Constitutive androstane receptor (CAR) functions as a tumor suppressor via regulating stemness in liver cancer

Abstract

Constitutive androstane receptor (CAR) is a xenosensor that is almost exclusively expressed in the liver. Studies in rodents suggest an oncogenic role for CAR in liver cancer, but its role in human liver cancer is unclear. We aimed to investigate the functional roles of CAR in human liver cancer with a focus on the liver cancer stem cells. We used bioinformatics to increase our understanding of CAR in human liver cancer and associated stem cell markers. We studied the functional roles of CAR in human liver cancer with a focus on the liver cancer stem cell using siRNA, modulation of CAR activity, and tumorsphere formation assays. We have revealed significant associations between CAR and a wide variety of signalling pathways including stemness signalling. Further in vitro studies have shown that activation of CAR significantly reduces cancer cell stemness and represses proliferation, migration, invasion, and the tumorsphere-forming abilities of liver cancer cells (p < 0.05). Our data demonstrates the unequivocal tumor-suppressive role of CAR in liver cancer. While more detailed mechanistic studies are warranted, the efficacy of CAR xeno-activators in the treatment of advanced hepatocellular carcinoma (HCC) may potentially open a new avenue for liver cancer therapy.

Keywords: CITCO; Constitutive androstane receptor (CAR); Hepatocellular carcinoma (HCC); Liver cancer; Liver cancer stem cells (LCSCs); Tumor suppressor.

Fig. 1

Correlation between CAR expression, gene signatures, and stem cell markers. Gene signatures were downloaded from MSigDB, the Human Protein Atlas, and cBioPortal. Spearman’s correlation analysis was performed using GEPIA2. CAR negatively correlated to genes that are over-expressed or involved in HCC growth and progression while the opposite trend was discovered with the gene signatures that are downregulated in high-grade HCC and those associated with better HCC survival (p < 0.05) (A). A significant negative correlation between CAR and CD24, CD44, CD133, and EpCAM can also be seen in the heatmap (p < 0.05) (B).

Fig. 2

CAR activation decreased the proliferation, migration, invasion, and tumorsphere formation of HCC cells. HCC cells were pre-treated with 1 µM of CITCO for 48 h, and then further cultured in the presence of 1 µM of CITCO for 24 h (for migration assay) or 48 h (for proliferation and invasion assay). A significant decrease in proliferation (A), migration, and invasion (B) was seen in Hep3B, Huh-7, and PLC/PRF/5 cells. (C) Representative images of migration and invasion of HCC cells treated with CITCO. (D) Quantitative analysis of the tumorsphere formation assay showing decreased sphere-forming ability in CITCO-treated cells. (E) Significant reductions in the gene expression of stem cell markers in CITCO-treated tumorspheres. *p < 0.05; **p < 0.01; ***p < 0.001. n = 3 per group. n = 3 per group. Data are represented as mean ± SEM.

Fig. 3

Effects of CAR knockdown on the proliferation, migration, invasion, and cell cycle distribution of HCC cells. Transient CAR knockdown by siCAR led to a significant increase in the proliferation of HCC cells (A). CAR knockdown also enhanced migration and invasion across all three cell lines (B). (C) Representative images of migration and invasion of HCC cells with CAR knockdown. (D) CAR knockdown resulted in a decreased proportion of cells in the G1 phase and a concomitant increase in the proportion of cells in the S and G2 phases. *p < 0.05; **p < 0.01; ***p < 0.001. n = 3 per group. Data are represented as mean ± SEM.