AKR1C1 overexpression leads to lenvatinib resistance in hepatocellular carcinoma

Abstract

Background: Lenvatinib is an orally administered drug that works as a multi-targeted tyrosine kinase inhibitor. It has been approved as a first-line drug after sorafenib in hepatocellular carcinoma (HCC). However, little is currently known about its treatment, targets, and possible resistance in HCC.

Methods: The proliferation of HCC cells was evaluated using colony formation, 5-ethynyl-2'-deoxyuridine (EDU), wound healing, cell counting kit-8 (CCK-8), and xenograft tumor assays. RNA sequencing (RNA-seq) was utilized to comprehensively examine variations in highly metastatic human liver cancer cells (MHCC-97H) cells (treated with various doses of lenvatinib) at the transcriptomic level. Protein interactions and functions were predicted using Cytoscape-generated networks and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, while the proportions of 22 immune cell types were examined with CIBERSORT. Aldo-keto reductase family 1 member C1 (AKR1C1) expression was verified by quantitative real time polymerase chain reaction (qRT-PCR) or immunohistochemistry in HCC cells and liver tissues. Micro ribonucleic acid (miRNAs) were predicted using online tools and potential drugs were screened using the Genomics of Drug Sensitivity in Cancer (GDSC) database.

Results: Lenvatinib inhibited the proliferation of HCC cells. The obtained results suggested that an elevated level of AKR1C1 expression was observed in lenvatinib-resistant (LR) cell lines and HCC tissues, whereas low AKR1C1 expression inhibited the proliferation of HCC cells. Circulating microRNA 4644 (miR-4644) was predicted to serve as a promising biomarker for the early diagnosis of lenvatinib resistance. Online data analysis of LR cells showed significant differences in the immune microenvironment and drug sensitivity compared with their parental counterparts.

Conclusions: Taken together, AKR1C1 may serve as a candidate therapeutic target for LR liver cancer patients.

Keywords: Aldo-keto reductase family 1 member C1 (AKR1C1); biomarker; drug sensitivity; hepatocellular carcinoma (HCC); lenvatinib resistance.

Figure 1

Inhibitory effect of lenvatinib on HCC cell proliferation. Different lenvatinib concentrations, i.e., 0, 20, 40, 60, 80, and 100 µM, and stain with crystal violet. Results display lenvatinib inhibited the proliferation of HCC cells. (A) Lenvatinib decreases the proliferation of LM3 cells at a low dose of 20 µM. (B) Lenvatinib decreases the proliferation of MHCC-97H cells at 40 µM. After methanol fixation, clones were stained with crystal violet. According to the CCK-8 assay, the proliferative rates of LM3 (C) and MHCC-97H (D) cells exposed to various concentrations of lenvatinib were evaluated. *P<0.05; **P<0.01. OD, optical density; HCC, hepatocellular carcinoma; DMSO, dimethyl sulfoxide.

Figure 2

Identification and analysis of GO and KEGG terms of differentially expressed genes following RNA-seq. Bubble diagram of the top 10 activated or suppressed terms in biological process (A), cellular component (B), molecular function (C), and Kyoto Encyclopedia of Genes and Genomes enrichment (D). GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3

AKR1C1 and SERPINE1 were the key differentially expressed genes of lenvatinib targets. (A) RNA-seq was used to examine the transcriptome alterations in MHCC-97H cells treated with varying doses of lenvatinib. Genes with high or low expression in the four treatment groups were simultaneously shown by a volcano plot. Blue represents downregulated genes, red represents upregulated genes, and grey represents no difference genes. (B) The PPI network of the hub genes from upregulated genes. (C) PPI network of the hub genes from downregulated genes. (D,E) High expressions of AKR1C1 and SERPINE1 predicted poor prognosis in HCC patients. (F) The KEGG analysis revealed that AKR1C1 is involved in the Wnt signaling pathway, calcium signaling pathway, gastric cancer, etc. (G) The KEGG analysis also showed that AKR1C1 is involved in the PI3K/AKT signaling pathway, the Wnt signaling pathway, the IL-17 signaling pathway, and other pathways. (H) According to the expression level of AKR1C1, the data were divided into two groups. The ratios of activated dendritic cells, macrophages (M0), activated memory CD4 T cells, and eosinophils were characterized in two groups. (I) Similarly, for SERPINE1, the ratios of the proportions of activated NK cells, neutrophils, macrophages (M0), resting memory CD4 T cell, resting mast cells, and activated mast cells showed marked changes. (J) AKR1C1 and SERPINE1 are significantly correlated with macrophages (M0) (red indicates that AKR1C1 and SERPINE1 expression are positively correlated with immune cells. Blue indicates that AKR1C1 and SERPINE1 expression are negatively correlated with immune cells. The larger the difference in the ratios of the proportions of immune cells, the larger the circle size). *P<0.05, **P<0.01, ***P<0.001. PPI, protein-protein interaction; KEGG, Kyoto Encyclopedia of Genes and Genomes; LIHC, liver cancer; HCC, hepatocellular carcinoma; NC, negative control; NS, no sense.

Figure 4

The AKR1C1 expression was elevated in LR-HCC cells. (A) Seven up-regulated genes in LR Hep3B and Huh7 cells. (B) Three down-regulated genes in LR Hep3B and Huh7 cells. AKR1C1 was increased in HCC cell lines that were resistant to lenvatinib compared with the control group, (C) Hep3B, and (D) Huh7, respectively. (E) AKR1C1 was elevated in HCC cells and LEN-HepG2 (LR-HepG2 cells). **P<0.01, ****P<0.0001. LR, lenvatinib-resistant; HCC, hepatocellular carcinoma; NC, negative control; LEN, lenvatinib; NS, no sense.

Figure 5

Analysis of AKR1C1 expression in human HCC tissues and the survival of HCC patients. (A) Expression of AKR1C1 in human HCC tissues and paracancerous tissues (IHC staining). (B) Staining scores of AKR1C1 expression in HCC tissue samples and surrounding noncancerous tissues (IHC staining). (C) The immunostaining scores of AKR1C1 in 90 pairs of human HCC tissues and paracancerous tissues (****P<0.0001). (D) Kaplan-Meier analysis of the overall survival outcomes for 90 pairs of human HCC tissues (P=0.0198). *P<0.05, **P<0.01. HCC, hepatocellular carcinoma; TNM, International Classification of Clinical Stages for Malignant Tumors; AFP, alpha-fetoprotein.

Figure 6

Knockdown of AKR1C1 inhibits the proliferation of LEN-HepG2 cells. (A) The mRNA expression level of AKR1C1 in LEN-HepG2 cells was determined by qRT-PCR analysis after transduction. The proliferation of LEN-HepG2 cells was assessed by CCK-8 assays (B), wound healing assay (C; ×100), and EdU (D; ×100), respectively. **P<0.01, ***P<0.001. NC, negative control; OD, optical density; EdU, 5-ethynyl-2'-deoxyuridine.

Figure 7

Knockdown of AKR1C1 inhibits the proliferation of MHCC-97H cells. (A) The mRNA expression level of AKR1C1 in MHCC-97H cells was determined by qRT-PCR analysis after transduction. The proliferation of MHCC-97H cells was assessed by CCK-8 assays (B), wound healing assay (C), and EdU (D), respectively (×20). **P<0.01, ***P<0.001, ****P<0.0001. NC, negative control; OD, optical density; EdU, 5-ethynyl-2'-deoxyuridine; DAPI, 4'-6-diamidino-2-phenylindole.

Figure 8

Knockdown of AKR1C1 inhibits the proliferation of HCC cells in vivo. (A) Images of tumors in the AKR1C1 knockdown and the control groups. (B,C) Knockdown of AKR1C1 causes a significant decrease in tumor weight and volume (**P<0.01). (D) IHC staining images of E-cadherin, N-cadherin, and PCNA in the xenograft tumor tissues from each group of mice. HCC, hepatocellular carcinoma; NC, negative control; IHC, immunohistochemistry; PCNA, proliferating cell nuclear antigen.

Figure 9

MiR-4644 may be a biomarker for the early diagnosis of LR in HCC. (A) The mirDIP and starBase databases predicted that miR-4644 may bind to AKR1C1; (B) in cells that have developed resistance to lenvatinib, miR-4644 expression was significantly higher than in their wild-type counterparts; (C) prediction of the lncRNA-miRNA network in HCC. *P<0.05. LR, lenvatinib-resistant; HCC, hepatocellular carcinoma.

Figure 10

Erastin or sorafenib suppressed AKR1C1 expression in HCC cells. (A) GSE104462 shows that erastin suppressed AKR1C1 expression in HepG2 cells. (B) Different concentrations of erastin were co-cultured with LEN-HepG2 cells for 24 h. According to the results, erastin suppressed AKR1C1 expression in LEN-HepG2 cells. (C) Sorafenib suppressed AKR1C1 expression in LEN-HepG2 cells after being co-cultured for 24 h. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. NC, negative control.

Figure 11

A drug sensitivity analysis. (A) Drug sensitivity analyses (IC₅₀<1). (B) Potential drugs with remarkable treatment variations between the high- and low-risk populations. The P value of each group is less than 0.05.