Hedgehog Signaling Pathway Regulates Autophagy in Human Hepatocellular Carcinoma Cells

Abstract

Hedgehog (Hh) signaling plays an important role in the regulation of cellular functions. Classical Hh signaling is characterized by Smoothened (Smo)‐dependent activation of Gli1 and Gli2. Aberrant activation of Hh signaling has been implicated in several human cancers including hepatocellular carcinoma. In this study we examined the molecular mechanisms of Hh signaling pathway in HCC cells. Treatment of cultured human HCC cells with the Hh signaling ligand (recombinant Shh) or agonist, SAG and purmorphamine, prevented autophagy. In contrast, GANT61 (a small molecule inhibitor of Gli1 and Gli2) induced autophagy, as determined by immunobloting for microtubule‐associated protein light chain 3 (LC3) and p62, GFP‐LC3 puncta, monodansylcadaverine (MDC) staining and transmission electron microscopy. Hh inhibition‐induced autophagy was associated with Bnip3 upregulation. Knockdown of Bnip3 by RNAi impaired GANT61‐induced autophagy. Furthermore, inhibition of Hh signaling increased HCC cell apoptosis and decreased cell viability. The inhibition of autophagy by 3‐methyladenine (3‐MA) or Beclin‐1 siRNA partially suppressed GANT61‐induced cell apoptosis and cytotoxicity. In a tumor xenograft model using SCID mice inoculated with Huh7 cells, administration of GANT61 inhibited tumor formation and decreased tumor volume; this effect was partially blocked by the autophagy inhibitor, 3‐MA. These findings illustrate that hedgehog inhibition induces autophagy through upregulation of Bnip3 and this mechanism contributes to apoptosis.