RESUMO
Combining RNAi in cultured cells and analysis of mutant animals, we probed the roles of known Piwi-interacting RNA (piRNA) pathway components in the initiation and effector phases of transposon silencing. Squash associated physically with Piwi, and reductions in its expression led to modest transposon derepression without effects on piRNAs, consistent with an effector role. Alterations in Zucchini or Armitage reduced both Piwi protein and piRNAs, indicating functions in the formation of a stable Piwi RISC (RNA-induced silencing complex). Notably, loss of Zucchini or mutations within its catalytic domain led to accumulation of unprocessed precursor transcripts from flamenco, consistent with a role for this putative nuclease in piRNA biogenesis.
Assuntos
Drosophila melanogaster/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Células Cultivadas , Elementos de DNA Transponíveis/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Feminino , Mutação , RNA Helicases/genética , RNA Helicases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N'-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-beta-mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-beta-dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-beta are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Interleucina-6/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Fator de Crescimento Transformador beta/metabolismo , Animais , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/genética , Cloridrato de Erlotinib , Gefitinibe , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/genética , Interleucina-6/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Camundongos Nus , Mutação , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths in the United States. The deoxynucleoside analogue gemcitabine is among the most effective therapies to treat PDAC, however, nearly all patients treated with gemcitabine either fail to respond or rapidly develop resistance. One hallmark of PDAC is a striking accumulation of stromal tissue surrounding the tumor, and this accumulation of stroma can contribute to therapy resistance. To better understand how stroma limits response to therapy, we investigated cell-extrinsic mechanisms of resistance to gemcitabine. Conditioned media from pancreatic stellate cells (PSC), as well as from other fibroblasts, protected PDAC cells from gemcitabine toxicity. The protective effect of PSC-conditioned media was mediated by secretion of deoxycytidine, but not other deoxynucleosides, through equilibrative nucleoside transporters. Deoxycytidine inhibited the processing of gemcitabine in PDAC cells, thus reducing the effect of gemcitabine and other nucleoside analogues on cancer cells. These results suggest that reducing deoxycytidine production in PSCs may increase the efficacy of nucleoside analog therapies. SIGNIFICANCE: This study provides important new insight into mechanisms that contribute to gemcitabine resistance in PDAC and suggests new avenues for improving gemcitabine efficacy.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Estreladas do Pâncreas/efeitos dos fármacos , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Gencitabina , Neoplasias PancreáticasRESUMO
Glioblastoma (GBM) is a devastating malignancy with few therapeutic options. We identify PRMT5 in an in vivo GBM shRNA screen and show that PRMT5 knockdown or inhibition potently suppresses in vivo GBM tumors, including patient-derived xenografts. Pathway analysis implicates splicing in cellular PRMT5 dependency, and we identify a biomarker that predicts sensitivity to PRMT5 inhibition. We find that PRMT5 deficiency primarily disrupts the removal of detained introns (DIs). This impaired DI splicing affects proliferation genes, whose downregulation coincides with cell cycle defects, senescence and/or apoptosis. We further show that DI programs are evolutionarily conserved and operate during neurogenesis, suggesting that they represent a physiological regulatory mechanism. Collectively, these findings reveal a PRMT5-regulated DI-splicing program as an exploitable cancer vulnerability.
Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Íntrons , Proteína-Arginina N-Metiltransferases/fisiologia , Animais , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/genética , Ensaios de Triagem em Larga Escala , Humanos , Isoquinolinas/farmacologia , Camundongos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Pirimidinas/farmacologia , Splicing de RNARESUMO
Targeting tyrosine kinase receptors (RTKs) with specific Abs is a promising therapeutic approach for cancer treatment, although the molecular mechanism(s) responsible for the Abs' biological activity are not completely known. We targeted the transmembrane RTK for hepatocyte growth factor (HGF) with a monoclonal Ab (DN30). In vitro, chronic treatment of carcinoma cell lines resulted in impairment of HGF-induced signal transduction, anchorage-independent growth, and invasiveness. In vivo, administration of DN30 inhibited growth and metastatic spread to the lung of neoplastic cells s.c. transplanted into immunodeficient nu/nu mice. This Ab efficiently down-regulates HGF receptor through a molecular mechanism involving a double proteolytic cleavage: (i) cleavage of the extracellular portion, resulting in "shedding" of the ectodomain, and (ii) cleavage of the intracellular domain, which is rapidly degraded by the proteasome. Interestingly, the "decoy effect" generated by the shed ectodomain, acting as a dominant negative molecule, enhanced the inhibitory effect of the Ab.