Abstract 1645: Identification of human hedgehog palmitoylacyltransferase inhibitors to block pancreatic cancer

Abstract

Abstract Pancreatic adenocarcinoma is among the leading causes of cancer-related death in the US. The low response to standard therapy, and the high recurrence rates following treatment, necessitates the better understanding of the molecular basis of the disease, as well as the development of novel therapies. Here, we propose Human Hedgehog Palmitoylacyltransferase (Hhat) as a novel candidate for successful treatment of pancreatic cancer. Hhat catalyzes attachment of the fatty acid palmitate to the N-terminus of Sonic Hedgehog (Shh). Shh is a member of the Hedgehog family of secreted proteins which regulate embryonic development. Abnormal overexpression of Shh in the adult has been implicated as an important contributor to pancreatic adenocarcinoma. Shh produced by pancreatic cancer cells drives tumor growth through an autocrine signaling mechanism, and indirectly, through paracrine signaling to the cells in the tumor microenvironment, which then signal back to the pancreatic cancer cells. Shh signaling during development is dependent on Hhat-mediated palmitoylation of Shh. Consequently, we propose that inhibiting Hhat could also attenuate Shh signaling in pancreatic tumors, and thus offer a way to both decrease pancreatic cancer cell proliferation, as well as disrupt the communication between these cells and the tumor stroma. To test the importance of Hhat in pancreatic cancer, we used shRNA-mediated knockdown to reduce Hhat expression in two human pancreatic adenocarcinoma cell lines, AsPC-1 and Panc1. Hhat-deficient AsPC-1 cells exhibited decreased autocrine Shh signaling, which resulted in strong decreases in anchorage-dependent (80%) and anchorage-independent (70%) cell proliferation. Additionally, Hhat-deficient Panc1 cells showed decreased paracrine Shh signaling, as monitored by a co-culture assay of Panc1 cells and C3H10T1/2 mouse fibroblast reporter cells. Similar effects were obtained in cells depleted of Shh, confirming the importance of the Hhat-catalyzed palmitoylation for Shh signaling. The effect of Hhat knockdown on paracrine Shh signaling, and ultimately tumor growth, is also being investigated in a Panc1-based xenograft mouse model. Based on our finding that Hhat plays an important role in pancreatic cancer, we set out to identify small molecule inhibitors of Hhat. We employed high-throughput screening (HTS) using an in vitro assay that monitors palmitoylation of a Shh peptide by Hhat. This assay was optimized for analysis by Scintillation Proximity Assay (SPA) technology; we achieved a signal:noise ratio of ∼4:1 with Z’ values >0.5. Potential “hits” from the HTS have been obtained and confirmed in secondary screens involving both the peptide-based assay, as well as a cell-based viability assay. We are currently selecting compounds with the greatest inhibitory properties as candidates for development into lead compounds that block pancreatic tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1645. doi:10.1158/1538-7445.AM2011-1645