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. 2019 Jun 24;10(17):3914-3925.
doi: 10.7150/jca.31448. eCollection 2019.

KPNA3 Confers Sorafenib Resistance to Advanced Hepatocellular Carcinoma via TWIST Regulated Epithelial-Mesenchymal Transition

Bo Hu  1 , Jian-Wen Cheng  1 , Jin-Wu Hu  1 , Hong Li  2 , Xiao-Lu Ma  3 , Wei-Guo Tang  1 , Yun-Fan Sun  1 , Wei Guo  3 , Ao Huang  1 , Kai-Qian Zhou  1 , Ping-Ting Gao  1 , Ya Cao  4 , Shuang-Jian Qiu  1 , Jian Zhou  1   5 , Jia Fan  1   5 , Xin-Rong Yang  1
Affiliations

KPNA3 Confers Sorafenib Resistance to Advanced Hepatocellular Carcinoma via TWIST Regulated Epithelial-Mesenchymal Transition

Bo Hu et al. J Cancer. .

Abstract

Sorafenib, a multikinase inhibitor, is a new standard treatment for patients with advanced hepatocellular carcinoma (HCC). However, resistance to this regimen is frequently observed in clinical practice, and the molecular basis of this resistance remains largely unknown. Herein, the antitumor activity of sorafenib was assessed in 16 patient-derived xenograft (PDX) models of HCC. Gene expression analysis was conducted to identify factors that promote sorafenib resistance. Quantitative RT-PCR and immunoblotting were used to determine gene expression and activation of signaling pathways. Cell proliferation, clone formation, and transwell assays were conducted to evaluate drug-sensitivity, proliferation, and invasiveness, respectively. Kaplan-Meier analysis was used to evaluate the predictive power of biomarkers for sorafenib response. Differential gene expression analysis suggested that sorafenib resistance correlated with high karyopherin subunit alpha 3 (KPNA3) expression. Overexpression of KPNA3 in HCC cells enhanced tumor cell growth and invasiveness. Interestingly, KPNA3 was found to trigger epithelial-mesenchymal transition (EMT), a key process mediating drug resistance. On a mechanistic level, KPNA3 increased phosphorylation of AKT, which then phosphorylated ERK, and ultimately promoted TWIST expression to induce EMT and sorafenib resistance. Moreover, retrospective analysis revealed that HCC patients with low KPNA3 expression had remarkably longer survival after sorafenib treatment. Finally, we have identified a novel KPNA3-AKT-ERK-TWIST signaling cascade that promotes EMT and mediates sorafenib resistance in HCC. These findings suggest that KPNA3 is a promising biomarker for predicting patient responsiveness to sorafenib. Targeting KPNA3 may also contribute to resolving sorafenib resistance in HCC.

Keywords: drug resistance; epithelial-mesenchymal transition; hepatocellular carcinoma; patient-derived xenograft; personalized medicine.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Identification of KPNA3 as a key regulator of sorafenib resistance. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. (B) The ras-related nuclear protein (RAN) signaling pathway analysis based on the KEGG database. (C) Hierarchical clustering analysis based on four candidate genes involved in RAN signaling. Red and green cells depict high and low expression levels, respectively. (D) Differential expression of the four candidate genes confirmed by qRT-PCR. (E) qRT-PCR assays for the four candidate genes involved in RAN signaling in sorafenib-resistant cells derived from Huh7 and HCCLM3 cell lines. (F) Apoptosis rates of Huh7-SR and HCCLM3-SR cells upon treatment with sorafenib after KPNA3 expression was knocked down. (G) KPNA3 expression in HCC cell lines. (H) Survival rates of Huh7 and HCCLM3 with modulated KPNA3 expression after 10 μM sorafenib treatment were evaluated by CCK8 assay. (I)The xenograft study confirmed that KPNA3-KD induced sensitization to sorafenib, whereas KPNA3-OE resulted in sorafenib resistance.
Figure 2
Figure 2
KPNA3 promotes growth and invasiveness of HCC. (A) Expression patterns of KPNA3 between HCC and normal liver tissues in different clinical cohorts. Datasets extracted from Oncomine (Chen, Roessler, and Wurmbach Liver). (B) mRNA expression levels of KPNA3 between tumor and adjacent normal liver tissues assessed via qRT-PCR. (C) Protein expression level of KPNA3 between tumor and adjacent normal liver tissues assessed by western blot. (D) Proliferation of Huh7 and HCCLM3 cells with modulated KPNA3 expression were evaluated by colony-formation assays. (E) Invasion of Huh7 and HCCLM3 cells with modulated KPNA3 expression were evaluated by transwell assays. (F) In vivo evaluation of the effect of KPNA3 on tumor proliferation.
Figure 3
Figure 3
KPNA3 induces EMT via increased TWIST expression to promote sorafenib resistance. (A) Evaluation of expression of epithelial and mesenchymal markers in Huh7 and HCCLM3 cells with modulated KPNA3 expression by qRT-PCR. (B) Validation of qRT-PCR results by western blot. (C) Evaluation of TWIST and SNAIL expression in Huh7 and HCCLM3 cells with modulated KPNA3 expression by qRT-PCR. (D) Evaluation of expression of epithelial and mesenchymal markers in HCCLM3 cells after TWIST knockdown by qRT-PCR (left) and western blot (right). (E) Survival rates of HCCLM3 cells with different modifications after 10 μM sorafenib treatment were evaluated by CCK8 assay.
Figure 4
Figure 4
KPNA3 promotes TWIST expression through activation of the AKT-ERK axis. (A, B) The downstream signaling of KPNA3 using a Cignal Finder Reporter Array for RTK signaling. (C) Effect of KPNA3 expression on the phosphorylation status of AKT and ERK were evaluated by western blot. (D) AKT and ERK inhibitors were used to evaluate their respective phosphorylation status in HCCLM3 cells with high KPNA3 expression. Experiments were conducted with or without 10 μM sorafenib treatment. (E) Activation status of AKT due to KPNA3 modulation under sorafenib treatment. (F) Effects of AKT inhibitor or combining AKT inhibitor with ERK activator on cell viability. (G) Twist transcriptional activities determined by dual fluorescence reporting system under different treatments. (H) Schematic depiction of the role of KPNA3 in sorafenib resistance.
Figure 5
Figure 5
KPNA3 correlates with response to sorafenib in HCC patients. (A) Kaplan-Meier analysis of OS of patients receiving sorafenib treatment for HCC stratified according to KPNA3 expression status (high or low). (B) Death rates of HCC patients receiving sorafenib treatment with high or low KPNA3 expression status.
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