Exportin-5 downregulation induces miRNA dysregulation and HDAC6 overexpression in cholangiocarcinoma

LORENZO PISARELLO, MARIA JOSE; HOWARD,BRYNN N.; TRUSSONI, CHRISTY; GRADILONE, SERGIO

Congreso; AACR 106th Annual Meeting 2015; 2015

holangiocarcinoma (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, 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.