Abstract
Sorafenib, an oral multi-tyrosine kinase inhibitor, has been the first-line therapy for the treatment of patients with advanced HCC, providing a survival benefit of only three months in approximately 30% of patients. Cancer stem cells (CSCs) are a rare tumour subpopulation with self-renewal and differentiation capabilities, and have been implicated in tumour growth, recurrence and drug resistance. The process of epithelial-to-mesenchymal transition (EMT) contributes to the generation and maintenance of the CSC population, resulting in immune evasion and therapy resistance in several cancers, including HCC. The aim of this study is to target the chemoresistant CSC population in HCC by assessing the effectiveness of a combination treatment approach with Sorafenib, an EMT inhibitor and an immune checkpoint inhibitor (ICI). A stem-cell-conditioned serum-free medium was utilised to enrich the CSC population from the human HCC cell lines Hep3B, PLC/PRF/5 and HepG2. The anchorage independent spheres were characterised for CSC features. The human HCC-derived spheres were assessed for EMT status and expression of immune checkpoint molecules. The effect of combination treatment with SB431542, an EMT inhibitor, and siRNA-mediated knockdown of programmed cell death protein ligand-1 (PD-L1) or CD73 along with Sorafenib on human HCC-derived CSCs was examined with cell viability and apoptosis assays. The three-dimensional spheres enriched from human HCC cell lines demonstrated CSC-like features. The human HCC-derived CSCs also exhibited the EMT phenotype along with the upregulation of immune checkpoint molecules. The combined treatment with SB431542 and siRNA-mediated PD-L1 or CD73 knockdown effectively enhanced the cytotoxicity of Sorafenib against the CSC population compared to Sorafenib alone, as evidenced by the reduced size and proliferation of spheres. Furthermore, the combination treatment of Sorafenib with SB431542 and PD-L1 or CD73 siRNA resulted in an increased proportion of an apoptotic population, as evidenced by flow cytometry analysis. In conclusion, the combined targeting of EMT and immune checkpoint molecules with Sorafenib can effectively target the CSC tumour subpopulation.
Highlights
The commonly reported mechanisms attributed to Sorafenib resistance are epithelial-to-mesenchymal transition (EMT) [12,13,14], cancer stem cells (CSCs) [15,16], autophagy [17,18], hypoxia [19,20] and deregulated signal transduction including EGFR
To explore the relationship between EMT and CSCs, we examined the EMT features related to Hepatocellular carcinoma (HCC)-derived CSCs
We observed that the percentage of apoptotic spheres increased with a combination therapy of SB431542 and immune checkpoint inhibitors (PD-L1 siRNA or CD73 siRNA) with Sorafenib compared to Sorafenib alone in both Hep3B (Figure 9A) and PLC/PRF/5-derived (Figure 9B) CSCs. These findings suggest that the combined targeting of EMT and immune checkpoint molecules can enhance the cytotoxic effects of Sorafenib or overcome the resistance of HCC-derived CSCs to Sorafenib, resulting in effective chemotherapeutic outcome in HCC
Summary
The. Food and Drug Administration (FDA) approved Sorafenib as the first drug for the treatment of unresectable advanced HCC [6]. Only onethird of advanced HCC patients respond to Sorafenib treatment due to the development of Sorafenib resistance [6,9,10]. Elucidating the mechanisms of Sorafenib resistance and exploring better treatment strategies to improve therapeutic outcomes in HCC is warranted. The commonly reported mechanisms attributed to Sorafenib resistance are epithelial-to-mesenchymal transition (EMT) [12,13,14], cancer stem cells (CSCs) [15,16], autophagy [17,18], hypoxia [19,20] and deregulated signal transduction including EGFR activation [21,22], c-Jun activation [23,24,25] and Akt activation [26,27]
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