Abstract 4619: Protein palmitoylation in non-small cell lung cancer (NSCLC): DHHC5 palmitoyltransferase as a potential therapeutic target

Abstract

Abstract Post-translational palmitoylation of intracellular proteins is mediated by protein palmitoyltransferases (PAT) belonging to the DHHC family, which share a common catalytic Asp-His-His-Cys (DHHC) motif. According to a genome-wide siRNA screen scoring for growth suppression in over 50 non-small cell lung cancer (NSCLC) cell lines, DHHC family members are strikingly over-represented. Among the tested DHHC palmitoyltransferases, knockdown of DHHC5 showed cell growth inhibition in many cell lines in the initial screen. To further investigate the growth suppression in response to DHHC5 depletion, transient and stable knockdowns of DHHC5 were undertaken in a dozen of NSCLC cell lines identified in the genome-wide screen using siRNAs and lentiviral shRNAs. Such knockdown was also performed in three immortalized human lung bronchial epithelial cell lines (HBECs). The gene expression and protein levels of DHHC5 were analyzed by RT-PCR and immunoblotting, both of which showed efficient knockdown levels. Cell proliferation and anchorage-dependent/independent colony formation assays indicated that DHHC5 knockdown led to cell growth arrest and decreased cell clonogenicity in most of the tested NSCLC cell lines in vitro, but not in the HBECs. H1299, H358 and H2009 cell lines were particularly dependent on DHHC5 expression and the growth-suppression phenotype of DHHC5 stable knockdown H1299/H358/H2009 cells was rescued by overexpression of DHHC5. The catalytically inactive DHHC5 mutant was not able to provide rescue. In vivo tumor formation assays were then carried out by injecting H1299 and H358 control cells and DHHC5 stable knockdown cells subcutaneously into NOD/SCID mice. DHHC5 knockdown led to absence or dramatically decreased tumor xenograft formation. Furthermore, a Tet-On inducible system with TRIPZ shRNA lentiviral delivery was established in H1299. Mouse xenografts using this H1299 Tet-on inducible cell line showed similar results. As DHHC5 function is needed for optimal lung cancer cell growth, it will be important to identify physiologically relevant substrates in order to develop inhibitors of DHHC5 function. Future plans include microarray and protein kinase array analysis to distinguish possible signaling pathways in which DHHC5 may be involved, as well as palmitoyl-protein profiling of normal and knockdown DHHC5 cell lines by mass spectrometry. Citation Format: Hui Tian, Jui-Yun Lu, Chunli Shao, Kenneth Huffman, Ryan Carstens, John D. Minna, Sandra L. Hofmann. Protein palmitoylation in non-small cell lung cancer (NSCLC): DHHC5 palmitoyltransferase as a potential therapeutic target. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4619. doi:10.1158/1538-7445.AM2014-4619