PERK/ATF4-Dependent ZFAS1 Upregulation Is Associated with Sorafenib Resistance in Hepatocellular Carcinoma Cells

Abstract

Sorafenib, a multi-kinase inhibitor, is the first-line treatment for advanced hepatocellular carcinoma (HCC) patients. However, this drug only provides a short improvement of patients' overall survival, and drug resistance is commonly developed. Thus, the identification of resistant factor(s) or biomarker(s) is needed to develop more efficient therapeutic strategies. Long, non-coding RNAs (lncRNAs) have recently been viewed as attractive cancer biomarkers and drive many important cancer phenotypes. A lncRNA, ZFAS1 (ZNFX1 antisense RNA 1) has been found to promote HCC metastasis. This study found that sorafenib induced ZFAS1 expression specifically in sorafenib-resistant HCC cells. Although ZFAS1 knockdown did not restore the sensitivity of HCC cells to sorafenib, its expression may act as a resistant biomarker for sorafenib therapy. Bioinformatics analysis predicted that sorafenib tended to induce pathways related to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in sorafenib-resistant HCC cells. In vitro experimental evidence suggested that sorafenib induced protein kinase RNA-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-dependent ZFAS1 expression, and sorafenib resistance could be overcome by PERK/ATF inhibitors. Therefore, PERK/ATF4/ZFAS1 signaling axis might be an attractive therapeutic and prognostic biomarker for sorafenib therapy in HCC.

Keywords: ER stress; drug resistance; hepatocellular carcinoma; long non-coding RNA; sorafenib; unfolded protein response.

Figure 1

Identification of sorafenib-induced lncRNAs in sorafenib-resistant HCC cells. (A) HepG2, HepG2-SR, and PLC5 cells were treated with 0–10 μM sorafenib for 48 h. The cell proliferation was examined by BrdU incorporation assay. ** p < 0.01 and *** p < 0.001 indicated the statistically significant difference compared to sorafenib-treated HepG2 cells. (B) HepG2, HepG2-SR, and PLC5 cells were treated with 5 μM sorafenib for 24 h. Total RNAs were subjected to RNA-sequencing. The altered lncRNAs were shown as a Venn diagram. Genes in red or blue indicated that they were upregulated or downregulated by sorafenib, respectively.

Figure 2

The expression of lncRNAs in HCC. The expressions of ZFAS1 (A), GAS5 (B), SNHG5 (C), and SNHG8 (D) in normal and cancer tissues (left part), and during tumor stage (right part) in HCC patients were obtained from the GEPIA database. * p < 0.05 indicated the statistically significant difference compared to the normal group.

Figure 3

The prognostic values of lncRNAs in HCC. The Kaplan–Meier plots for the relationship between lncRNA expression and overall/disease-free survivals were generated from the GEPIA database. The group cut-off value was set as “quartile”.

Figure 4

The effect of ZFAS1 knockdown on sorafenib resistance in HCC cells. (A) HepG2, HepG2-SR, and PLC5 cells were treated with 5 μM sorafenib for 18 h, and then ZFAS1 expression was analyzed by real-time qPCR. *** p < 0.001 indicated the statistically significant difference between sorafenib-treated and untreated cells. (B,C) HepG2, HepG2-SR, and PLC5 cells were transfected with si-ZFAS1 or its negative control siRNA (si-Cont) for 24 h, and then ZFAS1 expression was analyzed by real-time qPCR (B). The transfected cells were treated with 5 μM sorafenib for 48 h. The cell proliferation was examined by BrdU incorporation assay (C). * p < 0.05 and ** p < 0.01 indicated the statistically significant difference between sorafenib-treated and untreated cells. ^# p < 0.05 and ^## p < 0.01 indicated the statistically significant difference between si-Cont and si-ZFAS1-transfected cells. (D) The ZFAS1 mRNA expression levels in sorafenib responsive and non-responsive HCC patients were obtained from the microarray data set (GSE109211). *** p < 0.001 indicated the statistically significant difference between sorafenib responders and non-responders.

Figure 5

Pathway enrichment for ZFAS1-associated cancer hallmark. (A) The RNA-sequencing data in Table S1 were analyzed using the WebGestalt for cancer hallmark analysis. (B) The OncoPrint for ZFAS1 mRNA expression in TCGA-LIHC dataset. (C) The over- and under-expressed genes in ZFAS1-high expressing HCC patients were obtained from the cBioPortal website and these genes were analyzed using the WebGestalt for cancer hallmark analysis.

Figure 6

GSEA for UPR genes in HCC patients receiving sorafenib therapy. The microarray dataset (GSE109211) of sorafenib responsive and non-responsive HCC patients were analyzed by GSEA for the enrichment of the UPR cancer hallmark.

Figure 7

The effect of UPR inhibitors on sorafenib-induced ZFAS1. (A,B) PLC5 cells were pretreated with 10 μM GSK-2606414, 0.2 μM ISRIB, 10 μM 4μ8C, or 10 μM Ceapin-A7 for 0.5 h, and then exposed to 5 μM sorafenib for 18 h. ZFAS1 expression was analyzed by real-time qPCR. *** p < 0.001 indicated the statistically significant difference between sorafenib-treated and UPR inhibitor/sorafenib-treated cells. (C) HepG2, HepG2-SR, and PLC5 cells were treated with 5 μM sorafenib for 48 h in the absence or presence of 10 μM GSK-2606414, 10 μM 4μ8C, or 5 μM Ceapin-A7. The cell proliferation was examined by BrdU incorporation assay. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 indicated the statistically significant difference between untreated and UPR inhibitor-treated cells. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicated the statistically significant difference between sorafenib-treated and UPR inhibitor/sorafenib-treated cells.