Tumor-initiating cells contribute to acquired chemoresistance during anti-angiogenic therapies in hepatocellular carcinoma

Inhibition of neo-angiogenesis is an effective treatment strategy for advanced HCC. However, development of chemoresistance is observed in the majority of patients. Compelling evidence suggest that stem-like tumor-initiating cells (TICs) may contribute to the acquisition of resistant properties 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 to different anti-angiogenic therapies in HCC and define concomitant adaptive molecular changes. Several HCC cell lines and primary HCC isolates were exposed to sorafenib and sunitinib for total of 14 – 21 days. Treatment effects on TICs were estimated by sphere forming capacity and side-population (SP) approach. Expression levels of key oncogenic and TIC markers were assessed by qRT-PCR and flow cytometry. Whole transcriptome analyses were performed at different time points. Both treatments effectively reduced oncogenic properties in all investigated HCC cells. Sustained anti-proliferative effects were observed in only two cell lines whereas an initial treatment effect was subsequently followed by rapid re-growth in the majority of HCC cells thereby mimicking responses observed in patients. While anti-oncogenic effects in sensitive cell lines were associated with significant reduction sphere forming capacity, TIC markers as well as SP cells, resistant cell line showed transient increased in TIC properties. Acquired resistance to both drugs uniformly developed in the cell lines suggesting that common molecular mechanisms might be operative. These adaptive molecular changes involved signaling pathways known to be associated to cell survival (ERK, AKT, MYC), proliferation (TP53, CDKN1A) and angiogenesis (VEGF, PDGFR, HIF1a). The resistant cell lines showed compensatory upregulation of key oncogenic molecules such as EGFR as well as multidrug resistance ABC transporters. Our in vitro model recapitulates features of drug resistance observed in human HCC patients. Resistance to anti-angiogenic therapies might be fueled by transient expansion of TICs. Therefore, specific targeting of TICs as well as pro-oncogenic compensatory signaling pathways might be an effective therapeutic strategy to overcome resistance in HCC.