Abstract 3895: GLI1 induces the expression of bHLH type transcriptional repressor DEC2 in pancreatic cancer cells

Abstract

Abstract Pancreatic ductal adenocarcinoma (PDA), a major histological subtype of pancreatic cancer, is one of most highly lethal tumors because of its local invasion and distant metastasis during the early stage of disease. Therefore, it is desired to uncover the molecular characteristics of this cancer and to develop novel strategies for overcoming its worse prognosis. The activated Hedgehog (Hh) signaling due to the over-expression of Sonic hedgehog (Shh) is crucial for the cellular proliferation and the other malignant phenotypes in a variety of human cancers including PDA. The intracellular Hh signaling is mediated by zinc-finger type of transcriptional factors, GLI1-3, however, little is known about their target genes. Based on these circumstances, we newly established stable PDA cell lines to analyze the target genes of GLI1. We established Panc-1GLI1ER, a derivative of human PDA cell line Panc-1, which stably expresses GLI1 cDNA fused to AF2 domain of Estrogen Receptor (ER), and its control Panc-1ER expressing the fusion gene lacking GLI1. Using Agilent 44K cDNA expression microarray, we analyzed the gene expression status of these cell lines with or without β-estradiol (E2) induction. Genes were classified into eight groups according to its relative expression levels on the early (3hrs) and the late (24hrs) phases of E2 treatment. 277 genes which were up-regulated on the early phase in Panc-1GLI1ER but not Panc-1ER were determined as putative GLI1 direct targets. Among these genes, we isolated several bHLH type transcriptional repressors such as DEC2, HES1, HES5, and HEYL. In this report, we focused on the expression of DEC2 in PDA cells. As well as PTCH, one of the GLI1 direct targets, RT-PCR analysis showed DEC2-up-regulation on the early phase of E2 treatment only in Panc-1GLI1ER. Cyclophosphamide (CHX) treatment did not affect its up-regulation and double knockdown of GLI1 and GLI2 suppressed the endogenous expression in PDA cells, which supports the notion that DEC2 is one of the direct GLI1 target genes. For further investigation, we established tetracycline dependent GLI1 inducible cell lines PANC-1Tet/GLI1 and its control cells PANC-1Tet/Empty. Doxycycline (DOX) elimination from drinking water leads to GLI1 expression in KSN mouse-transplanted PANC-1Tet/GLI1. Immunohistochemical analysis of the tumor revealed that DEC2 was up-regulated by GLI1 induction in vivo. It was reported that HIF1α-DEC2 pathway represses MLH1 gene expression and leads to microsatellite instability. Indeed, immunohistochemical analysis of transplanted PANC-1Tet/GLI1 cells showed repression of MLH1 protein in association with GLI1-dependent DEC2 induction in vivo. There by, Shh-GLI1-DEC2 pathway represses MLH1 expression and could be inducing microsatellite instability in pancreatic carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3895.