Howard, Brynn N.

Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6‐targeting miRNAs, miR‐433 and miR‐22, are down‐regulated in CCA both in vitro and in vivo. Experimental restoration of these miRNAs in CCA cells reduced HDAC6 expression, induced ciliary restoration, and decreased the malignant phenotype. Furthermore, in contrast to the mature forms, levels of precursor forms of these miRNAs were higher in CCA compared to normal cholangiocytes and accumulated in the nuclei, suggesting a defective nuclear export. We assessed the expression of Exportin‐5, the protein responsible for transporting miRNA precursors out of the nucleus, and found it to be reduced by 50% in CCA compared to normal cholangiocytes. Experimental overexpression of Exportin‐5 in CCA cells restored precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6 and a decrease in the malignant phenotype. Conversely, short hairpin RNA (shRNA) depletion of Exportin‐5 in normal cholangiocytes resulted in increased nuclear retention of precursor miRNAs, decreased mature miRNAs, increased cell proliferation, and shorter cilia. Conclusion: These data suggest that down‐regulated Exportin‐5 impairs the nuclear export of miR‐433 and miR‐22 precursor forms, causing a decrease in levels of mature miR‐433 and miR‐22 forms, and leading to overexpression of HDAC6 and ciliary loss in CCA. (Hepatology 2018).

Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein–coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m‐tolyl 5‐chloro‐2‐[ethylsulfonyl] pyrimidine‐4‐carboxylate [SBI‐115]), and (3) a combination of SBI‐115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA‐treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5−/− ;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and Gαs (but not Gαi and Gαq) proteins was increased 2‐fold to 3‐fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by ∼40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5‐deficient TGR5−/−;Pkhd1del2/del2 mice. TGR5 expression and its colocalization with Gαs were increased ∼2‐fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by ∼30% in cystic cholangiocytes after treatment with SBI‐115 alone and by ∼50% when SBI‐115 was combined with pasireotide. Conclusion: TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197‐1218).

Abstract Cholangiocarcinoma (CCA) is a malignancy arising from the epithelial cells lining the biliary tract in the liver, the cholangiocytes. While cholangiocytes normally express cilia, we showed that they are significantly reduced in CCA by a mechanism involving overexpression of histone deacetylase 6 (HDAC6), a molecule that deacetylates the ciliary axoneme and promotes ciliary disassembly. Even though HDAC6 protein is overexpressed in CCA, we found no differences in its messenger RNA (mRNA) level, suggesting a post-transcriptional regulation, possibly involving microRNA (miRNA). Therefore, we hypothesized that decreased levels of specific miRNAs induce overexpression of HDAC6 in CCA. We compared miRNA levels between normal and CCA cholangiocyte cell lines and found that three miRNAs potentially targeting HDAC6 are downregulated (40-55% decrease) in CCA: miR-433, miR-22, and miR-200a. Experimental overexpression of miR-433, miR-22, or miR-200a in normal cholangiocytes decreased HDAC6 expression by 82%, 33%, and 39%, respectively. We selected miR-433 and miR-22 for further analysis and the downregulation of both miRNAs were confirmed by qRT-PCR on normal and six different CCA cell lines, and by in situ hybridization on control and CCA human liver samples. To further investigate the dysregulation of these miRNAs, we measured its precursor forms by qRT-PCR and, in contrast to the mature miR-433 and miR-22, the levels of the precursors were increased in malignant cells. In situ hybridization on human samples showed that the miR-433 probe was accumulated in the nucleus of malignant cholangiocytes. Taken together, these data suggested a potential impairment of nuclear export; therefore, we assessed the expression of the protein responsible for transporting microRNA precursors out of the nucleus, exportin-5. By western blotting, we found that exportin-5 is decreased by 50% in five different CCA cell lines compared to normal cholangiocyte cell lines. We confirmed the downregulation of exportin-5 protein by immunofluorescence of normal and CCA human liver samples. Finally, the experimental overexpression of exportin-5 in CCA cell lines restores the precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6, and decreased proliferation and anchorage independent growth of the malignant cells. In summary, these data suggest that downregulation of exportin-5, preventing the nuclear export of the precursor forms of miR-433 and miR-22, may be the underlying mechanism explaining the decrease of miR-433 and miR-22, and the overexpression of HDAC6 in CCA. Thus, our work further clarifies the molecular mechanisms accounting for the absence of cilia in CCA and also identifies multiple potential molecules for targeted therapies like HDAC6, miR-433, miR-22, or exportin-5. Citation Format: Maria J. Lorenzo Pisarello, Brynn N. Howard, Christy E. Trussoni, Sergio Gradilone. Exportin-5 downregulation induces miRNA dysregulation and HDAC6 overexpression in cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 192. doi:10.1158/1538-7445.AM2015-192