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 detecting cancerous cells in a first 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 sample compared to a normal tissue sample indicates the presence
of cancerous cells.
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. The method of claim 29 wherein said first tissue sample is
breast tissue, and said normal tissue is normal breast tissue.
35. The method of claim 30 wherein said first tissue sample is
non-breast tissue and said normal tissue is normal breast tissue.
36. A composition for inhibiting expression of protein by a mammary
carcinoma cell, said composition comprising the polynucleotide of
SEQ ID NO:4.
37. 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 is a continuation-in-part of U.S. patent
application Ser. No. 09/758,575, filed Jan. 9, 2001, now pending,
and claims priority from U.S. Patent Application No.60/175,462 filed
Jan. 10, 2000, which applications are incorporated herein by reference
in their entirety.
BACKGROUND OF THE INVENTION
 1. Field of the Invention
 This invention relates to methods for predicting the behavior
of tumors. More particularly, the invention relates to methods in
which tumor samples (primary and metastases) are examined for expression
of a specified gene.
 2. Description of the Related Art
 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.
 There is also a need in the art for markers of tumors, particularly
markers that may also be found in metastatic tumors.
BRIEF SUMMARY OF THE INVENTION
 It is an object of the present invention to provide markers
for detecting tumors, particularly those having a tendency to metastasize.
These and other objects of the invention are provided by one or
more of the embodiments described below.
 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 identifying 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 non-primary tumor tissue sample. A tissue sample which expresses
the product at a higher level than in a control sample is categorized
as being metastatic.
 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 further provides a population of antibodies
that can be used to detect breast cancer, wherein the antibodies
are contacted with primary breast cancer tissue, metastatic breast
cancer tissue, and/or a body fluid of a person suspected of having
breast cancer, thereby detecting a protein encoded by SEQ ID NO:1.
 The invention also provides a kit for use in diagnosing
breast cancer, comprising at least one ligand, such as an antibody,
capable of binding to a protein encoded by SEQ ID NO:1, wherein
the ligand is detectably labeled.
 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 SEVERAL VIEWS 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
(SEQ ID NO: 2) with the Drosophila OAF amino acid sequence (SEQ
ID NO: 7).
 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. 10 B 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 (SEQ ID NO: 9) (double underline), DNA sequence (SEQ ID NO:8).
 FIG. 12. FIGS. 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
 Metastasis of breast carcinomas and their proliferation
at distant loci (lung and bone, mainly) is one of the more severe
developments in patients with breast cancer. 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 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.
291(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 first
identified as being expressed in highly metastatic human breast
cancer cell lines. Antibodies to the protein were raised and immunohistochemical
staining of breast tumor samples was performed. The protein was
strongly expressed in 44/45 primary breast tumors, and in 26/26
metastasis. Thus, the protein is a marker for primary and metastatic
breast cancer. It may also play a role particular to tumors with
a tendency to metastasize. Because of its expression in primary
and metastatic breast cancer, the protein is useful in detecting
such cancer in body fluids including blood, which is consistent
with the secretory nature of the protein.
 The gene 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. 10(10):1260-70 (1996).
 This information can be utilized to make diagnostic reagents
specific for the expression products of the 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 (SEQ ID
NO: 10) (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 Ratio of Cell Line Pair 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 is reduced. 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) expressed in primary breast cancer and related 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. hsOAF gene is the focus of this invention as it encodes a
novel secreted protein, and is expressed in breast cancer tissue
and metastatic breast cancer tissue.
 To investigate the potential role 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, H. 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) led to recovery
of 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 involved in 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-231.
 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 hsOAFs. Reference
to hsOAF nucleotide or amino acid sequences includes variants which
have similar expression patterns in high metastatic relative to
non-metastatic or low metastatic cells. HsOAF polypeptides can differ
in length from full-length hsOAF 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 hsOAF 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.
HsOAF protein or polypeptide variants can be naturally or non-naturally
occurring. Naturally occurring hsOAF 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 hsOAF 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 hsOAF protein or polypeptide
variants are also included here. Preferably, naturally or non-naturally
occurring hsOAF 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 hsOAF 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 hsOAF protein
or polypeptide variant. Properties and functions of hsOAF protein
or polypeptide variants are of the same type as a hsOAF 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 hsOAF 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 hsOAF genes can be
used to detect expression of hsOAF proteins or variants thereof.
 HsOAF variants include glycosylated forms, aggregative conjugates
with other molecules, and covalent conjugates with unrelated chemical
moieties. HsOAF variants also include allelic variants, species
variants, and muteins. Truncations or deletions of regions which
do not affect the differential expression of hsOAF genes are also
hsOAF 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 hsOAF proteins can be extracted, using standard
biochemical methods, from hsOAF protein-producing human cells, such
as metastatic breast cancer cells. An isolated and purified hsOAF
protein or polypeptide is separated from other compounds which normally
associate with a hsOAF protein or polypeptide in a cell, such as
certain proteins, carbohydrates, lipids, or subcellular organelles.
A preparation of isolated and purified hsOAF 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 DNAcomprising 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.
 hsOAF proteins and polypeptides can also be produced by
recombinant DNA methods or by synthetic chemical methods. For production
of recombinant hsOAF proteins or polypeptides, coding sequences
selected from the nucleotide sequences shown in SEQ ID NO:1, or
variants of those sequences which encode hsOAF 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 hsOAF 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. hsOAF
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 hsOAF amino acids can also be
constructed. Human hsOAF fusion proteins are useful for generating
antibodies against hsOAF amino acid sequences and for use in various
assay systems. For example, hsOAF fusion proteins can be used to
identify proteins which interact with hsOAF 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 hsOAF 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 hsOAF
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 hsOAF protein.
 In one preferred embodiment, the first protein segment comprises
the polypeptide shown in SEQ ID NO:2. In avariation 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 hsOAF 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 hsOAF proteins, polypeptides, variants,
or fusion proteins can be used as immunogens, to obtain preparations
of antibodies which specifically bind to a hsOAF protein. The antibodies
can be used, interalia, to detect wild-type hsOAF proteins in human
tissue and fractions thereof. The antibodies can also be used to
detect the presence of mutations in hsOAF genes which result in
under- or over-expression of a hsOAF protein or in expression of
a hsOAF protein with altered size or electrophoretic mobility.
 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 hsOAF
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
hsOAF 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 hsOAF 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.
 HsOAF-specific antibodies specifically bind to epitopes
present in a full-length hsOAF protein having an amino acid sequence
encoded by a nucleotide sequence shown in SEQ ID NO:1, to hsOAF
polypeptides, or to hsOAF variants, either alone or as part of a
fusion protein. Preferably, hsOAF 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 acids.
 Antibodies which specifically bind to hsOAF 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 hsOAF
epitopes do not detect other proteins in immunochemical assays and
can immunoprecipitate a hsOAF protein, polypeptide, fusion protein,
or variant from solution. In a preferred method, hsOAF protein expression
is detected using an immunohistochemical staining kit, such as that
of BioGenex Laboratories, Inc. (San Ramon, Calif.).
 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 hsOAF 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 hsOAF subgenomic polynucleotides.
Typically, homologous hsOAF 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 hsOAF subgenomic polynucleotide comprising hsOAF protein
coding sequences can be used in an expression construct. Preferably,
the hsOAF subgenomic polynucleotide is inserted into an expression
plasmid (for example, the Ecdyson system, pIND, In vitro Gene).
HsOAF subgenomic polynucleotides can be propagated in vectors and
cell lines using techniques well known in the art. HsOAF 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 hsOAF expression construct can
then be used to express all or a portion of a hsOAF protein. Host
cells comprising hsOAF 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 hsOAF 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 phosphate-mediated transfection.
 A hsOAF 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 hsOAF 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
 Bacterial systems for expressing hsOAF 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 hsOAF 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 hsOAF 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 hsOAF 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 No. 30,985.
 Subgenomic polynucleotides of the invention can also be
used in gene delivery vehicles, for the purpose of delivering a
hsOAF mRNA or oligonucleotide (either with the sequence of native
hsOAF mRNA or its complement), full-length hsOAF protein, hsOAF
fusion protein, hsOAF polypeptide, or hsOAF-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 hsOAF subgenomic polynucleotide, or a hsOAF subgenomic polynucleotide
in conjunction with a liposome or a condensing agent.
 The invention provides a method of detecting hsOAF gene
expression in a biological sample. Detection of hsOAF 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 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 hsOAF protein. A hsOAF protein
comprises a sequence encoded by a nucleotide sequence shown in SEQ
ID NO:1 or its complement and can be detected using the hsOAF 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
hsOAF proteins can be assayed, for example, in tissue sections by
immunocytochemistry, or in lysates, using Western blotting, as is
known in the art.
 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 hsOAF 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 hsOAF-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 hsOAF 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 hsOAF 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
 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
hsOAF protein can also be used to alter hsOAF 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 hsOAF 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 hsOAF 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 hsOAF 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 hsOAF
proteins in a polyacrylamide gel. The amount of hsOAF proteins can
be detected, for example, using hsOAF protein-specific antibodies
of the invention in Western blots. The amount of the hsOAF 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 hsOAF protein synthesized with the amount of the hsOAF
proteins present in a standard curve.
 The effect of a test compound on hsOAF protein synthesis
can also be measured by Northern blot analysis, by measuring the
amount of hsOAF protein mRNA expression in response to the test
compound using hsOAF 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 decreases expression of a hsOAF protein by 60%,
75%, or 80%. More preferably, a decrease of 85%, 90%, 95%, or 98%
 The invention provides compositions for decreasing expression
of hsOAF protein. These compositions comprise polynucleotides encoding
all or at least a portion of a hsOAF 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 hsOAF protein which is effective to decrease metastatic
potential. Portions of hsOAF genes or proteins which are effective
to decrease metastatic potential of a cell can be determined, for
example, by introducing portions of hsOAF 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 can be used to assay the ability
of a portion of a hsOAF protein to increase expression of a hsOAF
 Typically, a therapeutic hsOAF 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 hsOAF 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 hsOAF 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 hsOAF composition directly to a specific site in the
 For treatment of tumors, including metastatic lesions, for
example, a therapeutic hsOAF 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 hsOAF 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 hsOAF proteins or polypeptide or a hsOAF subgenomic polynucleotide
and other therapeutic agents, can be administered simultaneously
 Alternatively, a hsOAF 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 hsOAF 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 therapeutic 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 hsOAF genes by 50%, 60%, 70%, or 80%.
Most preferably, expression of the hsOAF genes is decreased by 90%,
95%, 99%, or 100%. The effectiveness of the mechanism chosen to
alter expression of the hsOAF genes can be assessed using methods
well known in the art, such as hybridization of nucleotide probes
to mRNA of the hsOAF genes, quantitative RT-PCR, or detection of
the hsOAF proteins using specific antibodies of the invention.
 If the composition contains the hsOAF 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 .mu.g/kg.
 Therapeutic compositions containing hsOAF subgenomic polynucleotides,
such as antisense oligonucleotides, 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 hsOAF subgenomic polynucleotides. Where
greater expression is desired over a larger area of tissue, larger
amounts of hsOAF 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 effect.
 Expression of an endogenous hsOAF gene in a cell can also
be altered by introducing in frame with the endogenous hsOAF gene
a DNA construct comprising a hsOAF 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 hsOAF 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 hsOAF subgenomic polynucleotide can also be delivered
to subjects for the purpose of screening test compounds for those
which are useful for enhancing transfer of hsOAF subgenomic polynucleotides
to the cell or for enhancing subsequent biological effects of hsOAF
subgenomic polynucleotides within the cell. Such biological effects
include hybridization to complementary hsOAF mRNA and inhibition
of its translation, expression of a hsOAF subgenomic polynucleotide
to form hsOAF mRNA and/or hsOAF protein, and replication and integration
of a hsOAF 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
 Human tissues. Normal human tissues were obtained as Human
Total RNA Panels, Clontech. Tissue samples were also obtained from
breast cancer patients, and included primary breast tumors and metastases.
 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 lipitoid1 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.
 Preparation of hsOAF polyclonal antibody. hsOAF antisera
were generated in two rabbits immunized against the C-terminal peptide
(H-FYVPQ RQLCLWDEDPYPG-OHN, KLH conjugated, SEQ ID NO: 11), and
then affinity purification was conducted to obtain the hsOAF polyclonal
antibody (ResGen, an Invitrogen Corporation, Huntsville, Ala.).
The antibody preparation was titrated by ELISA assay.
 Immunohistochemical staining. Immunohistochemical staining
was performed to detect hsOAF protein expression in tissues with
the hsOAF polyclonal antibody using the immunohistochemical staining
kits from BioGenex Laboratories, Inc. (San Ramon, Calif.). All procedures
were carried out as instructed in the protocol provided by the manufacturer.
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.
Expression of hsOAF in Primary and Metastatic Breast Cancer Tissue
 To further understand the importance of hsOAF gene expression
in breast cancer, immunohistochemical staining in tissue samples
from breast cancer patient was conducted using the hsOAF polyclonal
antibody (FIG. 5). Strong hsOAF expression was detected not only
in all metastases (26/26) but also in almost all primary breast
tumors (44/45). Meanwhile, a weak hsOAF positive staining was observed
in 8 out of 24 normal breast tissue samples. These results suggest
that up-regulation of hsOAF gene expression may play important roles
in both mammary tumor formation and development.
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(12):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.