RESUMO

Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents, which participates in cell response to disease. We previously characterized VMP1 (Vacuole Membrane Protein 1) as an essential autophagy related protein that mediates autophagy in pancreatic diseases. We also demonstrated that VMP1-mediated autophagy is induced by HIF-1A (hypoxia inducible factor 1 subunit alpha) in colon-cancer tumor cell lines, conferring resistance to photodynamic treatment. Here we identify a new molecular pathway, mediated by VMP1, by which gemcitabine is able to trigger autophagy in human pancreatic tumor cell lines. We demonstrated that gemcitabine requires the VMP1 expression to induce autophagy in the highly resistant pancreatic cancer cells PANC-1 and MIAPaCa-2 that carry activated KRAS. E2F1 is a transcription factor that is regulated by the retinoblastoma pathway. We found that E2F1 is an effector of gemcitabine-induced autophagy and regulates the expression and promoter activity of VMP1. Chromatin immunoprecipitation assays demonstrated that E2F1 binds to the VMP1 promoter in PANC-1 cells. We have also identified the histone acetyltransferase EP300 as a modulator of VMP1 promoter activity. Our data showed that the E2F1-EP300 activator/co-activator complex is part of the regulatory pathway controlling the expression and promoter activity of VMP1 triggered by gemcitabine in PANC-1 cells. Finally, we found that neither VMP1 nor E2F1 are induced by gemcitabine treatment in BxPC-3 cells, which do not carry oncogenic KRAS and are sensitive to chemotherapy. In conclusion, we have identified the E2F1-EP300-VMP1 pathway that mediates gemcitabine-induced autophagy in pancreatic cancer cells. These results strongly support that VMP1-mediated autophagy may integrate the complex network of events involved in pancreatic ductal adenocarcinoma chemo-resistance. Our experimental findings point at E2F1 and VMP1 as novel potential therapeutic targets in precise treatment strategies for pancreatic cancer.

Assuntos

Autofagia , Desoxicitidina/análogos & derivados , Proteína p300 Associada a E1A/metabolismo , Fator de Transcrição E2F1/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Desoxicitidina/farmacologia , Proteína p300 Associada a E1A/genética , Fator de Transcrição E2F1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Células Tumorais Cultivadas , Gencitabina

RESUMO

During the last decade, autophagy has been pointed out as a central process in cellular homeostasis with the consequent implication in most cellular settings and human diseases pathology. At present, there is significant data available about molecular mechanisms that regulate autophagy. Nevertheless, autophagy pathway itself and its importance in different cellular aspects are still not completely clear. In this article, we are focused in four main aspects: (a) Induction of Autophagy: Autophagy is an evolutionarily conserved mechanism induced by nutrient starvation or lack of growth factors. In higher eukaryotes, autophagy is a cell response to stress which starts as a consequence of organelle damage, such as oxidative species and other stress conditions. (b) Initiation of Autophagy; The two major actors in this signaling process are mTOR and AMPK. These multitasking protein complexes are capable to summarize the whole environmental, nutritional, and energetic status of the cell and promote the autophagy induction by means of the ULK1-Complex, that is the first member in the autophagy initiation. (c) ULK1-Complex: This is a highly regulated complex responsible for the initiation of autophagosome formation. We review the post-transductional modifications of this complex, considering the targets of ULK1. (d)The mechanisms involved in autophagosome formation. In this section we discuss the main events that lead to the initial structures in autophagy. The BECN1-Complex with PI3K activity and the proper recognition of PI3P are one of these. Also, the transmembrane proteins, such as VMP1 and ATG9, are critically involved. The membrane origin and the cellular localization of autophagosome biogenesis will be also considered. Hence, in this article we present an overview of the current knowledge of the molecular mechanisms involved in the initial steps of mammalian cell autophagosome biogenesis.