Karabicici Mustafa, Azbazdar Yagmur, Ozhan Gunes, Senturk Serif, Firtina Karagonlar Zeynep, Erdal Esra
Izmir Biomedicine and Genome Center (IBG), Dokuz Eylul University Health Campus, Izmir, Turkey.
Izmir International Biomedicine and Genome Institute (IBG-Izmir), Dokuz Eylul University, Izmir, Turkey.
Front Cell Dev Biol. 2021 Aug 11;9:639779. doi: 10.3389/fcell.2021.639779. eCollection 2021.
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 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 and 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.
肝细胞癌（HCC）是一种侵袭性强、对化疗耐药的肿瘤，预后较差且治疗选择有限。探索药物治疗后激活的信号通路可用于发现更有效的抗癌药物，以克服治疗耐药性并改善晚期HCC患者的治疗效果。人肿瘤来源的细胞系概括了HCC的多样性，被广泛用于研究HCC中驱动耐药性的机制。在本研究中，我们发现瑞戈非尼治疗仅在肝母细胞样HCC细胞系中激活Wnt/β-连环蛋白信号通路，并诱导与肝干细胞/祖细胞相关的标志物富集。此外，Wnt/β-连环蛋白信号通路的激活——Wnt3a/R-Spo1治疗可保护这些细胞免受瑞戈非尼诱导的凋亡。另一方面，通过长期瑞戈非尼治疗建立的瑞戈非尼耐药细胞显示Wnt/β-连环蛋白信号活性降低，而这些细胞的TGF-β信号活性显著增强。瑞戈非尼耐药细胞（RRCs）还显示几种间充质基因的表达增加，同时诱导CD24和CD133癌症干细胞标志物。此外，瑞戈非尼耐药细胞还表现出显著增强的增殖和迁移能力，这可通过抑制TGF-β1型受体（TGFβ-R1）来逆转。当与瑞戈非尼治疗联合使用时，TGFβ-R1抑制也显著降低了耐药球体中的集落形成能力并增加了细胞死亡。重要的是，当我们使用慢病毒质粒敲低TGFβ-R1时，瑞戈非尼耐药细胞进入衰老状态，表明该信号通路对其存活很重要。用TGFβ-R1抑制剂和瑞戈非尼治疗RRCs显著消除了pSTAT3、pSMAD2和pERK（44/42）的表达，提示经典和非经典信号通路均参与其中。总之，我们的数据表明，Wnt/β-连环蛋白通路异常激活的HCC肿瘤可能具有更高的内在瑞戈非尼耐药性，在联合治疗中抑制该通路并给予瑞戈非尼可能会增加瑞戈非尼诱导的细胞死亡。然而，为了解决HCC患者中出现的获得性瑞戈非尼耐药性，联合使用TGF-β信号通路抑制剂和瑞戈非尼是一种有前景的方法，可增加瑞戈非尼敏感性并预防肿瘤复发。
