Gradilone, Sergio A.

Abstract Primary cilia are multisensory organelles recently found to be absent in some tumor cells, but the mechanisms of deciliation and the role of cilia in tumor biology remain unclear. Cholangiocytes, the epithelial cells lining the biliary tree, normally express primary cilia and their interaction with bile components regulates multiple processes, including proliferation and transport. Using cholangiocarcinoma as a model, we found that primary cilia are reduced in cholangiocarcinoma by a mechanism involving histone deacetylase 6 (HDAC6). The experimental deciliation of normal cholangiocyte cells increased the proliferation rate and induced anchorage-independent growth. Furthermore, deciliation induced the activation of mitogen-activated protein kinase and Hedgehog signaling, two important pathways involved in cholangiocarcinoma development. We found that HDAC6 is overexpressed in cholangiocarcinoma and overexpression of HDAC6 in normal cholangiocytes induced deciliation and increased both proliferation and anchorage-independent growth. To evaluate the effect of cilia restoration on tumor cells, we targeted HDAC6 by short hairpin RNA (shRNA) or by the pharmacologic inhibitor, tubastatin-A. Both approaches restored the expression of primary cilia in cholangiocarcinoma cell lines and decreased cell proliferation and anchorage-independent growth. The effects of tubastatin-A were abolished when cholangiocarcinoma cells were rendered unable to regenerate cilia by stable transfection of IFT88-shRNA. Finally, inhibition of HDAC6 by tubastatin-A also induced a significant decrease in tumor growth in a cholangiocarcinoma animal model. Our data support a key role for primary cilia in malignant transformation, provide a plausible mechanism for their involvement, and suggest that restoration of primary cilia in tumor cells by HDAC6 targeting may be a potential therapeutic approach for cholangiocarcinoma. Cancer Res; 73(7); 2259–70. ©2013 AACR.

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

Exosomes are small extracellular vesicles that are thought to participate in intercellular communication. Recent work from our laboratory suggests that, in normal and cystic liver, exosome-like vesicles accumulate in the lumen of intrahepatic bile ducts, presumably interacting with cholangiocyte cilia. However, direct evidence for exosome-ciliary interaction is limited and the physiological relevance of such interaction remains unknown. Thus, in this study, we tested the hypothesis that biliary exosomes are involved in intercellular communication by interacting with cholangiocyte cilia and inducing intracellular signaling and functional responses. Exosomes were isolated from rat bile by differential ultracentrifugation and characterized by scanning, transmission, and immunoelectron microscopy. The exosome-ciliary interaction and its effects on ERK1/2 signaling, expression of the microRNA, miR-15A, and cholangiocyte proliferation were studied on ciliated and deciliated cultured normal rat cholangiocytes. Our results show that bile contains vesicles identified as exosomes by their size, characteristic “saucer-shaped” morphology, and specific markers, CD63 and Tsg101. When NRCs were exposed to isolated biliary exosomes, the exosomes attached to cilia, inducing a decrease of the phosphorylated-to-total ERK1/2 ratio, an increase of miR-15A expression, and a decrease of cholangiocyte proliferation. All these effects of biliary exosomes were abolished by the pharmacological removal of cholangiocyte cilia. Our findings suggest that bile contains exosomes functioning as signaling nanovesicles and influencing intracellular regulatory mechanisms and cholangiocyte proliferation through interaction with primary cilia.

Cholangiocytes, epithelial cells lining the biliary tree, have primary cilia extending from their apical membrane into the ductal lumen. Although important in disease, cilia also play a vital role in normal cellular functions. We reported that cholangiocyte cilia are sensory organelles responding to mechanical stimuli (i.e., luminal fluid flow) by alterations in intracellular Ca 2+ and cAMP. Because cholangiocyte cilia are also ideally positioned to detect changes in composition and tonicity of bile, we hypothesized that cilia also function as osmosensors. TRPV4, a Ca 2+ -permeable ion channel, has been implicated in signal transduction of osmotic stimuli. Using purified rat cholangiocytes and perfused intrahepatic bile duct units (IBDUs), we found that TRPV4 is expressed on cholangiocyte cilia, and that hypotonicity induces an increase in intracellular Ca 2+ in a TRPV4-, ciliary-, and extracellular calcium-dependent manner. The osmosensation of luminal tonicity by ciliary TRPV4 induces bicarbonate secretion, the main determinant of ductal bile formation, by a mechanism involving apical ATP release. Furthermore, the activation of TRPV4 in vivo , by its specific agonist, 4αPDD, induces an increase in bile flow as well as ATP release and bicarbonate secretion. Our results suggest that cholangiocyte primary cilia play an important role in ductal bile formation by acting as osmosensors.