Abstract
Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAF(E600), but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression.
Highlights
Cases would be diagnosed, and 32,300 would die from the disease (American Cancer Society Cancer Facts and Figures 2006)
Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in pancreatic adenocarcinomas, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh signaling pathway in pancreatic ductal adenocarcinoma (PDA)
Our study shows that oncogenic transformation of human pancreatic ductal epithelial (HPDE) cells by oncogenic KRAS is accompanied by enhanced GLI activation and that specific down-regulation of oncogenic KRAS activity inhibits Hh signaling in PDA cell lines with KRAS mutations
Summary
Reagents—Wortmannin, PD98059, U0126, and MG132 were purchased from Calbiochem. S-Trans,transfarnesylthiosalicylic acid (FTS) was provided by Dr Victor J. Mouse antiKRAS antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Culture and Transfection—HPDE-c7, an immortalized pancreatic ductal epithelial cell line, was provided by Dr MingSound Tsao (University of Toronto, Canada) and cultured in keratinocyte serum-free (KSF) medium supplemented with bovine pituitary extract and epidermal growth factor (Invitrogen). Panc-1 and AsPC-1 cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium (AsPC-1) or Dulbecco’s modified Essential medium (Panc-1) supplemented with 10% fetal bovine serum (Invitrogen). Retroviral supernatants were collected, filtered, and incubated with the target cells in the presence of 4 g/ml Polybrene (Sigma). Quantitative Real-time PCR— Total RNA was isolated from cultured cells as previously described [33]. The amount of target (2Ϫ⌬⌬CT) was obtained by normalized to endogenous reference (18 S) and relative to a calibrator
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