The inhibition of autophagy and hedgehog pathway leads to a decrease in the proliferation of cholangiocarcinoma

Abstract

Cholangiocarcinoma (CCA) is the second most common liver cancer type. The median survival rate of CCA patients is really low. Aberrant signaling pathways such as PI3K/AKT/mTOR pathway could be main drivers in CCA pathogenesis. Hedgehog (Hh) pathway is also dysregulated in several carcinomas including CCA. It regulates and crosstalks with autophagy, which is a lysosomal degradation process. There is no study showing the crosstalk between Hh pathway and autophagy in the context of CCA. Since both autophagy and Hh pathways are dysregulated in CCA, better understanding of how they crosstalk with each other and contribute to CCA pathogenesis is important. Considering this crosstalk between Hh pathway and autophagy, we conducted a combination treatment comprising Hh and autophagy pathway inhibitors in EGI-1 and TFK-1 CCA cell lines. In our study, we firstly checked anti-proliferative effects of Hh pathway inhibitor, GANT61, and different autophagy blockers using MTT and Annexin V assay and cell cycle analysis. After determination of IC30 of GANT61 (15 uM), chloroquine (25 uM for TFK-1 and 50 uM for EGI-1), and nocodazole (0.2 uM for EGI-1 and 0.4 uM for TFK-1), we conducted combination experiments. When we inhibit Hh pathway with targeting different steps of autophagy, we observed that proliferation of both EGI-1 and TFK-1 cells decreased compared to single treatments. After that, we checked the expression of autophagy-related LC3B protein and Akt, a negative regulator of autophagy, using western blotting after single treatments and combinational treatments. Based on the change in LC3B and Akt expression, we also concluded that, inhibition of autophagy with Hh pathway either induce or inhibit autophagy depends on the administered treatments. This study highlights the importance of deciphering the exact mechanisms that control autophagy in CCA, thus leading to better treatment.