Cancer stem cells as significant drivers of sorafenib resistance in hepatocellular carcinoma

Development of chemoresistance is frequently observed in the majority of HCC patients. Evidence suggests that cancer stem cells (CSCs) may contribute to the acquisition of resistance in many solid tumors, but their exact role in this process for HCC remains to be defined.

Here, we evaluate the importance of TICs in the development of resistance and relapse formation after exposure to sorafenib in HCC and define concomitant adaptive molecular changes.

Four HCC cell lines and two primary HCC isolates were exposed to sorafenib for a total of 14 days. The treatment effects on CSCs were estimated by sphere forming capacity in vitro and tumor-initiating potential in vivo, as well as the side-population (SP) approach. Expression of key oncogenic and CSC markers, such as EpCAM, CD133 and ABCG2 transporter, were assessed by qRT-PCR and flow cytometry. Whole transcriptome analyses identified potential targets which were validated by western blot and administration of specific inhibitors.

Treatment effectively reduced oncogenic properties in all investigated HCC cells. However, sustained anti-proliferative effect after treatment was observed in three cell lines, while initial treatment effect in other lines was followed by rapid re-growth thereby mimicking responses observed in patients. While anti-oncogenic effects in sensitive cell lines were associated with significant reduction in sphere forming capacity, CSC marker EpCAM as well as SP cells, resistant cells showed increased CSC properties. Acquired resistance to the drug uniformly developed in cell lines suggesting that common molecular mechanisms might be operative. Adaptive molecular changes involved signaling pathways associated with cell survival, proliferation and cell cycle regulation (RAS, AKT, MYC, P53). Furthermore, the resistant cell lines showed compensatory upregulation of key oncogenic molecules such as EGFR, multidrug resistance ABC transporters as well as YAP. Conclusively, combined treatment including sorafenib and specific YAP inhibitor showed beneficial effects in resistant cell lines which resulted in complete response to the therapy.

Our model recapitulates features of drug resistance observed in human HCC patients. Resistance to sorafenib therapy might be fueled by transient expansion of CSCs. Therefore, specific targeting of CSCs as well as pro-oncogenic compensatory signaling pathways might be an effective therapeutic strategy to overcome resistance in HCC.