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. 2020 Sep 30:13:9721-9730.
doi: 10.2147/OTT.S262089. eCollection 2020.

IL-6/STAT3 Signaling Contributes to Sorafenib Resistance in Hepatocellular Carcinoma Through Targeting Cancer Stem Cells

Yu Li  1 , Gang Chen  1 , Zhijian Han  2 , Huijuan Cheng  2 , Liang Qiao  3 , Yumin Li  1   2
Affiliations

IL-6/STAT3 Signaling Contributes to Sorafenib Resistance in Hepatocellular Carcinoma Through Targeting Cancer Stem Cells

Yu Li et al. Onco Targets Ther. .

Erratum in

Retraction in

Abstract

Background: Sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), even though acquired resistance to sorafenib has been found in many HCC patients, resulting in poor prognosis. Accumulating evidence demonstrates that liver cancer stem cells (LCSCs) contribute to sorafenib resistance in HCC. The inflammatory factor interleukin 6 (IL-6) plays a role in sorafenib resistance in HCC. However, the mechanism by which IL-6 in LCSCs is involved in the process of HCC sorafenib resistance remains elusive.

Methods: In this study, the sorafenib-resistant cell line PLC/PRF/5-R was generated by the concentration gradient method, and cell viability was determined by the CCK-8 assay. LCSCs were isolated from the PLC/PRF/5-R cell line by flow cytometry, and tumorigenesis was confirmed in nude mice. Blockade of IL-6 cells was achieved by lentiviral-mediated interference. The protein levels of stem cell markers (EpCAM, CD44), stemness markers (Oct3/4, β-catenin), and hepatocyte differentiation markers (glucose-6-phosphate, AFP) were measured by Western blotting analysis. Finally, a xenograft model was used to evaluate the function of IL-6 in the sorafenib resistance of HCC.

Results: The stable sorafenib-resistant HCC cell line PLC/PRF/5-R was established and showed significant epithelial-mesenchymal transition (EMT) characteristics; the isolated resistant LCSCs from PLC/PRF/5-R were more tumorigenic than the control LCSCs. We showed that IL-6, IL-6R, STAT3 and GP130 expression were dramatically increased in resistant LCSCs compared to control LCSCs. Downregulation of IL-6 expression with short hairpin RNA (shRNA) restored sorafenib sensitivity in resistant LCSCs, suggesting the critical roles of IL-6/STAT3 in inducing sorafenib resistance. Furthermore, a xenograft tumor model showed that IL-6 downregulation improved the antitumor effect of sorafenib.

Conclusion: LCSCs play an important role in sorafenib-resistant HCC, and inhibition of the IL-6/STAT3 signaling pathway improves the antitumor effects of sorafenib against HCC in vitro and in vivo. These findings demonstrate that IL-6 in LCSCs may function as a novel target for combating sorafenib resistance in HCC.

Keywords: IL-6/STAT3 signaling; cancer stem cells; drug resistance; hepatocellular carcinoma; sorafenib.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
The sorafenib-resistant hepatocellular carcinoma cell line PLC/PRF/5-R was established. (A) Morphological characteristics of PLC/PRF/5 and PLC/PRF/5-R by microscopy (×100). (B) For PLC/PRF/5, the IC50 was approximately 5.464 μM; for the resistant cell lines, the IC50 shifted towards 12.18 μM. (C–E) MDR, E-cadherin and vimentin mRNA and protein expression levels in PLC/PRF/5 (PLC) and PLC/PRF/5-R (PLC-R) (**P<0.01, ***P<0.005, ****P<0.0001), PLC/PRF/5-R showed typical EMT characteristics.
Figure 2
Figure 2
LCSC-R isolation and identification. (A) FACS plots demonstrating the isolation of EpCAM+ and CD44+ resistant LCSCs from PLC/PRF/5-R cells. (B) Cell morphology of resistant LCSCs by microscope (×100). (C) The sorted positive and negative cells were assessed using qPCR. (D, E) Cell proliferation and apoptosis assays were performed to compare the positive and negative cells, but there were no statistically significant differences. (F) Nude mouse xenograft experiment (R+: positive (EpCAM+, CD44+) resistant LCSCs, R-: negative (EpCAM−, CD44−)) resistant LCSCs, B+: positive (EpCAM+, CD44+) LCSCs, B-: negative (EpCAM−, CD44−) LCSCs (***P<0.005, ****P<0.0001).
Figure 3
Figure 3
Blocking the IL-6/STAT3 signaling pathway in resistant LCSCs. (A) IL-6, IL-6R, STAT3 and GP130 mRNA levels in LCSCs and resistant LCSCs. (B, C) STAT3 protein expression in LCSCs and resistant LCSCs. (D–F) To confirm that IL-6 signaling was blocked in resistant LCSCs by qPCR and WB. (G–I) General stem cell markers (EpCAM and CD44), stemness markers (Oct3/4 and β-catenin), and hepatocyte differentiation markers (G-6-P and AFP) were detected using qPCR and WB (**P<0.01, ***P<0.005, ****P<0.0001), and there were significant differences.
Figure 4
Figure 4
Establishment of subcutaneous xenografts in nude mice using resistant LCSCs, which block the IL-6/STAT3 signaling pathway. (A) Tumor images of all subcutaneous xenografts in nude mice. Each indicated treatment group included 5 mouse tumors. (B) Growth curves of xenograft tumors from day 1 to day 11 in various treatment groups. (C, D) Analysis of IL-6R and STAT3 protein expression in xenograft tumors to confirm that the IL-6/STAT3 signaling pathway was blocked. (E, F) The LCSC markers (EpCAM and CD44), stemness markers (Oct3/4 and β-catenin) and angiogenic factors (VEGF and VEGFR) were assessed by WB (Sor-Block-IL-6): subcutaneous xenograft tumors in nude mice were established using resistant LCSCs, which blocked IL-6 signaling, treated with sorafenib, 100 mg/kg/d; sor: subcutaneous xenograft tumors in nude mice were established using resistant LCSCs, in which IL-6 signaling is NOT blocked, and sorafenib as the treatment, 100 mg/kg/d; PBS: subcutaneous xenograft tumors were established in nude mice using resistant LCSCs, in which IL-6 signaling is blocked, and PBS as the treatment (ns P>0.05, *P<0.05, **P<0.01, ***P<0.005).
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