Role of myofibroblasts in innate chemoresistance of pancreatic carcinoma—Epigenetic downregulation of caspases

Abstract

We recently reported on continuous tumor-stroma interactions essentially contributing to chemoresistance of pancreatic ductal adenocarinoma (PDAC) cells. As demonstrated here, long-term coculture with pancreatic myofibroblasts representing the main stromal compartment of PDAC resulted in a chemoresistant phenotype in the pancreatic ductal epithelial cell line H6c7 as well as in the chemosensitive PDAC cell line T3M4. This involved a reduced expression of caspases and the caspase inducing transcription factor STAT1, both caused by diminished gene transcription. The DNA-methylation inhibitor 5-azadeoxycytidine enhanced caspase and STAT1 expression in cocultured H6c7 and T3M4 cells along with an increased chemosensitivity, indicating a role for CpG DNA-hypermethylation in the downregulation of these crucial apoptosis mediators. Cocultured H6c7 and T3M4 cells exhibited elevated nuclear levels of DNA-methyltransferase-1 (DNMT1). Silencing of DNMT1 expression by siRNA increased expression of caspases and STAT1 and restored chemosensitivity. In SCID mice, tumors arising from coinoculated T3M4 cells and myofibroblasts (co-tumors) responded less towards chemotherapy than mono-tumors, exhibiting decreased apoptosis, no remission and reduced expression of caspases and STAT1. These data underscore the role of myofibroblasts in chemoresistance of PDAC and point to the importance of caspases as central target structures of epigenetic regulation in this scenario. Furthermore, an activated microenvironment might apparently promote the manifestation of chemoresistance already in premalignant precursor cells at early stages of PDAC tumorigenesis. ' 2008 Wiley-Liss, Inc.Key words: chemoresistance; pancreatic cancer; epigenetic gene silencing; myofibroblasts Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly malignant phenotype associated with rapid tumor progression and metastasis. 1,2 In Western countries, pancreatic cancer is 4th in the rank order of fatal tumor diseases exhibiting a still increasing prevalence. 3 The conventional therapy of PDAC still proves to be very difficult and poorly effective due to the preexisting (innate) or acquired resistance of PDAC cells towards chemotherapeutic drugs. One strategy by which tumor cells can adopt a chemoresistant phenotype is the protection from apoptosis. Because PDAC is largely composed of mesenchymal stromal cells, we recently started to investigate the impact of pancreatic myofibroblasts on the development of chemoresistance in these tumor cells. Using a transwell coculture model including murine pancreatic myofibroblasts and the chemosensitive human PDAC cell lines T3M4 and PT45-P1, we could show that during 1 week of coculture, these tumor cells become highly chemoresistant by an efficient protection from drug-induced apoptosis, apparently mediated by soluble factors released by the myofibroblasts. Since in this model system tumor and stromal cells originated from different species, we were able to distinguish the origin of all soluble factors. In this scenar...

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