Address reprint requeststo Dr. Ruth J.Muschel, Rm269 J. Morgan Building, Department of Pathology and Laboratory Medi- cine, University of Pennsylvania, ...
American Journal ofPathology, Vol. 149, No. 3, September 1996 Copyright © American Society for Investigative Pathology
Meeting Report Link Between Tumor Progression, Proteases, and Cell Motility Pathology B Workshop
Ruth J. Muschel,* Mark Tykocinski,t and Martin Padarathsingh* From the Department of Pathology and Laboratory Medicine,* University of Pennsylvania, Philadelphia, Pennsylvania; the Department of Pathologyt Case Western Reserve University School of Medicine, Cleveland, Ohio; and the Division of Research Grants,* National Institutes of Health, Bethesda, Maryland
The Pathology B study section sponsored a workshop to explore recent findings concerning tumor cell invasion and the participation of proteases in those processes. Lynn Matrisian (Vanderbilt University, Nashville, TN) opened the first session with a discussion of the role of matrix metalloproteinases in tumor progression. Although thought to be involved in basement membrane destruction during tumor invasion and metastasis, recent studies have expanded the view of the role of these enzymes and demonstrated effects on processes involved in earlier stages of tumor progression. For example, overexpression of an activated form of stromelysin-1 in the developing mammary gland of virgin transgenic mice revealed morphological alterations resembling those observed in pregnant females. The proliferative index, apopotic index, and differentiation features of the mammary epithelial cells were altered as a consequence of basement membrane degradation resulting from the mis-expression of metalloproteinase activity. These alterations altered the development of mammary neoplasias with no discernible effect on their ability to invade and metastasize. Additional studies presented by Dr. Matrisian pointed to a role of the metalloproteinase matrilysin in early stages of colorectal tumor progression. Matrilysin is expressed in approximately 50% of benign human colorectal tumors and in 88% of adenomas
arising in mice with an APC gene mutation giving rise to multiple intestinal neoplasias (Min mice). To determine whether matrilysin was contributing to intestinal tumor development, matrilysin knock-out mice were generated. Min/matrilysin-deficient mice developed 60% fewer intestinal tumors, suggesting that matrilysin plays a critical role in the development of benign lesions in this model system. These studies suggest a role for metalloproteinases in the early stages of tumor progression and suggest that inhibitors may be useful chemopreventive as well as therapeutic agents. Yves DeClerck (Children's Hospital of Los Angeles, Los Angeles, CA) discussed the family of inhibitors that control the activity of matrix metalloproteinases (MMPs), the tissue inhibitor of metalloproteinases (TIMP). Three members of this family have been described, and together the data suggest that TIMP-1 and TIMP-3 respond to alterations in the extracellular matrix, whereas a major function of TIMP-2 is to provide a stable, basal level of anti-MMP activity in tissues. Dr. DeClerck has explored the effect of TIMPs on tumor growth. TIMP-2 can exert a growth-promoting or a growth-inhibitory activity on tumor cells depending on its concentration and the presence of an intact extracellular matrix. At low concentrations, recombinant TIMP-2 exerted a growth-stimulatory effect on some This workshop took place on February 30 in Panama City, Florida, in association with the American Association for Cancer Research Scientific Conference entitled Proteases and Protease Inhibitors in Cancer, and supported by the Division of Research Grants, National Institutes of Health. Accepted for publication May 17, 1996. Address reprint requests to Dr. Ruth J. Muschel, Rm 269 J. Morgan Building, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104.
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cells, but a growth-inhibitory effect that was dependent culture in a three-dimensional collagen gel was also found. A similar growth-inhibitory effect was observed when these same cells were genetically manipulated to overexpress TIMP-2 and injected subcutaneously in immunodeficient mice. An attempt was made to influence the growth of cells in vivo with TIMP-2 by introducing a retroviral vector for TIMP-2 into cells and then irradiating the infected cells and co-injecting them with tumor cells into immunodeficient mice. A significant inhibition of growth was observed in these tumors. Despite a relatively low number of transduced tumor cells (13%), the tumors generated by cells co-injected with the TIMP-2 retrovirus producer cells were surrounded by a collagen-rich capsule and failed to invade the adjacent muscle. These data suggest that overexpression of TIMP-2 in a limited number of tumor cells in vivo may have a significant cytostatic and antiinvasive effect on tumors. Expression in malignancy of another family of proteases, the cathepsins, was evaluated by Dr. Bonnie Sloane (Wayne State University, Detroit, Ml). Her work has shown that cathepsin B levels are increased in many human cancers, and in colon cancers, increased expression of cathepsin B in tumor cells is predictive of shortened patient survival. Staining for cathepsin B mRNA and protein was highest at the invasive edge of a tumor leading to the suggestion that this enzyme participates in invasion and metastasis. Invasion of glioblastomas through Matrigel and infiltration of a spheroid of glioblastoma cells into normal brain aggregates is blocked by inhibitors of cathepsin B, supporting this hypothesis. The localization of cathepsin B within ras-transformed cells and tumor cells differed from that in normal or immortalized cells. In normal colonic mucosa, cathepsin B was observed in vesicles in the apical region of the cells, but in early carcinoma, cathepsin B was found only in vesicles at the extreme basal aspect. In vitro, the basal localization of cathepsin B occurs concomitantly with positioning at the external membrane. Thus, cathepsin B is ideally localized to participate in degradation of the basement membrane, a prerequisite to tumor motility and invasion. Of potential importance to tumor progression is the observation that cathepsin B becomes associated specifically with focal adhesions. The localization of proteases within the cell was the subject of study by Wen-Tien Chen (Georgetown University, Washington, DC). He showed evidence that many of the proteases induced during invasion can be found on plasma membrane protrusions, invadopodia, that contact and dissolve the extracellular matrix. Invasiveness can be induced by ligation of on
integrins, and the induced invasion is associated with an increase in localization of proteases at invadopodia but not at focal adhesions. These include serine integral membrane proteases (SIMP), including 1 70-kd and 200-kd seprases, which appear to be similar to the other integral membrane proteases, dipeptidyl peptidase IV and 95-kd fibroblast activation protein, and the membrane-type MMP (MTMMP) and MMP-2. The role of the transmembrane domain of MT-MMP in this localization was investigated using moderately invasive RPM17591 human melanoma cells transfected with chimeric MT-MMP plasmids. Expression of the transmembrane and cytoplasmic domains of MT-MMP in the fusion protein to TIMP-1 led to localization in invadopodia but absence of catalytic activity. The invadopodia localization and fibronectin degradation function of MT-MMP was abolished by truncation of the TM domain and by fusing the MT-MMP extracellular domain with the TM region of the interleukin-2 receptor -chain, although these MT-MMP mutants were still retained on the surface of cells, and the latter mutant could activate progelatinase A. Thus, the TM domain of MTMMP plays an essential role in melanoma cell invasion, probably directing its localization to invadopodia. These results demonstrate that MTMMP and seprases are localized to invadopodia and may require that localization to function in invasion. The remaining speakers dealt with cellular interaction required to coordinate tumor progression. Bodhan Wasylyk (INSERM, Strasbourg, France) summarized his work on the ets family involvement in ras action. Deregulation of the activities of transcription factors that are encoded by the ets and jun families of oncogenes is a common feature of Ras transformation. Blocking the activities of either ets or jun is sufficient to revert the Ras-transformed phenotype. Ras interacts through several members of the ets family. Net is particularly sensitive to Ras activation. Ras activates Net through the carboxy domain, a domain conserved in many ets family members. Although Ras can activate Net, under basal conditions, Net inhibits transcription. This repression is mediated by an inhibitory domain of the Net protein. The inhibitory domain is a potent inhibitor of Ras activation of transcription, suggesting that it may be a useful target for suppression of Ras transformation. To further explore how ras can influence ets activity, the Wasylyk lab has identified two Etsl interacting factors by a yeast two-hybrid system, EIF1 and SpG00. EIF1 is highly homologous to yeast ubiquitinconjugating enzymes, suggesting the possibility that EIF1 leads to mono-ubiquitination of Etsl, which may open its compact closed structure and facilitate in-
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teractions with DNA or other proteins. Sp100 is localized in nuclear punctate structures, binds to the amino-terminal region of Etsl, and forms a complex with Etsl when bound to DNA. Co-expression of Sp100 and Etsl in human cells results in a marked increase in the transcriptional activity. These results suggest that Sp100 is a co-activator of Etsl that is stored in nuclear punctate structures. The fundamental conserved role of Ets factors in Ras functions suggest that they are key effectors that may be an important target for developing anti-cancer therapies. The next speakers considered factors involved in regulation of cell motility. Tumor cell autocrine motility factor (AMF) was originally distinguished by its ability to induce the directed (chemotactic) and random (chemokinetic) migration of the AMF-producing tumor cells. Dr. Avraham Raz (Michigan Cancer Foundation, Detroit, MI) showed that AMF induces its cellular responses by binding to its receptor, a cell surface glycoprotein of 78 kd (gp78), and activating a signal transduction cascade. Significantly, highly metastatic cells that display migratory as well as altered mitogenic responses to AMF show signaling profiles that are absent from low metastatic cells, suggesting that differences in invasive and metastatic capabilities may be dictated in part by the ability to exploit responses to AMF in a more effective manner. AMF and its receptor have been postulated to play a role in the most devastating aspect of cancer, ie, tumor cell metastasis, based upon their motility-regulating effects. Recently, it was demonstrated that expression of the AMF receptor correlates with malignant potential in human bladder cell lines as well as disease stage and recurrence in human bladder cancer specimens. Up-regulation of gp78 expression concomitant with disease progression in bladder cancer is associated with downregulation of the calcium-dependent homotypic cell adhesion molecule E-cadherin, consistent with a shift from a sedentary to a motile (ie, invasive) cellular phenotype. The potential use of gp78 as a marker of loss of cell-cell contact regulation and tumor malignancy in general is bolstered by the findings presented here. Mary J. C. Hendrix (Saint Louis University, St. Louis, MO) addressed the significance of the intermediate filament expression in highly metastatic melanoma and breast carcinoma. Although vimentin is often found in mesenchymal cells and keratins are present in epithelial cells, the co-expression of vimentin and keratins 8 and 18 in tumors is associated with recurrent and metastatic disease. The importance of keratin expression for invasion was con-
firmed in that transfection of human melanoma cells with the expression vectors for keratins 8 and 18 led to an increase in their invasion of basement membrane matrix and migration through gelatin in vitro and lung colonization. However, no metastasis to the lung occurred after subcutaneous inoculation. Disruption of keratin in a second melanoma cell line that expressed keratin resulted in a three- to fourfold decrease in their invasive and migratory ability in vitro. To study the expression of vimentin in carcinoma cells, MCF-7 human breast cancer cells were engineered to express both vimentin and keratins. Overexpression of vimentin in these cells led to augmentation of motility and invasiveness in vitro but did not enhance metastatic potential. Motility and invasion could be transiently down-regulated by vimentin antisense oligonucleotides in cells that constitutively co-express keratins and vimentin intermediate filaments. Taken together, these studies suggest a direct link between keratin and vimentin intermediate filament co-expression and increased invasive behavior. Finally, the work of Candece Gladson (University of Alabama, Birmingham, AL) has addressed the special mechanisms that glioblastomas mobilize to facilitate invasion. The extracellular matrix of the normal brain and of malignant astrocytomas lacks expression of most adhesive glycoproteins, suggesting that astrocytoma cells must synthesize their own extracellular matrix to attach and invade. In fact, Dr. Gladson found that vitronectin was expressed in malignant astrocytomas, but not in low grade tumors or in normal adult brain. Furthermore, vitronectin was found in vivo only when the cells were grown intracerebrally but not when grown subcutaneously, indicating that the cerebral microenvironment was necessary for expression of the vitronectin gene in malignant astrocytoma. If the expression of vitronectin were important for astrocytomas, it would be predicted that these tumor cells would express receptors for vitronectin, and indeed, evidence for expression of the mRNA for the two vitronectin receptor integrins, avf33 and avf5, was found. The ,33 mRNA was detected among invading astrocytoma cells, and the 1B5 mRNA was detected throughout the tumor. In vitro, both integrin avf33 and av135 mediated vitronectin-dependent adhesion and migration of malignant astrocytoma cells, implicating these two receptors in malignant astrocytoma cell invasion in vivo. Thus, the synthesis of vitronectin allows malignant astrocytoma cells to overcome existing matrix regulatory controls and thus drive their own ability to invade.
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The studies presented at this workshop demonstrated the interconnection between tumor progression, protease expression, transcriptional activity, and cell motility. Proteases associated with malignancy appear to be involved in invasion, motility, and tumor cell growth. These proteases are sometimes
secreted but often must be precisely localized. Cell motility is triggered by signals at the cell surface and requires intracellular machinery that is only beginning to be understood. Elucidation of each of these steps provides a potential target for therapeutic intervention.
