Abstract LB-505: Molecular mechanisms of deciliation and malignant transformation in cholangiocarcinoma

Abstract

Abstract Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, the epithelial cells lining the biliary tree. Cholangiocytes normally express primary cilia extending from their apical plasma membrane into the ductal lumen. Our data show that CCA cells are devoid of primary cilia both in vivo and in vitro; therefore, ciliary loss may be associated with cancer development. Histone deacetylase 6 (HDAC6) is localized to the cell cytoplasm and functions as a tubulin deacetylase, and the activation of HDAC6 promotes ciliary disassembly. Therefore, we hypothesized that in CCA, overexpression of HDAC6 induces the resorption of primary cilia leading to derepression of tumorigenic signaling pathways and development of a malignant phenotype. To test this hypothesis, we analyzed the effect of pharmacologic and molecular deciliation on the phenotype of two normal human cholangiocyte cell lines (H69, NHC) using chloral hydrate treatment or shRNA silencing of IFT88, respectively. We found that deciliation increased the proliferation rate of cholangiocytes and induced anchorage independent growth and invasion. Furthermore, deciliation induced the activation of MAPK and Hh signaling pathways by 6- and 3-fold, respectively. To begin to understand why CCA cells lack cilia, we assessed expression of HDAC6 by western blot analysis on two different CCA cell lines (Hucct-1 and KMCH); we found increased expression (by 100%) of HDAC6 compared to normal cultured cholangiocytes (H69). Interestingly, overexpression of HDAC6 correlated with the decreased amount of its target, acetylated α-tubulin. Using confocal immunofluorescence microscopy, we also observed that HDAC6 was overexpressed in liver specimens from patients with CCA compared to normals. Furthermore, the in vitro overexpression of HDAC6 in normal cells induced deciliation, increased proliferation, anchorage independent growth, and invasion. Finally, to evaluate the effect of cilia restoration on tumor cells, we targeted HDAC6 by shRNA silencing or by the inhibitor tubastatin-A, and found that HDAC6 downregulation restored the expression of primary cilia in a cancer cell line, decreased cell proliferation (by 30%), and anchorage-independent growth (by 50%). When tumor cells unable to regenerate cilia by the stably transfection of IFT88 shRNA were used, the effects of tubastatin-A were abolished. In summary, expression of primary cilia is decreased in CCA by a mechanism involving the overexpression of HDAC6. Experimental deciliation of normal cholangiocytes induces a malignant phenotype as shown by increased cell proliferation, anchorage independent growth, invasion, and activation of Hh and MAPK signaling pathways. We speculate that the restoration of primary cilia in tumor cells by HDAC6 targeting may be a potential therapeutic approach for cholangiocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-505. doi:1538-7445.AM2012-LB-505