WO2002034282A2

WO2002034282A2 - Mammary secreted protein 36 (msp36) for cancer treatment and diagnosis

Info

Publication number: WO2002034282A2
Application number: PCT/US2001/045733
Authority: WO
Inventors: Sam W. Lee
Original assignee: Beth Israel Deaconess Medical Center Inc
Current assignee: Beth Israel Deaconess Medical Center Inc
Priority date: 2000-10-27
Filing date: 2001-10-23
Publication date: 2002-05-02
Anticipated expiration: 2003-04-27
Also published as: AU2002228730A1; WO2002034282A3

Abstract

Methods are provided for using msp36 molecule, particularly an msp36 nucleic acid or protein, for treating and diagnosing a subject having or at risk of developing a cancer, particularly breast cancer.

Description

MAMMARY SECRETED PROTEIN FOR CANCER TREATMENT AND

DIAGNOSIS

RELATED APPLICATIONS This application claims domestic priority under 35 U.S.C. § 1 19(e) to U.S.

Provisional Patent Application Serial No. 60/243,982, filed October 27,2000, incorporated herein in its entirety by reference.

FIELD OF THE INVENTION The invention relates to compositions and methods for treating and diagnosing cancers by administering and detecting, respectively, a mammary secreted protein. More particularly, the invention relates to methods for treating or diagnosing cancers, such as breast or prostate cancer, by administering to a patient or detecting an msp36 molecule.

BACKGROUND OF THE INVENTION

Cancer development is a multistage process that results from the step- wise acquisition of genetic alterations. These alterations may involve the dysregulation of a variety of normal cellular functions, leading to the initiation and progression of a tumor. Cancer cells bear an indefinite proliferative capacity being able to elude the commitment to terminal differentiation and quiescence that normally regulates tissue homeostasis. In most tumors, several regulatory circuits are altered during multistage tumor progression, most importantly, the control of proliferation, the balance between cell survival and programmed cell death (apoptosis), the communication with neighboring cells and the extracellular matrix, the induction of tumor neovascularization (angiogenesis) and finally tumor cell migration, invasion and metastatic dissemination (Browder 1^". et al., J Biol Chem. 2000, 27: 1521-4; Yancopoulos G.D. et al.. Cell 1998, 93: 661 -4; and Folkman J.. EXS Angiogenesis and angiogenesis inhibition: an overview. 1997, 79: 1 -8).

The role played by various growth factors and, FGF in particular, in cellular processes, is controversial. Normal mammary cells require growth factors for proliferation but their proliferation is under strict regulation. In view of the discrepancy in the art regarding the role played by various growth factors in cellular processes, a need exists to further understand the molecular changes mediating cancer cell genesis and proliferation.

SUMMARY OF THE INVENTION We have designed a differential screening experiment (a domain-specific differential display) to clone potential growth regulators. We have isolated a cDNA clone using primers that had been selected from highest homology region among the TGF-β family members (GDF 1, VG1 , VGR1 , OP 1 , BMP2, 4, 5, 60A and TGFβ) (McPherron, A., and Lee, S. .; J. Biol. Chem. 1993, 268: 3444-3449; Ozkaynak, E., et al., J. Biol. Chem. 1992, 267: 25220-25227). The recovered clone (msp36, 36-kDa mammary secreted protein) was originally reported as a heparin binding secreted molecule (Hbpl 7) (Wu, D. Kan, et al., J. Biol. Chem. 1991 , 266: 16778-16785). This gene product reportedly binds to acidic and basic FGFs in a non-covalent, reversible manner (Wu, D. et al., J. Biol. Chem. 1991 , 266: 16778-16785; and Lametsch R, et al., J Biol Chem. 2000, 275: 19469-74) and inhibits the mitogenic activities of FGF-2 preferentially (Wu, D. et al., J. Biol. Chem. 1991 , 266: 16778-16785; and Lametsch R. et al., J Biol Chem. 2000, 275: 19469-74). In contrast, others have reported that Hbp l 7/FGF-BP is expressed in several squamous cell carcinoma cell lines, and suggested that Hbpl7/FGF-BP-transfected squamous cell carcinoma cells may contribute more to a malignant phenotype and actually promote angiogenesis (Czubayko F, et al.. Nat Med. 1997, 3: 1 137-40). In view of such controversy in the art, we have designed and developed definitive assays to assess the role played by msp36 in cell growth regulation. The results of our experiments are presented in the Examples.

We have discovered, unexpectedly, that the msp gene is down-regulated in human breast cancer cells in vitro and in vivo as compared to their normal counterparts. (See, e.g., the Examples.) Accordingly, we believe that inducible ectopic expression may be an ideal approach for defining its precise biological role.

The invention is based, in part, on our discovery that expression of msp36 (alternatively referred to herein as "msp36^" or "mammary secreted protein-36^") was significantly reduced or lost in human breast cancer cell lines as well as in breast cancer patient tissue samples, as compared to samples from normal breast. We also found that exogenous msp36 expression, whether by transfection or by adenoviral infection of msp36, inhibited ( 1) tumor cell growth in vitro and //; vivo (xenografts studies), (2) invasion potential of breast cancer cells in vitro and (3) tumor angiogenesis in vivo. Moreover, treatment of invasive breast cancer cells with recombinant msp36 protein as w^rell as transfection studies blocked MMP-9 up-regulation. Our characterization of msp 6 as a regulator against breast cancer development provides important insights for potential therapeutic applications of this molecule and resolves the controversy in the art regarding the physical role played by FGF-BP in breast cancer development.

In view of these initial results, we have concentrated our efforts on ( 1) understanding of the molecular mechanisms responsible for the suppressive effects of msp36 through its functional interactions with growth factors including FGF, and (2) the role that an antagonist of growth factor activity plays in blocking invasion/metastasis and angiogenesis. We disclose herein results which support our contention that msp36 exerts broad effects on cancer cells which we believe regulates malignant potential including tumor angiogenesis. These and other results provide important information for more specific therapeutic approaches against breast cancer as well as other cancers, including prostate cancer.

The invention is based, in part, on our discovery that msp36 is a down regulated gene in human breast cancer cells in vitro and in vivo as compared to their normal counterparts. We also show, for the first time, that exogenous msp36 expression can inhibit tumor cell growth and invasive potential of breast cancer cells in vitro, and further tumor angiogenesis in vivo by matrix implant assay. Loss of msp36 represents a critical step in the induction of an invasive phenotype and/or angiogenesis in breast cancer as well as other types of cancer, including prostate cancer. Accordingly, the discoveries disclosed herein permit the design and development of msp36 as an anti-angiogenic and anti-invasive molecule in the prevention, treatment, diagnosis, and staging of mammalian cancers, particularly, breast cancer. According to a first aspect of the invention, a method for treating a subject having or at risk of developing a cancer is provided. Exemplary cancers include breast carcinomas, prostate carcinomas, bladder carcinomas, melanomas, esophageal carcinomas, lung carcinomas, head and neck carcinomas, colorectal carcinomas, myelomas, brain tumors, sarcomas, and renal carcinomas. The methods of the invention are particularly useful for treating any cancer which is characterized by reduced expression of msp36 compared to the expression of normal cells of the same tissue type.

The method involves administering to a subject in need of such treatment and free of indications otherwise calling for treatment with an msp36 molecule, an msp36 molecule in an amount effective to treat the cancer. As used herein, an msp36 molecule refers to an msp36 nucleic acid or an msp36 protein (the gene product of the msp36 gene), and functional fragments thereof. Functional fragment can be identified in screening assays which detect, e.g., tumor cell proliferation, invasion, angiogenesis (see also the Examples). The preferred msp36 molecule is the msp36 cDNA (SEQ ID NO: 1) or encoded msp36 protein (SEQ ID NO: 2) (alternatively referred to as the heparin-binding secreted protein) having GenBank Accession No. AF 149412.

In view of the discovery that msp36 is differentially expressed in breast cancer cells, the invention provides methods of treating a carcinoma (such as breast cancer), where the carcinoma is one in which expression of msp36 is decreased relative to normal cells of the tissue type from which the carcinoma cells were derived. In these methods, the patient is treated with an effective amount of a compound which increases the amount of msp36 in, or in the immediate vicinity of, his or her carcinoma cells. This compound could be. for example, msp36 or a biologically active fragment thereof: a nucleic acid encoding msp36 and having expression control elements permitting expression in the carcinoma cells: or an agent which increases the level of expression of a msp36 gene endogenous to the carcinoma cells (i.e., which up-regulates expression of the msp36 gene).

According to certain embodiments of this aspect of the invention, the method involves administering an msp36 molecule (e.g., an msp36 nucleic acid) to a subject having or at risk of developing a cancer and who is free of a condition for which an msp36 molecule would have been prescribed. In general, the subject is a mammal, preferably a human. According to certain preferred embodiments, the subject has a breast cancer or a prostate cancer. Optionally, the msp36 molecule is administered in conjunction with a second agent for treating or preventing the cancer. In certain embodiments the second agent for treating or preventing the cancer is a chemotherapeutic agent, or radiation therapy.

As used herein, an msp36 molecule is an msp nucleic acid or an msp36 protein, or a functional equivalent (e.g.. msp36 nucleic acid fragment), provided that the functional equivalent selectively interacts with, e.g.. binds (or encodes a protein that binds) to a growth factor (e.g., FGF, VEGF, EGF) and inhibits one or more of the following growth factor functional activities: (a) the ability to promote angiogenesis; (b) the ability to promote tumor cell growth; and (c) the ability to induce an invasive phenotype. According to certain preferred embodiments, the msp36 molecule (also referred to as "msp36^'") is an msp36 cDNA or encoded protein which, according to the Examples, is capable of inhibiting tumor cell growth, invasive potential, and/or angiogenesis of breast cancer cells in vitro, and in vivo, respectively. The preferred msp36 is described in GenBank Accession No. AF 149412. Either nucleic acid or polypeptide forms of msp36 can be administered in accordance with the methods of the invention. In another aspect, the invention provides a method for treating a subject having or at risk of developing a breast cancer. The method involves administering to a subject in need of such treatment and free of indications otherwise calling for treatment with msp36, msp36 in an amount effective to treat the breast cancer. The mode of administration and dosages are substantially the same for each of the therapeutic aspects of the invention.

According to yet another aspect of the invention, diagnostic methods for detecting carcinomas, or for staging a carcinoma, where the suspected carcinoma is derived from a type of cell which normally expresses the msp36 gene to a significant and easily detectable degree (e.g.. mammary epithelial cells) is prov ided. One such diagnostic method includes the steps of providing a test cell (e.g.. in the form of a tissue section or a cell preparation) from a given type of epithelial tissue; contacting the mRNA of the test cell with a nucleic acid probe containing a sequence antisense to (i.e.. complementary to the sense strand of) a segment the sequence encoding msp36 (SEQ ID NO: l ). which segment is at least 15 (preferably at least 20, more preferably at least 30, even more preferably at least 40, and most preferably at least 100) nucleotides in length; and comparing

(1) the amount of hybridization of the probe to the mRNA of the test cell, with

(2) the amount of hybridization of the probe to the mRNA of a normal control (i.e., non-cancerous) cell from the same type of epithelial tissue, wherein an amount of hybridization to the mRNA of the test cell substantially less than the amount obtained with the mRNA of the normal control cell (preferably less than about one-half, more preferably less than about one-third, and more preferably less than about one-tenth the control amount of hybridization) is an indication that the test cell is cancerous. An absence of hybridization with the mRNA of the test cell is an indication that the test cell is from an advanced, probably metastatic tumor, while an amount of hybridization that is detectable but substantially less (e.g., one-third or less) than that measured in a normal cell of the same tissue type is an indication that the test cell is from an early stage carcinoma that is probably not yet metastatic. The assay can be conveniently carried out using standard techniques of in situ hybridization or Northern analysis. The antibody-based assays of the invention are comparable to the above.

The proteins of the test cell, or from a fluid bathing the test cell, are contacted with an antibody (polyclonal or monoclonal) specific for msp36. and the amount of immunocomplex formed with such proteins is compared with the amount formed by the same antibody with the proteins of a normal control cell (or from a fluid bathing the normal control cell) from the same type of epithelial tissue as the test cell. An amount of immunocomplex observed with the proteins of the test cell substantially less than the amount observed with the proteins of the normal control cell (e.g.. less than about one-half, preferably less than about one- third, and more preferably less than about one-tenth) is an indication that the test cell is cancerous. The absence of consistently detectable immunocomplex formed with the proteins of the test cell is an indication that the test cell is from an advanced, probably metastatic tumor, while an amount of immunocomplex formation that is consistently detectable but less (e.g., one-third or less) than that measured in a normal cell of the same tissue type is an indication that the test cell is from an early stage carcinoma that is probably not yet metastatic. (By consistently detectable is meant that, in all or nearly all of repeated trials, an amount greater than the applicable background level is observed.)

The immunoassay of the invention alternatively can be carried out on a biological fluid, since msp36 protein is normally secreted by epithelial tissues such as mammary tissue. Such an assay would require obtaining a sample of a biological fluid (e.g., blood, serum, urine, saliva, milk, ductal fluid, tears, or semen) from an individual, which biological fluid would, in an individual free of carcinoma, contain a control amount of msp36 protein (mammary secreted protein-36). The sample, or protein derived from the sample, is contacted with the anti-msp36 protein antibody, and the amount of immunocomplex formed is determined. This amount indicates the concentration or amount of msp36 protein in the biological fluid. When compared to a sample previously or subsequently obtained from the same individual, this method provides a way to monitor the appearance, progress, or treatment of a carcinoma.

In another aspect, the invention features a method for screening candidate anticancer compounds, using as a screening tool cells (e.g., primary cells or an established cell line) from a carcinoma derived from a given tissue type in which the msp36 gene is intact but down-regulated: that is, the level of expression of msp36 in that carcinoma is significantly lower than (e.g.. less than one-third of) the level of expression in normal epithelial cells from that type of tissue. The tissue may be from a human or another animal, and is preferably mammary epithelium. It is preferred that there be no detectable expression of msp36 in the cells to be employed in the screen: i.e., the msp36 gene is entirely shut down. The screening method includes the step of providing two samples of the screening cells, one of which is treated with a candidate anticancer compound and the other of which serves as control. The level of expression of msp36 in the treated sample is compared with the level in the second sample, a higher level in the first sample being an indication that the candidate compound is a potential anticancer agent. The level of expression can be determined by use of hybridization methods or by immunoassay, as described herein.

As an alternative way of screening for potential anticancer agents, one can use any cell in which expression of msp36 is undetectable, but which contains an intact msp36 gene. This cell would be treated with a candidate anticancer compound, and a determination made of whether expression of msp36 is thereby increased in the cell. Such an increase of msp36 expression is an indication that the candidate compound is a potential anticancer agent. As above, the level of expression can be determined by use of hybridization methods or by immunoassay.

The invention also features methods for in vivo screening of candidate anticancer agents, or for determining whether a particular carcinoma, in which msp36 expression is down-regulated in comparison with normal cells of the same tissue type, is treatable with a given compound that increases expression of msp36. Such a method would include the steps of ( 1) introducing a carcinoma cell (e.g., a from a mammary carcinoma) into a severely immunodeficient animal (e.g. a nude mouse), the expression of msp36 (SEQ ID NO: l) in the cell being down-regulated in comparison with that in a normal cell of the same type of tissue as the carcinoma cell; (2) treating the animal with a compound which increases the concentration of msp36 in or around (i.e., in the immediate vicinity of) the carcinoma cell; and (3) determining whether this treatment affects the rate of proliferation or metastasis of the carcinoma cell in the animal, wherein a decrease in the rate of proliferation or metastasis in the presence of the compound is an indication that (a) the compound is potentially useful for treatment of carcinomas, and (b) the carcinoma from which the cell is derived is potentially treatable with the compound.

The invention also provides an in vitro assay for carcinoma cell invasive capacity based upon the assay described in detail below. Such an assay would include the steps of ( 1) providing a first and a second carcinoma cell, which cells express msp36 (SEQ ID NO: 1) to a degree substantially lower than (i.e.. less than one-third of, when measured by hybridization to cellular mRNA) that of a normal cell from the same type of tissue as said carcinoma cells: (2) treating the first cell with a compound which increases the concentration of msp36 (SEQ ID NO: l) in or around the first cell; and (3) comparing the invasive capacity of each of the first and second cells in an in vitro assay such as that described below, wherein a decrease in invasive capacity of the (treated) first cell relative to that of the (untreated) second cell is an indication that (a) the compound is potentially useful for treatment of carcinomas, and (b) the carcinoma from which the cells are derived is potentially treatable with the compound. This assay is also useful for detecting msp36 activity in a biological sample (e.g.. for testing the biological activity of msp36 fragments or derivatives, or for determining the presence of msp36 in a sample of blood, milk, or other biological fluid), wherein a decrease in invasive capacity of the (treated) first cell relative to that of the (untreated) second cell is an indication that msp36, or msp36 biological activity, is present in the sample.

Although the description of the invention is directed primarily to the use of msp36 molecules in the treatment and diagnosis of breast cancer, it is to be understood that the methods and compositions of the invention are useful for treating and diagnosing other types of cancers, (e.g.. by using a tissue source other than breast tissue, provided that the cancer cells from the alternative tissue source are characterized in having a reduction in msp36 expression compared to cells from the same tissue which are not cancerous.

These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES It is to be understood that the figures are provided for illustrative purposes only and are not required for understanding or practicing the invention.

Fig. 1. Growth rate decrease in an MDAMB435 cell line following transfection with Ad-msp36. Fig. 2. FACS cell cycle analysis of Ad-msp36-infectcd cell lines

(MDAMB435, Saos2 and MCI-7) as well as Ad-LacZ infected cells. Fig. 3. Inhibition of migration/invasiveness of MDAMB435 cells by msp36 and reversal of inhibition by treatment of anti-msp36 antibodies.

Fig. 4. Inhibition of tumor cell invasion by recombinant msp36 protein of E. coli origin (GST-msp36 fusion protein, 1 μg/ml). Fig. 5. Migration (haptotaxis) of msp36-transfected T47D mammary carcinoma cells on extracellular matrix proteins.

Fig. 6. Inhibition of angiogenesis by msp36 in vivo. Angiogenic response to tumor cell-Matrigel subcutaneous implants in nude mice after 10 days. Gross whole mount representations of photographs (A,C,E,G), and collagen IV immunostained sections (B,D,F,H). MDAMB435 control cell implants (A,B) show prominent blood vessel formation in both gross and collagen IV staining (arrow delineates vessel lumens). A significantly reduced angiogenic response was observed in msp36 expressing cells (CD). A more striking difference was found in the T47 control (E,F) compared to the msp36 expressing cells (G,H), confirming effective repression of angiogenesis by msp36 in vivo.

Fig. 7. Exogenous expression of msp36 reduces angiogenesis in vivo. Tumor cell/Matrigel-induced neovascularization was quantified at the skin/Matrigel interface. Significantly reduced vascular density was observed for both T47D and the MDAMB435 cells overexpressing msp36 compared to control cells. Results expressed as the mean vessels per microscopic field (20X)±SE (n=5); *denotes statistically significant from control, p 0.02.

ABBREVIATED SEQUENCE LISTING

SEQ ID NO: 1 is the cDNA sequence for msp36 (GenBank Accession No. AF 149412).

SEQ ID NO: 2 is the amino acid sequence for msp36 (GenBank Assession No. AF 149412).

SEQ ID NO: 3 is a portion of the I^'BFβ family conserved region.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the invention, a method is provided wherein msp36 molecules ("msp36^" are used for treating a subject afflicted by or susceptible to a cancer, particularly a breast cancer or a prostate cancer. The method involves administering to a subject having or at risk of developing a cancer a therapeutically effective amount of an msp36 molecule. Preferred subjects of the present invention do not have any other indication calling for treatment with an msp36 molecule.

A subject having a cancer is a subject with at least one identifiable sign, symptom, or laboratory finding sufficient to make a diagnosis of a cancer in accordance with clinical standards known in the art for identifying such disorder. Examples of such clinical standards can be found in Flarrisoif s Principles of Internal Medicine, 14th Ed., Fauci AS et al., eds., McGraw-Hill. New York.

1998. In some instances, a diagnosis of a cancer will include identification of a particular cancer antigen present in a body fluid or tissue obtained from the subject. Exemplary cancers include breast carcinomas, prostate carcinomas, bladder carcinomas, melanomas, esophageal carcinomas, lung carcinomas, head and neck carcinomas, colorectal carcinomas, myelomas, brain tumors, sarcomas, and renal carcinomas.

A subject at risk of developing a cancer is a subject with an identifiable risk factor for developing a cancer. For example, a subject at risk of developing a cancer can include an individual with a known or suspected exposure to a carcinogen (e.g., asbestos, cigarette smoke, radiation). Alternatively, a subject at risk of developing a cancer can include an individual with a known or suspected genetic predisposition to hav ing the cancer.

Thus, in one aspect the invention is useful whenever it is desirable to treat or prevent a cancer in a subject. ^'1 his includes prophylactic treatment to prevent cancer metastases, as well as in situations in which a subject, because of genetic or environmental reasons, has a propensity to developing a cancer.

A subject who has been diagnosed with a cancer also can be treated in accordance with the methods of the invention to prevent further a giogensis of a tumor associated with the cancer and/or further proliferation or inv asive penetration of the tumor.

According to this aspect of the invention, the msp36 molecules of the invention are placed in a pharmaceutically acceptable carrier and are delivered to a recipient subject (preferably a human) in accordance with known methods of drug delivery. The msp36 molecules may be administered alone or in combination with at least one other agent known or believed to be useful for treating a cancer. In general, the methods of the invention for delivering the msp36 molecules in vivo utilize art-recognized protocols for delivering nucleic acid or polypeptide based therapeutic agents.

As used herein, an msp36 molecule is an msp36 nucleic acid or an msp36 protein, or a functional equivalent (e.g., msp36 nucleic acid fragment), provided that the functional equivalent selectively interacts with, e.g., binds (or encodes a polypeptide that binds) to a growth factor (e.g., the acidic and basic forms of

FGF, VEGF, EGF, (see also Examples for additional growth factors) and inhibits one or more of the following growth factor functional activities: (a) the ability to promote angiogenesis; (b) the ability to promote tumor cell growth: and (c) the ability to induce an invasive phenotype. According to certain preferred embodiments, the msp36 molecule is a msp36 nucleic acid (e.g., msp36 cDNA) or an msp36 polypeptide (e.g., encoded by the msp36 cDNA) which, according to the Examples, is capable of inhibiting tumor cell growth, invasive potential, and/or angiogenesis of breast cancer cells in vitro, and in vivo, respectively. The preferred msp36 is described in GenBank Accession No. AF 149412 (cDNA and polypeptide sequences). Either nucleic acid or polypeptide forms of msp36 can be administered in accordance with the methods of the invention.

It will also be understood that the invention embraces the use of msp36 sequences in expression vectors including recombinant plasmids. phagemids. viruses and the like, as well as to transfect host cells and cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., dendritic cells, CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). The expression vectors require that the pertinent sequence, i.e., those described herein, be operably linked to a promoter. Delivery of expression vectors containing the msp36 sequence in vivo and/or in vitro can be via the use of nucleic acid delivery systems known in the art (see, e.g., Λllsopp et al.. Ear. J. Immunol. 26(8): 1951-1959. 1996). Recombinant vectors including viruses selected from the group consisting of adenoviruses, adeno-associated iruses. poxviruses including vaccinia viruses and attenuated poxviruses such as NYVAC, Semliki Forest virus, Venezuelan equine encephalitis virus, retroviruses, Sindbis virus, and Ty virus-like particle, plasmids (e.g. "naked^" DNA), bacteria (e.g. the bacterium Bacille Calmette Guerin, BCG), and the like can be used in such delivery. One can test the msp36 delivery systems in standard model systems such as mice to determine efficacy of the delivery system. The systems also can be tested in human clinical trials.

As used herein, a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids, bacteria and virus genomes as disclosed herein, such as adenovirus, poxvirus and BCG. A cloning vector is one which is able to replicate in a host cell or be replicated after its integration into the genome of a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.

Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase. luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they arc operably joined. As used herein, a coding sequence and regulatory sequences are said to be

"operably" joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5^" regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. In certain embodiments, the nucleic acids express only fragments of msp36 which include the amino acid sequence which binds to FGF. Such fragments can be determined, for example, in routine binding assays which detect the ability of an msp36 fragment to bind selectively to FGF or other growth factor.

The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5¹ non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box. capping sequence, CAAT sequence, and the like. Especially, such 5^" non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operablv joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See. e.g.. Sambrook et al.. Molecular Cloning: A Laboratory MamiaL Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding a msp36 which heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those such as pcDNA3.1 (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences. Additionally, suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element. Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor 1 Li, which stimulates efficiently transcription /;; vitro. The plasmid is described by Mishizuma and Nagata {Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferred expression vector is an adenovirus, described by Stratford-Perricaudet. which is defective for El and E3 proteins (J. Clin. Invest. 90:626-630. 1992). The use of the adenovirus to express proteins for immunization is disclosed by Warnier et al., in intradermal injection in mice for immunization against PI A (////. J. Cancer. 67:303-310. 1996).

The invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors. Such expression kits include at least separate portions of at least two of the previously discussed materials. Other components may be added, as desired. The methods for delivering a functional msp36 molecule for transcription and translation in vivo include the methods used to deliver a functional gene for gene therapy applications. For example, a procedure for performing ex vivo gene therapy is outlined in U.S. Patent 5,399.346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents. In general, the method involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject. The functional copy of the gene is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cell(s). Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654. In vivo gene therapy using vectors such as adenovirus. retroviruses, herpes virus, and targeted liposomes also is contemplated according to the invention. See, e.g., U.S. Patent Nos. 5,670,488, entitled "Adenovirus Vector for Gene Therapy", issued to Gregory et al., and 5,672,344, entitled "Viral-Mediated Gene Transfer System", issued to Kelley et al.

An expression vector encoding msp36 protein can be introduced into carcinoma cells, there by increasing the production of msp36 protein in the transfected cells, and decreasing the in vivo growth rate of tumors derived from these cells. The transfected cells are also shown to have a decreased invasive character, compared to untransfected controls. This evidence indicates that the msp36 DNA of the invention will be useful for genetic therapy to help control carcinomas characterized by down-regulated msp36 expression, or to ensure that early-stage carcinomas which have not yet lost the ability to manufacture msp36 do not progress through the progressively down-regulated stages. Standard methods of gene therapy may be employed: e.g., as described in Friedmann. Therapy for Genetic Disease. T. Friedman (ed.). Oxford Univ. Press. 1991. pp.105-121. Virus or plasmids containing a copy of the msp36 cDNA linked to expression control sequences which permit expression in the carcinoma cell would be introduced into the patient, either locally at the site of the tumor or systemically (in order to reach any tumor cells that may hav e metastasized to other sites). If the transfected DNA encoding msp36 is not stably incorporated into the genome of each of the targeted carcinoma cells, the treatment may have to be repeated periodically.

The pharmaceutical preparations disclosed herein are prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent or eliminate the condition (See, e.g.. Remington^'s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. and Goodman and Gilman"s The Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, N.Y., the contents of which are incorporated herein by reference, for a general description of the methods for administering various agents for human therapy). The msp36 molecules can be delivered using controlled or sustained release delivery systems (e.g., capsules, bioerodable matrices). Exemplary delayed release delivery systems for drug delivery that would be suitable for administration of the msp36 molecules are described in U.S. Patent Nos. L1S 5.990,092 (issued to Walsh); 5,039,660 (issued to Leonard): 4.452.775 (issued to Kent); and 3,854.480 (issued to Zaffaroni).

The pharmaceutically acceptable compositions of the present invention comprise one or more msp36 molecules in association with one or more nontoxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants and/or excipients, collectively referred to herein as "carrier" materials, and if desired other active ingredients.

The msp36 molecules of the present invention may be administered by any route, preferably in the form of a pharmaceutical composition adapted to such a route, and would be dependent on the condition being treated. The compounds and compositions may. for example, be administered orally. intravascularly, intramuscularly, subcutaneousl , intraperitoneally, or topically. Preferred routes of administration include oral and intravenous administration.

For oral administration, the msp36 molecules may be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a therapeutically effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, the tablets and capsules can contain, in addition to the active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, cellulose, glycine, lactose, maize-starch, mannitol, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica, or talc; disintegrants, for example potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid. The pharmaceutical compositions may also be administered via injection.

Formulations for parenteral administration may be in the form of aqueous or non- aqueous isotonic sterile injection solutions or suspensions. These solutions or suspensions may be prepared from sterile powders or granules having one or more of the carriers mentioned for use in the formulations for oral administration. The compounds may be dissolved in polyethylene glycol. propylene glycol. ethanol. corn oil, benzyl alcohol, sodium chloride, sterile w^rater, and/or various buffers.

For topical use the compounds of the present invention may also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and may take the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat paints. Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient. Suitable carriers for topical administration include oil-in-water or water-in-oil emulsions using mineral oils. petrolatum and the like, as well as gels such as hydrogel. Alternative topical formulations include shampoo preparations, oral pastes and mouthwash. For rectal administration the compounds of the present invention may be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or other glyceride.

Alternatively, the compounds of the present invention may be in powder form for reconstitution at the time of delivery.

The dosage regimen for treating a cancer with the msp36 molecules of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex and medical condition of the subject, the severity of the cancer, the route and frequency of administration, the renal and hepatic function of the subject, and the particular compound employed. An ordinarily skilled physician or clinician can readily determine and prescribe the effective amount of the drug required to treat a cancer. In general, dosages are determined in accordance with standard practice for optimizing the correct dosage for treating a cancer. The dosage regimen can be determined in part, for example, by following the response to the treatment in terms of vital signs. Examples of such vital signs are well known in the art, and they include the pulse, blood pressure, temperature, and respiratory rate. Harrison's Principles of Internal Medicine, 14th Ed.. Fauci AS et al., eds., McGraw-Hill, New York, 1998. In addition, because the msp36 molecules of the invention are believed to inhibit the proliferation of cancer cells (particularly breast cancer cells) in vivo, the dosage regimen can also be determined by culture of the breast cancer cells from a biopsy sample. Generally, a reduction in breast cancer cell proliferation or invasive potential following contact with the msp36 molecules of the invention, as a function of contact time and/or dosage, will indicate a response to treatment.

Typically dosages of the msp36 molecules will range from between 0.01 mg per kg of body weight per day (mg/kg/day) to about 10.0 mg/kg/day. Alternatively, the dosages of the msp36 molecule will range from between 0.01 micromole per kg of body weight per day (μmole/kg/day) to about 10 μmole/kg/day. Preferred oral dosages in humans may range from daily total dosages of about 1 -500 mg/day over the effective treatment period. Preferred intravenous dosages in humans may range from daily total dosages of about 1 - 100 mg/day over the effective treatment period.

Other agents which are known to be useful in the treatment of cancer or other proliferative disorders include ribavirin, amantadine. chemotherapeutic agents (e.g., Ta.xol, 5-fluorouracil and BCNU), radiation therapy, phototherapy, and cytokines, including IL-2, IL-12. and IFN-γ. Such agents can be administered to the patient receiving msp36 molecule therapy.

As used herein, the term coadminister refers to administering at least two agents in clinical association with one another. Coadministration can include administering the at least two agents together or sequentially. In a preferred embodiment, the msp36 molecule of the invention is administered either before, at the same time as, or after administering a further therapeutic agent for treating the cancer. The msp36 molecule of the invention and additional therapeutic agent also may be administered via different modes, for example such as administering the chemotherapeutic agent systemically or orally and administering the msp36 molecule locally.

In certain embodiments involving simultaneous coadministration, the msp36 molecule and additional therapeutic agent may be prepared as a single formulation. In other embodiments involving simultaneous coadministration, the msp36 molecule and the additional therapeutic agent may be prepared and administered separately. In this latter instance individual msp36 molecule and additional therapeutic agent formulations may be packaged together as a kit with instructions for simultaneous administration. Similarly, in embodiments calling for consecutive coadministration. individual msp36 molecule and additional therapeutic agent formulations may be packaged together as a kit with instructions for their sequential administration.

As used herein, administered locally refers to administration by a route that achieves a local concentration of msp36 molecule that exceeds the systemic concentration of msp36 molecule. For example, local administration to a particular lesion or organ could be accomplished by direct injection into the lesion or organ or by direct injection into an afferent blood vessel associated with and supplying the lesion or organ to be treated. In the example local administration to the liver, local administration can be accomplished by injection or infusion into the hepatic artery, the celiac artery, or the portal vein. Those skilled in the art will recognize which of the other agents to be administered in conjunction with the msp36 molecules are appropriate for treating a subject having or suspected of having a cancer.

The phrase "therapeutically effective amount" means that amount of a compound which prevents the onset of, alleviates the symptoms of. or stops the progression of a disorder or disease being treated. The phrase "therapeutically effective amount" means, with respect to a cancer, that amount of an msp36 molecule which prevents the onset of, alleviates the symptoms of, or stops the progression of the cancer. In general such symptoms are, at least in part, the result of unwanted proliferation of cancer cells in vivo. Thus, a "cancer" is a condition that is characterized by certain clinical features and which, it is generally believed, is associated with unwanted proliferation of cancer cells //; vivo. "Unwanted," with respect to proliferation of cancer cells in vivo, refers to ( 1) proliferation of cancer cells in vivo and/or (2) invasion of cells into adjacent or distant tissue (e.g., metastasis), and (3) which results in an adverse medical condition. Exemplary cancers include breast carcinomas, prostate carcinomas, bladder carcinomas, melanomas, esophageal carcinomas, lung carcinomas, head and neck carcinomas, colorectal carcinomas, myelomas, brain tumors, sarcomas, and renal carcinomas.

The term "treating" is defined as administering, to a subject, a therapeutically effective amount of an msp36 molecule (e.g., an msp36 nucleic acid) that is sufficient to prevent the onset of. alleviate the symptoms of, or stop the progression of a disorder or disease being treated (cancer). The term "subject." as described herein, is defined as a mammal. In a preferred embodiment, a subject is a human.

In a second aspect of the invention, a method is provided wherein an msp36 nucleic acid or protein is used as the msp36 molecule for treating a subject having or at risk of developing a cancer, particularly breast cancer. The method involves administering to a subject having or at risk of developing a cancer a therapeutically effective amount of the msp36 molecule. Subjects of this aspect of the present invention do not have any other indication calling for treatment with msp36. In accordance with this second aspect of the invention, a subject having or at risk of developing a cancer is as defined above.

According to this aspect of the invention, msp36 is administered to the subject as described above, i.e., msp36 is placed in a pharmaceutically acceptable carrier and is delivered to a recipient subject (preferably a human) in accordance with known methods of drug delivery. In general, the methods of the invention for delivering the msp36 /// vivo utilize art-recognized protocols for delivering msp36 or other like molecule. The msp36 may be administered alone or in combination with at least one other agent known or believed by the applicants to be useful for treating a cancer such as those described above. Those skilled in the art will recognize other agents that could be administered in conjunction with msp36 for treating a subject having a given suspected or identified cancer. According to yet another aspect of the invention, diagnostic methods for detecting carcinomas, or for staging a carcinoma, where the suspected carcinoma is derived from a type of cell which normally expresses the msp36 gene to a significant and easily detectable degree (e.g., mammary epithelial cells) is provided. One such diagnostic method includes the steps of providing a test cell (e.g., in the form of a tissue section or a cell preparation) from a given type of epithelial tissue: contacting the mRNA of the test cell with a nucleic acid probe containing a sequence antisense to (i.e., complementary to the sense strand of) a segment the sequence encoding msp36 (SEQ ID NO: 1), which segment is at least 15 (preferably at least 20, more preferably at least 30, even more preferably at least 40, and most preferably at least 100) nucleotides in length: and comparing

( 1) the amount of hybridization of the probe to the mRNA of the test cell, with

(2) the amount of hybridization of the probe to the mRNA of a normal control (i.e.. non-cancerous) cell from the same type of epithelial tissue, wherein an amount of hybridization to the mRNA of the test cell substantially less than the amount obtained with the mRNA of the normal control cell (preferably less than about one-half, more preferably less than about one-third, and most preferably less than about one-tenth the control amount of hvbridization) is an indication that the test cell is cancerous. An absence of hybridization with the mRNA of the test cell is an indication that the test cell is from an advanced, probably metastatic tumor, while an amount of hybridization that is detectable but substantially less (e.g., one-third or less) than that measured in a normal cell of the same tissue type is an indication that the test cell is from an early stage carcinoma that is probably not yet metastatic. The assay can be conveniently carried out using standard techniques of in situ hybridization or Northern analysis.

By "hybridizing conditions" is meant conditions under which the nucleic acid used as a probe in the method is able to specifically hybridize with RNA expressed from a candidate tumor suppressor gene (e.g., msp36) without significantly hybridizing to any other RNA expressed from either normal or cancerous human cells (e.g., conditions of high stringency, as described, for example, in Sambrook et al.. Molecular Cloning, a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989). In this way, hybridization of the RNA specifically indicates the presence or absence of a candidate tumor suppressor gene transcript (usually mRNA).

An exemplary assay for detecting expression of a tumor suppressor gene is described in U.S. 5,801 ,001 , issued to R. Sager, et al., entitled, "Method of Detecting Cancer" and is summarized below. The Sager et al. method can be adapted for use in detecting msp36 expression in the manner described below. Briefly, a nucleic acid probe containing some or all of the msp36- encoding sequence of the invention (SEQ ID NO: 1) can be used to detect msp36 mRNA in a sample of epithelial cells (e.g., a tissue section) suspected of being cancerous. The probe used is a single-stranded DNA or RNA (preferably DNA) antisense to the coding sequence of msp36. The probe can be produced by synthetic or recombinant DNA methods, and labeled with a radioactive tracer or other standard detecting means. The probe may include from 15 to the full 1 125 nucleotides of coding sequence, and preferably is at least 30 nucleotides long. The assay is carried out by standard methods of in situ hybridization or Northern analysis, using stringent h bridization conditions. Control hybridization assays can be run in parallel using normal epithelial cells or tissue sections from the same type of tissue as the test sample, and/or cells from a known carcinoma or carcinoma-derived cell line, or a cancer-containing tissue section. Cells which exhibit a substantially decreased level, or absence, of hybridization to the probe, compared to the level seen with normal epithelial cells, are likely to be cancerous. The amount of hybridization can be quantitated by standard methods, such as counting the grains of radioactivity exposed emulsion on an in situ hybridization assay of a biopsy slide, or by densitometrie scan of a Northern blot X-ray film. Alternatively, comparison of the test assay results with the results of the control assays can be relative rather than quantitative, particularly where the difference in levels of hybridization is dramatic. This assay is useful for detecting cancerous cells in breast epithelial tissue or in any other type of tissue in which msp36 is normally expressed.

The antibody-based assays of the invention are comparable to the above. The proteins of the test cell, or from a fluid bathing the test cell, are contacted with an antibody (polyclonal or monoclonal) specific for msp36, and the amount of immunocomplex formed with such proteins is compared with the amount formed by the same antibody with the proteins of a normal control cell (or from a fluid bathing the normal control cell) from the same type of epithelial tissue as the test cell. An amount of immunocomplex observed with the proteins of the test cell substantially less than the amount observed with the proteins of the normal control cell (e.g.. less than about one-half, preferably less than about one- third, and more preferably less than about one-tenth) is an indication that the test cell is cancerous. The absence of consistently detectable immunocomplex formed with the proteins of the test cell is an indication that the test cell is from an advanced, probably metastatic tumor, while an amount of immunocomplex formation that is consistently detectable but less (e.g., one-third or less) than that measured in a normal cell of the same tissue type is an indication that the test cell is from an early stage carcinoma that is probably not yet metastatic. (By consistently detectable is meant that, in all or nearly all of repeated trials, an amount greater than the applicable background level is observed.) ^'1 he immunoassay of the inv ention alternatively can be carried out on a biological fluid, since msp3ό protein is normally secreted by epithelial tissues such as mammary tissue. Such an assay would require obtaining a sample of a biological fluid (e.g., blood, serum, urine, saliva, milk, ductal fluid, tears, or semen) from an individual, which biological fluid would, in an individual free of carcinoma, contain a control amount of msp36 protein. I he sample, or protein derived from the sample, is contacted with the anti-msp protein antibody, and the amount of immunocomplex formed is determined. 1 his amount indicates the concentration or amount of msp36 in the biological fluid. When compared to a sample previously or subsequently obtained from the same individual, this method provides a way to monitor the appearance, progress, or treatment of a carcinoma. An antibody can be used to detect msp36 in a manner analogous to that described for detection of a different tumor suppressor in U.S. 5,801.001. Briefly, antibodies specific for msp36 can be generated by standard polyclonal or monoclonal methods, using as immunogen a purified, naturally-occurring msp36: recombinant msp36; or any antigenic fragment of msp36 which induces antibodies that react with naturally-occurring msp36. The latter fragment can be produced by synthetic or recombinant methods, or by proteolytic digestion of msp36. If desired, the antigenic fragment can be linked by standard methods to a molecule which increases the immunogenicity of the fragment, such as keyhole limpet hemocyanin in accordance with standard methods known to those of ordinary skill in the art. The polyclonal or monoclonal antibodies so produced can be screened using purified recombinant or naturally occurring msp36 to select those which form an immunocomplex with msp36 specifically.

The antibodies so produced are employed in diagnostic methods for detecting cells, tissues, or biological fluids in which the presence of msp36 is decreased relative to normal cells, an indication that the patient has a carcinoma. The sample tested may be a fixed section of a tissue biopsy, a preparation of cells obtained from a suspect tissue, or a sample of biological fluid, such as blood, serum, urine, sweat, tears, cerebrospinal fluid, milk, ductal fluid, or semen. Standard methods of immunoassay may be used, including those described above as well as sandwich HLISA. If the tested cells express no detectable msp36 protein in this assay, while normal cells of the same tissue type do express a detectable level of msp36 protein, the tested cells are likely to represent an advanced, metastatic carcinoma. If the tested cells express a decreased but consistently detectable level of msp36, the tested cells are probably from an early stage carcinoma that is not yet metastatic. Where the sample tested is a biological fluid into which msp36 would normally be secreted, the fluid may be directly contacted with the anti-msp protein antibody, or can be first partially processed (e.g., centrifuged, pre-cleared with other antibodies, dialyzed, or passed over a column) before using the anti-msp protein antibody. The amount of immunocomplex formed between the proteins of the sample and the anti-msp protein product antibody is then determined, and can be compared to a normal control run in parallel, or to a previously-determined standard.

As an alternative way of screening for potential anticancer agents, one can use any cell in which expression of msp36 is undetectable, but which contains an intact msp36 gene. This cell would be treated with a candidate anticancer compound, and a determination made of vvhether expression of msp36 is thereby increased in the cell. Such an increase of msp36 expression is an indication that the candidate compound is a potential anticancer agent. As above, the lev el of expression can be determined by use of hybridization methods or by immunoassay.

The invention also features methods for in vivo screening of candidate anticancer agents, or for determining whether a particular carcinoma, in which msp36 expression is down-regulated in comparison with normal cells of the same tissue type, is treatable with a given compound that increases expression of msp36. Such a method would include the steps of ( 1) introducing a carcinoma cell (e.g., a from a mammary carcinoma) into a severely immunodeficient animal (e.g. a nude mouse), the expression of msp36 in the cell being down-regulated in comparison with that in a normal cell of the same type of tissue as the carcinoma cell; (2) treating the animal with a compound which increases the concentration of msp36 in or around (i.e., in the immediate vicinity of) the carcinoma cell; and (3) determining whether this treatment affects the rate of proliferation or metastasis of the carcinoma cell in the animal, wherein a decrease in the rate of proliferation or metastasis in the presence of the compound is an indication that (a) the compound is potentially useful for treatment of carcinomas, and (b) the carcinoma from which the cell is derived is potentially treatable with the compound.

The invention also provides an in vitro assay for carcinoma cell invasive capacity based upon the assay described in detail below. Such an assay would include the steps of ( 1) providing a first and a second carcinoma cell, which cells express msp36 (SEQ ID NO: l) to a degree substantially lower than (i.e.. less than one-third of. when measured by hybridization to cellular mRNA) that of a normal cell from the same type of tissue as said carcinoma cells; (2) treating the first cell with a compound which increases the concentration of a msp36 molecule (e.g., SEQ ID NO: l) in or around the first cell: and (3) comparing the invasive capacity of each of the first and second cells in an in vitro assay such as that described below, wherein a decrease in invasive capacity of the (treated) first cell relative to that of the (untreated) second cell is an indication that (a) the compound is potentially useful for treatment of carcinomas, and (b) the carcinoma from which the cells are derived is potentially treatable with the compound. This assay is also useful for detecting msp36 activity in a biological sample (e.g., during the process of purification of msp36, or for testing the biological activity of msp36 fragments or derivatives, or for determining the presence of msp36 in a sample of blood, milk, or other biological fluid), wherein a decrease in invasive capacity of the (treated) first cell relative to that of the (untreated) second cell is an indication that msp36, or msp36 biological activity, is present in the sample.

The foregoing aspects of the invention can be practiced using animal models and in vitro assay systems such as those described in the Examples and in U.S. 5,801 ,001. For example, an in vivo assay, in which tumor growth is measured in severely immunodeficient mice (e.g.. nude mice), is useful in a number of applications concerning the present invention. For example, the assay can be used to determine (1) whether or not growth of a given carcinoma is inhibited by treatment either with msp36 or an agent which increases the concentration of msp36 in the carcinoma cells; or (2) whether or not a given candidate compound, which may be known to increase msp36 expression in carcinomas in which msp36 expression is down-regulated, can in fact inhibit growth of such carcinomas. The nude mice (or any other severely immunodeficient animal, such as a rat, rabbit, or other mammal) can also be adapted to study the effect of a given treatment on the rate of metastasis of the tumor, using standard methods of in vivo analysis of metastasis. A second type of assay described above, the in vitro assay of tumor cell invasion through reconstituted basement membrane matrix (e.g., MATRIGEL.RTM.), is also generally useful with respect to the present invention. Using this assay, the increase in invasive capacity of a given carcinoma over time, or of a series of carcinomas from different patients, can be correlated with the degree of inhibition of msp36 expression in each carcinoma sample. The assay can be used to screen various treatment protocols to determine whether a given msp36-increasing protocol is effective in reducing invasive capacity in a given carcinoma.

If expression of the msp36 tumor suppressor gene is down-regulated in the cells of a given carcinoma, but the gene remains present and intact in such cells, it is possible that the cells could be treated in a way that stimulates re- expression of the gene and thereby reverses or at least halts the progression of the carcinoma. This strategy would require affirmation that the gene remains intact and therefore available for up-regulation in the particular cancer cells to be treated. A Southern analysis of genomic DNA from the cancer cells and normal cells, such as described above, would provide evidence that the msp36 gene in the cancer cells is largely intact. Use of a battery of restriction enzymes would permit a more rigorous analysis of whether changes in the gene sequence had occurred in the cancer cells. One could use as hybridization probe full-length msp36 cDNA (SEQ ID NO: 1), msp36 genomic DNA, or a fragment of either. To obtain msp36 genomic DNA, a human genomic DNA library is probed with msp36 cDNA (SEQ ID NO: 1), using standard techniques such as described in Sambrook et al.. Molecular Cloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), herein incorporated by reference. The expression control elements of the naturally-occurring msp36 gene (e.g., promoters and enhancers usually located 5' to the transcription start site, or within one or more introns, but also possibly in the 3' untranslated region) are of particular interest, since down-regulation of transcription is associated with tumor progression.

Carcinoma or other cells in which the endogenous msp36 gene is present but down-regulated can be used as a screening tool to identity compounds or treatment strategies which induce re-expression of the msp36 gene. Re- expression of other down-regulated candidate tumor suppressor genes has been described: connexin 26, encoding a gap junction protein, by PMA (Lee et al., J. Cell Biol. 1 18: 1213, 1992), and a small calcium binding protein, CaN 19, by deoxyazacytidine (Lee et al., Proc. Natl. Acad. Sci. USA 89:2504. 1992). Of particular use in such a screen would be cell lines derived from an appropriate carcinoma, with a control being the same cells transfected with a vector encoding msp36 cDNA linked to expression control elements which permit constitutive expression of the cDNA (e.g.. the CMV promoter). However, other cell types with intact but unexpressed msp36 genes would also be potentially useful in this screening assay. I he cells would be treated in vitro with the candidate compounds, and the amount of msp36 expression determined using either a hybridization assay (e.g.. Northern analysis) or an immunoassay. The latter could be designed to detect intracellular msp36 or secreted msp36. or both. If a given compound is found to stimulate msp36 expression in the carcinoma cells, it could then be further tested to see whether treatment with the compound prevents carcinoma growth in the nude mouse model described above. A compound effective both in stimulating msp36 expression and in preventing carcinoma growth is a potential therapeutic useful for the treatment of carcinomas down- regulated in msp36 expression. Further evaluation of the clinical usefulness of such a compound would follow standard methods of evaluating toxicity and clinical effectiveness of anticancer agents.

EXAMPLES A. INTRODUCTION:

We have identified a cDNA clone (msp36, a 36kDa mammary secreted protein) with anti-proliferative properties on breast cancer cells. Its expression was significantly reduced in human breast cancer cells when compared with normal mammary cells, both in vitro and in vivo. This gene product, also named Hbpl 7/FGF-BP, was reported to bind basic FGF (bFGF, FGF-2) in a non- covalent, reversible manner and to inhibit the biological activity of bFGF. We have found that normal breast epithelium produces msp36 as an FGF-antagonist but that its expression is lost in breast carcinomas. We also have found that restoration of msp36 expression in several breast cancer cells resulted in inhibition of tumor growth, invasive potential and angiogenesis. The following experiments elucidate the molecular mechanisms responsible for the tumor suppressive effect of msp36 (hereafter abbreviated msp36), in vitro and in vivo. These experiments are useful for understanding the importance of msp36 for tumor growth, invasion, angiogenesis and metastasis, and elucidating the role of msp36 in affecting signaling pathways mediated through growth factors and their receptors. Based on these results, we believe that loss of msp36 expression represents a critical step in breast cancer progression and that stimulation of angiogenesis is one aspect of this progression.

Example 1. To characterize the role played by msp36 in inhibiting cancer growth, invasion, angiogenesis and metastasis. These experiments assess: the l iability of msp36 to impair tumor invasiveness /// vitro and in vivo: the ability of msp36 to inhibit angiogenesis induced by breast cancer cells in vivo: and the growth factors whose activities are inhibited by msp36. Growth factors to be tested include FGF- 1 , FGF-2, EGF. VEGF, P1GF-2. GF &TGF-α. We believe that msp36/Hbpl 7/FGF-BP modulates breast/mammary cancer growth, invasion, angiogenesis and metastasis. Since msp36/Hbpl 7 neutralizes the activities of FGF-2, we also believe that msp36 affects the activities of other growth factors important for breast cancer growth.

To address these questions, we overexpress msp36 in the MDAMB435+GFP invasive breast carcinoma cell line using a tet-regulatable expression system and recombinant adenovirus. We implant such cells orthotopically in the mammary fat pads of athymic mice and evaluate the effect of msp36 on tumor growth, invasion, angiogenesis and metastasis.

To characterize the signaling pathways affected by the anti-proliferative function of msp36. These experiments assess: the effects of msp36 on the FGF-2 down-stream signaling events; and the genes which are induced or repressed by msp36 that are responsible for tumor suppression. We believe that FGF-2- mediated cell growth signaling pathway is affected by msp36 expression.

The down-stream signaling events including MAPK and AKT that mediate FGF-2 induced proliferation and migration are analyzed in response to exogenous expression of msp36 using recombinant msp36, adenovirus and tet-inducible msp36. In addition, expression arrays are used to identify the genes which are specifically induced or repressed by msp36 in breast cancer cells.

The purpose of example 2 is to characterize the role of msp36 in vivo in mammary tumorigenesis by generating msp36- transgenic mice with Py VT (Polyoma virus middle T antigen) background. We believe that msp36 overexpression will delay mammary tumor development induced by the "polyoma virus middle 1^" antigen" oneogene. We use mouse strains with an oncogenic PyNT-transgene under the control of the MMTV promoter develop breast cancer. We address the effect of msp36 overexpression in vivo by targeting its expression to mammary tissue using transgenic technology. These studies allow us to determine vvhether the anti-tumor growth and/or anti-angiogenic effects oϊ msp36 overexpression predominate in the context of an in vivo model. Mammary tumors and associated angiogenesis are monitored by gross and microscopic analysis, combined with in situ hybridization and immunohistochemistry.

B. ANIMALS USED: The following animals are used in the proposed experiments: a. Nude immunodeficient mice, 6-8 w^reeks old, female, ~ 150/year for carrying transplants of human tumor cells are purchased from the National Cancer Institute. b. FVB mice, 7-8 wk, female, -300/year for generation and analysis of transgenic mice.

The animal species chosen are optimal for the studies proposed. FVB mice have been shown to be optimally suited for the generation of transgenic mice. Immunodeficient mice are required for studies of human tumor xenografts. The numbers of animals used are those required for obtaining statistically significant data. The experiments elucidate the mechanisms of angiogenesis. Only minor surgery is performed on mice: Biopsies of suspected mammary cell carcinomas (Aim 2, 3) using 4-mm punch biopsies. ^"I his surgery is performed under anesthesia (Avertin) to ensure that discomfort, distress and pain is limited. Injections of tumor cells involve no significant pain and only momentary discomfort. Mice are sacrificed by CO2 asphyxiation.

C. RESULTS:

MSP36 was isolated as a down-regulated gene in breast cancer cells in vitro and in vivo.

Although the clinical significance of high proliferative activity in breast carcinoma is widely recognized, the biological basis of proliferative activ ity is poorly understood. Considering the importance of growth factor regulation in bieast cancer, we designed a differential screening experiment to clone potential growth regulators. A domain-specific differential display experiment (DSDD- PCR) was performed using primers that had been selected from highest homology region among the TGF-β family members (GDF 1 , VG1. VGR1. OP1 , BMP2, 4. 5. 60A and DPP) (McPherron. A., and Lee, S.J.; J. Biol. Chem. 1993. 268: 3444-3449: Ozkaynak, E.. et al.. J. Biol. Chem. 1992. 267: 25220-25227). From the screening, we found a clone on the display gel whose expression was lost in the majority of human breast cancer cell lines. Msp36 exhibited significant levels of expression in normal mammary epithelial cells but not in human breast cancer cells or biopsy samples from breast cancer patients. Differential hybridization reaction was performed as follows to compare normal and cancer cells. Degenerate PCR primers based on the sequences of the regions exhibiting the conserved domains of the TGFβ family were designed to amplify cDNA fragments for differential-display PCR. A reverse 3' primer corresponding to a portion of the TGFβ family conserved region (TNHAIVQTL SEQ ID NO: 3) and an arbitrary primer (OPAl ; Operon Inc.) as the forward 5' primer were used for DD-PCR experiments. Differential expression of the msp36 gene on a display gel was performed using human normal mammary epithelial cells (hNMECs); immortalized human mammary epithelial cells; human breast cancer cells; hNMEC l ( 12N); hNMEC (15N); MCF10A; MCF7; T47D; SKBR3; BT20; MDAMB231 ; MDAMB435; and breast cancer tissue samples. This PCR based differential display using a conserved domain primer of known proteins (DS-DDPCR) from the TGFβ family produced only a few bands and more clearly resolved on the gel than conventional DD-PCR. Northern blot analysis showed an abundant expression of msp36 in human normal breast epithelial cells but no or little expression in a number of mammary cancer cell lines: hNMEC l(12N); hNMEC (17N); MCF 10A; MCF7; T47D; SKBR3; BT20; Hs578t; MDAMB435: and MDAMB231. A 36B4 probe was used as a loading control. 20 γg of total RNA was hybridized to ³ P-labeled msp36 cDNA probe. Msp36 expression in human normal prostate and cancer cell lines also was determined: normal prostate epithelial cells; LNCaP: Dul45: and ND1.

We then isolated a 460-bp cDNA fragment with high abundance in normal mammary cells. This fragment was used as a probe to clone the full- length cDNA clone from a normal human mammary cDNΛ library. Sequence comparison of the putative open reading frame with genes in the GenBank data base revealed a 100% sequence match to a reported gene. Hbpl 7, heparin- binding protein (Wu, D.. et al., J. Biol. Chem. 1991. 266: 16778-16785.). Sequencing of the msp36 cDNA indicates that the clone recovered was not a member of TGF-β superfamily, although a conserved sequence of the TGFβ family was found in the msp36 sequence. PCR amplification was probably attributable to similarity of degenerate oligonucleotide primers. Hbpl 7 was originally cloned as a secreted heparin-binding protein and binds specifically to the two fibroblast growth factors (acidic and basic FGF) in a non-covalent, reversible manner. In addition, the binding of this protein to FGFs (FGF-1 and -2) was reported to inhibit their biological activities (Kurtz A., et al., Oncogene. 1997, 14: 2671-81).

To further confirm the normal-specific expression of the recovered clone, Northern analysis was performed using RNA samples extracted from normal and tumor mammary epithelial cells. A transcript of -1.2 kb was detected in human normal breast epithelial cells. The expression of msp36 in human normal prostate and cancer cell lines was determined and the expression patterns were very close to that of the above-described differential display. Reduced msp36 expression in human breast cancer.

Comparison of msp36 mRNA levels in a variety of human normal and tumor cell lines revealed that msp36 expression was significantly diminished in tumor cell lines, including those derived from breast and prostate, when compared with normal cells. A polyclonal antibody was raised against msp36- specific peptide region (25 a. a, C-terminal region) and affinity-purified with peptide. This antibody was tested for specificity by Western blotting. A single band, approximately 36 kDa was detected from culture medium as well as cell lysates of normal mammary epithelial cells but not from breast tumor cells. Based on the amino acid sequence composition, the molecular mass was calculated to be 17 kDa (Wu, D.. et al.. J. Biol. Chem. 1991 , 266: 16778- 16785), whereas the protein migrates as a 36 kDa protein in SDS-polyacrylamide gels. The discrepancy in mass between the isolated and purified protein and the translated cDNA sequence is probably due to post-translation modifications such as glycosylation (Lametsch R., et al. J Biol Chem. 2000, 275: 1 469-74). Western blot analysis of msp36 expression in human normal mammary and cancer cells was performed as follows. Lysates were prepared from hNMECs and hTMECs and Western blotted using anti-msp36 antibodies against msp36^'-specific peptides. The blots showed that there was a secreted form of msp36 as well as a cytosolic or membrane-bound form of msp36. The following hNMEC strains, and hTMECs were used: 12N; 17N; 76N: MCF10A (immortalized); MCF7; T47D; SKBR3; BT20; MDAMB231 ; 21 MT2; Hs578t: and MDAMB435.

To assess differential expression of msp36 in vivo, we applied immunohistochemistry to a number of normal human breast (9 samples) and to preinvasive and invasive carcinomas. The expression pattern of msp36 in normal and benign samples was compared to those seen in both in situ carcinomas and infiltrating carcinomas. Normal and benign hyperplastic ductal or lobular breast elements were strongly positive. However, protein was undetectable in infiltrating carcinomas and adjacent carcinoma in situ. So far, we have examined 25 malignant breast tissue samples, all of which showed no or very little expression. Growth inhibitory effects of msp36 on human tumor cells.

To assess the impact of re-expressed msp36 on the malignant phenotype of breast cancer cells, T47D and MDAMB435 were transfected with an eukaryotic expression vector (pCDNA3) containing msp36 full-length cDNA under control of a CMV promoter. In addition, to examine the effect of exogenous msp36 overexpression on the proliferation of several cancer cell lines, cells were infected with a replication-defective adenovirus containing msp36 cDNA expression cassette (AdCMV-msp36). The effect of msp36 expression on human cancer cell lines was assessed by a cell proliferation assay, a soft-agar colony-forming assay, cell cycle analysis by flow cytometry and cell morphology changes.

Anchorage-independent growth of msp36- stable transfectants (T47D) was determined in soft-agarose for 2.5 weeks. msp36-transfected cells 12S. 20S versus control-transfected cells, C9. We have characterized msp36 transfected breast cancer cells (T47D+msp36) and control vector transfectants for changes in growth rate and colony formation in soft agarose. There was a significant difference in colony sizes. Msp36 overexpression resulted in substantial growth inhibition, as seen by roughly a 5-fold decrease in the size of colonies in soft agarose. To further determine the effects of re-expressing msp36 on tumor cell growth and to determine whether its expression was related to cell cycle arrest, we generated an adenoviral vector (He TC, et al. Proc Natl Acad Sci U S A. 1 98, 95: 2509-14.), Ad-msp36, and have infected it into several human cancer cell lines including two breast cancer cell lines (MDAMB435, MCF7) and the osteosarcoma cell line, Saos2. MDAMB435 and Saos2 cell lines both underwent morphological changes following infection with Ad-msp36 (100 moi), and by 30 hours were flattened and enlarged, resembling typical arrested cells. No noticeable change was observed with control Ad-LacZ infection ( 100 moi). Expression of msp36, confirmed by Western blot analysis, decreased the proliferation rate of MDAMB435 and Saos2 tumor cells by as much as 65-75° O (Fig. 1). MDAMB435 cells were plated in 6-well dishes ( 10* cells each) in triplicate, and incubated at 37°C. The following day, cells were infected with Ad- LacZ or Ad-msp36 at a MOI of 100, and cultured for 9 days. Every other day. one set of culture dishes was trypsinized. and cell numbers were measured by Coulter counting as well as with a hemocytometer.

In addition, flow cytometry revealed a Gl and G2/M arrest in cells infected with Ad-msp36 as compared to those infected with the control Ad-LacZ (Fig. 2). Exogenous expression of msp36 decreased the population of S-phase cells: from 47% to 26% in MDAMB435 cells and from 24% to 14% in Saos2 cells. In MCF7 cells infected with Ad-msp36, a significant population of cells underwent apoptosis (-28% increase of apoptotic cells compared to controls) but no sign of apoptosis was observed in Ad-LacZ-infected MCF7 cells. p21/Wafl , a potent cyclin-dependent kinase inhibitor, has been shown to be involved in both p53-dependent and independent control of cell proliferation, differentiation and apoptosis (Macleod KF, et al. Genes Dev. 1995, 9: 935-44; Harper, .W.. et al. Cell 1993, 75: 805-816). Cells were infected with Ad-LacZ or Ad-msp36 (MOI of 100) for 72 hours, stained with propidium iodide, and analyzed by FΛCScan (Becton Dickinson). We investigated whether p21 induction occurs following exogenous msp36 expression to gain insight into the mechanism of msp36-mediated growth arrest. Exogenous msp36 expression induced a significant rise in p21 protein at 24 hours after infection in MCF7 cells, demonstrating potential involvement of p21. MCF7 cells were infected with adenovirus (MOI of 100) expressing msp36 or lacZ as a control, for indicated times. p21 expression was measured by Western blot analysis, β-actin served as a loading control. Growth inhibition as well as the increase in p2 I induced by msp36 expression was more effective in MCF7 cells than MDAMB435 and Saos2 cells. The population of S phase MCF7 cells was significantly decreased from 32% to 1%. In addition, msp36 expression by Ad-msp36 infection induced a high level of apoptosis in MCF7 cells, with no sign of apoptosis in control cells (from 1% to 27% increase). Reduction of invasion potential in msp36 transfected human breast cells.

Since FGFs are reported to affect migration and invasion, we suspected that msp36 might inhibit cell migration (invasion) of cells in which it is expressed. Tumor progression is related to deregulated expression of a set of proteases as well as extracellular matrix proteins, and this may represent a fundamental step in the progressive expression of the invasive phenotype. Therefore, as an in vitro measure of invasive capacities, we tested the abilities of msp36 transfected human breast cancer cells (MDAMB435+msp3ό) as compared to control transfected cells to cross a Matrigel-coated membrane. As shown in Fig. 3, msp36 transfectants (stably transfected and isolated clones. MDAMB435- 14S and -6S) showed 60 - 70 % reduction of invasiveness in as early as 2 days as compared to control transfectants, the addition of msp36-specific antibodies completely abrogated their invasive suppression.

Invasion was measured using 24-well BioCoat Matrigel invasion chambers with an 8-μm pore size polycarbonate filter coated with Matrigel. Each sample was assayed in triplicate. MDAMB435 cells (1 x10^") were transfected with msp36 or control vectors and were plated in the upper compartment of Matrigel chambers for 48 hrs. The lower compartment contained DMEM+10% FBS or DMEM without FBS as a negative control. After incubation, filters were fixed with 3% glutaraldehyde and stained with Giemsa. The cells on the upper surface of the filter were removed by wiping, and migration was measured by counting the number of cells that had migrated to the lower side of the filter. Furthermore, we produced the recombinant GST+msp36 fusion protein in E. coli and treated invasive MDAMB435 cells ( 1 μg/ml). The treatment with recombinant GST/msp36 fusion proteins was very effective in blocking the invasiveness of both bFGF and EGF stimulated MDAMB435 cells in a Matrigel invasion assay, as compared to control GST protein (Fig. 4). These data provide evidence that msp36 acts as a invasion suppressor gene although its anti-invasive properties remain to be tested in other cell types and in an in vivo model. MDAMB435 cells were plated in Matrigel chambers and incubated with DMEM+bFGF or EGF (10 ng/ml) plus GST-msp36 ( 1 μg/ml), or GST (2μg/ml). Invasive cells were quantified as described above. msp36 protein blocked Matrigel invasion of both EGF or bFGF stimulated MDAMB435 cells.

We also investigated whether msp36 might be involved in extracellular matrix interactions with cells or if it might inhibit migration of the cells in which it is expressed. Tumor invasion requires interactions between invasive cells and the extracellular matrix, and these interactions are mediated predominantly by a large family of cell surface receptors, the integrins, which include multiple receptors for extracellular matrix and base membrane components (Starkey JR. Cancer Metastasis Rev. 1990, 9: 1 13-23.). We assayed the migration of msp36-transfected cells using a transwell migration assay (Haptotaxis) and membranes coated with a number of different matrices: fibronectin, collagen, laminin, vitronectin and hemoglobin (as a control). As shown in Fig. 5, exogenous expression of msp36 in human breast cancer cells, T47D, resulted in a strong decrease in migration compared with control T47D cells.

Down-regulation of MMP-9 production by msp36. Changes in cancer cell adhesion or locomotion alone are not sufficient for the active penetration of tissue barriers. To infiltrate host tissues, cancer cells must elaborate proteolytic enzymes ( Starkey JR. Cancer Metastasis Rev. 1990. 9: 1 13-23; Liotta LA. et al. Cancer Res. 1991 , 51 : 5054s-5059s: Liotta LA, et al. Semin Cancer Biol. 1991. 2: 1 1 1 -4; Liotta LA, et al. Cell. 1991. 64: 327-36. Among the most studied are urokinase plasminogen activator (uPA) generated plasmin and matrix metalloproteinases (MMPs) (Liotta LA. et al. Cell. 1991 , 64: 327-36; Vu TH and Werb Z. Matrix .Genes Dev. 2000. 14: 2123-33). MMPs are a large family of ECM-degrading enzymes, which include collagenases, gelatinases, stromelysins, and MT-MMPs (Vu TH and Werb Z. Matrix, Genes Dev. 2000, 14: 2123-33.), many of which have been reported to have a role in tumor angiogenesis, invasion and metastasis ( Vu TH and Werb Z. Matrix, Genes Dev. 2000, 14: 2123-33; Zhou Z, et al. Proc Natl Acad Sci U S A. 2000, 97: 4052-7; Nelson AR, et al. J Clin Oncol. 2000, 18: 1 135-49). Several MMPs. including gelatinase B/MMP-9 (Vu TH and Werb Z. Matrix, Genes Dev. 2000, 14: 2123-33), have been reported to be directly involved in cancer invasion and are believed to be predominantly, but not exclusively, produced by stromal cells. This prompted us to examine the effect of msp36 on the induction of MMP(s) in an invasive breast cancer line, MDAMB435. Our data show an inhibition of MMP-9 secretion in these cells in response to treatment of cells with GST+msp36 fusion protein (2 μg/ml), while control GST at the same concentration (2μg/ml) did not have any effect. In addition, msp36-transfected MDAMB435 cells decreased MMP9 production as compared to control- transfectants. MDAMB435 cells were treated with either 2 μg/ml of GST-msp36 or GST alone for 24 hours. Conditioned medium from these cells was analyzed by zymography. Msp36 transfected MDAMB435 cell were also analyzed by zymography.

Inhibition of angiogenesis by msp36 expression in vivo.

The growth of tumors beyond a minimal size has been hypothesized to be dependent upon the induction of new blood vessel growth or angiogenesis, which in turn supplies needed nutrients to rapidly dividing tumor cells. Since numerous angiogenic cytokines such as bFGF and VEGF may be elicited from tumors during the initial growth phase, the function of these factors in overall tumor growth and survival is critical. However, these pro-angiogenic growth factors can be offset by anti-angiogenic factors which modulate their activities. In an attempt to evaluate whether exogenous expression of msp36 would alter the ability of cytokines to promote the angiogenic pathway, we utilized two breast cancer cell lines, 147D and MDAMB435 control cells, and msp36-transfected I^'47D and MDA435 cells in a quantitative mouse angiogenesis model, a Matrigel implant assay.

Two different msp36-transfected breast cancer cell lines (T47D+msp36 and MDAMB435+msp36) as well as control cells (T47D+pcDNA3 and MDAMB435+pcDNA3) were implanted into the subcutaneous space of athymic nude mice encapsulated in a proteinaceous Matrigel plug. The angiogenic response was visualized by photography and defined in more detail by collagen IV immunostaining of vessel basement membranes which are formed at the skin Matrigel interface. As shown in Fig. 6, the implants of both msp36 expressing breast cancer cells showed a significantly reduced response, as compared to implants of control cells. To evaluate the scale of angiogenic response, the direct quantitation of the defined neovascularization was determined by counting the number of Collagen IV-stained luminal vessels in the skin/Matrigel interface. Note that there is a significant reduction in blood vessel formation for each of the cell lines which are expressing msp36 protein. At least a 50% inhibition of the angiogenic response was observed. Fig. 7 shows the data for the angiogenic response quantitated for each of the tumor/Matrigel implanted group.

In summary, using a domain-specific DD-PCR we identified a cDNA clone, msp36, that is preferentially expressed in normal human mammary epithelial cells in vitro and in vivo as compared to breast tumor cells, suggesting that it may play a general role in regulating normal cell growth. It appears that exogenous msp36 expression inhibits tumor cell growth with increase of p21/Wafl expression and invasion potential of breast cancer cells in vitro, and tumor angiogenesis in vivo. Therefore, msp36 functions to inhibit cancer progression, metastasis and angiogenesis of breast as well as other cancers.

We believe that msp36 modulates one or more of the following effects on mammary cancers: inhibition of tumor cell growth, invasion, angiogenesis and metastasis. Our preliminary studies indicated that msp36 expression was significantly reduced in human breast cancer cell lines and also in breast cancer patient tissue samples, compared with normal breast cells. msp36 binds to and inhibits the biological activities of FGF-1 and FGF-2. We also have accumulated evidence that exogenous msp36 expression inhibits tumor cell growth and the invasive potential of breast cancer cells /// vitro: it also inhibits tumor angiogenesis as measured by the /// vivo matrix implant (Matrigel) assay.

Detection of MDAMB435-GFP tagged cells by confocal microscopy in lymph node and lung tissues of nude mice.

Human breast cancer cells (MDAMB435) were implanted orthotopically in the mammary fat pads of athymic, nude mice in order to investigate the effects of msp36 on tumor growth, invasion, induction of angiogenesis, and possible metastasis. To facilitate detection of small numbers of tumor cells, MDAMB435 cells are transfected with green fluorescent protein and the resulting cells are named MDAMB435+GFP.

Determine the ability of msp36 to impair tumor cell invasiveness and metastasis in vitro & in vivo. In our initial transfection experiments msp36 was found to inhibit tumor cell growth and invasion in breast cancer cells in vitro. We extended these studies to other invasive breast cancer cells and also to studies performed in vivo. Using a highly metastatic GFP (Green Fluorescence ProteinVexpressing subclone of the MDA-MB-435 cell line, we determined the effect of msp36 on the invasive phenotype in vitro and in vivo.

We used a new, tetracycline-inducible vector system, the T-REx system (Invitrogen), to transduce msp36 into tumor cells. Both the TetR, repressor expression vector, and the T-REx CMV with double TetR binding domains downstream of the TATA box provide continual blocking of transcription in the absence of tetracycline or its analogue, doxycvcline. Both vectors or TetR v ector alone are transfected into the MDA-435-GFP cell line and selected for zeocin resistance for the CMV expression/Tet-inducible cDNA expression vector and blasticidin for the TetR expression vector. Inducibilitv and repression of msp36- induction is assayed with/without tetracycline //; vitro. When these function properly. msp36 is expressed only in cells exposed to tetracycline. The cells then are used for /// vivo studies of tumor invasion, angiogenesis and metastasis as described above. GPP-expressing MDAMB435+GFP cells have the advantage of being detectable at the single cell level in tissue sections, facilitating studies of both invasion and metastasis.

In order to determine the effect of msp36 overexpression, we perform the following analyses on cultured MDAMB435+GFP cells in the presence or absence of tet: cell cycle analysis by FACS, soft-agar assay, cell proliferation assay using BrdU (Oncogene), MMPs modulation by Zymographic assay, and the Matrigel-invasion assay (Lee, S.W.. et al. Carcinogenesis 1998, 19: 1 157-1 1 59). If, as is likely, we find significant change(s) in any of these assays upon msp36 induction (1-5 days), we repeat the experiments with two other msp36-inducible JMDAMB435-GFP clones.

In addition, we repeat the above in vitro experiments with recombinant msp36 (GST-fusion protein) that was produced in E. coli and introduced into MDAMB435+GFP cells, thus testing the effect of introducing msp36 from another source. These studies test the ability of msp36 to inhibit the growth, invasion and metastatic capacity of breast cancer cells in vivo as well as to induce apoptosis. Ten female nu/nu mice are injected with a stable clone of MDAMB435+GFP tumor cells with tetracycline-inducible msp36 expression. 3x 10^(l cells are injected into each of two mammary fat pads. Doxycycline is delivered in drinking water (500 μg/ml doxycycline in 1% sucrose). Animal weights and external caliper measurements of primary tumor growth are performed every other day. After the primary tumors grow to a palpable size (- 100 mm³), typically 7-10 days post- implantation, induction of msp36 expression is initiated in half of the mice and the experiment is continued for up to 5-7 weeks. Animals are sacrificed at appropriate intervals for gross and microscopic study of the primary tumors, axillary lymph nodes and lungs (to which these tumors metastasizc).

Metastasis is evaluated in both the draining lymph nodes and lungs. Axillary lymph nodes are dissected from the flank adjacent to the primary mammary tumor, fixed in 3.7% paraformaldehyde for 15 min. washed in PBS. sliced, equilibrated with PBS:0.3M sucrose overnight, and frozen in OCT tissue embedding compound. Frozen sections are then cut (7 μm) and digital fluorescent microscopy for GFP-containing cells performed. Fluorescent threshold analysis using an image processing package (ImageProPlus, Media Cybernetics) defines a value for fluorescence per field which is averaged and compared to other treatment groups by statistical analyses.

Quantification of tumor metastasis to the lung is performed in a similar manner. Lungs are fixed by perfusion of the bronchi with 3.7% paraibrmaldehvde in PBS (5ml), clamping the trachea with a hemostat to prevent efflux. The lungs are then removed, rinsed thoroughly in PBS and fixed for an additional hour before transferring them to PBS. Wet mounted lung slices are then examined by fluorescence microscopy for GFP-expressing tumor nodules (typically fi e cross- sections per lobe, each approx. 2-3 mm thick). The number of colonies per lung is counted. In addition, to take into account the size of individual metastases, we measure the cross-sectional area of lung occupied by tumor with image analysis software (ImageProPlus, Media Cybernetics, Inc). The data from different groups of animals is analyzed statistically (Mann- Whitney test). The in vitro and in vivo experiments provide important confirmatory information as to the biological effects of msp36 on tumor cell growth, invasiveness, angiogenesis and metastasis. Because the FGFs act on many different cell types, it is not surprising that the effects of msp36 are multi- factorial, i.e., affected both tumor cell behavior and that of stromal cells and angiogenesis (see below). Optionally, we transduce MDAMB435+GFP cells with an adenovirus we have engineered to express msp36; this vector also can be used in experiments outlined above. Immortalized human breast epithelial cell lines including 184B5 and MCF10 continue to express measurable levels of msp36 and it therefore is important to assess the levels expressed in cells that have not been transduced with this protein. Alternatively, we include groups of animals that have been transfected with antisense msp36 constructs to drive endogenous lev els to undetectable levels. As discussed above, we have produced recombinant msp36 as a GST fusion in E. coli. and the treatment of recombinant GST-msp36 fusion protein was very effective in blocking the invasiveness of MDAMB435 cells in the Matrigel invasion assay. Therefore, anti-proliferative and/or anti- invasive abilities of the protein can be tested in several different breast cancer cell lines in the assa s described above. As the role of msp36 has until now been evaluated primarily in tissue culture cells, the generation and characterization of an in vivo tumor model with metastatic potential provides important evidence supporting therapeutic applications of msp36 in treating breast cancer.

Evaluation of the anti-invasive effect of recombinant msp36 on a series of invasive human breast cancer cell lines: Five invasive human breast cancer cell lines, MDAMB436. MDAMB 157, BT549, HS578t and SUM 159, are used for this study. As discussed above, we have produced recombinant msp36 as a GST fusion in E. coli, and the treatment of recombinant GST-msp36 fusion protein was very effective in blocking the invasiveness of MDAMB435 cells in the Matrigel invasion assay. Therefore, anti-invasive activity of the protein can be tested in several different breast or other tissue cancer cell lines in the assays described above. In order to establish the optimum dose for anti-invasion and anti-proliferative activity of recombinant msp36 in breast cancer cells // vitro, various concentration of recombinant msp36 (lOng, l OOng, 1 μg, 2.5 μg, and 5 μg/ml of medium) are added to MDAMB435 cells in Matrigel invasion assays using lxlO⁵ MDAMB435 cells in the presence or absence of FGF-2 (10 ng/ml). After the optimum dose for Matrigel invasion is determined with MDAMB435 cells, a series of invasive human breast cancer cells is tested for the anti-invasive and anti-proliferative effects of msp36. msp36 expression in baculovirus: In some of the above experiments and in those of subsequent Examples, an additional source of msp36, namely, msp36 expressed in baculovirus, is used.

Many factors such as solubility, disulfide bond formation, and post- translational modification can influence the choice of one expression vector over another. The baculovirus expression system in insect cell cultures offers significant advantages over prokaryotic and other eukaryotic systems for production of many proteins: i) high yields of expression: ii) very low endotoxin level from purification procedure and iii) glycosylation; post-translational modifications such as myristylation. phosphorylation. etc. We utilize the Bac-to- Bac baculovirus expression system (Life ^"fech.) which is uniquely designed to allow the rapid and efficient generation of recombinant baculovirus DNAs by site-specific transposition in E.coli. rather than homologous recombination. The pFastBacHTa vector includes a 6Xhis affinity tag and TEV protease cleavage site upstream of the multicloning site so that heterologous proteins expressed in insect cells (Sf9 cells) can be easily purified using Ni-resin. The expression and purification of msp36 in the Bac-to-Bac system is performed as described in the vendor's protocol. Optionally, we use the pTriEx system (Novagen) to express msp36 in mammalian cells.

Baculovirus-produced msp36 is tested for its activity to inhibit tumor invasion in the Matrigel assay.

Assess the ability of msp36 to inhibit angiogenesis induced by breast cancer cells in vivo.

Loss or substantial reduction of msp36 expression is a characteristic feature of breast cancer cell lines and correlates with their ability to form invasive tumors in vivo. Such cell lines also induced a strong angiogenic response in the Matrigel assay. This experiment is used to confirm the hypothesis that exogenous expression of msp36 inhibits the capacity of these cells to induce angiogenesis in vivo. Therefore, this experiment directly confirms our belief that msp36 plays an important role as an endogenous inhibitor of breast tumor growth and angiogenesis.

The human mammary carcinoma line MDAMB435 has been well characterized for its tumorigenicity and ability to induce a strong angiogenic response. It possesses distinctive growth and differentiation characteristics in vitro and growth patterns /// vivo after orthotopic transplantation (mammary fat pads) into nude mice, producing rapidly growing tumors which become apparent within 2 weeks. Tumor diameters increase to 10 to 20 mm 4 weeks after inoculation. For angiogenesis studies, we use the human breast cancer cell line MDAMB435+GFP. stably transfected with a GFP expression vector and lines described above that have been additionally transfected with msp36 under tet regulation. This allows us to confirm the role of msp36 in angiogenesis. Two types of experiment are performed, using stably transfected cell lines: Angiogenic response induced by tumor xenotransplant and Matrigel implant angiogenesis assav. Preparation of stably transfected cell lines: Two different expression systems are used to express msp36 in MDAMB435 cell line: constitutive msp36 expression (pcDNA3 hyg^R) and tetracycline-regulatable expression.

Constitutive msp36 expression. Stable transfection is achieved using the calcium phosphate method and hygromycin selection. Following clonal selection, more than 20 clones of each construct are characterized with regard to mRNA expression by Northern analysis and for protein secretion by Western blot. Four clones that show the best expression of msp36 are used for further studies. We have already isolated several msp36 expressing clones and are currently characterizing them. Cells transfected with vector alone and the parent cell line serve as matched controls.

Tetracycline-regulatable expression: These are prepared as described above. msp36 expression is assessed and quantitated at the RNA level by Northern analysis; culture supematants are assessed for msp36 protein by Western blot. Northern analyses makes use of the human msp36 cDNA.

Angiogenic response induced by tumor xenotransplants. 2 x 10⁶ tumor cells (stable transfectants, controls) suspended in 0.1 ml culture medium are injected into each of two mammary fat pads of immunologically compromised virgin female nude (nu/nu) mice in groups of at least 5 animals. Tumor growth is assessed twice weekly, and tumors are harvested when they reach a size of 20 mm diameter (largest diameter) or no later than 8 weeks. Tumors are measured for size, msp36 expression (ISH, IH through the Morphology core), and vascularity and the character of vessels induced is assessed for msp36 expression and angiogenesis. In situ hybridization (ISH) and immunohistochemistry (IFI) are performed to confirm expression of msp36. Tissues are processed and studied as described above. Sense riboprobes serve as negative controls.

The methodology for ISH is described herein. In brief, tissues are fixed for 1 hr in cold (4°C) 4% paraformaldehyde in PBS, stored thereafter in cold PBS for up to 2 days, and processed for either frozen section or paraffin embedding. At all stages precautions are taken to avoid contamination with ubiquitous RNAses (e.g.. DEPC-treated water used in fixative and all other solutions). Subsequent tissue processing, cutting of sections and probe preparation follow published protocols (Streit M, et al. EMBO J. 2000, 19: 3272-82; Streit M. et al. Am J Pathol. 1999, 155: 441 -52). Transcription reactions are carried out using a Riboprobe Gemini II kit (Promega) in the presence of ³⁵S UTP. Cell proliferation is assessed with the BrdU technique to quantitate the percentage of proliferating tumor cells and vascular cells. BrdU (Sigma) is injected intraperitoneally (250 mg per kilogram of body weight), and mice are sacrificed 2 hours after injection. Skin samples are fixed, paraffin embedded, and 5 μm sections are stained with a fluorescein isothiocyanate-conjugated antibody to BrdU (Becton-Dickinson). 100-500 vascular endothelial cells are counted and data are expressed as percentage of BrdU-positive endothelial cells per field ( Streit M, et al. Proc Natl Acad Sci U S A. 1999, 96: 14888-93; Pike SE, et al. J Exp Med. 1998 ,188: 2349-56).

In addition, a variety of other studies are performed, as determined to be necessary in individual cases, to assess the angiogenic response. These studies are described in accordance with standard procedures and include: ( 1) Vascular pattern, size and shape of individual vessels; (2) Expression of growth factors, growth factor receptors, endothelial cell and pericyte markers: (3) Apoptosis; (4) Vascular basement membranes, extracellular matrices; (5) Proteolytic enzymes: and (6) Micro vascular permeability.

Matrigel implant angiogenesis assay. The Matrigel assay is performed as described above, using 4-7 weeks old female immunodeficient mice (athymic NCr-nu/nu). Three volumes of Matrigel with protein concentrations of 15mg/ml and endotoxin less than 1 EU/ml (Becton Dickson Labware) are mixed with cell suspension at 4°C yielding a final cell concentration of 1 x 10 cells/ml. Animals are anesthetized with avertin and tumor cell (MDΛMB435+FGF-BP)-Matrigel mixtures (0.3 ml) are injected in the subcutaneous space with a pre-chilled tuberculin syringe (27G needle) at the dorsal midline of the back. Ten days later, animals arc euthanized with C02 and the dorsal skin resected retaining the Matrigel implant. Gross photographs are obtained of the Matrigel implant with a dissecting photomicroscope using fiberoptic illumination. Matrigel implants then are fixed with skin attached using 4% paraformaldehyde:PBS for 2 hr at ambient temperature. Matrigel and adjoining skin then are serially sliced into four- sections with a razor blade approximately 2-3 mm thick, processed for paraffin embedding and histological sectioning. Standard hematoxylin and eosin (H&E) stain are used to define skin, angiogenic stromal interface and tumor cell- Matrigel features. Newly formed vessels within the skin/Matrigel interface is defined as luminal structures staining with anti-collagen IV antibodies (BioDesign Int.) detected with DAB substrate using ABC detection kit (Vector Laboratories). At least three complete Matrigel/skin cross-sections are manually counted from each implant using a 20x objectives and the number of vessels per field averaged for at least 5 animals per group.

Determine the growth factors whose activities are inhibited by msp36. Our data indicate that msp36-transfected breast cancer cells show significant reduction of invasiveness and that addition of msp36-specific antibodies abrogated the invasion-suppressive activity of msp36 when FGF-2 was used as a stimulant. In addition, we have demonstrated that exogenous expression of msp36 in the T47D human breast cancer cell line significantly inhibited tumor cell growth, indicating that msp36 inhibits tumor cell growth as well as invasion. We believe that the activities of growth factors other than FGF-2 are affected by msp36. Accordingly, tet-regulatable MDAMB435 cell lines as well as recombinant msp36 protein and adenovirus expressing msp36 as described above are used to evaluate inhibitory function of msp36 against other growth factors including: FGF-1. FGF-2. EGF, VEGF-A, P1GF, KGF, and TGF- . The interaction between FGF and heparan sulfate (HS) stabilizes FGF and the growth- stimulatory activities of the FGFs are HS-dependent. Most mammary cancer cells including MCF7 and MDAMB435 cell lines produce excess HS and so FGFs in the medium are expected to be active (Rahmoune I I, et al. J Biol Chem. 1998, 273: 7303-10.). msp36-mediated anti-proliferative and anti-invasive activities is likely to be HS-independent. This experiment assesses the relationship between msp36 and HS-stimulated bFGF and the effect of msp36 on the interaction of HS with bFGF in the serum-free medium.

We determine the extent to which msp36 affects the stimulation of cell proliferation by growth factors other than FGF-2. MDAMB435 cell lines and recombinant msp36 protein are used to test the inhibitory function of msp36 against other growth factors. Our data indicates that msp36 transfected MDAMB435 cells significantly reduced invasiveness and that the addition of msp36-specific antibody abrogated the invasion-suppressive activity of msp36 when EGF as well as FGF-2 were used as stimulants, respectively.

Assays to determine the extent to which msp36 affects the stimulation of cell proliferation by these other growth features include the following: Matrigel invasion assay, cell migration (haptotaxis) assay, and cell proliferation assays. In addition, the Miles assay is used to determine the extent to which msp36 inhibits the vascular permeability enhancing activity of VEGF-A.

Msp36-transfected MDAMB435 cells are stimulated with FGF- 1, FGF-2, HGF, EGF, TGF-α and KGF. Vascular endothelial cells are treated with VEGF- A. The following biological assays are performed: Matrigel invasion assay, cell migration assay, soft agar assay, and cell growth rate by H thvmidine incorporation, in the presence or absence of tetracycline (2ug/ml). Recombinant msp36 is added to cultured endothelial cells. Differences detected in these experiments using tet-inducible MDAMB435 cells are confirmed with recombinant msp36 treatment. A range of concentrations is tested: 500 ng, 1 μg, 2.5 μg, 5 μg, or 10 μg/ml in these assays. The effect of msp36 on proliferation and migration of endothelial cells also is studied. Human microvascular endothelial cells (passage 2-4) are cultured in medium alone or in medium supplemented with bFGF (50ng/ml), P1GF-2 (50ng/ml), or VEGF-A (50ng/ml). Recombinant adenovirus expressing msp36 or LacZ is used to infect these cultures. Cell proliferation is measured by cell counts every other day up to 7-9 days or by ^~H thvmidine incorporation during the final hours of cultures.

In summary, these experiments further confirm the functions of msp36 and also reveal important insights into the molecular events that regulate tumor cell growth, cell-cell adhesion and tumor metastasis, results which provide a promising new reagent for breast and other cancer therapy. Example 2. To elucidate the signaling pathways affected by the anti- proliferative function of msp36.

Characterize the effects of msp36 on the FGF-2 down-stream signaling events. We believe that msp36 affects growth factor-mediated signaling pathways. FGF stimulates cell migration, invasion and angiogenesis as well as MMP up-regulation. FGFs transduce their signals by binding to specific cell surface tyrosine kinase receptors. Although the mechanism of FGF-induced signal transduction is not well defined, it is clear that FGF-2 induces rapid FGFR- activation, phosphorylation of PI3 kinase, sustained phosphorylation of MAP kinases (ERK) and p38 MAPK, along with stimulation of DNA synthesis (Tan Y, et al. EMBO J. 1996, 15: 4629-42; Gerber HP, et al. J Biol Chem. 1998, 273: 30336-43; Chen Y, et al. Oncogene. 2000,19: 3750-6; Maher P. J Biol Chem. 1999, 274: 17491-8). msp36 reportedly can bind to both acidic and basic FGFs in a non-covalent, reversible manner (Wu, D., et al., J. Biol. Chem. 1991 , 266: 16778-16785.) and inhibit the mitogenic activities of FGFs (Wu, D., et al., J. Biol. Chem. 1991 , 266: 16778- 16785). Our data indicate that msp36 inhibits cell proliferation of breast cancer cells. In addition, ectopic expression of msp36 inhibited MAPK (ERK) activation as well as AKT in a FGF-2 stimulated breast cancer cell line (MCF7). Furthermore, we found that MAPK inhibition by msp36 is restricted to ERK pathway since no effect was observed on p38 MAPK activation in response to FGF-2. This differential effect on ERK and p38 MAPK indicates that some of the action of msp36 is at the level of intracellular signal transduction pathways. Therefore, these data suggest that the functional antagonistic interaction between FGF-2 and msp36 plays an important role in the transduction of FGF signals supporting proliferation, cell migration and invasion. Using our system, we further elucidate the molecular basis for modulation of FGF-2 downstream signaling by msp36. In this Example we incorporate several approaches to determine the molecular basis for msp36-mediated inhibition of cell proliferation. These experiments define the cascade of events that mediate the msp36-induced anti-proliferation and anti-invasion. msp36-mediated inhibition of activated forms of MAPK (ERK) and AKT in bFGF-stimulated MCF7 cells. Recombinant adenovirus (100 moi), Ad- LacZ or Ad-msp36, was infected into MCF7 cells, 24 hours after infection, infected MCF7 cells were starved for serum (FBS) for 24 hours and stimulated with bFGF for the indicated times. Western blot analysis was performed with msp36, phospho-specific ERK, and phospho-PKB, phospho-p38 and total p38 antibodies to measure activated forms of MAPK and PKB (phospho-MAPK and pho-PKB-Ser473). β-actin antibodies were used as a loading control. Decreased expression of phosphorylated PKB and MAPK (ERK) was detected in response to bFGF stimulation in msp36-expressing cells, but control cells showed increased phosphorylation of PKB and MAPK (ERK) in response to bFGF. FIow^rever, no inhibition of the activation levels of p38 (p-p38) MAPK in Ad- msp36 infected cells in response to bFGF.

Two types of experiments are performed: Sustained MAPK activation in msp36-expressing cells. We define fiirther the role played by msp36-mediated MAPK inhibition in msp36-mediated anti-proliferation as well as anti-invasion by using a constitutively active MEK1 mutant.

In this Example, we further characterize the extent to which sustained MAPK activity is required for cell proliferation and the extent to which msp36 inhibits ERK MAPK levels. First, we overexpress a constitutively active MEK mutant (MEK-EL) in breast cancer cells (MDAMB435 and MCF7) and endothelial cells by using a tetracycline-inducible or retroviral expression system. We have successfully expressed constitutively active MEK mutant in human fibroblasts as well as human cancer cells by retroviral expression and tet- regulatable system. The cells containing sustained MAPK (ERK) activation are transfected or infected with msp36 constructs to specifically examine the extent to which anti-proliferative function by msp36 is impaired. In addition, a constitutive active AKT mutant is used by the same approach as above. To determine the level and time course of MAPK activation, we employ the phospho-specific MAPK antibodies (anti-P-ERK. NEB) as well as an /// vitro assay that measures phosphorylation of an exogenous myelin basic protein (MBP) substrate by activated MAPK (Lee, S.W., et al. Proc. Natl. Acad. Sci. 2000, 97: 8302-8305).

Expression of activated MEK/MAPK in human NDFs (501T) by infection of retroviral stocks (~10^ft moi) containing MEK-EL or empty vector (pBabe). Expression of eetopic MEK-EL in retrovirally infected 50 I T cells was analyzed by Western blot analysis at day 7 post-selection. Overexpression of MEK-EL induced activation of ERK, p21 and pi 6.

Effect on FGFR function. We believe that msp36 affects FGF receptor activity by decreasing receptor levels or FGFR binding affinity to FGF-2. thus leading to decreased activity and altering down-stream signaling including

MAPK and P13K activation. Therefore, we assess the effect on FGFR function in relation to msp36. To further characterize this interaction, we use FGFR Scatchard analysis to determine the number and affinity of receptors for FGF-2 (Spivak-Kroizman T, et al. Cell. 1994, 79: 1015-24.). MCF7 human breast cancer cell line as well as FGFR-1 and -2 transfected, overexpressing NIH3T3 cells (Miki T, et al. Proc Natl Acad Sci U S A. 1992, 89: 246-50) are used for this study. MCF7 cell lines were chosen for this study because they overexpress both FGFR-1 and -2 receptors (Adnane J, et al. Oncogene. 1991 , 6: 659-63; Dionne CA, et al. EMBO J. 1990 9: 2685-92). We compare the values for FGFR receptor number and affinity obtained by Scatchard analysis with levels of FGFR message and protein obtained by Northern and Western blotting. In addition, the activation status of FGFR in the presence and absence of exogenous msp36 is determined by immunoblotting of total cell lysates or FGFR (Santa Cruz antibodies) immunoprecipitates with anti-phosphotyrosine antibodies. We compare FGF binding to MCF7 cells with/without msp36. We radioiodinate commercially available FGF-2 (Repro Lab.) using the Enzymobeads (BioRad) protocol. MCF7 cells infected with adenovirus expressing msp36 or control adenovirus LacZ are plated in 24 well plates, washed in binding buffer, and incubated on ice for 1 hour with serial dilutions of '"I-FGF-2. Cells are then washed and solubilized in 0.3N NaOH and counted. Non-specific binding is determined in the presence of 100-fold molar excess of unlabeled FGF-2. The number of FGFR and their affinity in both msp36 expressing cells and control cells is determined.

Our data provide evidence that MAPK and AKT activities are decreased by msp36. These further experiments establish the extent to which modulation of MAPK phosphorylation results from msp36 expression through an inhibition of FGFR signaling. The use of the MCF7 cells as well as NIH3T3 cells overexpressing FGFR-1 and -2 provide a series of controls. The extent to which the PI3 kinase pathway, known to be involved in cell survival and migration, is activated by FGFs also is determined. We first establish the extent to which msp36 expression influences PI3-K activation and down-stream targets such as AKT/PKB or Rac/Rho molecules known to mediate cell survival, invasion or migration (Clark EA, et al. Nature. 2000, 406: 532-5; Ridley A. Nature. 2000, 406: 466-7). We believe that msp36 inhibits cell proliferation, cell survival and migration/invasion, thus preventing tumor cells expressing msp36 from being able to induce angiogenesis and metastasize. Although this Example is designed primarily to further characterize the MAPK pathway, it can be easily modified to characterize other signal transduction pathways.

Identify the genes which are induced or repressed by msp36 that are responsible for tumor suppression. The availability of cells in which the invasive phenotype can be suppressed by a growth inhibitor, msp36, provides an opportunity to dissect effectors that may be specific and those that may be common factors in anti- proliferation, anti-invasion and anti-angiogenesis. Accurately defining the sets of genes involved in these processes and recovering the corresponding transcripts increase our understanding. Gene array technology has recently been shown to be a powerful approach in defining the multiple proteins involved in different cell functions. We analyze the gene expression profiles of msp36-expressing breast cancer cells (MDAMB435+msp36) and compare them to control MDAMB435 cells using high-density cDNA filter arrays or DNA chip arrays. We believe that this approach prov ides important molecular insights into the genes responsible for the invasive and pro-angiogenic phenotypes of breast cancer cells. The relevance of such genes to breast cancer invasion, angiogenesis and metastasis is subsequently tested /; vitro and in vivo.

We initially compare hybridization signals obtained when high density filter arrays or DNA chips that are hybridized with cDNAs derived from MDAMB435 with/without msp36 (tetracycline-regulatable msp36 from Example 1). In our previous experience with cDNA filter assays for differential screening, we used the Atlas Array (Clontech), which contains - 1 ,500 known cDNA clones, and high density arrays of EST-cDNAs obtained from Research Genetics Inc., to identify a series of genes induced by the p53 tumor suppressor gene. The filters from Research Genetics seemed to give better results by Northern blotting. In addition, we use DNA chips for expression profiling (Affymetrix).

Candidate clones are confirmed by Northern blot analysis. Clones which are identified as responsive transcripts are further characterized by sequencing and Northern blot analysis to determine expression patterns in a series of normal human breast and cancer cells.

Probes are labeled by fluorescence for DNA chips or with radioactivity for DNA filter arrays. RNA is extracted from cell cultures, using the guanidinium isothiocyanate method. Fluorescently labeled cRNAs or radioactively labeled cDNAs are prepared from mRNA by oligo dT-primed polymerization using superscript II reverse transcriptase. The pool of nucleotides in the labeling reaction is 0.5 mM dGTP, dATP and dCTP and 0.2 mM dTTP. Fluorescent nucleotides, Rhodamine 1 10 dUTP (Perkin Elmer) or Cy3 dUTP (Amersham), are present at 0.1 mM in the fluorescent labeling reactions. Alternatively. "P dCTP is used for the filter arrays. The probes are purified by gel chromatography and ethanol precipitation. Prior to hybridization (at 62 C in a water-bath), the probes are boiled for 2-3 min. then allowed to cool to room temperature.

We verify the candidate clones by Northern blot analysis using RNA samples from msp36 induced or non-induced MDAMB435 cells as well as a number of different invasive and non-invasive breast cancer cells as well as primary normal mammarv epithelial cells. We sort out all positive clones and determine those known by homology and function by searching the GenBank data base (Blast program). Unknown clones are further sequenccd. ^"1 he full- length cDNA clones are isolated from commercially available or existing cDNA libraries. These experiments provide us with new insights in understanding molecular mechanisms of the evolution of breast cancer progression.

We investigate further candidate genes of interest that appear to have roles regulating cell proliferation, migration, angiogenesis and metastasis; these are transfected back into the original breast carcinoma cells in vectors that either overexpress copies of the genes or express antisense messages.

Example 3. To further characterize the role of msp36 in vivo in mammary tumorigenesis by generating msp36-transgenic mice with P VT (Polyoma virus middle T antigen) background.

We have presented evidence that msp36 is involved in breast cancer. Tissue culture models as well as the xenograft studies described above provide a powerful tool for elucidating the functions of this gene. Transgenic models for tumor initiation and progression offer the possibility of confirming the hypothesis that tumor evolution requires inactivation of the tumor suppression program induced by gene(s) of interest. Down-regulation of msp36 was a frequent occurrence in human breast cancer specimens as compared to normal breast tissue specimens. This finding suggests that altered expression of msp36 contributes to the malignant phenotype of breast cancer cells. Mouse strains with an oncogenic PyV transgene under the control of the MMTV promoter develop breast cancer (adenocarcinomas). Tumors were multifocal, highly fibrotic, and involved the entire mammary fat pad (Vomachka AJ. et al. Oncogene. 2000, 19: 1077-84: Guy CT, et al. Mol Cell Biol. 1992, 12:954-61). Pulmonary metastases were observed in 80-94% of tumor-bearing lemale mice. We believe that mammary-specific msp36 overexpression delays mammarv' tumor development in response to "polyoma virus middle T antigen" oncogene. We further characterization the effect of msp36 overexpression in vivo by targeting its expression to mammary tissue using transgenic technology. This set of studies is used to assess the extent to which anti-tumor growth and/or the anti-angiogenic effects of msp36 overexpression predominate in the context of an in vivo model. The onset of primary mammary tumors and angiogenesis is monitored by gross and microscopic analysis and by histologic evaluation using histology, in situ hybridization and immunohistochemistry.

Our strategy involves generation of constructs containing the full-length msp36 cDNA fused to Flag epitope tag linked by an internal-ribosomal-entry site (IRES site) to generate a bi-cistronic transcript carrying GFP to track transgene expression using a hormone-regulated promoter, the MMTV promoter. We initially test the effects of hydrocortisone-regulated expression in tissue culture following transfection into NIH3T3 cells. DNA preparation involves plasmid isolation using the Qiagen plasmid maxi kit. Following restriction endonuclease digestion to liberate the DNA construct for oocycte injection, samples are run on an agarose gel, and the appropriate band excised and then the DNA is purified using the Qiagex II gel extraction kit. Transgenic mice are produced, for example, by micro-injection of the DNA fragments carrying the msp36 expression unit into pronuclear stage zygotes of the FVM mouse strain, reimplanting the resulting embryos, and maintaining msp36-transgenic mouse lines. After injection and reimplantation. pregnant females are housed and monitored closely. Founder mice are analyzed by PCR and Southern blot hybridization with tail-derived DNA. msp36 primers are used for the PCR reactions. To obtain stable lines, mice are crossed and litters evaluated by tail blot. Prior to expanding colonies, animals are checked to determine if the gene is expressed in the expected target tissues, e.g., mammary glands.

Transgene expression initially is assessed by analysis of target tissues for GFP expression, a rapid screen. msp36 transgene expression then is assessed by RT-PCR and Northern blot analysis. Positive tissues are analyzed using anti-Flag antibody and confirmed by msp36-specifιc polyclonal antibodies using immunohistochemistry. When expression is demonstrable, histological histology analysis of mammary gland and other tissues is performed at serial time points to assess abnormalities in development or differentiation. Tissues are obtained from the mammary gland and other tissues and fixed in ZX-fiix neutral buffered formalin or Bouin^'s fixative (sigma). Paraffin sections are stained for histological analysis. For in vivo cell cycle analysis, mice receive an intraperitoneal injection of BrdU (BMB) at 120 ug/kg body weight 1 - 1.5 hour before being sacrificed in order to label cells in S phase. For BrdU. and PCNA staining, each section is placed on ProbeOn slides (Fisher). Sections are incubated with an antibody to BrdU or PCNA. The primary antibodies are detected using the Vectastatin ABC immunoperoxidase system. The next phase involves breeding of msp36 transgenic mice into a transgenic strain with an oncogene Py VT (Polyoma virus middle T antigen). These onco-mice are commercially available (Jackson Labs) and have been developed in the FVM mouse strain (Vomachka AJ, et al. Oncogene. 2000, 19: 1077-84; Guy CT, et al. Mol Cell Biol. 1992, 12:954-61). This transgene is uniformly expressed in mammary tissue and induced the formation of multi-focal tumors in the mammary epithelium by -100 days postpartum. In addition to mammary tissue, Polyoma virus middle T antigen transcripts were also detected in the Harderian-lacrimal gland and to lesser extent in lung and salivary glands, suggesting that the MMTV promoter is transcriptionally active in these tissues (Vomachka AJ, et al. Oncogene. 2000, 19: 1077-84; Guy CT. et al. Mol Cell Biol. 1992, 12:954-61). We believe that exogenous msp36 expression provides an additional barrier to the development of mammary tumors by oncogene. If tumor incidence is in fact reduced, we analyze mammary tissues as described above, including angiogenesis assays, and also observe evidence of decreased S- phase or increased apoptotic cells as compared to the oncogene transgene alone. Msp36 transgenic mice (wild-type FVM) and msp36/A' VT crossed mice are closely monitored for lifespan and we determine tumor incidence with age. Tumor tissue specimens are dissected for histology and immunohistochemistry. The generation of transgenic mice provides a genetic model to further characterize the role of msp36 in the course of mammary carcinogenesis with/without mammary tumor-promoting oncogenes. We characterize the effect of msp/FGF-BP overexpression in vivo in mammary tissue in ErbB2 or Ras overexpressing mammary tumor mice, genes which are known to play important roles in mammary tumorigcnesis. This set of studies allows us to determine the extent to which anti-tumor growth and/or anti-angiogenic effects of msp36 overexpression predominate in the context of an /^"// vivo mammary tumor model. We have found that expression of msp36/FGF-BP in human microvascular endothelial cells resulted in decreased cell proliferation measured by BrdU proliferation and growth rate assays. Using the above-described experimental design, we also have observed that exogenouse expression of msp36 in MCF7 cells modulated cell migration when fibronectin and collagens were used as matrix molecules. In addition, adhesion assays showed that msp 6 expression increased cell-extracellular matrix interations. Exogenous expresion of msp36 reduced FGF-mediated FGFR (FLG) activation, suggesting that msp36- induced inhibition of MAPK resulted from down-regulation of FGF-induced FGFR activation. We also found that msp36/FGF-BP modulated the activation of a lipid-anchored docking protein, FRS2, that targets signaling molecules to the plasma membrane in response to FGF stimulation. msp36 expression in Saos2, osteosarcoma cells, induced a significant rise of p21/Wafl protein, which is a potent cell cycle inhibitor, consistent to induction of p21 in breast cancer cells by msp36. The data demonstrate that the p21 increase is probably responsible for msp36 mediated tumor cell growth inhibition.

In summary, these studies provide definitive in vivo confirmation of our results obtained in cellular assays and nude mice, and are used to further characterize the sequential changes that occur during mammary epithelial carcinoma progression.

All of the references, patents and patent publications identified or cited herein are incorporated, in their entirety, by reference.

Although this invention has been described with respect to specific embodiments, the details of these embodiments are not to be construed as limitations. Various equivalents, changes and modifications may be made without departing from the spirit and scope of this invention, and it is understood that such equivalent embodiments are part of this invention.

We claim:

Claims

CLA1MS

1. A method for treating a subject having or at risk of developing a cancer, comprising

5 administering to a subject in need of such treatment and free of indications otherwise calling for treatment with an msp36 molecule, an msp36 molecule in an amount effective to treat the cancer.

2. The method of claim 1 , wherein the cancer comprises a breast cancer. 10

3. The method of claim 1 , wherein the cancer comprises a prostate cancer.

4. The method of claim 1 , wherein the msp36 molecule is administered in conjunction with an agent for treating or preventing the cancer.

15

5. The method of claim 4, wherein the agent for treating or preventing a cancer is a chemotherapeutic agent or radiation.

6. The method of claim 1 , wherein the msp36 molecule is an msp36 nucleic 20 acid.

7. The method of claim 1 , wherein the msp36 molecule is an msp36 protein.

8. A method for treating a subject having or at risk of developing breast 25 cancer, comprising administering to a subject in need of such treatment and free of indications otherwise calling for treatment with msp36, msp36 in an amount effective to treat the breast cancer.

_> 0 9. 1 he method of claim 8, wherein the msp36 is administered in conjunction with an agent for treating or preventing the breast cancer.

10. The method of claim 9, wherein the agent for treating or preventing a cancer is a chemotherapeutic agent or radiation.

1 1. A method of determining, by in situ hybridization, whether a sample containing a test cell from human breast tissue is (a) normal, or (b) cancerous, said method comprising the steps of: contacting the mRNA of said test cell in situ with a nucleic acid probe comprising a nucleotide sequence at least 15 nucleotides in length which is complementary to a portion of the coding sequence of msp36; and determining whether said probe hybridizes to said mRNA under high stringency conditions selected to permit hybridization of said probe to normal cells of human breast tissue, wherein the failure to hybridize to said mRNA is an indication that said test cell is cancerous.

12. A method of confirming, by in situ hybridization, a diagnosis that a given fraction of cells in a human breast tissue sample are cancerous, said method comprising the steps of: contacting the mRNA of said sample in situ with a nucleic acid probe comprising a nucleotide sequence at least nucleotides in length which is complementary to a portion of the coding sequence of a msp36 gene, said gene being one which is expressed in normal cells of human breast tissue at a given control level; and determining whether the fraction of cells in said sample which, under high stringency hybridization conditions selected to permit hybridization to normal cells of human breast tissue, fail to exhibit detectable hybridization to said probe is approximately equivalent to said given fraction of cells previously diagnosed as being cancerous, said equivalence of fractions being a confirmation of said diagnosis.

13. A method for determining the presence of cancerous cells in the breast tissue of a patient, which method comprises the steps of: providing a nucleic acid probe comprising a nucleotide sequence at least 15 nucleotides in length which is complementary to a portion of the coding sequence of a msp36 gene; obtaining from a patient a first sample of breast tissue, said first sample potentially comprising cancerous cells; providing a second sample of breast tissue, substantially all of the cells of said second sample being non-cancerous; contacting in situ said nucleic acid probe under stringent hybridizing conditions with RNA of each of said first and second tissue samples, said hybridization conditions being selected to permit hybridization of said probe to non-cancerous cells of breast tissue; and comparing (a) the in situ hybridization of said nucleic acid probe w^rith said first tissue sample, with (b) the in situ hybridization of said nucleic acid probe with said second tissue sample, wherein hybridization with said second tissue sample but not with said first tissue sample indicates the presence of cancerous cells in said first tissue sample.

14. A method of determining, by in situ hybridization, whether a sample containing a test cell from human breast tissue is (a) normal, or (b) cancerous, said method comprising the steps of: contacting the mRNA of said test cell in situ with a nucleic acid probe comprising a nucleotide sequence at least 15 nucleotides in length which is complementary to a portion of the coding sequence of a msp36 gene, said gene being one which is normally expressed in normal cells of human breast tissue; and determining whether said probe hybridizes to said mRNA under high stringency conditions selected to permit hybridization to normal cells of human breast tissue, wherein the failure to hybridize to said mRNA is an indication that said test cell is cancerous.

15. A method of confirming, by in situ hybridization, a diagnosis that a given fraction of cells in a human breast tissue sample are cancerous, said method comprising the steps of: contacting the mRNA of said sample in situ with a nucleic acid probe comprising a nucleotide sequence at least 15 nucleotides in length which is complementary to a portion of the coding sequence of a msp36, said gene being one which is expressed in normal cells of human breast tissue at a given control level; and determining whether the fraction of cells in said sample which, under high stringency hybridization conditions selected to permit hybridization to normal cells of human breast tissue, fail to exhibit detectable hybridization to said probe is approximately equivalent to said given fraction of cells previously diagnosed as being cancerous, said equivalence of fractions being a confirmation of said diagnosis.

16. A method of determining, by in situ hybridization, whether a sample containing a test cell from human tissue is (a) normal, or (b) cancerous, said method comprising the steps of: contacting the mRNA of said test cell in situ with a nucleic acid probe comprising a nucleotide sequence at least 15 nucleotides in length which is complementary to a portion of the coding sequence of msp36; and determining whether said probe hybridizes to said mRNA under high stringency conditions selected to permit hybridization of said probe to normal cells of the same human tissue as the test cell, wherein the failure to hybridize to said mRNA is an indication that said test cell is cancerous.