M. Sharon Stack

Proinvasive Properties of Ovarian Cancer Ascites-Derived Membrane Vesicles

Malignant ovarian ascites are rich in cellular components, membrane-bound vesicles, and soluble proteins. This study focused on the structure of membrane-bound vesicles and their ability to promote invasion in cultured malignant ovarian epithelium. Membrane vesicles were derived from women with stage I–IV malignant ovarian ascites and from nonmalignant gynecologic ascites. Isolated vesicles were characterized by immunofluorescence and Western blot analysis. Using gel zymography for matrix metalloproteinase (MMP) detection and a colorimetric assay for urokinase-type plasminogen activator (uPA) analysis, we analyzed the proteinase activities of MMP-2, MMP-9, and uPA from the prepared vesicles, whole cells isolated from ascites, and the cell-free ultracentrifuged supernatant. The invasiveness of established cultured malignant ovarian epithelium on addition of ascites-derived vesicles was tested using a Matrigel-based invasion assay. Fractionation of malignant ascites revealed that extracellular matrix-degrading proteinases including MMPs and uPA are localized preferentially in membrane vesicles. All malignant vesicles tested, regardless of cancer stage, stimulated invasion. Furthermore, the combination of ovarian cancer cells and membrane vesicles resulted in greater uPA activation than that of cells or vesicles alone. Membrane vesicles from malignant ascites were also found to contain activated MMP-2, MMP-9, and uPA. Our data suggest that vesicle-stimulated proteinase activation leads to increased extracellular matrix degradation, which may facilitate tumor cell invasion and metastasis.

Engagement of Collagen-Binding Integrins Promotes Matrix Metalloproteinase-9–Dependent E-Cadherin Ectodomain Shedding in Ovarian Carcinoma Cells

Reversible modulation of cell-cell adhesion, cell-matrix adhesion, and proteolytic activity plays a critical role in remodeling of the neoplastic ovarian epithelium during metastasis, implicating cadherins, integrins, and proteinases in i.p. metastatic dissemination of epithelial ovarian carcinoma (EOC). Aberrant epithelial differentiation is an early event in ovarian carcinogenesis; thus, in contrast to most carcinomas that lose E-cadherin expression with progression, E-cadherin is abundant in primary EOC. Metastasizing EOCs engage in integrin-mediated adhesion to submesothelial interstitial collagens and express matrix metalloproteinases (MMP) that facilitate collagen invasion, thereby anchoring secondary lesions in the submesothelial matrix. As metalloproteinases have also been implicated in E-cadherin ectodomain shedding, the current study was undertaken to model the effects of matrix-induced integrin clustering on proteinase-catalyzed E-cadherin ectodomain shedding. Aggregation of collagen-binding integrins induced shedding of an 80-kDa E-cadherin ectodomain [soluble E-cadherin (sE-cad)] in a MMP- and Src kinase-dependent manner, and sE-cad was prevalent in ascites from ovarian cancer patients. Expression of MMP-9 was elevated by integrin aggregation, integrin-mediated ectodomain shedding was inhibited by a MMP-9 function blocking antibody, and incubation of cells with exogenous MMP-9 catalyzed E-cadherin ectodomain shedding. In contrast to other tumors wherein sE-cad is released into the circulation, EOC tumors maintain direct contact with sE-cad-rich ascites at high concentration, and incubation of EOC cells with physiologically relevant concentrations of recombinant sE-cad disrupted adherens junctions. These data support a novel mechanism for posttranslational modification of E-cadherin function via MMP-9 induction initiated by cell-matrix contact and suggest a mechanism for promotion of EOC metastatic dissemination.

Cyclooxygenase-2 Functions as a Downstream Mediator of Lysophosphatidic Acid to Promote Aggressive Behavior in Ovarian Carcinoma Cells

Elevated levels of the bioactive lipid lysophosphatidic acid (LPA) are detectable in the majority of patients with both early- and late-stage ovarian cancer, suggesting that LPA promotes early events in ovarian carcinoma dissemination. LPA contributes to the development, progression, and metastasis of ovarian cancer in part by inducing the expression of genes that contribute to proliferation, survival, or invasion, including cyclooxgenase-2 (COX-2) and matrix metalloproteinase-2 (MMP-2). We have previously shown that LPA promotes proMMP-2 activation and MMP-2-dependent migration and invasion in ovarian cancer cells. The purpose of the current study was to determine whether the effect of LPA on acquisition of the metastatic phenotype in ovarian cancer cells is mediated via a COX-2-dependent mechanism. Immunohistochemical analysis of 173 ovarian tumors showed strong COX-2 immunoreactivity in 63% of tumor specimens, including 50% of borderline tumors. LPA increased COX-2 protein expression in a time- and concentration-dependent manner in two of three immortalized borderline ovarian epithelial cells as well as in four of six ovarian cancer cell lines. This was accomplished by both activation of the Edg/LPA receptor and LPA-mediated transactivation of the epidermal growth factor receptor, which increased COX-2 expression via the Ras/mitogen-activated protein kinase pathway. COX-2 also played a role in LPA-induced invasion and migration, as treatment with the COX-2 specific inhibitor NS-398 reduced LPA-induced proMMP-2 protein expression and activation and blocked MMP-dependent motility and invasive activity. These data show that COX-2 functions as a downstream mediator of LPA to potentiate aggressive cellular behavior.

Metastatic Dissemination of Human Ovarian Epithelial Carcinoma Is Promoted by α2β1-Integrin-Mediated Interaction with Type I Collagen

Metastatic dissemination of epithelial ovarian carcinoma is thought to be mediated via tumor cell exfoliation into the peritoneal cavity, followed by adhesion to and invasion through the mesothelium which overlies the contents of the peritoneal cavity. In this study, we have utilized short-term primary cultures to analyze the effect of specific extracellular matrix proteins on properties of human ovarian epithelial carcinoma cells which contribute to the invasive phenotype. Analysis of cell:matrix adhesive profiles indicated that ovarian carcinoma cells adhere preferentially to type I collagen. Immunoprecipitation analyses demonstrated the presence of the collagen-binding α2β1 integrin in biotin-labeled ovarian carcinoma cell membranes, and cellular adhesion was inhibited by blocking antibodies directed against the α2 and β1 integrin subunits. The α2β1-binding peptide Asp-Gly-Glu-Ala (DGEA) was also moderately effective at blocking adhesion to collagen relative to the control peptide Ala-Gly-Glu-Ala (AGEA). Analysis of cell motility on protein-coated colloidal gold coverslips demonstrated that ovarian carcinoma cells migrate preferentially on type I collagen coated surfaces. Type I collagen promoted migration in a concentration-dependent, saturable manner, with maximal migration observed at a collagen-coating concentration of 50 μg/ml. Migration on collagen was inhibited by antibodies directed against the α2 and β1 integrin subunits and by DGEA peptide, providing evidence for the role of the α2β1 integrin in ovarian carcinoma cell motility. Culturing ovarian carcinoma cells on type I collagen gels led to a significant increase in conversion of the matrix metalloproteinase 2 zymogen to the 66-kD form, suggesting that adhesion to collagen also influences matrix-degrading proteinases. These data suggest that α2β1-integrin-mediated interaction of ovarian carcinoma cells with type I collagen, a protein prevalent both in the mesothelial extracellular matrix and in the peritoneal cavity of ovarian carcinoma patients, may function on multiple levels to promote metastatic dissemination of ovarian carcinoma cells.

Functional relevance of urinary-type plasminogen activator receptor-α3β1 integrin association in proteinase regulatory pathways

Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin α3β1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of α3β1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of α3β1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/α3β1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (–1967) site in the uPA promoter blocked the uPAR/integrin-mediated transcriptional activation. Because uPAR is redistributed to clustered α3β1 integrins, the requirement for uPAR/α3β1 interaction in uPA regulation was assessed. Clustering of α3β1 in the presence of a peptide (α325) that disrupts uPAR/α3β1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin α3β1 clustering. These results were confirmed using a genetic strategy in which α3 null epithelial cells reconstituted with wild type α3 integrin, but not a mutant α3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/α3β1 binding using peptide α325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering ofα3β1 integrin promotes uPAR/α3β1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.

Down-regulation of Integrin α2 Surface Expression by Mutant Epidermal Growth Factor Receptor (EGFRvIII) Induces Aberrant Cell Spreading and Focal Adhesion Formation

Elevated expression or activity of the epidermal growth factor receptor (EGFR) is common in ovarian cancer and is associated with poor patient prognosis. A naturally occurring EGFR mutation termed variant III (EGFRvIII) has been detected in many human tumors, including those of the ovary. This mutant receptor does not bind EGF; however, it is constitutively active as detected by receptor dimerization, autophosphorylation, and stimulation of signal transduction cascades. To identify the consequences of EGFRvIII expression in ovarian tumor cells, we introduced EGFRvIII into the epithelial ovarian cancer cell line OVCA 433. The EGFRvIII-transfected cells displayed a motile phenotype, defects in cell spreading, and decreased integrin alpha2 protein expression as detected by Western blot analysis and flow cytometry. Inhibition of EGFRvIII catalytic activity using the EGFR-selective tyrphostin AG1478 restored integrin alpha2 expression within 4 to 8 hours after treatment. The modulation of integrin alpha2 expression corresponded to marked changes in the actin cytoskeleton as detected by redistribution of filamentous-actin. Furthermore, focal adhesions were evident only when EGFRvIII activity was inhibited. Together, these findings suggest that expression of the constitutively active mutant EGFRvIII promotes changes in cell shape and focal adhesion formation, mediated in part through specific modulation of integrin alpha2 expression and function. We conclude that EGFR-activating mutations, such as EGFRvIII, in ovarian cancer may contribute to a more aggressive disease.

Targeting the EGF Receptor for Ovarian Cancer Therapy

Ovarian carcinoma is the leading cause of death from gynecologic malignancy in the US. Factors such as the molecular heterogeneity of ovarian tumors and frequent diagnosis at advanced stages hamper effective disease treatment. There is growing emphasis on the identification and development of targeted therapies to disrupt molecular pathways in cancer. The epidermal growth factor (EGF) receptor is one such protein target with potential utility in the management of ovarian cancer. This paper will discuss contributions of EGF receptor activation to ovarian cancer pathogenesis and the status of EGF receptor inhibitors and EGF receptor targeted therapies in ovarian cancer treatment.

Virology and molecular pathogenesis of HPV (human papillomavirus)- associated oropharyngeal squamous cell carcinoma

The current literature fully supports HPV (human papillomavirus)-associated OPSCC (oropharyngeal squamous cell carcinoma) as a unique clinical entity. It affects an unambiguous patient population with defined risk factors, has a genetic expression pattern more similar to cervical squamous cell carcinoma than non-HPV-associated HNSCC (head and neck squamous cell carcinoma), and may warrant divergent clinical management compared with HNSCC associated with traditional risk factors. However, a detailed understanding of the molecular mechanisms driving these differences and the ability to exploit this knowledge to improve clinical management of OPSCC has not yet come to fruition. The present review summarizes the aetiology of HPV-positive (HPV+) OPSCC and provides a detailed overview of HPV virology and molecular pathogenesis relevant to infection of oropharyngeal tissues. Methods of detection and differential gene expression analyses are also summarized. Future research into mechanisms that mediate tropism of HPV to oropharyngeal tissues, improved detection strategies and the pathophysiological significance of altered gene and microRNA expression profiles is warranted.

Lysophosphatidic acid down-regulates stress fibers and up-regulates pro-matrix metalloproteinase-2 activation in ovarian cancer cells.

Epithelial ovarian cancer (EOC) is asymptomatic at early stages and is often diagnosed late when tumor cells are highly metastatic. Lysophosphatidic acid (LPA) has been implicated in ovarian oncogenesis as levels of this lipid are elevated in patient ascites and plasma. Because the underlying mechanism governing LPA regulation of matrix metalloproteinase-2 (MMP-2) activation remains undefined, we investigated the relationship between LPA-induced changes in actin microfilament organization and MMP-2 enzymatic activity. We report that when cells were cultured at a high density, LPA mediated stress fiber and focal adhesion disassembly and significantly repressed RhoA activity in EOC cells. Inhibition of Rho-kinase/ROCK enhanced both LPA-stimulated loss of stress fibers and pro–MMP-2 activation. In contrast, expression of the constitutively active RhoA(G14V) mutant diminished LPA-induced pro–MMP-2 activation. LPA had no effects on membrane type 1–MMP or tissue inhibitor of metalloproteinase-2 expression, but up-regulated MMP-2 levels, contributing to the induction of MMP-2 activation. Interestingly, when cells were cultured at a low density, stress fibers were present after LPA stimulation, and ROCK activity was required for EOC cell migration. Collectively, these results were consistent with a model in which LPA stimulates the metastatic dissemination of EOC cells by initiating loss of adhesion and metalloproteinase activation. (Mol Cancer Res 2007;5(2):121–31)

Microenvironmental Regulation of Ovarian Cancer Metastasis

Tumors arising from the ovarian surface epithelium (OSE) account for the vast majority of ovarian malignancies; however, the etiology of epithelial ovarian cancer (EOC) remains poorly understood, and the analysis of early events in ovarian carcinogenesis is limited by the relative lack of early-stage tumors for study. The normal OSE is a single layer of mesodermally derived cells that exhibit the remarkable ability to transition between epithelial and fibroblastic phenotypes in response to microenvironmental cues. Such phenotypic plasticity is usually limited to immature, regenerating, or neoplastic epithelium. Unlike most carcinomas that initially de-differentiate during neoplastic progression, ovarian carcinomas undergo a mesenchymal-epithelial transition and acquire a more differentiated epithelial phenotype resulting in significant morphologic heterogeneity as tumors acquire increasingly complex differentiation reminiscent of the highly specialized epithelia of Mullerian duct origin. Differentiated primary ovarian tumors acquire morphologic characteristics of the fallopian tube (serous carcinoma), endometrium (endometrioid carcinoma), endocervix (mucinous carcinoma), and vagina (clear cell carcinoma). More recently, classification of ovarian tumors into low-grade (type I) versus highgrade (type II) malignancies has been proposed based on presumed pathways leading to tumorigenesis, rather than histopathologic characteristics. Lowgrade carcinomas are more indolent, develop from a recognized precursor lesion, and are often confined to the ovary at diagnosis. In contrast, highgrade tumors are clinically aggressive at initial presentation, are not associated