RESUMO
We developed surface proteome signatures (SPS) for identification of new biomarkers playing a role in cancer drug resistance. SPS compares surface antigen expression of different cell lines by immunocytochemistry of a phage display antibody library directed to surface antigens of HT1080 fibrosarcoma cells. We applied SPS to compare the surface proteomes of two epithelial derived cancer cell lines, MCF7 and NCI/ADR-RES, which is drug resistant because of overexpression of the P-glycoprotein (P-gp) drug efflux pump. Surface proteomic profiling identified CD44 as an additional biomarker that distinguishes between these two cell lines. CD44 immunohistochemistry can distinguish between tumors derived from these lines and predict tumor response to doxorubicin in vivo. We further show that CD44 plays a role in drug resistance, independently of P-gp, in NCI/ADR-RES cells and increases expression of the antiapoptotic protein Bcl-xL. Our findings illustrate the utility of SPS to distinguish between cancer cell lines and their derived tumors and identify novel biomarkers involved in drug resistance.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Receptores de Hialuronatos/metabolismo , Proteoma/análise , Proteína bcl-X/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Humanos , Receptores de Hialuronatos/genética , Camundongos , Camundongos SCID , Proteoma/genética , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Neoplasias Encefálicas/patologia , Movimento Celular/fisiologia , Glioma/patologia , Proteínas de Membrana/fisiologia , Receptores Virais/fisiologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Adesões Focais/fisiologia , Glioma/genética , Glioma/metabolismo , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Ratos , Receptores Virais/biossíntese , Receptores Virais/deficiência , Receptores Virais/genética , Transdução de Sinais , TransfecçãoRESUMO
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.
Assuntos
Movimento Celular , Proteínas de Membrana/análise , Invasividade Neoplásica , Proteínas de Neoplasias/análise , Receptores Virais/análise , Linhagem Celular Tumoral , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Fibrossarcoma/secundário , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/secundário , Humanos , Imunoprecipitação/métodos , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Proteômica/métodos , Receptores Virais/fisiologiaRESUMO
Tumour cell invasiveness is crucial for cancer metastasis and is not yet understood. Here we describe two functional screens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shock protein 90 (hsp90). The hsp90 alpha isoform, but not hsp90 beta, is expressed extracellularly where it interacts with the matrix metalloproteinase 2 (MMP2). Inhibition of extracellular hsp90 alpha decreases both MMP2 activity and invasiveness. This role for extracellular hsp90 alpha in MMP2 activation indicates that cell-impermeant anti-hsp90 drugs might decrease invasiveness without the concerns inherent in inhibiting intracellular hsp90.
