STAT3 mediates cancer stem-like tumorsphere formation and PD-L1 expression to contribute radioresistance in HBV-positive hepatocellular carcinoma

Abstract

We proposed that cancer stem cells (CSCs) survived and presented resistance to radiotherapy (RT) in hepatocellular carcinoma (HCC) cells. Interleukin 6 (IL-6) has been reported to be particularly involved in HCC tumorigenesis. Therefore, we intended to validate that IL-6 downstream STAT3-mediated CSCs formation and immune checkpoint PD-L1 expression in HCC, thus contributing to radioresistance. HBV-positive HCC tumorspheres were formed and exposed with X-ray irradiation, cell viability of which was measured consequently. Specific inhibitors targeting EGFR (by gefitinib), STAT3 (by BBI608), and HCC-targeted therapy sorafenib were investigated to suppress tumorsphere formation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used for detecting STAT3-downstream PD-L1 and anti-apoptosis MCL1 and BCL2 gene expression in the PLC5-derived tumorspheres and STAT3-knockdown PLC5. We found that RT significantly inhibited HBV-positive Hep3B and PLC5 cell viability but not for HCC-derived stem-like tumorspheres cultured by EGF, IL-6, bFGF, and HGF. It revealed that tumorspheres presented radioresistance compared with the parental cells. Specifically, RT induces IFNs, EGF, and IL-6 expression, resulting in STAT3 phosphorylation. Kaplan–Meier plotter indicated that highly EGF (p = .0024), IL-6 (p = .12), and FGF2 (p = .0041) were associated with poor survival probability in patients with HBV-positive HCC. We further demonstrated that BBI608 and sorafenib significantly suppressed PLC5 cell viability and PLC5-derived tumorsphere formation. To investigate the mechanism of CSC-presented radioresistance, STAT3 and STAT3-downstream genes, including PD-L1 and anti-apoptosis MCL1 and BCL2, were detected using qPCR. We demonstrated higher STAT3, PD-L1, MCL1, and BCL2 in Hep3B- and PLC5-derived CSCs compared to PLC5. In addition, knockdown of STAT3 reduced cell proliferation in PLC5 cells, resulting in down-regulation of IL-6-mediated PD-L1 and BCL-2. Meanwhile, we found that knockdown of STAT3 significantly improved RT-mediated suppression of tumorsphere formation. In conclusion, we found that CSCs presented radioresistance and figured out which may be mediated by STAT3 in HBV-positive HCC.