Abstract B11: Porcupine inhibition stimulates osteosarcoma growth and non-canonical Wnt signaling

Abstract

Abstract Purpose: The objective of this study is to characterize the function of porcupine inhibitors in osteosarcoma and to determine the contribution of the porcupine enzyme to osteosarcoma cell growth. Each year, approximately 1000 children and adults in the United States are diagnosed with osteosarcoma (OS), the most common primary bone malignancy among children. Although overall survival is relatively good in OS patients, the need for aggressive surgery (often involving limb amputation) to prevent relapse significantly impacts the quality of life of survivors. OS has been well characterized to display aberrantly activated Wnt signaling. The necessity for active Wnt signaling pathways in OS patient tissue and cultured cell lines has been well established. Recently, a group of Wnt pathway inhibitors targeting the porcupine enzyme have been developed and used in treatment of pancreatic adenocarcinoma, BRAF mutant colorectal cancer and other tumors reported to exhibit aberrant Wnt signaling. Porcupine is a membrane bound O-acyl transferase which lipid-modifies the Wnt proteins prior to their secretion. This processing step regulates their release and paracrine or autocrine activity in stimulating both canonical and non-canonical Wnt signaling. These drugs have proven effective in initial studies which have applied them in vitro, in mouse models of Wnt- dependent cancers, and a recent phase 1 clinical trial. Our data show the first report of increased porcupine enzyme expression in human OS in vitro cell lines, compared to primary human osteoblasts. These findings have been confirmed by quantitative real-time PCR (qRT-PCR) analysis of PPN mRNA expression. Surprisingly, we found that inhibition of the porcupine enzyme with newly developed porcupine inhibitors significantly increased OS cell viability. While we observed only slight decreases in canonical Wnt pathway signaling with PPN inhibitor treatment (as measured by TOPflash reporter assay), we found that both NFATc1 and Cyclin D1 mRNA expression was upregulated. These data indicate that treatment with PPN inhibitors may stimulate a pro-tumorigenic response that is mediated primarily through non-canonical Wnt signaling. Continuing work is being done to establish the mechanism of action of these drugs, as well as unique roles that PPN-mediated non-canonical Wnt signaling may play in promoting OS cell growth. In conclusion, this study is aimed to provide insight into the function of a group of novel Wnt-targeting therapies in osteosarcoma, a disease which is well established to exhibit aberrant Wnt signaling. We aim to utilize these insights to rationally exploit Wnt signaling as a therapeutic measure for osteosarcoma. Methods: Western blotting was performed according to accepted methods using BioRad Criterion™ 10% gel and an antibody polyclonal to known porcupine splice variants (anti-rabbit) obtained from GeneTex. Cell viability was determined by CellTiter 96 ® (Promega) proliferation assay. C59 (Novartis) was purchased from Cayman Chemical and reconstituted in DMSO. Quantitative PCR performed using an Applied Biosystems ABI 7900HT Fast Real Time PCR system. FuGENE® HD utilized for transfection of TOPflash components. Citation Format: Carl T. Gustafson, Avudai Maran, Michael J. Yaszemski. Porcupine inhibition stimulates osteosarcoma growth and non-canonical Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B11.