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 antibody that binds specifically to an isolated
polypeptide comprising one of the amino acid sequences selected
from the group consisting of: (a) amino acids from about 1 to about
273 of SEQ ID NO:2; (b) amino acids from about 2 to about 273 of
SEQ ID NO:2; and (c) amino acids from 26 to 273 of SEQ ID NO:2.
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
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
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:
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 tissues.
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 (1998).
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:
TABLE-US-00001 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. 5).
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
247:1306 1310 (1990).
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
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
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
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 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 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 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
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
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% is achieved.
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 or sequentially.
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
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 were boiled.
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
MDA-MB-435, derived from estrogen receptor-negative human breast
MCF-7, derived from non-metastatic human breast cancer cells; and
ZR-75-1, derived from estrogen receptor-positive human breast carcinoma
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
TABLE-US-00002 TABLE 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
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.
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-TAVector (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.
TABLE-US-00003 TABLE 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 herein.