The present invention provides methods and compositions for the diagnosis and treatment of cells lacking normal growth arresting characteristic. The present invention demonstrates that many tumor cells lack normal cell surface .alpha.-dystroglycan and thereby lack dystroglycan function. Dystroglycan can be lost from the cell surface by proteolytic shedding of a fragment of .alpha.-dystroglycan into the surrounding medium. Upon restoration of dystroglycan function and over-expression of the dystroglycan gene, the once tumorigenic cells revert to non-tumorigenic cells which polarize and arrest cell growth in the presence of basement membrane proteins, demonstrating that dystroglycan functions as a tumor marker and suppressor.

Description of the Invention

BACKGROUND OF THE INVENTION

Cell growth is highly regulated in normal tissues by a variety of mechanisms in order to guide normal tissue development and homeostasis. A cell's response to the "microenvironment" is a major portion of the growth regulatory machinery. The microenvironment consists of soluble factors, adjacent cell surfaces and molecules of the extracellular matrix (ECM). Information within the microenvironment is primarily detected by cell surface receptors that bind specific molecules found in the micro environment and elicit varied cell responses for growth, morphogenesis or differentiation.

The work reported here focuses on cell interactions with the ECM in general and a specialized form of ECM, called the basement membrane (BM). This specialized extracellular matrix serves not only as a barrier between cell layers, but also as an active signaling substrate that regulates epithelial cell growth, differentiation and tissue architecture. Key signaling components of the BM are the laminin glycoproteins. Laminin-1 alone can induce cell shape changes, growth arrest, and functional differentiation when added to cultured mammary epithelial cells (MECs). Signals from laminin are mediated by direct binding to multiple cell-surface receptors whose individual functions are not completely defined. It has been hypothesized that the aberrant behavior of tumor cells arises, in part, from alterations in cell-BM interactions. In support of this model, tumor cells frequently demonstrate altered responsiveness to BM proteins, indicating changes in BM receptor functions. Significantly, the laboratory of Dr. Mina Bissell has demonstrated that functionally normal MECs can be distinguished from tumorigenic MECs by their growth characteristics when cultured within a 3-dimensional gel of BM proteins (3D-BM assay); functionally normal MECs cultured within Matrigel will grow from single cells to form multi-cellular, polarized acinar structures that arrest growth, whereas tumorigenic MECs grow as disorganized cell masses with unregulated cell growth. The 3-D basement membrane assays distinguish between normal and tumorigenic mammary epithelial cell behavior. Normal cells growth arrest as acinar structures, whereas tumor cells do not growth arrest. This tumor cell characteristic is referred to as a "tumorigenic phenotype". This growth difference has been described in U.S. Pat. No. 5,846,536 incorporated by reference herein. Although it is evident that the cellular machinery that senses the BM is altered in tumorigenic epithelial cells, it is less certain where the critical changes occur. Studies of cell-BM interactions have largely focused on the integrins, an extensively characterized family of heterodimeric receptors. However, integrin signaling generally favors tumor cell growth and metastasis, and no integrin has been unambiguously assigned the role of tumor suppressor, leaving the possibility that other important receptors may still need to be investigated. The present invention relates to the characterization of one such receptor, dystroglycan (DG).

Accordingly, it is an object of the present invention to provide an assay of dystroglycan expression. This assay may be used to show that the laminin binding portion of a-dystroglycan is lost in tumor cells.

It is another aspect of the invention to provide an assay of dystroglycan proteolysis and shedding through the detection of cell-surface .alpha.-dystroglycan. This assay focuses on the relative ratio of .alpha.- to .beta.-dystroglycan at the surface of cells, as compared to cells like the BT474 cells (FIG. 2, Lane 2, see Original Patent) which shed little or no .alpha.-dystroglycan.

It is another aspect of the invention to provide an assay for the generation of dystroglycan fragments that can be used to assay for inhibitors of the metaloproteinase(s) cleaving and shedding .alpha.-dystroglycan.

It is yet another aspect of the invention to provide an assay that utilizes .alpha.-dystroglycan protein, or derivative thereof, as a substrate for a cell-free assay measuring the activity of the protease(s) cleaving it. The substrate consists of either the full-length .alpha.-dystroglycan molecule, a fragment thereof, or a synthetic peptide capable of being recognized and cleaved by the enzyme cleaving .alpha.-dystroglycan.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for the diagnosis and treatment of cells lacking normal growth arresting characteristics. This characteristic is referred to as "tumorigenicity," which means the properties of a cell normally associated with tumor forming properties, especially, growth arresting properties, normal cell arrest, and appearance in the 3D-BM assay. Normal, non-tumorigenic cells will be polarized and, in the case of mammary epithelial cells, form acini with regulated growth properties. In the case of tumorigenic cells, the cells are disorganized and sometimes invasive, and exhibit abnormal growth.

It has been found that many tumor cells lack normal cell surface .alpha.-dystroglycan and thereby lack dystroglycan function. Re-establishment of dystroglycan function has been achieved in one cell line by transfection and over-expression of the dystroglycan gene. By re-establishing dystroglycan function, the once tumorigenic cells reverted to non-tumorigenic cells which polarized and arrested growth in the presence of basement membrane proteins, demonstrating that dystroglycan functions as a tumor suppressor. Loss of a tumor suppressor function, like that of dystroglycan, facilitates the development of tumors, therefore, cells lacking a tumor suppressor are said to have a higher "potential tumorigenicity." In some cases, loss of a single tumor suppressor, like dystroglycan, can indicate a tumorigenic state, and in other cases additional changes to the cell are required before it becomes capable of forming tumors. For the purpose of this application, either case is described as a higher potential tumorigenicity.

Most importantly, it has been found that dystroglycan can be lost from the cell surface by proteolytic shedding; some tumors cells shed a fragment of .alpha.-dystroglycan into the surrounding medium. These forms of .alpha.-dystroglycan are distinguishable because normal .alpha.-dystroglycan has a molecular weight of .about.180 kD, while the shed fragment has a molecular weight (Mr) of 120-130 kD (FIG. 1A, see Original Patent). As is known in the field, the term "Mr" refers to relative mobility on electrophoretic gels. This shedding is inhibited by the presence of metaloproteinase inhibitors (FIGS. 1B and 1C, see Original Patent).

The present assays may be carried out on tissue samples, the cells themselves, or on the surrounding medium. In vivo, the surrounding medium will comprise the blood and its serum.

Using the above information, one can measure the potential tumorigenicity of cells by assaying for the presence of a fragment of .alpha.-dystroglycan in medium, particularly fragments having an Mr of 120-130 kD. Identifying the presence of the .alpha.-dystroglycan fragment indicates a higher potential tumorigenicity.

Using the above information, one can also measure the potential tumorigenicity of cells by assaying to determine the ratio of the total amount of .alpha.-dystroglycan present in a cell sample relative to the amount of .beta.-dystroglycan present in the sample. A ratio showing a deficiency of .alpha.-dystroglycan relative to .beta.-dystroglycan indicates .alpha.-dystroglycan shedding.

A correlation between tumorigenicity and the loss of .alpha.-dystroglycan through proteolysis has been shown. Treatment of the tumorigenic cells with a metalloprotease inhibitor, at concentrations that inhibit dystroglycan shedding, reverses the tumorigenic phenotype (FIG. 3, see Original Patent). Furthermore, treatment of cells with a genetic construct for .alpha.-dystroglycan also reverses the tumorigenic phenotype.

The present invention also provides an assay for identifying compounds which can inhibit the cleavage of .alpha.-dystroglycan by the endogenous protease that cleaves .alpha.-dystroglycan on the surface of cells. The assay comprises the steps of providing test cells, preferably tumor cells, more preferably mammary epithelial tumor cells; adding test inhibitors, along with positive and negative controls; growing the cells; and observing the resultant cell phenotype, i.e., growth arrested (normal phenotype) and tumorigenic phenotype. In cells normally having polarity, the normal phenotype will also involve polarity.

The present invention also provides an assay for identifying compounds that can inhibit the cleavage of .alpha.-dystroglycan by the creation of an in vitro assay of dystroglycan proteolysis. The assay comprises the addition of the protease, in a crude protein mixture or in pure form, with a substrate. The substrate consists of either the full-length .alpha.-dystroglycan molecule, a fragment thereof, or a synthetic peptide capable of being recognized and cleaved by the enzyme cleaving .alpha.-dystroglycan.

One can also use the above information to develop an assay of proteolysed .alpha.-dystroglycan fragments in blood serum. This assay would add a labeled antibody specific for an .alpha.-dystroglycan or a fragment thereof, and assaying for the amount of bound label present in the serum. As an aspect of this assay, one would look for .alpha.-dystroglycan fragments having a Mr of approximately 120 kD.

The present invention also provides a method for suppressing the abnormal growth of tumor cells, or, in effect causing reversion of tumorigenic cells to a normal phenotype. This method involves the addition of a protease inhibitor to the cells, specifically a metalloproteinase inhibitor. The amount of inhibitor to be added can be determined by routine experimentation, in view of the examples provided herein. Metalloproteinase inhibitors may be selected from the group consisting of TAPI, GM6001 or a pharmaceutically acceptable salt thereof, or an ARAM's family protease inhibitor or pharmaceutically acceptable salts thereof. Simply stated, the "effective amount" of metalloproteinase (or protease) inhibitor is reached when the cells to be treated, when grown in culture, specifically the 3D-BM culture system, show normal phenotype and growth arrest, polarity, and secondary organization, such as acini in the case of some mammary epithelial cells.

Finally, one could also use the present invention to restore normal dystroglycan function to a mammalian cell having an abnormal dystroglycan function by contacting a cell with an adenovirus transfection agent containing a normal mammalian dystroglycan gene and a cationic agent which interacts with cell surfaces or nucleic acids so as to result in a cell with said normal functioning dystroglycan gene therein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Dystroglycan Function

Through assays of normal cell function, we have identified dystroglycan as a laminin receptor signaling cytoskeletal and cell shape changes, and cell growth arrest in normal breast epithelial cells. Dystroglycan is a known transmembrane laminin receptor composed of two non-covalently linked portions: .alpha.-dystroglycan and .beta.-dystroglycan; see U.S. Pat. No. 5,449,616 hereby incorporated by reference. These originate from a single protein that is post-ranslationally cleaved. .beta.-dystroglycan is imbedded in the cell membrane. The extracellular chain, .alpha.-dystroglycan, binds to laminin. We have shown that inhibition of dystroglycan binding to laminin permits cell spreading and growth in the presence of laminin, conditions where cells would normally round-up and growth arrest. Results suggest a model whereby dystroglycan operates as a co-receptor, which organizes the laminin in the BM and facilitates signaling through other BM receptors. But, dystroglycan is shown to mediate shape changes and growth control without help from .beta.1 and .beta.4 integrins. K. Campbell et al. in Pat. No. 5,449,616 identified dystroglycan using four overlapping clones (designated HD-1 to HD-4) covering the entire mRNA were completely sequenced. The full-length human cDNA consists of 5510 nucleotides (SEQ ID NO: 7, but herein included as SEQ ID NO: 1), of which 2685 nucleotides represent an open reading frame. A polyadenylation sequence and poly(A) tail were also identified. The deduced amino acid sequence (SEQ ID NOS: 7 and 8, herein included as SEQ ID NOS: 1 and 2) predicts a polypeptide of a calculated Mr of 97,552 with a signal sequence of 27 amino acids, a single transmembrane domain close to the C-terminal region, four potential N-glycosylation sites and many potential sites for O-glycosylation.

Alignment of amino acid sequences for human and rabbit dystroglycan demonstrate that both proteins contain 895 amino acids with overall sequence identity of 93%. Ninety percent of the amino acid substitutions are conservative. The transmembrane domain of human dystroglycan is identical to that of rabbit dystroglycan. The intracellular C-terminal region of human and rabbit dystroglycan is highly conserved and is enriched in proline (23%). Both proteins have identical consensus sites for N-glycosylation and have high content of threonine and serine as potential sites for O-glycosylation. High homology between rabbit and human dystroglycan suggests its functional importance, especially in terms of carbohydrate chain attachment sites, since carbohydrates may play an important role in laminin binding.

Because dystroglycan is found to regulate cell growth and cytoskeletal architecture in response to laminin in normal tissues, we have compared these signaling mechanisms in normal and malignant cells in order to ask whether dystroglycan might be altered in tumor cells. Although the .beta.-dystroglycan protein is detected in all tumor cells, the laminin binding portion, .alpha.-dystroglycan, was found to be greatly reduced or undetectable in the majority (5 of 8). Loss of .alpha.-dystroglycan in these tumor cells was reflected by both the loss of antibody detection and loss of laminin binding ability. Therefore, within this survey .alpha.-dystroglycan was functionally absent from 5 of 8 tumor cell lines. As predicted, only those cell lines possessing adequate levels of .alpha.-dystroglycan on the cell surface were able to undergo cell rounding in response to laminin. The presence of .alpha.-dystroglycan also corresponded with the growth characteristics of tumor cells cultured within a 3D basement membrane. As described earlier, this assay has been employed to distinguish the behavior of tumor cells and normal cell in response to the BM.

Also, as described in Example 5, we have demonstrated that restoration of dystroglycan function to tumorigenic cells can revert the tumorigenic behavior of these cells, restoring normal tissue structure, differentiation potential and growth control. Re establishment of dystroglycan function was achieved in one cell line by transfection and over-expression of the dystroglycan gene. By re-establishing dystroglycan function, the once tumorigenic cells reverted to non-tumorigenic cells which polarized and arrested growth in the presence of basement membrane proteins. Cells over-expressing the dystroglycan gene no longer form tumors after injection in nude mice. This reversion of the tumorigenic phenotype demonstrates that dystroglycan functions as a tumor suppressor.

Receptor Shedding

Because .alpha.- and .beta.-dystroglycan are translated originally as a single polypeptide, it was surprising that .alpha.-dystroglycan was not detected on the cell surface of many cells when .beta.-dystroglycan was present. We concluded that, by some mechanism, .alpha.-dystroglycan was being shed from the cell surface. Shedding could occur by two mechanisms: 1) simple detachment from the cell surface (because .alpha.-dystroglycan is non-covalently linked), or 2) shedding induced by proteolytic cleavage of .alpha.-dystroglycan or some component attaching .alpha.-dystroglycan to the cell surface.

To test these possibilities we looked for the presence of a-dystroglycan in the culture medium of cells which shed the protein and asked if it was proteolytically cleaved. In one mammary carcinoma cell line SCg6, .alpha.-dystroglycan was detectable both on the cell surface and in the cell culture medium. Detection was achieved with an anti-.alpha.-dystroglycan antibody. One such antibody is described by Durbeej M., Campbell K. P., J. Biol. Chem. 1999; 274(37): 26609-16. Laminin binding may also be used in place of an antibody since .alpha.-dystroglycan binds specifically to laminin. However, the .alpha.-dystroglycan detected in the medium was approximately .about.60 kD smaller than that on the cell surface. This suggested that .alpha.-dystroglycan was proteolytically cleaved either before or after shedding. To ask if shedding was induced by the proteolysis, we treated the cells with a general matrix metaloproteinase (MW) inhibitor, GM6001, to see if .alpha.-dystroglycan shedding was inhibited. With cells cultured in the presence of 40 .mu.M GM6001, the proteolysed form of dystroglycan was no longer detected in the culture medium (FIG. IB, see Original Patent), A control analog, C1004, had no effect at the same concentration.

Therefore, loss of .alpha.-dystroglycan from the cell surface is induced by metalloprotease-induced shedding. Titration of GM6001 showed a pKi of approximately 10 .mu.M, and a nearly complete inhibition over 25 .mu.M (FIG. 1C, see Original Patent). This represents an unusually high pKi for this inhibitor of metalloproteinases. Most MMPs are inhibited with pKi's of GM6001 below 1.0 .mu.M (Galardy et al., Ann. N. Y. Acad. Sci., 1994. 732: p. 315-23). The results in FIG. 1C (see Original Patent) indicate that the protease cleaving .alpha.-dystroglycan is not among the majority of well-characterized proteases. The best candidates currently are among the ADAMs family of proteases, which are so far the only metaloproteases known to require high concentrations of GM6001 for inhibition. The ADAMs (A Disintegrin And Metalloprotease) are a recently discovered group of multidomain cell surface proteins postulated to play important roles in cell-cell and cell-matrix interactions. For example, ADAM 12 is upregulated in breast and colon cancer, and ADAM 12 supports tumor cell adhesion. Most ADAMs have no assigned substrate and the family is rapidly growing.

The treatment of cells with a matrix metalloprotease inhibitor can inhibit .alpha.-dystroglycan shedding and thereby increase .alpha.-dystroglycan levels at the cell surface. In turn, as previously demonstrated by gene transfection, restoration of dystroglycan function to the cell surface can restore a normal response to the BM, (i.e. organized cell structure and growth arrest).

Dystroglycan is expressed in all cells of the body, therefore, dystroglycan function and shedding is likely to play an important role in the growth and differentiation of virtually all cells. This suggests that inhibition of dystroglycan shedding may inhibit growth of any cell type, including those contacting the BM such as epithelial and endothelial cells (blood vessels). Because inhibition of endothelial cell growth is an effective therapy against tumor growth itself, an inhibitor of a-dystroglycan shedding will not only revert the tumorigenic characteristics of a tumor cell but also act against tumor growth by inhibiting angiogenesis.

Screens for Therapeutic Compounds

Recognizing that a protease sheds .alpha.-dystroglycan from the surface, this protease becomes the target for the action of therapeutic compounds to inhibit the shedding of a-dystroglycan. The use of GM6001 and TAPI to revert the tumorigenic phenotype has demonstrated proof of principle that such compounds can be therapeutic. Therefore, an assay is created for the activity of this protease using as a substrate a peptide containing the cleavage recognition sequences of this metalloprotease.

In one assay, a full-length human .alpha.-dystroglycan molecule is added to a physiological solution containing a human protease that cleaves the protein. Cleavage products are detected by separating solution components by size, e.g. through gel electrophoresis, size exclusion chromatography, etc. Test inhibitors are added to the solution and their effect on the creation of fragments by the protease are measured.

Assay for The Detection of Tissue Re-Organization and Cell Growth

We believe .alpha.-dystroglycan shedding occurs principally in cells that are reorganizing and growing. Little of such activity occurs in adult tissues, except in cases like the normal processes of mammary gland development, and perhaps angiogenesis. However, such activity would occur on a large scale during hyperplasia or tumor cell growth and the accompanying angiogenesis. .alpha.-dystroglycan is shed in two forms, one which binds laminin and a smaller portion with no known binding activity.

Any assay that detects .alpha.-dystroglycan proteolysis would be an assay for the detection of tissue re-organization and cell growth. Assays have been created to test for a-dystroglycan proteolysis in cultured cells, tissue sections, and in blood serum. Assays in cell culture include detection of shed a-dystroglycan fragments in the culture medium, and measurement of the ratio of .alpha.-dystroglycan to .beta.-dystroglycan on the cell surface. Assays in tissue samples include detection of proteolysed .alpha.-dystroglycan fragments by immunoblotting extracted tissues or immunostaining of "nouveau antigens" created by dystroglycan proteolysis. Assays in blood serum include immunologic detection of dystroglycan fragments or nouveau antigens in serum samples.

Normal MEC Function

Using assays of normal MEC function, we divided laminin signaling functions among three different receptor systems, the .beta.1 integrins, .alpha.6 .beta.4 integrin, and a yet to be identified "B3 laminin receptor". Most importantly, these results suggested that a non integrin laminin receptor, binding to the E3 domain of laminin, is a critical mediator of cell morphogenesis and growth control in MECs. We now have direct evidence that the "E3 laminin receptor" is dystroglycan. First identified in muscle cells, dystroglycan is now recognized as a laminin receptor expressed in virtually all cell types, including epithelia. We have shown that over-expression of the dystroglycan gene in HMT-3522-T4 cells (T4 cells), which do not respond to laminin in morphogenesis assays, restored correct responsiveness of these cells to laminin. Moreover, these once tumorigenic cells now formed polarized, growth arrested acinar structures in 3D-BM assays, and no longer produced tumors upon injection in nude mice. The reversion of the tumorigenic phenotype of T4 cells by dystroglycan over-expression demonstrates that restoration of dystroglycan function to breast tumor cells can reduce or eliminate their tumorigenic potential, suggesting novel approaches to the treatment of cancer. The role of dystroglycan as a tumor suppressor was, until now, entirely uninvestigated.

Assays in Breast Tumor Cell Lines

Assays of dystroglycan expression in several breast tumor cell lines showed that the laminin binding portion of dystroglycan was lost in the majority of tumor cells. Dystroglycan is composed of two subunits, .alpha. and .beta., which are the product of a single gene that is post-translationally cleaved. Immunoblots showed that the .beta.-dystroglycan subunit was present in all breast tumor cell lines tested, but that the .alpha.-dystroglycan subunit, which binds laminin, was greatly diminished or absent in 5 of 8 (FIG. 2, see Original Patent). Evidently the .alpha.-dystroglycan subunit was shed from the cell surface. Loss of a-dystroglycan in these cell lines correlated with loss of organization in the 3D BM assay and correlated with more aggressive tumor cell behavior in vivo. The ratio of .alpha.-dystroglycan to .beta.-dystroglycan is higher in the BT474 cell line, FIG. 2 (see Original Patent), lane 2, than any other cell line or in normal cells, suggesting that some degree of shedding occurs in all cells, but that shedding is low or absent in BT474s.

In addition, we have demonstrated that shedding of .alpha.-dystroglycan can be caused by proteolysis. The .alpha.-dystroglycan molecule is detected in the supernatant of some tumor cells, but is smaller than the molecule detected at the cell surface. Shedding of cell surface molecules is most often attributed to cleavage by the ADAM subfamily of metalloproteinases (MPs). Indeed, the action of the hydroxamate MP inhibitor GM6001 implicates an ADAM; shedding of .alpha.-dystroglycan is inhibited by GM6001 at a Ki of .about.10 .mu.M (FIG. 1C, see Original Patent). This Ki is characteristic for some ADAMs but not for other MP's which are generally inhibited by GM6001 concentrations below 30 .mu.M. In addition, the enhanced .alpha.-dystroglycan shedding was not detected after conditioned medium from shedding cells was placed on T47D and BT474 cells, again indicating a cell surface-bound MP. Therefore, there is good evidence that .alpha.-dystroglycan is shed by the activity of an ADAM or similar MP.

The model drawn above predicts that increasing dystroglycan levels at the cell surface, through inhibition of proteolytic shedding, can revert the tumorigenic phenotype of T4 cells and inhibit the growth of other tumor cells. As shown in FIGS. 3B and 317 (see Original Patent), treatment with GM6001 at 2 .mu..M had no effect on tumor cell growth characteristics in the 3D-BM assay, even though this concentration is sufficient to inhibit most MPs. However, GM6001 concentrations over 20 .mu.M, (sufficient to inhibit dystroglycan shedding) reverted the T4 cells, which formed polarized and growth-arrested acini, and dramatically reduced the growth and invasion of MDA-MD-231 cells (FIGS. 3C and 3G (see Original Patent)).

Claim 1 of 5 Claims

1. A method of restoring dystroglycan function in a tumorigenic mammalian epithelial cell having a loss of dystroglycan function which comprises (a) obtaining an epithelial cell sample suspected of being tumorigenic; (b) identifying cells that have shed a 120-130 kD fragment of .alpha.-dystroglycan, whereby the presence of the fragment indicates that said cells are tumorigenic; (c) contacting said cell with a transfection agent containing a mammalian dystroglycan gene SEQ ID NO: 1, whereby the cell comprises a transfected dystroglycan gene therein, and dystroglycan function is restored and the tumorigenicity of said mammalian cell is reduced or reversed.

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