RESUMEN
We recently identified the immunoglobulin-CAM CD155/PVR (the poliovirus receptor) as a regulator of cancer invasiveness and glioma migration, but the mechanism through which CD155/PVR controls these processes is unknown. Here, we show that expression of CD155/PVR in rat glioma cells that normally lack this protein enhances their dispersal both in vitro and on primary brain tissue. CD155/PVR expression also reduced substrate adhesion, cell spreading, focal adhesion density, and the number of actin stress fibers in a substrate-dependent manner. Furthermore, we found that expression of CD155/PVR increased Src/focal adhesion kinase signaling in a substrate-dependent manner, enhancing the adhesion-induced activation of paxillin and p130Cas in cells adhering to vitronectin. Conversely, depletion of endogenous CD155/PVR from human glioma cells inhibited their migration, increased cell spreading, and down-regulated the same signaling pathway. These findings implicate CD155/PVR as a regulator of adhesion signaling and suggest a pathway through which glioma and other cancer cells may acquire a dispersive phenotype.
Asunto(s)
Neoplasias Encefálicas/patología , Movimiento Celular/fisiología , Glioma/patología , Proteínas de la Membrana/fisiología , Receptores Virales/fisiología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Adhesión Celular/fisiología , Línea Celular Tumoral , Adhesiones Focales/fisiología , Glioma/genética , Glioma/metabolismo , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratas , Receptores Virales/biosíntesis , Receptores Virales/deficiencia , Receptores Virales/genética , Transducción de Señal , TransfecciónRESUMEN
Apoptotic evasion is a hallmark of cancer and its resistance to chemotherapeutic drugs. Identification of cellular proteins that mediate apoptotic programs is a critical step toward the development of therapeutics aimed at overcoming apoptosis resistance. We developed an innovative high-throughput screen to identify proteins that modulate Fas ligand-mediated apoptosis using fluorophore-assisted light inactivation (HTS-FALIpop). The FALI protein knockdown strategy was coupled to a caspase activity assay with the ability to detect both proapoptotic and antiapoptotic surface molecules expressed by HT-1080 human fibrosarcoma cells. FALI of the Fas receptor (Fas/CD95) using a fluorescein-conjugated anti-Fas antibody abrogated Fas ligand-mediated caspase activation. Ninety-six single-chain variable fragment antibodies (scFv), selected for binding to the surface of HT-1080 cells, were screened by HTS-FALIpop. Three of the scFvs caused decreases in caspase induction after FALI of their protein targets. One of the targets of these positive scFvs was identified as CD44 and was validated by performing FALI using a CD44-specific monoclonal antibody, which resulted in similar protection from Fas apoptosis. CD44-targeted FALI was antiapoptotic in multiple human cancer cell lines, including both Fas signaling type I and II cells, and was also protective against other ligands of the tumor necrosis factor death receptor family. FALI of CD44 inhibited formation and activation of the death-inducing signaling complex, suggesting that CD44 regulates Fas at the cell surface. This mechanism of death receptor regulation represents a novel means of apoptosis modulation that could be exploited by pharmacologic agents.
Asunto(s)
Apoptosis , Receptores de Hialuranos/metabolismo , Fragmentos de Inmunoglobulinas/inmunología , Proteómica , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptor fas/metabolismo , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Caspasas/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte , Activación Enzimática , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Humanos , Receptores de Hialuranos/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Espectrometría de Masas , Ratones , Células Tumorales Cultivadas , Receptor fas/genéticaRESUMEN
BACKGROUND: Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration. METHODS: We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion. RESULTS: Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and alphav-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate. CONCLUSION: These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis.