ in degree. Whether an amino acid change results in a metastatic
marker protein or polypeptide variant with the appropriate differential
expression pattern can readily be determined. For example, nucleotide
probes can be selected from the marker gene sequences disclosed
herein and used to detect marker gene mRNA in Northern blots or
in tissue sections, as is known in the art. Alternatively, antibodies
which specifically bind to protein products of metastatic marker
genes can be used to detect expression of metastatic marker proteins
or variants thereof.
 Metastatic marker variants include glycosylated forms, aggregative
conjugates with other molecules, and covalent conjugates with unrelated
chemical moieties. Metastatic marker variants also include allelic
variants, species variants, and muteins. Truncations or deletions
of regions which do not affect the differential expression of metastatic
marker genes are also metastatic marker variants. Covalent variants
can be prepared by linking functionalities to groups which are found
in the amino acid chain or at the N- or C-terminal residue, as is
known in the art.
 It will be recognized in the art that some amino acid sequence
of the polypeptide of the invention can be varied without significant
effect on the structure or function of the protein. If such differences
in sequence are contemplated, it should be remembered that there
are critical areas on the protein which determine activity. In general,
it is possible to replace residues that form the tertiary structure,
provided that residues performing a similar function are used. In
other instances, the type of residue may be completely unimportant
if the alteration occurs at a non-critical region of the protein.
The replacement of amino acids can also change the selectivity of
binding to cell surface receptors. Ostade et al., Nature 361:266-268
(1993) describes certain mutations resulting in selective binding
of TNF-alpha to only one of the two known types of TNF receptors.
Thus, the polypeptides of the present invention may include one
or more amino acid substitutions, deletions or additions, either
from natural mutations or human manipulation.
 The invention further includes variations of the disclosed
polypeptide which show comparable expression patterns or which include
antigenic regions. Such mutants include deletions, insertions, inversions,
repeats, and type substitutions. Guidance concerning which amino
acid changes are likely to be phenotypically silent can be found
in Bowie, J. U., et al., "Deciphering the Message in Protein
Sequences: Tolerance to Amino Acid Substitutions," Science
 Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or negatively
charged amino acids. The latter results in proteins with reduced
positive charge to improve the characteristics of the disclosed
protein. The prevention of aggregation is highly desirable. Aggregation
of proteins not only results in a loss of activity but can also
be problematic when preparing pharmaceutical formulations, because
they can be immunogenic. (Pinckard et al., Clin. Exp. Immunol. 2:331-340
(1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al.,
Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).
 Amino acids in the polypeptides of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as receptor binding, or in vitro
proliferative activity. Sites that are critical for ligand-receptor
binding can also be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al.,
J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312
 As indicated, changes are preferably of a minor nature,
such as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein. Of course, the number
of amino acid substitutions a skilled artisan would make depends
on many factors, including those described above. Generally speaking,
the number of substitutions for any given polypeptide will not be
more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.
 Full-length metastatic marker proteins can be extracted,
using standard biochemical methods, from metastatic marker protein-producing
human cells, such as metastatic breast cancer cells. An isolated
and purified metastatic marker protein or polypeptide is separated
from other compounds which normally associate with a metastatic
marker protein or polypeptide in a cell, such as certain proteins,
carbohydrates, lipids, or subcellular organelles. A preparation
of isolated and purified metastatic marker proteins or polypeptides
is at least 80% pure; preferably, the preparations are 90%, 95%,
or 99% pure.
 A human gene encoding SEQ ID NO:2 can be identified and
isolated using methods know in the art. According to one method,
SEQ ID NO:1 is prepared in a computer-readable format. The sequence
is compared with polynucleotide sequences of a human genome, and
one or more human genome sequences having at least 95% sequence
identity to SEQ ID NO:1 are identified, for example by using the
Smith-Waterman algorithm using an affine gap search with a gap open
penalty of 12 and a gap extension penalty of 1 as parameters. Probes
based on the regions of homology between SEQ ID NO:1 and the human
genome sequences are prepared and used to isolate polynucleotides
from human genomic DNA, using methods known in the art. As of the
filing date a human polynucleotide corresponding to the full polynucleotide
of SEQ ID NO:1 was not identified in the public databases. Thus,
the invention includes human genomic DNA comprising the coding region
of SEQ ID NO:1 and any untranslated regions which do not share homology
with SEQ ID NO:1 but which are contiguous with homologous regions.
Such genomic DNA includes but is not limited to introns, promoters,
and other regulatory regions functionally associated with a human
gene having a region encoding SEQ ID NO:2.
 Metastatic marker proteins and polypeptides can also be
produced by recombinant DNA methods or by synthetic chemical methods.
For production of recombinant metastatic marker proteins or polypeptides,
coding sequences selected from the nucleotide sequences shown in
SEQ ID NO:1, or variants of those sequences which encode metastatic
marker proteins, can be expressed in known prokaryotic or eukaryotic
expression systems (see below). Bacterial, yeast, insect, or mammalian
expression systems can be used, as is known in the art.
 Alternatively, synthetic chemical methods, such as solid
phase peptide synthesis, can be used to synthesize a metastatic
marker protein or polypeptide. General means for the production
of peptides, analogs or derivatives are outlined in CHEMISTRY AND
BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS--A SURVEY OF
RECENT DEVELOPMENTS, Weinstein, B. ed., Marcell Dekker, Inc., publ.,
New York (1983). Moreover, substitution of D-amino acids for the
normal L-stereoisomer can be carried out to increase the half-life
of the molecule. Metastatic marker variants can be similarly produced.
 Non-naturally occurring fusion proteins comprising at least
6, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260,
265, 270 or 271 or more contiguous metastatic marker amino acids
can also be constructed. Human metastatic marker fusion proteins
are useful for generating antibodies against metastatic marker amino
acid sequences and for use in various assay systems. For example,
metastatic marker fusion proteins can be used to identify proteins
which interact with metastatic marker proteins and influence their
functions. Physical methods, such as protein affinity chromatography,
or library-based assays for protein-protein interactions, such as
the yeast two-hybrid or phage display systems, can also be used
for this purpose. Such methods are well known in the art and can
also be used as drug screens.
 A metastatic marker fusion protein comprises two protein
segments fused together by means of a peptide bond. The first protein
segment comprises at least 6, 8, 10, 12, 15, 18, 20, 25, 30, 35,
40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160,
180, 200, 220, 240, 250, 260, 265, 270 or 271 or more contiguous
amino acids of a metastatic marker protein. The amino acids can
be selected from the amino acid sequences encoded by the nucleotide
sequence shown in SEQ ID NO:1 or from variants of the sequence,
such as those described above. The first protein segment can also
comprise a full-length metastatic marker protein.
 In one preferred embodiment, the first protein segment comprises
the polypeptide shown in SEQ ID NO:2. In a variation of this embodiment,
the first protein segment consists of amino acids 31-287 of SEQ
ID NO:2. This fusion protein lacks the signal peptide of SEQ ID
NO:2 and would be suitable for retention of the expressed fusion
protein inside the cell.
 The second protein segment can be a full-length protein
or a protein fragment or polypeptide not found adjacent to the first
protein segment in the native protein encoded by SEQ ID NO:1. The
fusion protein can be labeled with a detectable marker, as is known
in the art, such as a radioactive, fluorescent, chemiluminescent,
or biotinylated marker. The second protein segment can be an enzyme
which will generate a detectable product, such as .beta.-galactosidase.
The first protein segment can be N-terminal or C-terminal, as is
 Techniques for making fusion proteins, either recombinantly
or by covalently linking two protein segments, are also well known.
Recombinant DNA methods can be used to prepare metastatic marker
fusion proteins, for example, by making a DNA construct which comprises
coding sequences of SEQ ID NO:1 in proper reading frame with nucleotides
encoding the second protein segment and expressing the DNA construct
in a host cell, as described below. The open reading frame of SEQ
ID NO:1 is shown in FIG. 4.
 Isolated and purified metastatic marker proteins, polypeptides,
variants, or fusion proteins can be used as immunogens, to obtain
preparations of antibodies which specifically bind to a metastatic
marker protein. The antibodies can be used, inter alia, to detect
wild-type metastatic marker proteins in human tissue and fractions
thereof. The antibodies can also be used to detect the presence
of mutations in metastatic marker genes which result in under- or
over-expression of a metastatic marker protein or in expression
of a metastatic marker protein with altered size or electrophoretic
 Preparations of polyclonal or monoclonal antibodies can
be made using standard methods. Single-chain antibodies can also
be prepared. A preferred immunogen is a polypeptide comprising SEQ
ID NO:2. Single-chain antibodies which specifically bind to metastatic
marker proteins, polypeptides, variants, or fusion proteins can
be isolated, for example, from single-chain immunoglobulin display
libraries, as is known in the art. The library is "panned"
against metastatic marker protein amino acid sequences of SEQ ID
NO:2, and a number of single chain antibodies which bind with high-affinity
to different epitopes of metastatic marker proteins can be isolated.
Hayashi et al., 1995, Gene 160:129-30. Single-chain antibodies can
also be constructed using a DNA amplification method, such as the
polymerase chain reaction (PCR), using hybridoma cDNA as a template.
Thirion et al., 1996, Eur. J. Cancer Prev. 5:507-11.
 Metastatic marker-specific antibodies specifically bind
to epitopes present in a full-length metastatic marker protein having
an amino acid sequence encoded by a nucleotide sequence shown in
SEQ ID NO:1, to metastatic marker polypeptides, or to metastatic
marker variants, either alone or as part of a fusion protein. Preferably,
metastatic marker epitopes are not present in other human proteins.
Typically, at least 6, 8, 10, or 12 contiguous amino acids are required
to form an epitope. However, epitopes which involve non-contiguous
amino acids may require more, e.g., at least 15, 25, or 50 amino
 Antibodies which specifically bind to metastatic marker
proteins, polypeptides, fusion proteins, or variants provide a detection
signal at least 5-, 10-, or 20-fold higher than a detection signal
provided with other proteins when used in Western blots or other
immunochemical assays. Preferably, antibodies which specifically
bind to metastatic marker epitopes do not detect other proteins
in immunochemical assays and can immunoprecipitate a metastatic
marker protein, polypeptide, fusion protein, or variant from solution.
 Subgenomic polynucleotides contain less than a whole chromosome.
Preferably, the polynucleotides are intron-free. In a preferred
embodiment, the polynucleotide molecules comprise a contiguous sequence
of 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500,
600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2100, 2200, 2300 or 2350 nucleotides from SEQ
ID NO:1 or the complements thereof. The complement of a nucleotide
sequence shown in SEQ ID NO:1 is a contiguous nucleotide sequence
which forms Watson-Crick base pairs with a contiguous nucleotide
sequence shown in SEQ ID NO:1.
 Degenerate nucleotide sequences encoding amino acid sequences
of metastatic marker protein or variants, as well as homologous
nucleotide sequences which comprise a polynucleotide at least 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to
the coding region of the nucleotide sequence shown in SEQ ID NO:1,
are also metastatic marker subgenomic polynucleotides. Typically,
homologous metastatic marker subgenomic polynucleotide sequences
can be confirmed by hybridization under stringent conditions, as
is known in the art. Percent sequence identity between wild-type
and homologous variant sequences is determined by aligning the wild-type
polynucleotide with the variant to obtain the greatest number of
nucleotide matches, as is known in the art, counting the number
of nucleotide matches between the wild-type and the variant, and
dividing the total number of matches by the total number of nucleotides
of the wild-type sequence. A preferred algorithm for calculating
percent identity is the Smith-Waterman homology search algorithm
as implemented in MPSRCH program (Oxford Molecular) using an affine
gap search with the following search parameters: gap open penalty
of 12, and gap extension penalty of 1.
 A metastatic marker subgenomic polynucleotide comprising
metastatic marker protein coding sequences can be used in an expression
construct. Preferably, the metastatic marker subgenomic polynucleotide
is inserted into an expression plasmid (for example, the Ecdyson
system, pIND, In vitro Gene). Metastatic marker subgenomic polynucleotides
can be propagated in vectors and cell lines using techniques well
known in the art. Metastatic marker subgenomic polynucleotides can
be on linear or circular molecules. They can be on autonomously
replicating molecules or on molecules without replication sequences.
They can be regulated by their own or by other regulatory sequences,
as are known in the art.
 A host cell comprising a metastatic marker expression construct
can then be used to express all or a portion of a metastatic marker
protein. Host cells comprising metastatic marker expression constructs
can be prokaryotic or eukaryotic. A variety of host cells are available
for use in bacterial, yeast, insect, and human expression systems
and can be used to express or to propagate metastatic marker expression
constructs (see below). Expression constructs can be introduced
into host cells using any technique known in the art. These techniques
include transferrin-polycation-mediated DNA transfer, transfection
with naked or encapsulated nucleic acids, liposome-mediated cellular
fusion, intracellular transportation of DNA-coated latex beads,
protoplast fusion, viral infection, electroporation, and calcium
 A metastatic marker expression construct comprises a promoter
which is functional in a chosen host cell. The skilled artisan can
readily select an appropriate promoter from the large number of
cell type-specific promoters known and used in the art. The expression
construct can also contain a transcription terminator which is functional
in the host cell. The expression construct comprises a polynucleotide
segment which encodes all or a portion of the metastatic marker
protein, variant, fusion protein, antibody, or ribozyme. The polynucleotide
segment is located downstream from the promoter. Transcription of
the polynucleotide segment initiates at the promoter. The expression
construct can be linear or circular and can contain sequences, if
desired, for autonomous replication.
 Bacterial systems for expressing metastatic marker expression
constructs include those described in Chang et al., Nature (1978)
275:615, Goeddel et al., Nature (1979) 281:544, Goeddel et al.,
Nucleic Acids Res. (1980) 8:4057, EP 36,776, U.S. Pat. No. 4,551,433,
deBoer et al., Proc. Nat'l Acad. Sci. USA (1983) 80:21-25, and Siebenlist
et al., Cell (1980) 20:269.
 Expression systems in yeast include those described in Hinnen
et al., Proc. Nat'l Acad. Sci. USA (1978) 75:1929; Ito et al., J.
Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986)
6:142; Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson
et al., J. Gen. Microbiol. (1986) 132:3459, Roggenkamp et al., Mol.
Gen. Genet. (1986) 202:302) Das et al., J. Bacteriol. (1984) 158:1165;
De Louvencourt et al., J. Bacteriol. (1983) 154:737, Van den Berg
et al., Bio/Technology (1990) 8:135; Kunze et al., J. Basic Microbiol.
(1985) 25:141; Cregg et al., Mol. Cell. Biol. (1985) 5:3376, U.S.
Pat. Nos. 4,837,148, 4,929,555; Beach and Nurse, Nature (1981) 300:706;
Davidow et al., Curr. Genet. (1985) 10:380, Gaillardin et al., Curr.
Genet. (1985) 10:49, Ballance et al., Biochem. Biophys. Res. Commun.
(1983) 112:284-289; Tilburn et al., Gene (1983) 26:205-221, Yelton
et al., Proc. Nat'l Acad. Sci. USA (1984) 81:1470-1474, Kelly and
Hynes,EMBO J. (1985) 4:475479; EP 244,234, and WO 91/00357.
 Expression of metastatic marker expression constructs in
insects can be carried out as described in U.S. Pat. No. 4,745,051,
Friesen et al. (1986) "The Regulation of Baculovirus Gene Expression"
in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.), EP
127,839, EP 155,476, and Vlak et al., J. Gen. Virol. (1988) 69:765-776,
Miller et al., Ann. Rev. Microbiol. (1988) 42:177, Carbonell et
al., Gene (1988) 73:409, Maeda et al., Nature (1985) 315:592-594,
Lebacq-Verheyden et al., Mol. Cell. Biol. (1988) 8:3129; Smith et
al., Proc. Nat'l Acad. Sci. USA (1985) 82:8404, Miyajima et al.,
Gene (1987) 58:273; and Martin et al., DNA (1988) 7:99. Numerous
baculoviral strains and variants and corresponding permissive insect
host cells from hosts are described in Luckow et al., Bio/Technology
(1988) 6:47-55, Miller et al., in GENETIC ENGINEERING (Setlow, J.
K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279,
and Maeda et al., Nature, (1985) 315:592-594.
 Mammalian expression of metastatic marker expression constructs
can be achieved as described in Dijkema et al., EMBO J. (1985) 4:761,
Gorman et al., Proc. Nat'l Acad. Sci. USA (1982b) 79:6777, Boshart
et al., Cell (1985) 41:521 and U.S. Pat. No. 4,399,216. Other features
of mammalian expression of metastatic marker expression constructs
can be facilitated as described in Ham and Wallace, Meth. Enz. (1979)
58:44, Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat.
Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO
87/00195, and U.S. RE 30,985.
 Subgenomic polynucleotides of the invention can also be
used in gene delivery vehicles, for the purpose of delivering a
metastatic marker mRNA or oligonucleotide (either with the sequence
of native metastatic marker mRNA or its complement), full-length
metastatic marker protein, metastatic marker fusion protein, metastatic
marker polypeptide, or metastatic marker-specific ribozyme or single-chain
antibody, into a cell, preferably a eukaryotic cell. According to
the present invention, a gene delivery vehicle can be, for example,
naked plasmid DNA, a viral expression vector comprising a metastatic
marker subgenomic polynucleotide, or a metastatic marker subgenomic
polynucleotide in conjunction with a liposome or a condensing agent.
 The invention provides a method of detecting metastatic
marker gene expression in a biological sample. Detection of metastatic
marker gene expression is useful, for example, for identifying metastases
or for determining metastatic potential in a tissue sample, preferably
a tumor. Appropriate treatment regimens can then be designed for
patients who are at risk for developing metastatic cancers in other
organs of the body.
 The body sample can be, for example, a solid tissue or a
fluid sample. The native polypeptide encoded by SEQ ID NO:1 is a
putative secreted protein, and is likely to be detected in body
fluids including blood and lymphatic fluid, particularly those draining
from tumor sites in the body. Protein or nucleic acid expression
products can be detected in the body sample. In one embodiment,
the body sample is assayed for the presence of a metastatic marker
protein. A metastatic marker protein comprises a sequence encoded
by a nucleotide sequence shown in SEQ ID NO:1 or its complement
and can be detected using the marker protein-specific antibodies
of the present invention. The antibodies can be labeled, for example,
with a radioactive, fluorescent, biotinylated, or enzymatic tag
and detected directly, or can be detected using indirect immunochemical
methods, using a labeled secondary antibody. The presence of the
metastatic marker proteins can be assayed, for example, in tissue
sections by immunocytochemistry, or in lysates, using Western blotting,
as is known in the art. Presence of the marker protein is indicative
that the tissue sample is metastatic.
 In another embodiment, the body sample is assayed for the
presence of marker protein mRNA. A sample can be contacted with
a nucleic acid hybridization probe capable of hybridizing with the
mRNA corresponding the selected polypeptide. Still further, the
sample can be subjected to a Northern blotting technique to detect
mRNA, indicating expression of the polypeptide. For those techniques
in which mRNA is detected, the sample can be subjected to a nucleic
acid amplification process whereby the mRNA molecule or a selected
part thereof is amplified using appropriate nucleotide primers.
Other RNA detection techniques can also be used, including, but
not limited to, in situ hybridization.
 Marker protein-specific probes can be generated using the
cDNA sequence disclosed in SEQ ID NO:1. The probes are preferably
at least 15 to 50 nucleotides in length, although they can be at
least 8, 10, 11, 12, 20, 25, 30, 35, 40, 45, 60, 75, or 100 or more
nucleotides in length. A preferable region for selecting probes
is within nucleotide positions 446-1173 of SEQ ID NO:1. The probes
can be synthesized chemically or can be generated from longer polynucleotides
using restriction enzymes. The probes can be labeled, for example,
with a radioactive, biotinylated, or fluorescent tag.
 Optionally, the level of a particular metastatic marker
expression product in a body sample can be quantitated. Quantitation
can be accomplished, for example, by comparing the level of expression
product detected in the body sample with the amounts of product
present in a standard curve. A comparison can be made visually or
using a technique such as densitometry, with or without computerized
assistance. For use as controls, body samples can be isolated from
other humans, other non-cancerous organs of the patient being tested,
or non-metastatic breast cancer from the patient being tested. As
indicated by the results herein, expression of SEQ ID NO:1 in low-metastatic
or non-metastatic breast cancer cells is between 3% and 44% of the
expression levels in highly-metastatic breast cancer cells. If expression
in a test sample is at least 2-fold greater than in a suitable control
sample, this is indicative of metastatic cells.
 Polynucleotides encoding metastatic marker-specific reagents
of the invention, such as antibodies and nucleotide probes, can
be supplied in a kit for detecting marker gene expression products
in a biological sample. The kit can also contain buffers or labeling
components, as well as instructions for using the reagents to detect
the marker expression products in the biological sample.
 Expression of a metastatic marker gene can be altered using
an antisense oligonucleotide sequence. The antisense sequence is
complementary to at least a portion of the coding sequence (nucleotides
365-1173) of a metastatic marker gene having a nucleotide sequence
shown in SEQ ID NO:1. Preferably, the antisense oligonucleotide
sequence is at least six nucleotides in length, but can be at least
about 8, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides long.
Longer sequences can also be used. Antisense oligonucleotide molecules
can be provided in a DNA construct and introduced into cells whose
division is to be decreased. Such cells include highly-metastatic
breast cancer cells.
 Antisense oligonucleotides can comprise deoxyribonucleotides,
ribonucleotides, or a combination of both. Oligonucleotides can
be synthesized manually or by an automated synthesizer, by covalently
linking the 5' end of one nucleotide with the 3' end of another
nucleotide with non-phosphodiester internucleotide linkages such
alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates,
alkylphosphonates, phosphoramidates, phosphate esters, carbamates,
acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
See Brown, 1994, Meth. Mol. Biol. 20:1-8; Sonveaux, 1994, Meth.
Mol. Biol. 26:1-72; Uhlmann et al., 1990, Chem. Rev. 90:543-583.
 Antibodies of the invention which specifically bind to a
metastatic marker protein can also be used to alter metastatic marker
gene expression. By antibodies is meant antibodies and parts or
derivatives thereof, such as single chain antibodies, that retain
specific binding for the protein. Specific antibodies bind to metastatic
marker proteins and prevent the proteins from functioning in the
cell. Polynucleotides encoding specific antibodies of the invention
can be introduced into cells, as described above.
 Marker proteins of the present invention can be used to
screen for drugs which have a therapeutic anti-metastatic effect.
The effect of a test compound on metastatic marker protein synthesis
can also be used to identify test compounds which modulate metastasis.
Test compounds which can be screened include any substances, whether
natural products or synthetic, which can be administered to the
subject. Libraries or mixtures of compounds can be tested. The compounds
or substances can be those for which a pharmaceutical effect is
previously known or unknown.
 Synthesis of metastatic marker proteins can be measured
by any means for measuring protein synthesis known in the art, such
as incorporation of labeled amino acids into proteins and detection
of labeled metastatic marker proteins in a polyacrylamide gel. The
amount of metastatic marker proteins can be detected, for example,
using metastatic marker protein-specific antibodies of the invention
in Western blots. The amount of the metastatic marker proteins synthesized
in the presence or absence of a test compound can be determined
by any means known in the art, such as comparison of the amount
of metastatic marker protein synthesized with the amount of the
metastatic marker proteins present in a standard curve.
 The effect of a test compound on metastatic marker protein
synthesis can also be measured by Northern blot analysis, by measuring
the amount of metastatic marker protein mRNA expression in response
to the test compound using metastatic marker protein specific nucleotide
probes of the invention, as is known in the art.
 Typically, a biological sample is contacted with a range
of concentrations of the test compound, such as 1.0 nM, 5.0 nM,
10 nM, 50 nM, 100 nM, 500 nM, 1 mM, 10 mM, 50 mM, and 100 mM. Preferably,
the test compound increases or decreases expression of a metastatic
marker protein by 60%, 75%, or 80%. More preferably, an increase
or decrease of 85%, 90%, 95%, or 98% is achieved.
 The invention provides compositions for increasing or decreasing
expression of metastatic marker protein. These compositions comprise
polynucleotides encoding all or at least a portion of a metastatic
marker protein gene expression product. Preferably, the therapeutic
composition contains an expression construct comprising a promoter
and a polynucleotide segment encoding at least a portion of the
metastatic marker protein which is effective to decrease metastatic
potential. Portions of metastatic marker genes or proteins which
are effective to decrease metastatic potential of a cell can be
determined, for example, by introducing portions of metastatic marker
genes or polypeptides into metastatic cell lines, such as MDA-MB-231,
MDA-MB-435, Km12C, or Km12L4, and assaying the division rate of
the cells or the ability of the cells to form metastases when implanted
in vivo, as is known in the art. Non-metastatic cell lines, such
as MCF-7, can be used to assay the ability of a portion of a metastatic
marker protein to increase expression of a metastatic marker gene.
 Typically, a therapeutic metastatic marker composition is
prepared as an injectable, either as a liquid solution or suspension;
however, solid forms suitable for solution in, or suspension in,
liquid vehicles prior to injection can also be prepared. A metastatic
marker composition can also be formulated into an enteric coated
tablet or gel capsule according to known methods in the art, such
as those described in U.S. Pat. No. 4,853,230, EP 225,189, AU 9,224,296,
and AU 9,230,801.
 Administration of the metastatic marker therapeutic agents
of the invention can include local or systemic administration, including
injection, oral administration, particle gun, or catheterized administration,
and topical administration. Various methods can be used to administer
a therapeutic metastatic marker composition directly to a specific
site in the body.
 For treatment of tumors, including metastatic lesions, for
example, a therapeutic metastatic marker composition can be injected
several times in several different locations within the body of
tumor. Alternatively, arteries which serve a tumor can be identified,
and a therapeutic composition injected into such an artery, in order
to deliver the composition directly into the tumor.
 A tumor which has a necrotic center can be aspirated and
the composition injected directly into the now empty center of the
tumor. A therapeutic metastatic marker composition can be directly
administered to the surface of a tumor, for example, by topical
application of the composition. X-ray imaging can be used to assist
in certain of the above delivery methods. Combination therapeutic
agents, including a metastatic marker proteins or polypeptide or
a metastatic marker subgenomic polynucleotide and other therapeutic
agents, can be administered simultaneously or sequentially.
 Alternatively, a metastatic marker therapeutic composition
can be introduced into human cells ex vivo, and the cells then replaced
into the human. Cells can be removed from a variety of locations
including, for example, from a selected tumor or from an affected
organ. In addition, a therapeutic composition can be inserted into
non-affected cells, for example, dermal fibroblasts or peripheral
blood leukocytes. If desired, particular fractions of cells such
as a T cell subset or stem cells can also be specifically removed
from the blood (see, for example, PCT WO 91/16116). The removed
cells can then be contacted with a metastatic marker therapeutic
composition utilizing any of the above-described techniques, followed
by the return of the cells to the human, preferably to or within
the vicinity of a tumor or other site to be treated. The methods
described above can additionally comprise the steps of depleting
fibroblasts or other non-contaminating tumor cells subsequent to
removing tumor cells from a human, and/or the step of inactivating
the cells, for example, by irradiation.
 Both the dose of a metastatic marker composition and the
means of administration can be determined based on the specific
qualities of the therapeutic composition, the condition, age, and
weight of the patient, the progression of the disease, and other
relevant factors. Preferably, a therapeutic composition of the invention
decreases expression of the metastatic marker genes by 50%, 60%,
70%, or 80%. Most preferably, expression of the metastatic marker
genes is decreased by 90%, 95%, 99%, or 100%. The effectiveness
of the mechanism chosen to alter expression of the metastatic marker
genes can be assessed using methods well known in the art, such
as hybridization of nucleotide probes to mRNA of the metastatic
marker genes, quantitative RT-PCR, or detection of the metastatic
marker proteins using specific antibodies of the invention.
 If the composition contains the metastatic marker proteins,
polypeptide, or antibody, effective dosages of the composition are
in the range of about 5 .mu.g to about 50 .mu.g/kg of patient body
weight, about 50 .mu.g to about 5 mg/kg, about 100 .mu.g to about
500 .mu.g/kg of patient body weight, and about 200 to about 250
 Therapeutic compositions containing metastatic marker subgenomic
polynucleotides can be administered in a range of about 100 ng to
about 200 mg of DNA for local administration. Concentration ranges
of about 500 ng to about 50 mg, about 1 .mu.g to about 2 mg, about
5 .mu.g to about 500 .mu.g, and about 20 .mu.g to about 100 .mu.g
of DNA can also be used during a gene therapy protocol. Factors
such as method of action and efficacy of transformation and expression
are considerations that will affect the dosage required for ultimate
efficacy of the metastatic marker subgenomic polynucleotides. Where
greater expression is desired over a larger area of tissue, larger
amounts of metastatic marker subgenomic polynucleotides or the same
amounts readministered in a successive protocol of administrations,
or several administrations to different adjacent or close tissue
portions of, for example, a tumor site, can be required to effect
a positive therapeutic outcome. In all cases, routine experimentation
in clinical trials will determine specific ranges for optimal therapeutic
 Expression of an endogenous metastatic marker gene in a
cell can also be altered by introducing in frame with the endogenous
metastatic marker gene a DNA construct comprising a metastatic marker
protein targeting sequence, a regulatory sequence, an exon, and
an unpaired splice donor site by homologous recombination, such
that a homologously recombinant cell comprising the DNA construct
is formed. The new transcription unit can be used to turn the metastatic
marker gene on or off as desired. This method of affecting endogenous
gene expression is taught in U.S. Pat. No. 5,641,670, which is incorporated
herein by reference.
 A metastatic marker subgenomic polynucleotide can also be
delivered to subjects for the purpose of screening test compounds
for those which are useful for enhancing transfer of metastatic
marker subgenomic polynucleotides to the cell or for enhancing subsequent
biological effects of metastatic marker subgenomic polynucleotides
within the cell. Such biological effects include hybridization to
complementary metastatic marker mRNA and inhibition of its translation,
expression of a metastatic marker subgenomic polynucleotide to form
metastatic marker mRNA and/or metastatic marker protein, and replication
and integration of a metastatic marker subgenomic polynucleotide.
The subject can be a cell culture or an animal, preferably a mammal,
more preferably a human.
 The above disclosure generally describes the present invention.
A more complete understanding can be obtained by reference to the
following specific examples which are provided herein for purposes
of illustration only, and are not intended to limit the scope of
Materials and Methods
 Cell culture. MDA-MB-435, MDA-MB-231, ALAB, MDA-MB-468,
MDA-MB-361, ZR-75-1, MCF-7, MDA-MB-453 and SK-BR-3 human breast
cancer cell lines (obtained from Chiron Master Culture Collection,
Chiron Corporation) were grown at 37.degree. C. in 5% CO.sub.2 in
DMEM+HAM'S F-12 (1:1) (Bio*Whittaker, Walkersville, Md.) containing
2 mM L-Glutamine, 1 mM Sodium Pyruvate, 100 U/ml Penicillin and
100 .mu.g/ml Streptomycin (Bio*Whittaker, Walkersville, Md), 1.times.Vitamin
Solution, 1.times.Non-Essential Amino Acids (Irvine Scientific,
Santa Ana, Calif.), and 10% heat-inactivated fetal bovine serum
(Life Technologies, Rockville, Md). COS-7 cells were obtained from
ATCC and grown at 37.degree. C. in 5% CO.sub.2 in DMEM with 10%
heat-inactivated fetal bovine serum (Life Technologies).
 Concentration of Opti-MEM1 supernatant. Opti-MEM1 (Life
Technologies) culture media were concentrated through Centricon
YM-10 and/or Microcon YM-10 columns (Millipore Corporation, Bedford,
Mass.). SDS-PAGE sample loading buffer was then added and the samples
 Northern blot hybridization. Total RNAs were prepared from
cultured breast cancer cell lines and tumor tissues of SCID mice
transplanted with breast cancer cell lines with RNeasy Maxi Kit
(Qiagen, Valencia, Calif.). Approximately 20 .mu.g of total RNA
per lane was loaded onto a formaldehyde/agarose gel for electrophoresis,
then transferred to a Hybond-N+nylon membrane (Amersham Life Science,
Little Chalfont, England). The blot was fixed by UV irradiation.
Rapid-Hyb buffer (Amersham Life Science) with 5 mg/ml denatured
single stranded sperm DNA was pre-warmed to 65.degree. C. and the
blot was pre-hybridized in the buffer with shaking at 65.degree.
C. for 30 minutes. A hsOAF cDNA fragment or a .beta.-actin cDNA
fragment as probe labeled with [.alpha.-.sup.32P]dCTP (3000 Ci/mmol,
Amersham Pharmacia Biotech Inc., Piscataway, N.J.) (Prime-It RmT
Kit, Stratagene, La Jolla, Calif.) and purified with ProbeQuant.TM.
G-50 Micro Column (Amersham Pharmacia Biotech Inc.) was added and
hybridized to the blot with shaking at 65.degree. C. for overnight.
The blot was washed in 2.times.SSC, 0.1%(w/v) SDS at room temperature
for 20 minutes, twice in 1.times.SSC, 0.1%(w/v) SDS at 65.degree.
C. for 15 minutes, then exposed to Hyperfilms (Amersham Life Science).
 Immunoblotting. Protein samples were subjected to electrophoresis
on 10-20% SDS-PAGE gels then transferred to PVDF membranes (0.2
.mu.m) by electroblotting in 25 mM Tris, 192 mM glycine, 20% (v/v)
methanol, pH 8.3. Membranes were blocked in TBST (pH 7.5) containing
10% non-fat milk, then blotted in PBS (pH 7.4) containing 1% BSA
with a rabbit anti-hsOAF serum (1:1000), followed by probing with
a secondary antibody alkaline phosphatase-conjugated goat anti-rabbit
IgG (1:2000) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.
). Protein bands were then visualized by NBT/BCIP reagent (Boehringer
 Transient transfection. The coding region (356-1174) of
hsOAF cDNA was cloned into a modified expression vector pRetro-On
(Clontech, Palo Alto, Calif.). The pRetro-On vector harboring hsOAF
or the control pRetro-On vector with GFP was transfected into COS-7
cells on a 100 mm culture plate using Effectene.TM. Transfection
Reagent Kit (Qiagen) as instructed in the protocol provided by the
manufacturer. Cells were recovered in DMEM with 10% FBS for overnight
then switched to Opti-MEM1. After two more days, the supernatant
was collected and concentrated for western blot analysis.
 Antisense oligo transfection. MDA-MB-435 cells were seeded
on 6-well culture plates one day before transfection to yield a
90% density at transfection. 100 .mu.M antisense or reverse control
oligo was diluted to 2 .mu.M in Opti-MEM1 for transfection. 0.5
mM sterile lipitoidl was diluted to a ratio of 1.5 nmol lipitoid1:
1 .mu.g oligo in the same volume of Opti-MEM1. The diluted oligo
and the diluted lipitoid1 were mixed and immediately added to cells
in culture media to a final concentration of 100, 200, or 300 nM
oligo. After 6 hrs, the transfection mixture was replaced with normal
culture media and cells were incubated for recovery for overnight.
The sequence of the antisense oligo is AGCTGCGGATGCCACACTTGTAGG
(SEQ ID NO:4) and the sequence of the reverse control oligo is GGATGTTCACACCGTAGGCGTCGA
(SEQ ID NO:5).
 Matrigel invasion assay. Cells were trypsinized, washed,
and resuspended in media for counting. 4.times.10.sup.4 cells were
washed and resuspended in 100 .mu.l media on ice. 200 .mu.l Matrigel
(Collaborative Biomedical Products, Bedford, Mass.) was added to
the cells on ice. The Matrigel and the cells were carefully mixed
then dispensed into a well of 24-well culture plate and solidified
at 37.degree. C. for 30 min. The Matrigel-cell mixture was topped
with 0.5 ml medium and incubated at 37.degree. C. in 5% CO.sub.2
for 6 days. The medium was replenished every 2 days.
 Proliferation assay. Cells were trypsinized, washed, and
resuspended in media for counting. Cells were then transferred into
96-well plates (5000 cells/well) for incubation. Cell numbers were
measured with Quantos.TM. Cell Proliferation Assay Kit (Stratagene,
La Jolla, Calif.) every day.
Identification of a Human cDNA Sequence
 DNA encoding a putative human homologue of the Drosophila
Out at First (oaf) gene is shown in SEQ ID NO:1. An alignment of
hsOAF and Drosophilia OAF is shown in FIG. 7. The polynucleotide
comprises 2366 base pairs, and an open reading frame is identified.
A translation of the ORF, a polypeptide of 273 amino acids, is shown
in SEQ ID NO:2. FIG. 4 provides the DNA and amino acid sequences,
indicating the position of the ORF. The first 30 amino acids form
a signal peptide, indicating that the protein may be secreted. The
amino acid sequence of the signal peptide is: MRLPGVPLARPALLLLLPLLAPLLG#TGAPA
(SEQ ID NO:3). "#" indicates the location of the predicted
protease cut site.
Differential Expression of SEQ ID NO:1 In Breast Cancer Cell Lines
 Expression of SEQ ID NO:1 in the following human breast
cancer cell lines was compared:
 MDA-MB-361, derived from human breast adenocarcinoma;
 MDA-MB-231, derived from human breast cancer cells metastatic
to bone and/or lung;
 MDA-MB-468, derived from estrogen receptor-negative human
breast cancer cells;
 MDA-MB-435, derived from estrogen receptor-negative human
breast carcinoma cells;
 MCF-7, derived from non-metastatic human breast cancer cells;
 ZR-75-1, derived from estrogen receptor-positive human breast
 Expression of SEQ ID NO:1 was measured in the highly metastatic
breast cancer cell lines MDA-MB 231 and MDA-MB-435, and compared
with low-metastatic or non-metastatic breast cancer cell lines.
Expression in MDA-MB-361 was 11% of the level in MDA-MB-231; expression
in MDA-MD-468 was 44% of the level in MDA-MB-231; expression in
MCF-7 was 17% of the level in MDA-MB-231; and expression in ZR-75-1
was 12% of the level in MDA-MB-231.
 Expression in MDA-MB-361 was 6% of the level in MDA-MB-435;
expression in MDA-MB-468 was 36% of the level in MDA-MB-435; and
expression in MCF-7 was 3% of the level in MDA-MB-435. Thus, as
shown in Table 2, there is a clear trend of increased expression
of SEQ ID NO:1 in breast cancer cell lines derived from human tumors
with high metastatic potential.
2TABLE 2 Low Metastatic Cell Lines: High Metastatic % Expression
Relative to High Metastatic Cell Line Cell Line MDA-MB-361 MDA-MB-468
MCF-7 ZR-75-1 MDA-MB-231 11% 44% 17% 12% MDA-MB-435 6% 36% 3% ND
 A similar expression pattern of this gene remained in tumor
tissue samples from SCID mice transplanted with tumorigenic mammary
carcinoma cell lines. (FIG. 6.)
HSOAF Encodes a Secreted Protein and HSOAF Protein Secretion Levels
are Consistent with HSOAF mRNA Expression Levels of Mammary Carcinoma
 A predicted signal peptide sequence is located at the N-terminus
of the deduced amino acid sequence of hsOAF gene (FIG. 3). To verify
the secretion of hsOAF protein, transient transfection of COS-7
cells and MCF-7 cells was performed with vector pRetro-On harboring
hsOAF cDNA. Meanwhile, vector pRetro-On harboring GFP was used as
control. Using a hsOAF rabbit antiserum, secreted hsOAF protein
was detected in Opti-MEM1 culture media of both cell lines after
transfection with hsOAF by immunoblotting (FIG. 8A). Secreted hsOAF
protein was probably glycosylated since multiple bands with higher
apparent molecular weights were seen (the predicted MW of secreted
hsOAF protein is 28 Kda). The same hsOAF antiserum was used to detect
the secretion of hsOAF protein by various mammary carcinoma cell
lines. The secretion levels of hsOAF protein were consistent with
the hsOAF mRNA expression levels among these cell lines overall:
highly metastatic cell lines showed much stronger hsOAF secretion
than low metastatic/nonmetastatic cell lines (FIG. 8B). MDA-MB-435
had the strongest hsOAF protein secretion.
Knockout of HSOAF Expression in MDA-MB-435 Cells by Antisense Oligo
Caused Morphological Change, Reduced Cell Invasiveness and Slower
 To determine if high level of hsOAF gene expression is essential
for the metastatic potential of human mammary carcinoma cells, antisense
oligo technology was used to knock out hsOAF expression, then the
consequent effects were observed. MDA-MB-435 was chosen since this
highly metastatic cell line showed the strongest hsOAF protein secretion
among all of the breast cancer cell lines examined. Several pairs
of hsOAF antisense (AS) and reverse control (RC) oligos were chosen
to test for their ability to shut down hsOAF gene expression at
the mRNA level. Real-time quantitative RT-PCR analysis in Lightcycler
(Roche Diagnostics, Indianapolis, Ind.) was performed to measure
hsOAF mRNA levels in cells. Kang, S. et al., Cancer Research 60(18):5296-5302
(2000). The best pair was then selected for the titration of oligo
working concentration. Low oligo concentration is preferred to reduce
potential oligo toxicity to cells. The results indicated that treatment
with 100 nM of the antisense oligo was sufficient to significantly
reduce hsOAF protein secretion of MDA-MB-435 cells. (FIG. 12). This
pair of oligos (SEQ ID NO:4 (AS) and 5 (RC)) at 100 nM working concentration
was used for all the following experiments.
 After treatment of MDA-MB-435 cells with hsOAF antisense
oligo, dramatic morphological alteration of cells was observed along
with reduced hsOAF protein secretion (FIG. 10A). Cells became more
spherical and lost their spreading protrusions. Meanwhile, cells
treated with reverse control oligo remained similar to the normal
tissue cultured MDA-MB-435 cells. Furthermore, culture medium of
normal MDA-MB-435 cells containing high level of hsOAF protein as
the conditioned medium added to cells treated with antisense oligo
was able to prevent this morphological change, though not completely.
This alteration of cell shape may be an indication of reduced invasion
ability of cells.
 Matrigel invasion assay was then performed to estimate the
invasiveness of cells. It has been reported that a stellate, invasive
morphology of breast cancer cells embedded in matrigel correlates
with their metastatic potential (Thompson, E. W., et al., J. Cell
Physiol. 150(3):534-44 (1992); Sugiura, T., et al. J. Cell Biol,
146(6):1375-89 (1999); Albini, A., et al., Cancer Res. 47(f2):3239-45
(1987); and Kramer, R. H., et al., Cancer Res. 46(4 Pt 2):1980-89
(1986)) and this was confirmed with various breast cancer cell lines
grown in matrigel. Cells were trypsinized, counted, and mixed with
matrigel. Media were then topped on the cell-matrigel mixture. After
6 days of incubation, cell invasion was examined (FIG. 10B). The
results showed that cells treated with hsOAF reverse control oligo
formed penetrating, invasive, network-like three-dimensional structures,
as the normal MDA-MB-435 cells did; on the other hand, cells treated
with hsOAF antisense oligo only formed smooth, spherical colonies.
Again, penetrating colonies were also observed in hsOAF antisense
oligo-treated cells incubated in the conditional medium. These data
demonstrate that secreted hsOAF protein is required for the invasiveness
and metastatic potential of MDA-MB-435 cells.
 Additional experiments were performed to examine if secreted
hsOAF protein was involved in MDA-MB-435 cell growth. Cell proliferation
assay results indicated that knockout of hsOAF protein secretion
indeed slowed down proliferation rate of MDA-MB-435 cells, though
the change was moderate.
Northern Blot Analysis of RNA Expression in Human Breast Cancer
Cell Lines and in Human Tissues
 As shown in FIG. 5, mRNA expression was upregulated in metastatic
cell lines MDA-MB-231 and MDA-MB-435. Total RNA was prepared using
the RNeasy Kit from Quiagen. Northern blot analysis was performed
using 20-30 .mu.g total RNA isolated by guanidinium thiocyanate/phenol
chloroform extraction from cell lines, from primary tumors, or from
metastases in lung. Primary tumors and lung metastasis were developed
from cell lines injected into scid mice according to methods well
known in the art. Plasmids containing partial cDNA clones of hOAF
cloned into pCR2.0-TA Vector (In vitrogen) were radiolabeled and
hybridized at 65.degree. C. in Express-hyb (Clontech). Among all
the tissues examined, liver, pancreas, spleen, ovary and small intestine
showed significant hsOAF expression. HsOAF mRNA expression was also
detected in heart, skeletal muscle, kidney, prostate, colon and
bone marrow. (FIG. 9).
 Table 3 shows the percentage of hsOAF positives in a variety
of tumors and normal tissues.
3TABLE 3 Immunohistochemistry: Percentage of hsOAF positives Tumor
Normal Pancreas 9/11 0/9 Esophagus 5/8 0/1 Liver 3/6 0/13 Stomach
6/7 6/10 Breast 1/1 Hodgkin's 1/8
Soft Agar Assay
 Soft Agar Assay: The bottom layer consisted of 2 ml of 0.6%
agar in media plated fresh within a few hours of layering on the
cells. For the cell layer, MDA-MB-435 cells as described above were
removed from the plate in 0.05% trypsin and washed twice in media.
Cells were counted in coulter counter, and resuspended to 106 per
ml in media. 10 ml aliquots were placed with media in 96-well plates
(to check counting with WST1), or diluted further for soft agar
assay. 2000 cells were diluted in 800 ml 0.4% agar in duplicate
wells above 0.6% agar bottom layer.
 Media layer: After the cell layer agar solidified, 2 ml
of media was bled on top and antisense or reverse control oligo
was added without delivery vehicles. Fresh media and oligos were
added every 3-4 days.
 Colonies were counted in 10 days to 3 weeks. Fields of colonies
were counted by eye. Wst-1 metabolism values were used to compensate
for small differences in starting cell number. Larger fields can
be scanned for visual record of differences. The results are shown
in FIG. 6, in which MDA-MB-435 cells treated with antisense formed
fewer colonies compared to cells exposed to the control oligonucleotide.
 Those skilled in the art will recognize, or be able to ascertain,
using not more than routine experimentation, many equivalents to
the specific embodiments of the invention described herein. Such
specific embodiments and equivalents are intended to be encompassed
by the following claims.
 All patents, published patent applications, and publications
cited herein are incorporated by reference as if set forth fully