Abstract
Hepatocellular carcinoma (HCC) is an aggressive, chemo resistant neoplasm with poor prognosis and limited treatment options. Exploring activated pathways upon drug treatment can be used to discover more effective anticancer agents to overcome therapy resistance and enhance therapeutic outcomes for patients with advanced HCC. Human tumor-derived cell lines recapitulate HCC diversity and are widely used for studying mechanisms that drive drug resistance in HCC. In this study, we show that regorafenib treatment activates Wnt/β-catenin signaling only in hepatoblast-like HCC cell lines and induces enrichment of markers associated with hepatic stem/progenitor cells. Moreover, activation of Wnt/β-catenin signaling via Wnt3a/R-Spo1 treatment protects these cells from regorafenib induced apoptosis. On the other hand, regorafenib resistant cells established by long-term regorafenib treatment demonstrate diminished Wnt/β-catenin signaling activity while TGF-β signaling activity of these cells is significantly enhanced. Regorafenib resistant cells (RRCs) also show increased expression of several mesenchymal genes along with an induction of CD24 and CD133 cancer stem cell markers. Moreover, regorafenib resistant cells also exhibit significantly augmented in vitro and in vivo migration capacity which could be reversed by TGF-β type 1 receptor (TGFb -R1) inhibition. When combined with regorafenib treatment, TGFβ-R1 inhibition also significantly decreased colony formation ability and augmented cell death in resistant spheroids. Importantly, when we knocked down TGFβ-R1 using a lentiviral plasmid, regorafenib resistant cells entered senescence indicating that this pathway is important for their survival. Treatment of RRCs with TGFβ-R1 inhibitor and regorafenib significantly abolished pSTAT3, pSMAD2 and pERK (44/42) expression suggesting the involvement of both canonical and non-canonical pathways. In conclusion, our data suggest that HCC tumors with aberrant activation in the Wnt/β-catenin pathway, might have higher intrinsic regorafenib resistance and the inhibition of this pathway along with regorafenib administration might increase regorafenib-induced cell death in combinational therapies. However, to resolve acquired regorafenib resistance developed in HCC patients, the combined use of TGF-β pathway inhibitors and Regorafenib constitute a promising approach that can increase regorafenib sensitization and prevent tumor recurrence.
Highlights
Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of cancer-related deaths worldwide (Bertuccio et al, 2017; Savitha et al, 2017; Yang et al, 2019; Pérez et al, 2020)
We show that regorafenib treatment activates Wnt/β-catenin signaling only in hepatoblast-like HCC cell lines and induces enrichment of markers associated with hepatic stem/progenitor cells
We showed that the sorafenib resistant clones had acquired regorafenib resistance while regorafenib resistant clones had become sorafenib resistant (Figure 1A)
Summary
Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of cancer-related deaths worldwide (Bertuccio et al, 2017; Savitha et al, 2017; Yang et al, 2019; Pérez et al, 2020). Multi-kinase inhibitors Sorafenib and Lenvatinib, are the two systemic therapies approved first-line for unresectable HCC (Kudo et al, 2018; Xie et al, 2018; Personeni et al, 2019). Since majority of Phase 3 trials are done after sorafenib failure, sorafenib still remains the globally accepted first-line treatment for advanced HCC despite its poor therapeutic response and high rates of resistance (Kudo et al, 2018; Personeni et al, 2019; Kim et al, 2020). There are currently no molecular markers for currently used systemic treatments (sorafenib, lenvatinib, regorafenib, and others) in HCC which could be used in therapeutic decision-making to improve patient outcomes.
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