WO1997031653A1

WO1997031653A1 - Diagnosis and treatment of metastic breast carcinoma

Info

Publication number: WO1997031653A1
Application number: PCT/US1996/013691
Authority: WO
Inventors: Art Mercurio; Leslie Shaw; Ulla M. Wewer
Original assignee: Beth Israel Deaconess Medical Center
Priority date: 1996-03-01
Filing date: 1996-08-23
Publication date: 1997-09-04

Abstract

The invention provides a method of detecting an increased likelihood of metastic breast cancer by detecting the levels of α6β1 integrin. The invention also provides methods for the prophylaxis or treatment of metastic breast cancer by blocking α6β1 function.

Description

DIAGNOSIS AND TREATMENT OF METASTIC BREAST CARCINOMA The field of the invention is cancer diagnosis and therapy.

Summary of the Invention We have discovered that a6βl integrin plays an essential role in breast cancer metastisis and that methods of blocking α601 function block the metastatic process. The presence of α6£l allows me astis; blocking of c.6/31 function prevents metastisis. Accordingly, the invention provides methods of predicting the likelihood of metastic breast carcinom and methods for predicting the prognosis for patients diagnosed with breast carcinoma. The invention also provides methods for preventing and treating breast carcinoma by blocking a6βl function.

In general, the invention features a method of diagnosing a mammal for the presence of a metastatic breast cancer or an increased likelihood of developing metastatic breast cancer. The method involves measuring α6/3l integrin in a breast tissue sample from the mammal, with an increase in α6 l levels relative to a sample from an unaffected individual being an indication that the mammal has a malignancy or has an increased likelihood of developing a malignancy.

In preferred embodiments, the increase in α6/3l integrin levels is two-fold or greater.

Kits for carrying out the above methods are also included in the invention. Such kits preferably include a substantially pure antibody that specifically recognizes and binds a α601 polypeptide, and may also include means for detecting and guantitating antibody binding. Alternatively, the kit may include all or a fragment of a α63l nucleic acid sequence useful for hybridization purposes, and may also include means for detecting and quantitating α6j91 RNA hybridization.

The invention also features a method of treating a mammal with a α60l-associated malignancy involving administering to the mammal a α631 antibody in an amount sufficient to inhibit growth of the malignancy, and further features a therapeutic composition having as an active ingredient a o6/31 antibody, formulated in a physiologically-acceptable carrier. Preferably, the antibody is directed to the extracellular domain of α6/ϊl integrin.

In two other related aspects, the invention features methods of identifying α631 inhibitory compounds. The first method involves the identification of inhibitory compounds that are capable of decreasing the expression of a α631 gene, involving (a) providing a cell expressing the α601 gene; and (b) contacting the cell with a candidate compound, an increase in α601 expression following contact with the candidate compound identifying a modulatory compound. The second method involves the identification of modulatory compounds which are capable of decreasing α631 function, involving (a) providing a cell expressing the a6βl protease; and (b) contacting the cell with a candidate compound, an decrease in otββl activity following contact with the candidate compound identifying a modulatory compound. In a related aspect, the invention features a method of treating a mammal with a disease involving increased presence of a βjSl, involving administering to the patient a modulatory compound (for example, identified according to the above methods) in an amount effective to reduce the symptoms of the disease in the mammal.

By "α601 polypeptide" is meant an amino acid sequence which is an integrin laminim receptor as described, for example, in Mercurio, Trends Cell Biol. 5:419-423, 1993, whose expression correlates with the presence of malignant breast carcinoma. Preferably, such a polypeptide has an amino acid sequence which is at least 45%, preferably 60%, and most preferably 85% or even 95% identical to the amino acid sequence referenced in Mercurio (1993) , supra.

By a "substantially identical" polypeptide sequence is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, e.g. , as described herein) .

Preferably, such a sequence is at least 85%, more preferably 90%, and most preferably 95% identical at the amino acid level to the sequence of Fig. 10 (SEQ ID NO: 1) . For polypeptides, the length of comparison sequences will generally be at least 15 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. Homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) . Such software matches similar sequences by assigning degrees of ho ology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By "protein" or "polypeptide" is meant any chain of amino acids, regardless of length or post- translational modification (e.g., glycosylation or phosphorylation) .

By "substantially pure" is meant a preparation which is at least 60% by weight (dry weight) the compound of interest, e.g., the α6,91 polypeptide or α6,91-specific antibody. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate method, e.g. , column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

By "purified DNA" is meant DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally-occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plas id or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. By a "substantially identical" nucleic acid is meant a nucleic acid sequence which encodes a polypeptide differing only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g. , valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, e.g., as described herein) . Preferably, the encoded sequence is at least 45%, more preferably 60%, and most preferably 85% identical at the amino acid level to the sequence of Fig. 10 (SEQ ID NO: 1) . If nucleic acid sequences are compared a "substantially identical" nucleic acid sequence is one which is at least 85%, more preferably 90%, and most preferably 95% identical to the sequence of Fig. 11 (SEQ ID NO: 2) . The length of nucleic acid sequence comparison will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides. Again, homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) . By "purified antibody" is meant antibody which is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, antibody.

By "specifically binds" is meant an antibody which recognizes and binds a α6/3l polypeptide but which does not substantially recognize and bind other molecules in a sample (e.g., a biological sample) which naturally includes α691 polypeptide. An antibody which

"specifically binds" α69l is sufficient to detect a α6,91 protein product in such a biological sample using one or more of the standard immunological techniques available to those in the art (for example, Western blotting or immunoprecipitation) . By "malignancy" is meant any abnormal tissue that grows by cellular proliferation more rapidly than normal or that continues to grow after growth stimuli cease.

Most malignancies show partial or complete lack of structural organization or functional coordination with surrounding normal tissue. A malignancy according to the invention is generally either locally invasive or metastatic.

By "relative to a wild-type sample" is meant either (a) relative to an equivalent tissue sample from an unaffected individual or (b) relative to an unaffected sample of similar tissue type from the mammal being diagnosed.

By "immunological methods" is meant any assay involving antibody-based detection techniques including, without limitation, Western blotting, immunoprecipitation, and direct and competitive ELISA and

RIA techniques.

By "means for detecting" is meant any one or a series of components that sufficiently indicate a detection event of interest. Such means involve at least one label that may be assayed or observed, including, without limitation, radioactive, fluorescent, and chemiluminescent labels. By "c.6,91 RNA" is meant messenger RNA transcribed from a α6,9l DNA sequence.

By "hybridization techniques" is meant any detection assay involving specific interactions (based on complementarity) between nucleic acid strands, including DNA-DNA, RNA-RNA, and DNA-RNA interactions. Such hybridization techniques may, if desired, include a PCR amplification step.

By a "modulatory compound", as used herein, is meant any compound capable of either decreasing α6/31 expression (i.e., at the level of transcription. translation, or post-translation) or decreasing α6Sl protein activity (i.e., the amount of activity per unit of C-6/31 protein) .

By a "differentiation agent" is meant any compound which, when added to cells An vitro or introduced into a mammal, result in a change in the phenotype of a cell or tissue, including the expression of one or more markers indicative of a particular stage in the cell's or tissue's life cycle. Differentiation agents include, without limitation, retinoic acid and cyclic AMP.

Detailed Description of the Drawings Fig. 1 is a graph of surface expression of integrin subunits on MDA MB-435 cells. MDA MB-435 cells were analyzed by flow cytometry using MAbs specific for the indicated integrin subunits. The left scan in each profile was obtained using a nonspecific mouse IgG, and the right scan corresponds to the expression levels of the specified integrin subunits.

Figs. 2A and 2B are graphs of mAb inhibition of MDA MD-435 adhesion and migration on laminin and collagen I. 2A shows adhesion to tissue culture wells were coated with laminin (20 μg/ml) or collagen 1 (20 μg/ml) . MDS MB-435 cells were preincubated with antibodies to specific integrin subunits before addition to the protein-coated wells. After a 30 min. incubation at 37°C, nonadherent cells were removed by washing, and adherent cells were fixed, stained, and quantitated as described below. The data shown are the mean values +SEM (bars) of three experiments done in duplicate. Fig. 2B shows migration data for MDA MB-435 cells preincubated with antibodies to specific integrin subunits before addition to Transwell chambers. Media containing either laminin (15 μg/ml) or collagen I (μg/ml) were added to the bottom wells. After a 4 hr. incubation at 37^βC, the cells that had not migrated were removed, and the cells that had migrated onto the lower surface of the filter were fixed, stained, and quantitated. The data shown are the mean values +SEM (bars) from two experiments done in duplicate, laminin; collagen I.

Figs. 3A-3C depict expression of the truncated B4- ΔCYT integrin subunits in MDA MB-435 cells. Fig. 3A is a schematic demonstration of the strategy used to knock out α6,Sl. This strategy is based on the finding that α6 associates preferentially with ,94 in comparison with ,31. Thus, expression of the /94-ΔCYT subunit will result in formation of the α6,94-ΔCYT heterodimer at the expense of c.6/31 association. Because of its cytoplasmic domain deletion, the 6/34-ΔCYT integrin should be a null receptor unable to mediate laminin interactions.

Presumably, the excess βl that arises from this process can associate with α subunits. Fig. 3B show profiles obtained when a population of transfected MDA MB- 35 cells expressing ,94-ΔCYT on the cell surface were isolated by FACS using UM-A9, a mAb specific for the βl integrin subunit. The resulting cells were analysed by flow cytometry. The left scan in each profile was obtained using a nonspecific mouse IgG, and the right scan corresponds to the expression levels of the specified integrin subunits, Mock, MDA MB-435 cells transfected with the vector alone: ,54-ΔCYT, MDA MB-435 cells transfected with the 04-ΔCYT cDNA. Fig. 3C shows immunoprecipitation of transfected cells. The cells were surface labeled with biotin, and aliquots of detergent extracts from equal numbers of cells were immunoprecipitated with either the 2B7 or UM-A9 Abs. Immunoprecipicies were resolved by 8% SDS-PAGE under reducing conditions and transferred to nitrocellulose filters. Proteins were visualized with streptavidin conjugated to horseradish peroxidase and enhanced chemiluminescence. Lane l, mock transfectants immunoprecipitated with 2B7; Lane 2, 94-ΔCYT transfectants immunoprecipitated with UM-A9; Lane 3, mock transfectants immunoprecipitated sequentially with UM-A9 and then with 2B7; Lane 4, 34-ΔCYT transfectants immunoprecipitated sequentially with UM-A9 and then 2B7. The migration positions of the ,91, α6 and 94-ΔCYT subunits are shown in the left margin. The molecular mass markers are indicated in the right margin. Figs. 4A-C show functional assays using the 04- ΔCYT MDA MB-435 transfectants. Fig. 4A is a graph of adhesion of MDA MB-435 transfectants. Cells were assayed for their ability to adhere to laminine and collagen I subunits. Transfected cells (109) were added to laminin- coated (20 μg/ml) and collagen I-coated (20 μg/ml) wells and incubated for 30 min. at 37°C. Nonadherent cells were removed by washing, and subsequent cells were fixed, stained and quantitated. The data shown are the mean values +SEM (bars) of these experiments done in triplicate. Fig. 4B is a graph of migration of MDA MB- 435 transfectants. Cells were assayed for their ability to migrate toward laminin and collagen I. Media containing either laminin (15 μg/ml) or collagen I (15 μg/ml) were added to the bottom wells of Traswell chambers and the transfectants (10⁵) were added to the top wells. After a 4 hr. incubation at 37°C, the cells that had not migrated were removed, and the cells that had migrated onto the lower surface of the filter were fixed, stained and quantitated. The data shown are the mean values +SEM (bars) from two experiments done in duplicate. Fig. 4C is a graph of invasion by MDA MB-435 transfectants. Cells were assayed for their ability to invade Marrigel. Marrigel was diluted in cold distilled water, added to the upper well of Transwell chambers and dried under a sterile hood. The Marrigel was reconstructed with medium, and the transfectants (2 x 109) were added to each well. Conditioned NIH-3T3 medium was added to the bottom wells of the chambers. After 5-7 hr. at 37°C, the cells that had not invaded were removed, and the cells that had invaded to the lower surface of the filters were fixed, stained and quantitated as described in "Materials and Methods".

Detailed Description Introduction The involvement of the α601 integrin in breast carcinoma progression has been the focus of many recent studies. However, a survey of literature in this area reveals that the potential involvement of this laminin receptor in breast carcinoma remains the subject of considerable debate. Several immunohistochemical studies have concluded that the expression of α601 is diminished in primary breast carcinomas. Such studies, although potentially interesting, are limited in their significance by a number of factors. For example, a common observation is that in normal mammary glands, the cells with the strongest α6 immunoreactivity are myoepithelial cells and not the ductal epithelial and acinar cells that give rise to the majority of breast cancers. Thus, the conclusion that the ct6 integrin is diminished in breast cancers may be misleading, because it is based on comparing the staining intensity of diverse populations of cells. A second factor is that the pathological specimens used in such studies are often chosen at random, with no reference to either disease stage or other clinical parameters.

The above observations emphasize the need for functional studies on the role of the o601 integrin in breast carcinoma progression. To address this issue, we have assessed the expression and function of this integrin in human breast carcinoma. Here we provide data showing that a human breast carcinoma cell line, MDA-MB- 435, known to be highly invasive and metastatic (Price et al.. Cancer Res . , 50:717-721, 1990), expresses three potent integrin laminin receptors: 201, α301, and α601 (Mercurio, Trends Cell Biol . 5:419-423, 1993). Antibody inhibition studies, however, revealed that these cells use only α601 to mediate adhesion and migration on laminin matrices. To investigate further whether α601 may be involved in the aggressive behavior of MDA-MB-435 cells, we developed a dominant-negative strategy for knocking out α601 function in these cells. This strategy involves expression of a cytoplasmic domain deletion mutant of the 04 integrin subunit by cDNA transfection. Stable transfectants of MDA-MB-435 cells that expressed this mutant 04 subunit were inhibited dramatically in their ability to adhere and migrate on laminin matrices. In addition, the capacity of these cells to invade through a basement membrane matrix was also reduced significantly. Because in vitro invasive properties correlate strongly with in vivo behavior of carcinoma cell lines (Albini et al., Cancer Res . 47:3239-3245, 1987) , these findings provide functional data to support the idea that the o601 integrin is important for breast cancer progression. Moreover, this dominant-negative approach is a powerful method that should be useful in assessing the role of the 601 integrin in other cell types.

In addition to our in vitro results, we have used the MDA-MB-435 cell line expressing the cytoplasmic domain deletion mutant of the 04 integrin subunit to demonstrate loss of metastic potential in vivo.

Our results indicate that α604 integrin can be used as a diagnostic and prognostic antibody. In addition, therapies which eliminate 601 from the cell surface or block 601 function may be used to slow, halt, or prevent metastisis of breast cancer. The following examples are provided to illustrate the invention. They are not intended to limit the scope of the invention. Anti-α6?l Antibodies

To generate α601-specific antibodies, a 601 coding sequence (i.e., the whole protein or the ctyoplasmic region may be expressed as a C-terminal fusion with glutathione S-transferase (GST) (Smith et al., Gene 67:31-40, 1988). The fusion protein may be purified on glutathione-Sepharose beads, eluted with glutathione cleaved with thrombin (at the engineered cleavage site) , and purified to the degree necessary for immunization of rabbits. Primary immunizations may be carried out with Freund's complete adjuvant and subsequent immunizations with Freund's incomplete adjuvant. Antibody titres are monitored by Western blot and immunoprecipitation analyses using the thrombin- cleaved α601 protein fragment of the GST-α601 fusion protein. Immune sera may be affinity purified using CNBr-Sepharose-coupled α601 protein. An iserum specificity is determined using a panel of unrelated GST proteins (e.g., GSTp53, Rb, HPV-16 E6, and E6-AP) and GST-trypsin (which may be generated by PCR using known sequences) .

As an alternate or adjunct immunogen to GST fusion proteins, peptides corresponding to relatively unique hydrophilic regions of α601 may be generated and coupled to keyhole limpet hemocyanin (KLH) through an introduced c-terminal lysine. Antiserum to each of these peptides is similarly affinity purified on peptides conjugated to BSA, and specificity tested in ELISA and Western blots using peptide conjugates, and by Western blot and immunoprecipitation using 601 expressed as a GST fusion protein. Alternatively, monoclonal antibodies may be prepared using the α601 proteins described above and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al.. In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981; Ausubel et al., supra) . Once produced, monoclonal antibodies are also tested for specific o601 recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra) . Antibodies which specifically recognize α601 are considered to be useful in the invention; such antibodies may be used, e.g., in an immunoassay to monitor the level of 601 produced by a mammal (for example, to determine the amount or location of α601) .

Preferably, antibodies of the invention are produced using fragments of the α601 protein which lie outside highly conserved regions and appear likely to be antigenic, by criteria such as high frequency of charged residues. In one specific example, such fragments are generated by standard techniques of PCR and cloned into the pGEX expression vector (Ausubel et al., supraI . Fusion proteins are expressed in E___ coli and purified using a glutathione agarose affinity matrix as described in Ausubel et al. (supra) . To attempt to minimize the potential problems of low affinity or specificity of antisera, two or three such fusions are generated for each protein, and each fusion is injected into at least two rabbits. Antisera are raised by injections in a series, preferably including at least three booster injections. Identification and Administration of Molecules that Modulate a6fll Protein Expression

Use of the 601 cDNA facilitates the identification of molecules which increase or decrease α601 expression. According to one approach, candidate molecules are added at varying concentrations to the culture medium of cells expressing 601. α601 expression is then measured, for example, by standard Northern blot analysis (Ausubel et al. , supra) using a α60l cDNA (or cDNA fragment) as a hybridization probe. The level of α601 expression in the presence of the candidate molecule is compared to the level measured for the same cells in the same culture medium but in the absence of the candidate molecule. If desired, the effect of candidate modulators on expression may, in the alternative, be measured at the level of α601 protein production using the same general approach and standard immunological detection techniques, such as Western blotting or immunoprecipitation with a o601-specific antibody (for example, the α601 antibody described herein) .

Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells; Ausubel et al., su^pra) . In a mixed compound assay, α60l expression is tested against progressively smaller subsets of the candidate compound pool (e.g., produced by standard purification techniques, e.g., HPLC or FPLC) until a single compound or minimal compound mixture is demonstrated to modulate 601 expression.

Candidate o601 modulators include peptide as well as non-peptide molecules (e.g., peptide or non-peptide molecules found, e.g., in a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured) .

A molecule which promotes a decrease in α601 expression or α601 activity is considered particularly useful in the invention; such a molecule may be used, for example, as a therapeutic to dencrease cellular levels of cc601.

Modulators found to be effective at the level of α601 expression or activity may be confirmed as useful in animal models and, if successful, may be used as anti- cancer therapeutics.

A o601 modulator may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer 601 to patients suffering from or presymptomatic for a o601- associated carcinoma. Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital , ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for o601 modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, i plantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

If desired, treatment with a 601 modulatory compound may be combined with more traditional cancer therapies such as surgery, radiation, or chemotherapy. Detection of A Malignant Condition

C.601 polypeptides and nucleic acid sequences find diagnostic use in the detection or monitoring of breast carcinoma. In particular, because α601 is involved in the control of cell division and because the absence of 601 correlates with the development of carcinomas in humans, a increase in the level of α601 production provides an indication of a malignant or pre- alignant condition. Levels of 601 expression may be assayed by any standard technique. For example, its expression in a biological sample (e.g., a biopsy) may be monitored by standard Western blot analysis or may be aided by PCR (see, e.g., Ausubel et al., supra; PCR Technology: Principles and Applications for DNA Amplification, ed. , H.A. Ehrlich, Stockton Press, NY; and Yap and McGee, Nucl. Acids. Res. 19:4294, 1991).

In yet another approach, immunoassays are used to detect or monitor α601 protein in a biological sample. α601-specific polyclonal or monoclonal antibodies (produced as described above) may be used in any standard immunoassay format (e.g. , ELISA, Western blot, or RIA assay) to measure α601 polypeptide levels; again comparison is to wild-type α601 levels, and a decrease in α601 production is indicative of a malignant condition. Examples of immunoassays are described, e.g., in Ausubel et al., supra. Immunohistochemical techniques may also be utilized for α601 detection. For example, a tissue sample may be obtained from a patient, and a section stained for the presence of 601 using an anti-o601 antibody and any standard detection system (e.g., one which includes a secondary antibody conjugated to horseradish peroxidase) . General guidance regarding such techniques can be found in, e.g., Bancroft and Stevens (Theory and Practice of Histological Technicrues. Churchill Livingstone, 1982) and Ausubel et al. (supra) . α6 1 Therapy

The methods of the instant invention may be used to reduce or diagnose the disorders described herein in any mammal, for example, humans, domestic pets, or livestock. Where a non-human mammal is treated or diagnosed, the α60l polypeptide, nucleic acid, or antibody employed is preferably specific for that species.

Examples I. Materials and Methods

Cells and Antibodies . The MDA-MB-435 breast carcinoma cell line was obtained from the Lombardi Breast Cancer Depository (Georgetown University) . The cells were grown in DMEM (GIBCO) supplemented with 10% FCS (GIBCO) and 1% penicillin-streptomycin (GIBCO) . The antibodies specific for integrin subunits were as follows: mAb³ 13 (01); UM-A9 (04); IIE10 (α2) ; IVA5 (α3) ; and 2B7 (α6) (Shaw et al., J. Biol . Chem . 268:11401-11408, 1993); and Mouse IgG was obtained from Sigma Chemical Co.

Trans fections . The construction of the 04 cytoplasmic deletion mutant cDNA and its insertion into the pcDNA3 eukaryotic expression vector have been described previously (Clarke et al., J. Biol . Chem. 270:22673-22676, 1995). The pcDNA3 vector containing the mutant 04 cDNA, as well as the vector alone, were transfected into the MDA-MB-435 cell line using lipofectin (GIBCO) according to the instructions of the manufacturer. Neomycin-resistant cells were isolated by selective growth in medium containing G418 (0.6 mg/ml; GIBCO) . The stable transfectants were pooled, and a population of cells that expressed the human 04 subunit on the cell surface was isolated by FACS. A human 04 integrin-specific mAB, UM-A9, was listed for this sorting and for subsequent analysis of the transfectants (Clarke et al., J. Biol . Chem . 270:22673-22676, 1995). The sorting was repeated sequentially to enrich for homogeneous populations of cells expressing high levels of the transfected 04-ΔCYT subunit on the cell surface.

Flow Cymmetry. Transfected MDA-MB-435 cells were washed twice with RPMI 1640 media containing 25 mm HEPES (RPMI-H) and 0.2% BSA (RH/BSA). Aliqouts of cells (5 X 10^s) were incubated for 45-60 min at room temperature with RH/BSA containing UM-A9 (3 μg/ml) . The cells were washed two times with RH/BSA and then incubated with goat F(ab')₂ antimouse IgG coupled to FITC (Iago) for 45-60 min at room temperature. After washing two times with RH/BSA, the cells were resuspended in the same buffer and analyzed using a FACScm (Becton Dickinson) .

Functional Assays . Adhesion assays were performed as described previously (Shaw et al., J. Biol . Chem . 268:11401-11408, 1993). Briefly, multiwell tissue culture plates (11.3-mm diameter) were coated overnight at 4°C with 0.2 ml PBS (20 μg/ml) containing either murine Engelbreth-Holm-Swain laminin or rat collagen I. The wells were then washed with PBS, and 10⁵ cells in RPMI-H containing 1% DSA were added. After a 30-min incubation at 37°C, the wells were washed three times with RPMI-H at 37^βC, fixed for 15 min with methanol and stained with a 0.2% solution of crystal violet in 2% ethanol. The crystal violet strain was solubilized with a 1% solution of SDS, and adhesion was quantitated by measuring the A at 600 run. To examine inhibition of adhesion, cells were preincubated for 30 min at room temperature in the presence of antibodies before addition to the assay wells.

Cell migration assays were performed using 6.5-mm Transwell chambers (8-μm pore size; Costar) as described previously (Shaw and Mercurio, Mol . Biol . Cell 5:679-690, 1994). Briefly, RPMI-H containing 15 μg/ml laminin (0.6 ml) was added to the bottom well, and the filters were coated for -30 min at 37°C. Cells were resuspended in the appropriate buffer at a concentration of 10⁵/ml, and 10⁵ cells were added to the top well of the Transwell chambers. After a 4-h incubation, the cells that had not migrated were removed from the upper face of the filters using cotton swabs, and the cells that had migrated to the lower surface of the filters were fixed in methanol and stained with a 0.2% solution of crystal violet in 2% ethanol. Migration was quantitated by counting using bright-field optics with a Nikon Diaphot microscope equipped with a 16-square reticle. The surface area of this grid was determined to be 1 mm². Five separate fields were counted for each filter. To examiner the inhibition of migration, cells were preincubated for 30 min at room temperature in the presence of antibodies before addition to the assay wells. - 20 -

Matrigel invasion assays were performed as described (Albini et al.. Cancer Res . 47:3239-3245, 1987) using 6.5-mm Transwell chambers (8-μm pore size). Matrigel, purified from the Engelbreth-Holm-Swam tumor (Kleinman et al., Biochemistry 25:312-318, 1986), was diluted in cold distilled water, added to the Transwells (6.25-12.5 μg/well) , and dried under a sterile hood. The Marigel was then reconstituted with medium for 2 h at 37°C before the addition of cells. Cells were resuspended in serum-free medium containing 0.1% BSA, and 2 x 10⁵ cells were added to each well. Conditioned NIH- 373 medium was added to the bottom wells of the chambers. After 5-7 h, the cells that had not invaded were removed from the upper face of the filters using cotton swabs, and the cells that had invaded to the lower surface of the filters were fixed in methanol and stained with a 0.2% solution of crystal violet in 2% ethanol. Invasion was quantitated as described above for migration assays. Surface Labeling and Immunoprecipitation . Cells were trypsinized from their dishes and washed twice with PBS. After washing, the cells were resuspended in the same buffer at a concentration of 5 x 10⁵ cells/ml, NHS- LC-biotin (Pierce Chemical Co., Rockville, IL) was resuspended in DMSO and added to the cells at a concentration of 0.1 mg/ml. Cells were incubated in the presence of biotin for 1 hour at room temperature with gentle shaking. Subsequently, the cells were washed several times with PBS containing 50 mM NH₄C1 to remove unincorporated biotin. Surface biotinylated cells were solubilized at 4°C for 15 min in 10 mM Tris buffer (pH 8) containing 0.15 M NaCl, 1% Triton-X-100, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin and pepstatin, and 50 μg/ml leupeptin. Nuclei were removed by centrifugation at 12.000 x g for 10 min. Aliquots of labeled cell extracts from an equivalent number of cells - 21 - were incubated for 1 h at 4°C with α6-or 04-specific antibodies. Immune complexes were recovered with protein G agarose (Pharmacia) . The agarose beads were added for 1 h at 4°C with constant agitation. For immunodepletion experiments, the cell extracts were immunoprecipitated initially with 04-specific antibodies, and the supernatant was immunoprecipitated subsequently with α6- specific antibodies. The beads were washed two times with 50 mM Tris buffer (pH 7.5) containing 0.15M NaCl and 0.1% Triton X-100. two times with the same buffer containing 0.5 M NaCl, and one time with 0.05 M Tris (pH 6.8). Laemmeli sample buffer was added to the samples, which were then incubated at 100°C for 4 min. Surface biotinylated immunoprecipitates were resolved by SDS-Page (8%) and transferred to ainocellulose filters. The filters were blocked for 30 min. using 50 mM Tris buffer (pH 7.5) containing 0.5 M MaCl, 0.1% Tween 20, and 5% (w/v) Carnation dry milk. The filters were incubated for 1 h in the same buffer containing streptavidin conjugated to horseradish peroxidase (2 μg/ml; Pierce) . After three 10-min washes in blocking buffer lacking dry milt, protein was detected by enhanced chemiluminescence (Amersham) . II. Integrin Laminin Receptor Expression and Usage in MDA-MB 435 Cells.

MDA-MB-435 cells were analyzed by flow cytometry to determine the cell surface expression of integrin laminin receptor subunits. As shown in Fig. 1, these cells express the α2, α3, α6, and 01 subunits, but they lack expression of the 04 subunit. These data indicate that three possible integrin laminin receptors are expressed on the cell surface by this cell line: α201, C.301, and α601. However, expression of an integrin receptor on the cell surface does not always correlate with functional activity (Mercurio, Trends. Cell Biol. 5:419-423, 1995). To assess whether these receptors participated in mediating laminin interactions, adhesion assays were performed in the presence of inhibitory antibodies specific for each of the integrin subunits. Antibodies that recognize the 6 and 01 subunits inhibited adhesion to laminin almost completely (Fig. 2A) . In contrast, function-blocking antibodies specific for the α2 and α3 subunits had no inhibitory effect on laminin adhesion compared with the control mouse IgG antibody. The α2-specific MAb did inhibit adhesion to collagen I, however (Fig. 2A) .

One property considered important for metastatic cells is their ability to migrate on laminin matrices (?? et al., Amer. Rev. Biochem. 55:1037-1057, 1986). As shown in Fig. 2B, MDA-MB-435 cells exhibited significant migration toward a laminin gradient in a 4-h modified Boyden chamber assay. In fact, this rate of migration is considerably faster than the rates of many other carcinoma cell lines we have examined. Function-blocking experiments revealed that only 06- and 01-specific antibodies inhibited this migration on laminin (Fig. 2B) . Taken together, these data demonstrate that the α60i integrin is essential for mediating both the adhesion and migration of MDA-MB-435 cells on laminin. III. Use of a Dominant-Negative Transfection Strategy to Knock our αβøl Function in MDA-MB- 35 Cells.

To assess the contribution for the laminin receptor function of α601 to the behavior of MDA-MB-435 cells more rigorously, we designed a dominant-negative approach to eliminate, or knock out, the expression of this receptor. Our strategy was based on the findings that the 04 subunit associates exclusively with the α6 subunit, and α6 associates preferentially with 04 in comparison with 01 (He ler et al., J. Biol . Chem . 264: 6529-6535, 1989). Thus, we hypothesized that if a 04 subunit lacking its cytoplasmic domain (04-ΔCYT) was expressed in these cells, it would associate with the α6 subunit on the cell surface and create a nonfunctional receptor (OE604-ΔCYT) . This expression also should decrease the amount of βøl on the cell surface, eliminating the only functional laminin receptor in these cells (Fig. 3A) .

MDA-MB-435 cells were transfected with the 04-ΔCYT cDNA and a population of stably transfected cells that expressed high surface levels of 04 were obtained by FACS (Fig. 3B) . The 04-ΔCYT subunit associated with the α6 subunit on the cell surface as determined by surface biotinylation and immunoprecipitation with antibodies specific for α6 and 04 (Fig. 3C) . The total level of 06 on the cell surface was not altered in the 04-ACYT transfectants (Fig. 3B) . However, the amount of α601 receptor present on the cell surface was decreased by the expression of the 04-ΔCYT CDNA, as shown in Fig. 3C, after removing 04 from cell lysates by immunodepletion, very little α6 remained in the 04-ΔCYT lysates in comparison to the amount obtained from the mock transfectant lysates. The small amount of α6 that is seen in the 04-ΔCYT transfectant lysates may be residual α604-ΔCYT, because some 04-ΔCYT is present in this 06 immunoprecipitation. By FACS analysis, the level of 201 on the surface of the 04-ΔCYT transfectants was comparable to the mock transfectants, whereas an increase in α301 surface expression was observed (data not shown) . MDA-MB-435 cells that had been transfected with either the vector alone or the 04-fjCYT cDNA were analyzed for their ability to adhere and migrate on laminin. As shown in Figs. 4A and 4B, cells that expressed the truncated 04 subunit were inhibited almost completely in their ability to both adhere and migrate on laminin. In contrast, their adhesion and migration on collagen I did not differ significantly from the mock transfectants, indicating that the inhibition wa specific for laminin. In control experiments, the mock and 04-ΔCYT transfectants also adhered comparably to uncoated Transwell filters. It can be concluded from these data that the α604-ΔCYT receptor cannot mediate normal adhesive interactions with a laminin substratum, and that the 04-ΔCYT subunit is functioning in a dominant-negative manner for α601. To begin to assess how the loss of laminin-binding function might influence the invasive properties of this metastatic cell line, in vitro invasion assays were performed. The mock and 04-ΔCYT transfectants were assayed for their ability to invade through Transwell filters that had been coated on the upper surface with Matrigel. As shown in Fig. 4C, the mock transfectants invaded through the matrix at levels that have been demonstrated previously for the parental MDA-MB-435 cell line (Brunner et al., Eur. J. Cancer, 28A:1989-1995, 1992) . However, expression of the 04-ΔCYT subunit in these cells reduced Matrigel invasion by -68% (Fig. 4C) . We conclude from these data that α601 is critical for the invasive behavior of these cells.

IV. Loss of MDA-MB-435 Metastic Potential In Vivo

TAB E I MDA-435 BREAST CARCINOMA CELLS INITIAL NUDE MICE DATA

Subclone Ascites Degree of Peritoneal Spread

(extensive/ oderate/negative)

Mock-Bl 5/5 5/5 0/5 0/5

Mock-B6 4/7 4/7 2/7 1/7

TOTAL MOCK 9/12 9/12 2/12 1/12

04-ΔCYT-1A2 0/3 0/3 1/3 2/3

04-ΔCYT-3C12 1/7 0/7 2/7 5/7

TOTAL 04-ΔCYT 1/10 0/10 3/10 7/10

-Extensive Metastatic Foci Evident in Lungs and Liver of

Mock Transfectants

-Few, if any, Foci Evident in 04-ΔCYT Transfectants

V. Discussion of Experimental Results Our results provide evidence to support the involvement of the integrin α601 in the aggressive behavior of breast carcinoma cells. Specifically, we observed that MDA-MB-435 cells, a highly invasive and metastatic breast carcinoma cell line, use only 601 to interact with laminin matrices, although they express other potential integrin laminin receptors. More convincingly, we developed a strategy to knock out α601 function in these cells by expression of a dominant- negative receptor (O604-ΔCYT) . MDA-MB-435 cells that expressed this dominant-negative receptor were inhibited drastically in their ability to adhere and migrate on laminin matrices. In addition, such cells invaded Matrigel substantially less well than either wild-type cells or mock transfectants. This in vitro invasion assay has been shown to provide a strong indication of invasive potential in vivo (Albini et al., Cancer Res . , 47: 3239-3245, 1987).

In its capacity as a laminin receptor, a role for the α601 integrin in the invasive processes of carcinoma cells appears quite feasible. It has been known for some time that laminin plays a key role in carcinoma invasion and metastasis (Kleinman et al., Biochemistry 25:312-318, 1986; Albrechtsen et al., Pathol. Annu. 21:251-276, 1986; Kleinman et al..The Laminins: 47:161-86, 1993). Earlier studies emphasized the need for carcinoma cells to penetrate epithelial basement membranes as a prelude to stro al invasion, and more recent studies have highlighted the possibility that invasive carcinoma cells may deposit their own laminin matrix to promote their egress (Miyazaki, et al., Proc. Natl . Acad. Sci . USA, 90: 11767-11771, 1993). Within this context, our data on the expression and function of α601 in metastatic breast carcinoma cells support a key role for this integrin in these laminin mediated invasive processes.

Most likely, the marked diminution in laminin adhesion, migration, and invasion observed in the 04-ΔCYT transfectants can be attributed to the elimination of α601 expression on the cell surface. This assumption is based on our finding that the parental MDA-435 cells use only α601 to interact with laminin, although they express other potential laminin receptors, including α201 and α30l. For this reason, it does not seem likely that the overexpression of α301 that we observed in the 04-ΔCYT transfectants is directly responsible for the inhibition of laminin-mediated interactions. The possibility exists, however that in addition to the elimination of α601 expression, the expression of the 04-ΔCYT CDNA could influence other unknown factors (e.g., interacting molecules) that might contribute to the functional differences observed in the transfectants. In any case, it is apparent that neither α604-ΔCYT nor α301 has the ability to mediate interactions with laminin based on our functional data.

Aside from its relevance to breast carcinoma, the dominant-negative transfection strategy described in this study should be a powerful tool for knocking out the function of α601 specifically in other cell types that do not express α604. Such an approach could be extremely useful, because α601 has been implicated in a myriad of biological and pathological phenomena (Mercurio et al., Trends, Cell Biol . , 5:419-423, 1995). Moreover, expression of this dominant-negative receptor can be used to dissect the functions of α601 in cells that express multiple integrin laminin receptors. For example, RKO rectal carcinoma cells use both the α601 and α201 receptors for mediating laminin interactions. Expression of the truncated 04 subunit in the RKO cell line does not inhibit their adhesion to laminin completely because of the presence of α201. However, it does decrease their ability to migrate on laminin markedly, an observation that supports the notion that these cells use primarily c-601 for migration.

In addition to use of antibodies to full-length polypeptides, the invention also includes use of antibodies to α601 polypeptide fragments. As used herein, the term "fragment," means at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids. Fragments of α601 polypeptides can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g. , removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events) . Preferable fragments or analogs according to the invention are those which facilitate specific detection of a α601 polypeptide or polypeptide levels in a sample to be diagnosed.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

Other embodiments are within the following claims.

What is claimed is:

Claims

1. A method of detecting an increased likelihood of metastic breast cancer in a patient, said method comprising comparing the levels of α601 integrin present in the breast cells of said patient to the levels of α601 integrin in the breast cells from an unaffected individual, and increase in said α601 integrin levels in said cell of said patient relative to said levels in cells from said unaffected individual indicating an increased likelihood of metastic breast cancer.

2. The method of claim 1, wherein said increase in α601 integrin levels is two-fold or greater.

3. A method of prophylaxis or treatment of breast cancer in a patient, said method comprising inhibition of of α601 integrin function.

4. The method of claim 3, wherein said inhibition is by administering an antibody which binds the extracellular domain of α601 integrin.

5. The method of claim 3, wherein said inhibition is by administering a dominant negative nucleic acid construct.

6. The method of claim 5, wherein said construct encodes an α601 polypeptide comprising the transmembrane and extracellular domains of 601, said polypeptide having a deletion of at least a portion of the cytoplasmic domain.

7. The method of claim 3, wherein said inhibition is by administering a polypeptide comprising the α601 transmembrane and cytoplasmic domains.

8. The method of claim 3, wherein said inhibiting is by the administering of laminin or a fragment thereof.

9. The method of claim 8, wherein said fragment is from the G domain of laminin.

10. A kit for diagnosing a mammal for the presence of breast carcinoma or an increased likelihood of developing a breast carcinoma, said kit comprising a substantially pure antibody that specifically recognizes and binds a α601 polypeptide.

11. The kit of claim 10, further comprising means for detecting said binding of said antibody to said α601 polypeptide.

12. A kit for diagnosing a mammal for the presence of a malignancy or an increased likelihood of developing a malignancy, said kit comprising a substantially pure wild-type α601 polypeptide.

13. A method of identifying a modulatory compound which is capable of decreasing α601 levels, comprising (a) providing a cell expressing said o601 polypeptide; and (b) contacting said cell with a candidate compound, a decrease in said α601 protein levels following contact with said candidate compound identifying a modulatory compound.

14. A method of treating a mammal with a disease involving increased expression of a 601, said method comprising administering to said patient a modulatory compound that is capable of decreasing α60l protein levels, in an amount sufficient to reduce the symptoms of said disease in said mammal.

15. The use of an antibody which specifically binds α601 integrin for the manufacture of a kit for the diagnosis of malignant breast carcinoma.