Inhibition of HDAC6 and autophagy reverts ciliary loss and decreases proliferation rates in cholangiocarcinoma cells

ESTANISLAO PEIXOTO; STEPHANIE HOLTORF; KRISTEN M. THELEN; MARIA LORENZO PISARELLO; NICHOLAS F. LARUSSO; SUJEONG JIN; SERGIO A. GRADILONE

Washigton DC

Congreso; Liver Meeting2017-American Association for study of liver disease; 2017

Abstract Body: Background: Primary cilia are microtubule based organelles that sense and transduce multiple extracellular signals to the interior of the cell through several pathways. Decreased ciliary expression has been associated with different types of cancer. We previously demonstrated that HDAC6 is overexpressed in cholangiocarcinoma (CCA) and is involved in ciliary loss, but the mechanisms remain unknown. In this context, autophagy is dysregulated in CCA. We propose a novel mechanism for tumor cells ciliary loss that involves HDAC6- dependent autophagy, i.e. ciliophagy. Therefore, our aim is to study the relationship between autophagy and ciliary disassembly through HDAC6 activity.Methods: We assessed the number of autophagosomes by IEM of human liver samples and by LC3 immunofluorescence in CCA cell lines. GFP-LC3 distribution and ciliary protein colocalizations were assessed with confocal immunofluorescence. In addition, LC3 accumulation was measured using western blotting and immunofluorescence analysis. Immunoprecipitations were performed with an LC3B specific antibody and interactions were revealed by western blots. SiRNAs were used to inhibit mRNA expression of the autophagy cargo receptor proteins CalCOCO2, OPTN, NBR1 and SQSTM1 in a normal cholangiocyte cell line. Silenced cells were assessed for cilia length using gamma and acetylated alpha tubulin immunofluorescence. Cell proliferation was assessed by MTS.Results: The number of autophagosomes is increased in cholangiocarcinoma tissue of patients and in cultured malignant cholangiocytes. Confocal microscopic analysis revealed LC3 juxtaposition and colocalization with the ciliary proteins IFT88 and ARL13B. LC3 accumulation was induced after HDAC6 inhibition in cholangiocytes, suggesting an inhibition of the autophagy flux. The immunoprecipitation of LC3 showed interaction with HDAC6 on the pulled-down complex. Cells with NBR1 downregulation had significantly longer cilia than control cells. Finally, a reduction of proliferation rates and an increase in the number of cilia after HDAC6 and/or autophagy inhibitions was observed.Conclusion: Our data suggests that HDAC6 overexpression in CCA cells induces ciliophagy, likely through a mechanism involving the autophagy cargo receptor protein NBR1, leading autophagosomes to target ciliary components. Our results, showing that the inhibition of HDAC6 and autophagy can reduce proliferation rates while restoring ciliary structures, can be further investigated and explored as a potential therapeutic approach.