RESUMO
Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer death in the western world, with a 5-year survival rate below 5%. Murine double minute 2 (Mdm2) is an important negative regulator of the tumor suppressor p53. Reactivation of wild-type p53 is a promising treatment strategy, and inhibitors of Mdm2 have already entered clinical trials. To investigate the effects of Mdm2 inhibitors in PDAC, we used a murine cell line platform with a genetically defined status of p53. Here, we describe that Mdm2 inhibitors can act on a subset of murine PDAC cell lines p53 independently. Furthermore, we observed that Mdm2 inhibitors increase the sensitivity of murine PDAC cell lines toward topoisomerase II inhibitors by inducing effector caspase-independent cell death. The combination of Mdm2 inhibitors with topoisomerase II inhibitors acts independent of the survival factor NFκB/RelA. Mechanistically, Mdm2 inhibitors increase topoisomerase II inhibitor-induced DNA double-strand breaks. We show that Mdm2 binds to Nbs1 of the Mre11-Rad50-Nijmegen breakage syndrome (Nbs) 1 DNA repair complex. In addition, we provide evidence that Mdm2 inhibitors delay DNA repair. These findings may help to design novel therapeutic strategies to overcome therapeutic resistance of PDAC.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Indóis/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirrolidinonas/farmacologia , Inibidores da Topoisomerase II/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Western Blotting , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Etoposídeo/farmacologia , Imidazóis/farmacologia , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Neoplasias Pancreáticas/metabolismo , Piperazinas/farmacologia , GencitabinaRESUMO
A placebo-controlled phase 3 trial demonstrated that the epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine was especially efficient in a pancreatic ductal adenocarcinoma (PDAC) subgroup of patients developing skin toxicity. However, EGFR expression was not predictive for response, and markers to characterize an erlotinib-responding PDAC group are currently missing. In this work, we observed high erlotinib IC50 values in a panel of human and murine PDAC cell lines. Using EGFR small interfering RNA, we detected that the erlotinib response was marginally influenced by EGFR. To find novel EGFR targets, we used an unbiased chemical proteomics approach for target identification and quality-controlled target affinity determination combined with quantitative mass spectrometry based on stable isotope labeling by amino acids in cell culture. In contrast to gefitinib, we observed a broad target profile of erlotinib in PDAC cells by quantitative proteomics. Six protein kinases bind to erlotinib with similar or higher affinity (K(d) = 0.09-0.358 µM) than the EGFR (K(d) 0.434 µM). We provide evidence that one of the novel erlotinib targets, ARG, contributes in part to the erlotinib response in a PDAC cell line. Our data show that erlotinib is a multikinase inhibitor, which can act independent of EGFR in PDAC. These findings may help to monitor future erlotinib trials in the clinic.