Hedgehog signaling pathway affects the sensitivity of hepatoma cells to drug therapy through the ABCC1 transporter

The poor response to drug therapy often seen in hepatocellular carcinoma requires insight into the molecular interplay responsible for intrinsic or acquired drug resistance. We previously demonstrated that the CD133^-/EpCAM^- subpopulation of the Huh-7 hepatoma cell line features aberrant activation of the hedgehog signaling (Hh) pathway and chemoresistance. The prevailing hypothesis of the present study is that hedgehog signaling may govern expression of ATP-binding cassette (ABC) transporters, which are responsible for drug resistance in the CD133^-/EpCAM^- subpopulation. Our aim is to reveal the molecular interplay in the mediation of drug resistance with a newly established Huh-7 subpopulation featuring high Hh signaling activity and drug resistance. In this study, chemoresistance was determined in a newly established Huh-7-DN subpopulation featuring the CD133^-/EpCAM^- surface marker profile, aberrant expression of Hh pathway, and epithelial-mesenchymal transition (EMT). Expression of ABC transporter proteins (ABCB1, ABCC1, and ABCG2) and Hh transcription factor Gli-1/2 was evaluated with and without Hh signaling antagonists LDE225 or itraconazole. We found that hedgehog signaling activity as determined by transfection with a Gli-Lux reporter cassette and gene expression levels tended to increase from Huh-7 CD133⁺/EpCAM⁺ to CD133^-/EpCAM^-, and the highest levels were found in Huh-7-DN cells. The Huh-7-DN subpopulation exhibited characteristics of EMT as evidenced by increased expression of vimentin and loss of E-cadherin. Sorafenib significantly inhibited the viability of all subpopulations except the Huh-7-DN subpopulation. Compared with other sorafenib-sensitive subpopulations, the Huh-7-DN subpopulation showed enhanced expression of Hh transcription factor Gli-2 and ABCC1 transporter protein. Silencing Gli-2 by lentivirus harboring shRNA against Gli-2 or LDE225 significantly suppressed expression of Gli-2 and ABCC1 genes in Huh-7-DN subpopulation. In conclusion, aberrant hedgehog signaling activation is linked to poor differentiation, epithelial-mesenchymal transition, and chemoresistance in the Huh-7-DN subpopulation. Hedgehog signaling transcription factor Gli-2 appears to be the primary regulator for drug sensitivity of hepatoma through the ABCC1 transporter.