RESUMO
Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.
Assuntos
Carcinoma Hepatocelular/genética , Genoma Humano , Neoplasias Hepáticas/genética , Mutação , Sequência de Aminoácidos , Carcinoma Hepatocelular/virologia , DNA Viral/genética , Feminino , Vírus da Hepatite B/genética , Humanos , Janus Quinase 1/genética , Neoplasias Hepáticas/virologia , Masculino , Dados de Sequência Molecular , Fatores de Transcrição STAT/genética , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genética , Integração Viral , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
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.
Assuntos
Apoptose , Receptores de Hialuronatos/metabolismo , Fragmentos de Imunoglobulinas/imunologia , Proteômica , Receptores do Fator de Necrose Tumoral/metabolismo , Receptor fas/metabolismo , Animais , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Caspases/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Ativação Enzimática , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Humanos , Receptores de Hialuronatos/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Espectrometria de Massas , Camundongos , Células Tumorais Cultivadas , Receptor fas/genéticaRESUMO
There is a critical need for global methods that allow for high-throughput assessment of cellular function for clinical and basic scientists working in both academia and the pharmaceutical industry. These methods typically couple systematic inactivation strategies with high-throughput cell-based assays that facilitate rapid target validation. We present here a survey of these technologies and their applications. We discuss their promise and limitations in addressing the vast number of candidate molecules of disease relevance that are emerging from genomics and proteomics.
Assuntos
Fenômenos Fisiológicos Celulares , Programas de Rastreamento/métodos , Animais , Testes Genéticos , HumanosRESUMO
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.