RESUMO
Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anticancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here, we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways and, consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo.
Assuntos
Transformação Celular Neoplásica/patologia , Dano ao DNA/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Animais , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas ras/metabolismoRESUMO
The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins.
