RESEARCH PLAN, M.SHARON STACK, PH.D.

REGULATION OF
METASTASIS-ASSOCIATED
PROTEINASES

The ability to invade host tissues and metastasize is the major cause of cancer-related death. During tumor invasion, metastasizing cells disrupt normal cell-cell and cell-matrix contacts and acquire a migratory, invasive phenotype. Subsequent alterations in cellular architecture mediated by modified extracellular matrix (ECM) attachments induce expression of proteinases that degrade ECM proteins, facilitating migration through the modified tissue to establish metastatic foci. Although malignant cells produce a spectrum of matrix-degrading enzymes, predominant among these proteinases are enzymes in the plasminogen activator (PA) and matrix metalloproteinase (MMP) families. Current research centers on two model systems: epithelial ovarian carcinoma and squamous cell carcinoma of the oral cavity (OSCC).

Epithelial ovarian carcinoma is the leading cause of death from gynecologic malignancy, as 75% of women with this disease succumb to complications resulting from disseminated intra-peritoneal metastasis. Thus, strategies aimed at prevention of metastasis would generate immediate clinical impact. In the human adult, the normal ovarian epithelium is a single cell layer separated from the underlying stroma, the tunica albuginea, which is comprised of dense collagenous connective tissue. The mesodermally-derived normal ovarian epithelium displays both epithelial and mesenchymal characteristics in situ and reversible modulation of ovarian epithelial cells to a fibroblastic form occurs during post-ovulatory repair of the epithelium. Such unique phenotypic plasticity suggests that ovarian epithelium adapts to changes in the cellular microenvironment by transition between epithelial and fibroblastic phenotypes, a characteristic usually limited to immature, regenerating, or neoplastic epithelia. The initiating events in the development of ovarian carcinoma are poorly understood. However, ovarian carcinoma has an unique metastatic mechanism requiring reversible modulation of cell-cell and cell-ECM contacts that involves shedding of cells from the primary tumor followed by subsequent intra-peritoneal adhesion, invasion and proliferation [Fig. 1]. Unlike most highly metastatic tumors, the majority of women with advanced intraperitoneal disease have no clinically apparent lymphatic or hematogenous metastases, implying that a novel mechanism for metastasis is operative in ovarian cancer. Further, as metastases are largely confined to the peritoneal cavity, microenvironmental factors that modulate intraperitoneal adhesion, motility and invasion play a predominant role in ovarian pathobiology.

Fig. 1. Working Model for Ovarian Cancer Metastasis. The initiating events in development of ovarian cancer are poorly understood. In addition to genetic mutations, the progression to neoplasia results in part from changes in the response of ovarian epithelium to mitogenic and motogenic growth factors due to aberrant expression or activity of growth factors or their receptors. Clinically, tumors often involve the ovary and omentum, with diffuse intraperitoneal metastases and malignant ascites. Reversible modulation of cell-cell and cell-matrix adhesive contacts likely plays a critical role in remodeling of the ovarian surface epithelium during tumor progression, resulting in shedding of tumor cells from the ovary and intraperitoneal metastasis, invasion and spread. Metastatic dissemination is believed to occur by direct extension or shedding of cells into the peritoneal cavity. Initial distribution of exfoliated malignant cells or multi-cellular aggregates (MCAs) is facilitated by the peritoneal fluid. Proteinase-induced changes in normal cell-cell and cell-matrix contacts play a critical role in the remodeling of ovarian epithelia that occurs during tumor progression. The ovarian tumor mileu is rich in a number of extracellular proteinases produced by the tumor cells with contributions from stromal elements including mesothelium and inflammatory cells. Following intra-peritoneal adhesion and mesothelial cell retraction, proteolytic activity is required for motility and invasion, two cellular behaviors that facilitate metastasis. Cell-matrix adhesive interactions may regulate subsequent metastatic behavior by controlling proteinase expression and trafficking. Ovarian tumors exist in an unique microenvironment wherein the primary tumor often maintains direct contact with malignant ascites, providing the opportunity for persistent exposure to soluble factors capable of modulating ovarian tumor behavior.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity, causing more deaths than any other oral disease. The 5-year survival rate has not improved appreciably in over 20 years, remaining at a low 50%. These statistics reflect our limited understanding of the molecular events that govern disease progression. Treatment of advanced OSCC [Fig. 2] is associated with high mortality, resulting from local, regional and distant metastasis; however the cellular and biochemical factors that underlie OSCC dissemination are poorly understood. In the absence of reliable molecular markers for use in early detection or as prognostic indicators, treatment failures remain difficult to predict. Thus, a more detailed analysis of the molecular events that contribute to OSCC metastasis are a necessary prerequisite to the development of novel early detection and treatment strategies that favorably impact survival of OSCC patients. To this end, recent studies have utilized cDNA microarray analysis for genome-wide monitoring of genetic and epigenetic changes associated with OSCC primary tumors and lymph node metastases. These studies have identified loss of E-cadherin and enhanced expression of the proteinase urinary-type plasminogen activator (uPA, urokinase) and the cell-matrix adhesion molecule α3 integrin as key candidate biomarkers for prediction of poor disease outcome.

Fig. 2. Human and murine OSCC lesions. Left panel: Immunohistochemical analysis of a well-differentiated SCC of the human tongue stained for α3 integrin (A,B) and uPAR (C,D). Magnification 4X (A,C), 20X (B,D). Courtesy of Dr. Mark Lingen, Univ. of Chicago. Right panel: Orthotopic murine model of SCC of the oral tongue. Mice were injected in the anterior tongue with wild type SCC25 cells, pooled clones of uPAR-knockdown (SCC25-uPAR-KD), or pooled clones of uPAR-overexpressing (SCC25-uPAR/f) cells. Lesions produced by wild type SCC25 cells at 9 weeks appear multicentric with focal keratin formation at low power [2A]. Lesions are well-circumscribed and cuffed by inflammatory cells. At higher magnification, high vascularity and myxoid change is present in the adjacent stroma [2D]. In tumors formed by SCC25-uPAR-KD cells, low power evaluation demonstrates a fairly well-circumscribed tumor [2B]. Higher power examination [2E] reveals that the tumor has features of a well-differentiated squamous cell carcinoma, such as numerous aggregates of keratin, low mitotic index and minimal pleomorphism. In contrast, low power examination of uPAR-overexpressing tumors reveals an infiltrative lesion with ill-defined borders [2C]. Higher power evaluation shows that the tumor is composed of clusters of hyperchromatic cells with a high nuclear to cytoplasm ratio [2F]. There is cytologic atypia and mitotic figures are easily identified. Unlike uPAR-KD tumors, only focal keratin production is evident. Tumor pathology was evaluated by Dr. Susan Crawford, Northwestern Univ.

Ongoing research, summarized briefly below, utilizes an integrative approach involving examination of 2-dimensional (2D) and 3D tissue culture systems complemented by murine tumor models and analyses of human tumors.

Understanding the molecular mechanisms by which tumor cells orchestrate multiple microenvironmental cues to regulate the expression and activity of metastasis-associated proteinases is the major focus of the laboratory.

Regulation of pericellular proteolysis through matrix and mechanotransduction. Integrins are heterodimeric adhesion molecules that mediate cell-matrix interactions [Fig. 3]. Because integrins influence diverse processes including cell adhesion, transmembrane signal transduction, and cytoskeletal organization, a complex variety of cellular behaviors are influenced by integrin engagement including adhesion, migration, proliferation, differentiation and apoptosis. Integrin binding events which result in receptor occupancy, receptor aggregation, or both (occupancy and aggregation) induce distinct subsets of cellular events which are differentiated based on the physical nature of the specific ligand-receptor interaction. Thus, integrin-mediated ECM adhesion is inherently a mechanosensory process.

Fig. 3. Matrix status influences proteinase expression.

Left: Model for integrin-matrix interactions. The physical nature of the integrin-ligand interaction dictates the formation of specific signal transduction complexes on a cytoskeletal framework. (A, C) Low valency integrin occupancy can be induced by matrix protein fragments (A) or soluble integrin subunit-specific antibodies (C) and results in redistribution of the integrin to focal adhesions without activation of tyrosine kinase signaling or accumulation of cytoskeletal components. (B,D) Integrin occupancy by a multivalent ligand such as presented by intact extracellular matrix (B) leads to a more robust cytoplasmic response characterized by the accumulation of a large variety of cytoskeletal (e.g., talin, α-actinin, paxillin) and signaling (e.g., Src, ERK, JNK) molecules at the cytoplasmic face of the integrin. This response can be mimiced by integrin subunit-specific antibodies immobilized on beads (D). [adapted from Yamada, K. M. and Miyamoto, S. Curr Opin Cell Biol, 7: 681-689, 1995]

Right: Response of ovarian cancer cells to integrin engagement. upper panel: Cells in contact with a 2D collagen surface (‘thin deposit’ or ‘gelatin’) do not exhibit an altered proteinase response (appearance of active proteinase designated “MMP-2”); while cells cultured in 3D collagen gels (‘type I collagen’ or ‘type I CR collagen’) exhibit enhanced proMMP-2 activation (zymogram upper right) mediated via increased expression and activity of MT1-MMP (not shown). Lower panels. Use of integrin subunit-specific antibodies to mimic matrix status. Soluble antibodies ligate integrins (similar to 2D matrix) while bead-immobilized antibodes ligate and aggregate integrins. While soluble antibodies do not induce proMMP-2 activation (zymogram middle), cells respond to bead-immobilized α3- or β1-antibodies by enhanced proMMP-2 activation (zymogram lower right) mediated via increased expression and activity of MT1-MMP (not shown).

We are evaluating the role of matrix status in regulation of proteinase expression and acquisition of altered pericellular proteolytic potential. Our results suggest that multivalent integrin aggregation induces expression of several proteinases including the transmembrane enzyme membrane type 1 (MT1-)MMP and the secreted proteinases MMP-9 and urinary type plasminogen activator (uPA). MT1-MMP membrane dynamics are also influenced by integrin signaling. Further, the GPI-anchored uPA receptor (uPAR) can function as a lateral integrin ligand to modulate the magnitude and duration of integrin signaling, leading to potentiated upregulation of proteinase expression [Fig. 4]. Ongoing studies are aimed at evaluating uPAR/α3β1 interaction, lipid raft redistribution of the complex and the organization of the Src/MEK/ERK signaling complex. Additional experiments focus on the relationship between ECM status, mechanical loading and changes in proteinase expression together with mechanistic analysis of post-translational acquisition of pericellular proteolytic potential to provide a more detailed understanding of the role of these epignetic factors in tumor progression.

Fig. 4. Model for Modulation of Integrin Signaling by uPA/R. A metastasizing cell encounters a 3D ECM barrier, leading to multivalent integrin clustering. This activates a signal transduction pathway leading to transcriptional activation of the uPA promoter, secretion of the proteinase, and interaction with surface-associated uPAR. Membrane re-distribution of ligand-bound uPAR and integrins leads to lateral association of uPAR with α3β1 integrin, potentially altering the magnitude and duration of integrin signaling. Our studies indicate that highly metastatic OSCC may lose the capacity for matrix-regulated proteinase expression, resulting in consititutively high pericellular proteolysis and enhanced invasive capacity.

Receptor cross-talk in metastatic dissemination. Cell-cell and cell-matrix adhesion are essential for tissue cohesion during epithelial remodeling. Dysregulation of adhesion, via alteration in expression and/or function of adhesion molecules, has been implicated in loss of proliferation control and acquisition of a motile, invasive phenotype. Thus reversible modulation of cell-cell and cell-matrix adhesive events likely plays a critical role in remodeling during tumor progression. E-cadherin is a transmembrane cell-cell adhesion molecule that binds to cytoplasmic catenins, mediating association with the actin cytoskeleton. Disruption of adherens junctions releases β-catenin for degradation or translocation to the nucleus where it participates in transcriptional regulation of Tcf/Lef-responsive genes. Epidermal growth factor receptors are frequently overexpressed or constitutively activated in tumors. Aberrant EGFR activation may play a critical role in tumor progression and metastasis through regulation of cell-cell and cell-matrix interactions, cell migration, and induction of matrix degrading proteinases [Fig. 5]. As indicated above, integrins are also signal transducing molecules, and convergence between EGFR and integrin signaling pathways has been demonstrated in multiple cell types. We are currently testing the hypothesis that cross-talk between cadherin-, integrin- and EGFR-mediated signaling plays an active role in metastatic dissemination.

Fig. 5. Receptor cross-talk in metastatic dissemination. In a “resting” cell, integrins are engaged, cadherin-mediated adhesion is intact and proteinase expression is low. Several key events occur in an “activated” cell (for example, ligand or mutational activation of EGFR signaling, or other mechanisms to stimulate cadherin or integrin-mediated signal transduction). Disruption of cadherin-based cell adhesion and/or integrin clustering initiate signaling cascades that lead to increased proteinase production and activation. Secreted and membrane-associated proteinases may then participate in continued disruption of cadherin function through cleavage protein domains and generation of the soluble E-cadherin ectodomain. The shed E-cadherin ectodomain may potentiate junction dissolution through protein:protein interactions with endogenous E-cadherin. Subsequent acquisition of the invasive phenotype accompanies dissemination of cells from the primary tumor, potentiating intraperitoneal metastasis. A more detailed understanding of these processes may lead to novel strategies for therapeutic intervention(s) that prevent the establishment of metastases.

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