RESUMO
The CDC73 tumor suppressor gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. Its product, the Cdc73 protein, is a component of the RNA polymerase II and chromatin-associated human Paf1 complex (Paf1C). Here, we show that Cdc73 physically associates with the cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF) complexes that are required for the maturation of mRNA 3' ends in the cell nucleus. Immunodepletion experiments indicate that the Cdc73-CPSF-CstF complex is necessary for 3' mRNA processing in vitro. Microarray analysis of CDC73 siRNA-treated cells revealed INTS6, a gene encoding a subunit of the Integrator complex, as an in vivo Cdc73 target. Cdc73 depletion by siRNA resulted in decreased INTS6 mRNA abundance, and decreased association of CPSF and CstF subunits with the INTS6 locus. Our results suggest that Cdc73 facilitates association of 3' mRNA processing factors with actively-transcribed chromatin and support the importance of links between tumor suppression and mRNA maturation.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/fisiologia , Fator Estimulador de Clivagem/fisiologia , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator Estimulador de Clivagem/química , Humanos , Proteínas de Ligação a RNA , Proteínas Ribossômicas/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Targeted cancer therapies impede cancer cell growth by inhibiting the function of activated oncogene products. Patients with non-small cell lung cancer and somatic mutations of EGFR can have a dramatic response to treatment with erlotinib and gefitinib; different somatic mutations are associated with different times to progression and survival. In this study, the relative and absolute potencies of two distinct EGFR tyrosine kinase inhibitors, erlotinib and an investigational irreversible inhibitor, HKI-272, were found to vary significantly in a panel of Ba/F3 cells transformed by representative EGFR somatic mutations. HKI-272 more potently inhibited the primary exon 20 insertion mutants, the secondary erlotinib-resistance mutants including T790M and many erlotinib-sensitive mutants including L858R. In contrast, erlotinib is a more potent inhibitor of the major exon 19 deletion mutants than is HKI-272. Analyses of EGFR autophosphorylation patterns confirmed the mutation-specific variation in relative potency of these tyrosine kinase inhibitors. Our finding that distinct EGFR inhibitors are more effective in vitro for different mutant forms of the protein suggests that tyrosine kinase inhibitor treatment could be tailored to specific EGFR mutations. More broadly, these results imply that the development and deployment of targeted therapies should focus on inhibition of specific cancer-causing mutations, not only on the mutated target.
Assuntos
Alelos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Quinolinas/farmacologia , Substituição de Aminoácidos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Primers do DNA/química , Cloridrato de Erlotinib , Humanos , Camundongos , Fragmentos de Peptídeos/química , Fosforilação , Mutação Puntual , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Retroviridae/genética , Deleção de SequênciaRESUMO
BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.
Assuntos
Receptores ErbB/genética , Mutação de Sentido Incorreto , Quinazolinas/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Receptores ErbB/química , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Células NIH 3T3 , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Quinazolinas/química , Quinazolinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Mutation-specific cancer therapy has shown promising clinical efficacy. In non-small-cell lung cancer (NSCLC), the presence of mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase correlates with clinical response to small-molecule tyrosine kinase inhibitors. Here, we show that cells harboring the G776insV_G/C mutation in the related ERBB2 tyrosine kinase (also known as HER2 or Neu), present in a small percentage of NSCLCs, are sensitive to HKI-272, an irreversible dual-specific kinase inhibitor targeting both EGFR and ERBB2. In the ERBB2-mutant NCI-H1781 cell line, HKI-272 treatment inhibited proliferation by induction of G(1) arrest and apoptotic cell death. Furthermore, HKI-272 abrogated autophosphorylation of both ERBB2 and EGFR. Finally, Ba/F3 murine pro-B cells, engineered to express mutant ERBB2, became independent of interleukin-3 and sensitive to HKI-272. Thus, the subset of NSCLC patients with tumors carrying the ERBB2 G776insV_G/C mutation may benefit from treatment with HKI-272.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Quinolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Cloridrato de Erlotinib , Humanos , Interleucina-3/fisiologia , Neoplasias Pulmonares/genética , Quinazolinas/farmacologiaRESUMO
The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.
Assuntos
Mapeamento Cromossômico/métodos , DNA de Neoplasias/genética , Mutação , Neoplasias/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Neoplasias/diagnóstico , Sensibilidade e EspecificidadeRESUMO
The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation.