Abstract 36: Human ecdysoneless protein: Regulator of cholesterol homeostasis in pancreatic cancer

Abstract

Abstract The human Ecdysoneless protein (hEcd) is a human orthologue of the D.melanogaster ecdysoneless protein, which is highly conserved from insects to mammals and identified as one of the factors regulating the synthesis of the steroid hormone ecdysone. Ecd is required autonomously throughout the body and its deficiency can lead to reproductive or developmental defects. hEcd, also known as human suppressor of GCR2 gene (hSGT) since it can rescue growth defects in S.cerevisiae mutants with a deletion of the growth control regulatory genes 2 (GCR2), a transcriptional activator of the glycolytic gene expression. Recent investigations revealed that hEcd interacts with murine double minute-2 and stabilizes p53 by inhibiting mdm-2 mediated degradation of p53. Another study done on MEFs generated from Ecd conditional knockout mice showed evidence that Ecd interacts with retinoblastoma protein at the pocket domain and competes with E2F for association with hypophosphorylated Rb, thereby regulating E2F target gene expression and cell cycle progression. In this study, we have analyzed the expression of Ecd in PanIN and Pancreatic adenocarcinoma tissues as well as pancreatic cancer cell lines. The studies show that Ecd is overexpressed in pancreatic cancer samples as compared to normal pancreas. Therefore based on its role as a cell cycle regulator and its increased expression in pancreatic tumors, we hypothesized that Ecd might act as a potential modulator contributing to the pathogenesis of pancreatic cancer cells. Stable knockdown clones of Ecd were established in CD/HPAF pancreatic cancer cell line using two different shRNA constructs, which led to reduced proliferation rate in vitro and decreased tumor weight in vivo. Further, microarray analysis of genes in Ecd knocked-down vs scrambled cells followed by bioinformatics analysis revealed that the major pathway affected due to Ecd downregulation is the cholesterol biosynthetic and regulatory pathway. The genes which were affected by more than two-fold in Ecd knocked-down cells, encoded key cholesterol biosynthetic pathway enzymes such as FDFT1, NSDHL, DHCR7 and CYP51A1 along with INSIG1, a protein which acts as a cholesterol level sensor in cells. This was further confirmed by Quantitative Real-Time PCR analysis. Flow-cytometric analysis for cholesterol-specific filipin staining showed increased cholesterol content in Ecd-kd cells vs the scrambled cells. Additionally, insulin stimulated GLUT-4 membrane translocation, one of the cholesterol-dependent pathways was also found to be affected with Ecd knockdown cells showing higher glucose uptake than the scrambled cells. Cholesterol being a major component of cells and a mediator of endocytic and cell signaling pathways, we conclude that Ecd contributes to pancreatic cancer pathogenesis by disrupting multiple cholesterol-dependent pathways. 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 36.