Human constitutive androstane receptor represses liver cancer development and hepatoma cell proliferation by inhibiting erythropoietin signaling

Abstract

The constitutive androstane receptor (CAR) is a nuclear receptor that plays a crucial role in regulating xenobiotic metabolism and detoxification, energy homeostasis, and cell proliferation by modulating the transcription of numerous target genes. CAR activation has been established as the mode of action by which phenobarbital-like nongenotoxic carcinogens promote liver tumor formation in rodents. This paradigm, however, appears to be unrelated to the function of human CAR (hCAR) in hepatocellular carcinoma (HCC), which remains poorly understood. Here, we show that hCAR expression is significantly lower in HCC than that in adjacent nontumor tissues and, importantly, reduced hCAR expression is associated with a worse HCC prognosis. We also show overexpression of hCAR in human hepatoma cells (HepG2 and Hep3B) profoundly suppressed cell proliferation, cell cycle progression, soft-agar colony formation, and the growth of xenografts in nude mice. RNA-Seq analysis revealed that the expression of erythropoietin (EPO), a pleiotropic growth factor, was markedly repressed by hCAR in hepatoma cells. Addition of recombinant EPO in HepG2 cells partially rescued hCAR-suppressed cell viability. Mechanistically, we showed that overexpressing hCAR repressed mitogenic EPO-EPO receptor signaling through dephosphorylation of signal transducer and activator of transcription 3, AKT, and extracellular signal-regulated kinase 1/2. Furthermore, we found that hCAR downregulates EPO expression by repressing the expression and activity of hepatocyte nuclear factor 4 alpha, a key transcription factor regulating EPO expression. Collectively, our results suggest that hCAR plays a tumor suppressive role in HCC development, which differs from that of rodent CAR and offers insight into the hCAR-hepatocyte nuclear factor 4 alpha-EPO axis in human liver tumorigenesis.

Keywords: EPO; HCC; HNF4α; hepatoma cell proliferation; human CAR.

Figure 1

CAR downregulation correlates with poor survival in patients with liver cancer.A, the microarray analysis of hCAR expression in 247 HCC and 239 nontumor liver tissues reveals expression of hCAR is significantly lower in HCC. B and C, HCC samples were further divided into high and low hCAR clusters, and both have lower levels of hCAR in comparison to their paired controls. D, HCC patients with relatively low levels of hCAR are associated with poor survival (log-rank test, p < 0.05; n = 116). E and F, the analysis of hCAR expression and survival using TCGA datasets demonstrated that low expression of hCAR is associated with poor overall survival. CAR, constitutive androstane receptor; hCAR, human CAR; HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas.

Figure 2

Overexpression of CAR suppresses the viability of hepatoma cells.A, endogenous mRNA expression of hCAR was measured using RT–PCR in human primary hepatocytes (three liver donors) and liver cancer cells (HepG2, Hep3B, and Huh7). B, schematic illustration of the generation of Tet-On-based hCAR-inducible hepatoma cell line. C, mRNA expression of hCAR was analyzed in HepG2-hCAR and Hep3B-hCAR cell lines as well as normal HepG2 and Hep3B cells treated with vehicle control (Con) or Dox (1 μg/ml). D, protein expression of hCAR in these cell lines is consistent with the mRNA changes 72 h after Dox or vehicle control treatment. E, overexpression of hCAR in HepG2-hCAR and Hep3B-hCAR cells results in suppression of cell growth in a time-dependent manner through 8 days of Dox treatment, whereas the growth of normal HepG2 and Hep3B was not significantly affected. F, HepG2 cells were transfected with the CYP2B6 luciferase reporter construct in the presence of hCAR or hCAR3 expression vector. Relative luciferase activity was measured using the Promega dual-luc reagent. In a separate experiment, (G) overexpression of hCAR or hCAR3 in HepG2 and Hep3B cells was achieved by infection of lentivirus-expressing hCAR or hCAR3 as detailed in the “Experimental procedures” section. H, relative cell viability in HepG2 and Hep3B cells infected with lentivirus-expressing empty vector (Con), hCAR, or hCAR3 was monitored for 8 days using CCK-8 reagents. Relative blot densitometry was quantified using ImageJ from three separately prepared cell experiments and normalized to the density of the loading control. Results are expressed as mean ± SD from at least three independent experiments. ∗p < 0.05 and ∗∗p < 0.01. CAR, constitutive androstane receptor; CCK-8, Cell Counting Kit-8; Dox, doxycycline; hCAR, human CAR.

Figure 3

Overexpression of CAR inhibits cell cycle progression in HepG2 and Hep3B cells. Cell cycle progression was analyzed in HepG2-hCAR and Hep3B-hCAR cells 3 days after treatment with vehicle control or Dox (1 μg/ml). The percentage of cells in different cell cycle phases was compared between vehicle control and Dox-treated HepG2-hCAR (A), Hep3B-hCAR (B), as well as normal HepG2 and Hep3B (C) cells. D, Western blotting was carried out to measure the protein levels of p21 in HepG2-hCAR, Hep3B-hCAR, HepG2, and Hep3B cells treated with vehicle control or Dox as detailed in the “Experimental procedures” section. Relative blot densitometry was quantified using ImageJ from three separately prepared cell experiments and normalized to the density of the loading control. Data were expressed as mean ± SD (n = 3). ∗∗p < 0.01. CAR, constitutive androstane receptor; Dox, doxycycline; hCAR, human CAR.

Figure 4

Overexpression of hCAR inhibits colony formation of hepatoma cells in vitro and the growth of hepatoma xenograft in vivo. Normal and hCAR-inducible HepG2 and Hep3B cells were treated with Dox (1 μg/ml) or vehicle control for 72 h followed by a colony formation in soft-agar assay as described in the “Experimental procedures” section. Representative colony images and quantification colony formation are shown in A and B. The histograms represent mean ± SD of the cloning efficiency (%). Data presented are representative images from at least three independent experiments in A and B. For the xenograft experiment, 3 to 5 × 10⁶ HepG2-hCAR or Hep3B-hCAR cells were inoculated into the nude mice for xenograft formation as detailed under the “Experimental procedures” section. The tumor growth curves of HepG2-hCAR (C) and Hep3B-hCAR xenografts (F) were measured in the control-diet and Dox-diet feeding groups during the experimental periods. RT–PCR and Western blotting were used to measure the mRNA and protein expressions of hCAR in the xenograft tumors (D and G). Relative blot densitometry was quantified using ImageJ from three separately prepared cell experiments and normalized to the density of the loading control. Expression of Ki67 was examined by immunohistochemistry (E and H); the scale bar represents 100 μm. Ki67 intensity was quantified using ImageJ and was expressed as mean ± SD (n = 9). ∗p < 0.05 and ∗∗p < 0.01. Dox, doxycycline; hCAR, human constitutive androstane receptor.

Figure 5

RNA-Seq analysis identifies EPO as a novel hCAR target gene. The total RNA from HepG2-hCAR was prepared 72 h after Dox (1 μg/ml) or vehicle control treatment for RNA-Seq analysis as detailed under the “Experimental procedures” section. A, the volcano plot of RNA-Seq data showed 374 upregulated and 348 downregulated genes in HepG2-hCAR cells after hCAR induction. B, heatmaps illustrate 10 selected upregulated and downregulated genes with high fold changes in HepG2-hCAR cells treated with Dox or vehicle control. RT–PCR validation of RNA-Seq analysis results for the 10 upregulated and 10 downregulated genes in HepG2-hCAR (C), Hep3B-hCAR (D), HepaRG WT and hCAR-KO cells (E), respectively. Data were collected from three independent experiments and expressed as mean ± SD in C–E. ∗p < 0.05 and ∗∗p < 0.01. Dox, doxycycline; EPO, erythropoietin; hCAR, human constitutive androstane receptor; ND, not detected.

Figure 6

EPO is inversely correlated to hCAR expression in HCC and promotes proliferation of HepG2 and Hep3B cells.A, hCAR and EPO gene expression was inversely correlated in patients with HCC. A total of 381 HCC samples were analyzed from a publicly accessible TCGA database through SurvExpress, and the overall survival was compared between low-hCAR–expressing (123), medium-hCAR–expressing (232), and high-hCAR expressing (26) groups. B, correlation analysis between hCAR and EPO reveal a Spearman’s correlation coefficient of −0.20 (p < 0.01). C, the pair of low hCAR with high EPO is associated with poor survival. D, in HepG2-hCAR and Hep3B-hCAR cells, treatment with Dox resulted in increased expression of hCAR and decreased expression of EPO in a concentration-dependent manner. E, expression of EPO in HepG2 cells was efficiently abolished after EPO knockdown via lentivirus shRNA targeting different regions of EPO (shEPO 1 to shEPO 5). F, depletion of EPO in HepG2 and Hep3B cells results in suppression of cell growth in a time-dependent manner. HepG2 and Hep3B cells were also visualized by Coomassie blue staining 8 days after infection with lentivirus shEPO 1, shEPO 3, or shCon. G, relative cell growth rate of HepG2-hCAR cells treated with Dox was partially rescued by addition of the recombinant human EPO treatment. Results are expressed as mean ± SD from three independent experiments in D–G. ^##p < 0.01; ∗p < 0.05; and ∗∗p < 0.01. Dox, doxycycline; EPO, erythropoietin; hCAR, human constitutive androstane receptor; HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas.

Figure 7

Overexpression of hCAR represses EPO downstream signaling in HepG2 and Hep3B cells.A, schematic illustration of the EPO–EPOR signaling pathway. B and C, HepG2-hCAR and Hep3B-hCAR cells were treated with vehicle control or Dox (1 μg/ml) for 72 h before Western blotting analysis of total and phosphorylation of STAT3 (p-STAT3-Y705), AKT (p-AKT-S473), and ERK1/2 (p-ERK1/2-T183/Y185). D, relative blot densitometry was quantified using ImageJ from three separately prepared cell experiments and normalized to the density of the loading control. Data are expressed as mean ± SD (n = 3). ∗p < 0.05 and ∗∗p < 0.01. Dox, doxycycline; EPO, erythropoietin; EPOR, EPO receptor; ERK1/2, extracellular signal–regulated kinase 1/2; hCAR, human constitutive androstane receptor; STAT3, signal transducer and activator of transcription 3.

Figure 8

hCAR downregulates EPO expression through suppression of HNF4α.A, RT–PCR was used to analyze expression of hCAR, EPO, HNF4α, and HIF1β in HepG2-hCAR and Hep3B-hCAR cells 72 h after treatment with vehicle control or Dox (1 μg/ml) as detailed in the “Experimental procedures” section. B, the protein levels of HNF4α were analyzed by immunoblotting in HepG2-hCAR, Hep3B-hCAR, as well as normal HepG2 and Hep3B cells treated with vehicle control or Dox for 72 h. Relative blot densitometry was quantified using ImageJ from three separately prepared cell experiments and normalized to the density of the loading control. C, knockdown of HNF4α expression in HepG2 and Hep3B cells via lentivirus shRNA targeting different regions of HNF4α (shHNF4α-1 and shHNF4α-2) resulted in downregulation of EPO expression in these cells. D, overexpression of HNF4α partially rescues hCAR-mediated downregulation of EPO expression in HepG2-hCAR and Hep3B-hCAR stable cell lines. E, EMSA was performed to detect interaction between the EPO–DR2 motif and nuclear extract samples isolated from HepG2-hCAR and Hep3B-hCAR cells treated with vehicle control or Dox for 72 h. HNF4α antibody was used for measuring specific supershift of the HNF4α/DR2 band. 1: NE of HepG2-hCAR-control; 2: NE of HepG2-hCAR-Dox; 3: NE of Hep3B-hCAR-control; and 4: NE of Hep3B-hCAR-Dox. F, HEK293T cells were transfected with EPO-3′UTR-enhancer reporter construct in the presence of HNF4α and/or hCAR expression vectors as indicated, and luciferase activities were determined 48 h after transfection. Three independent measures from each group were analyzed and expressed as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01. Dox, doxycycline; DR2, direct repeat 2; EPO, erythropoietin; hCAR, human constitutive androstane receptor; HEK293T, human embryonic kidney 293T cell line; HIF1β, hypoxia-inducible factor 1β; HNF4α, hepatocyte nuclear factor 4 alpha.