Colon cancer is the third most frequent cancer and the fourth most common cause of cancer-related mortality worldwide and the standard therapy is surgical resection plus adjuvant chemotherapy. Photodynamic therapy (PDT) has been proposed as an adjuvant therapy because it can prevent the tumor recurrence after surgical excision in colon cancer patients. Hypoxia is a common feature in solid tumors and leads to chemo/radioresistance. Recently, it has been shown that in response to hypoxia, cells can induce HIF-1α-mediated autophagy to survive in this hostile microenvironment. Moreover, hypoxia and autophagy have been implicated in the resistance to antitumor PDT. However, the molecular signals by which HIF-1α induces autophagy in the PDT context have not been studied yet. Here we evaluate the interplay between HIF-1α and autophagy as well as the underlying mechanism in the PDT resistance of colon cancer cells. Our study demonstrates that HIF-1α stabilization significantly increases VMP1-related autophagy through binding to hypoxia responsive elements in the VMP1 promoter. We show that HIF-1α-induced autophagy increases colon cancer cell survival as well as decreases cell death after PDT. Moreover, here we demonstrate that HIF-1α-induced autophagy is mediated by VMP1 expression, since the downregulation of VMP1 by the RNA interference strategy reduces HIF-1α-induced autophagy and cell survival after PDT. In conclusion, PDT induces autophagy as a survival mechanism and the induction of the novel HIF-1α/VMP1-autophagic pathway may explain, at least in part, the resistance of colon cancer cells to PDT. The knowledge of the molecular mechanisms involved in PDT resistance may lead to more accurate therapeutic strategies.

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.

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