Abstract 5773: 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 rates to standard therapy, and the high recurrence rates following treatment, necessitate having 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 their proliferation, and is involved in paracrine signaling to the cells in the tumor microenvironment. Shh signaling is dependent on Hhat-mediated palmitoylation of Shh. Consequently, inhibiting Hhat could attenuate Shh signaling and offer a way to both decrease pancreatic cancer cell proliferation and disrupt the communication between these cells and the tumor stroma. We have taken two approaches to inhibit Hhat. First, levels of Shh or Hhat in two pancreatic adenocarcinoma cell lines, Panc05.04 and AsPC-1, were reduced using shRNA-mediated knockdown. qPCR analysis verified that specific decreases of 40-80% in Shh and Hhat mRNAs occurred with the respective shRNAs. Knockdown of each gene resulted in decreased Gli1 mRNA levels, confirming that Shh signaling was impaired. Importantly, both Hhat and Shh deficient cells exhibited decreases in anchorage-dependent (40-70%) and anchorage-independent (55-70%) cell proliferation. This suggests that Hhat activity is indeed necessary for Shh signaling and for cell growth and metastasis in pancreatic cancer cells. Our second approach for Hhat inhibition is based on our finding that Hhat is required for the proliferation of pancreatic cancer cells. We developed an in vitro assay to monitor palmitoylation of a Shh peptide by Hhat. This assay was then miniaturized and optimized for analysis by Scintillation Proximity Assay (SPA) technology. We have achieved a signal: noise ratio of ∼4:1 with Z’ values >0.5. We are currently performing high-throughput screening (HTS) to identify small molecule inhibitors of Hhat. Our HTS libraries contain over 80,000 compounds including natural and synthetic compounds, marketed drugs, and scaffolded pharmacophores and templates designed for rapid re-synthesis and analog generation. Potential “hits” from the HTS have already been obtained. Our goal is to choose 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 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 5773.