e21035 Background: EMT renders neoplastic cancer cells the ability to migrate and to invade distant organs. The hallmark of EMT is the loss of E-cadherin, which is a prerequisite for epithelial tumor cell invasion. In pancreatic cancer, loss of tumor E-cadherin is an independent predictor of poor outcome. Aims: To analyze the effect of pancreatic fibroblasts (PF) on inducing EMT in pancreatic cancer cells and to identify the transcription factors (Snail, Slug, ZEB1, ZEB2) that mediate EMT process. Methods: Human PFs were isolated from human pancreatic specimens obtained from chronic pancreatitis and from unaffected margins of pancreatic adenocarcinoma and serous cistoadenoma. PF were cultured until complete cellular activation, as assessed by expression of α-smooth muscle actin, vimentin and fibronectin. Human pancreatic cancer cells Panc-1 were exposed to PF conditioned medium (PF-CM) and EMT analyzed by cell morphology, migration, and E-cadherin expression (quantitative RT-PCR and immunoblot). Gene expression of Snail, Slug, ZEB1, and ZEB2 was analyzed by quantitative RT-PCR, and their activity modulated by siRNA Results: Conditioned media from all types of activated PFs induced EMT changes in Panc-1 cells, as shown by 1) morphological transition from cobblestone shaped to fibroblast-like cells, 2) stimulation of cell migration, and 3) E-cadherin down–regulation; mRNA expression of Snail transiently increased at 30 min after exposure to PF returning to basal levels afterwards; mRNA levels of ZEB1 were not up-regulated upon exposure to PF-CM. However, ZEB1 protein greatly accumulated after 48h incubation with PF-CM, suggesting that PF prevent ZEB1 degradation in Panc-1 cells. Combined RNA downregulation of ZEB1 and ZEB2, but not of Snail and/or Slug, suppressed E-cadherin repression induced by PF. Conclusions: Activated PFs promote the invasive phenotype of pancreatic cancer cells through ZEB1 and ZEB2 activation.
Src kinase inhibition restores E-cadherin expression in dasatinib-sensitive pancreatic cancer cells
