Loss of PGRMC1 Delays the Progression of Hepatocellular Carcinoma via Suppression of Pro-Inflammatory Immune Responses

Abstract

Pgrmc1 is a non-canonical progesterone receptor related to the lethality of various types of cancer. PGRMC1 has been reported to exist in co-precipitated protein complexes with epidermal growth factor receptor (EGFR), which is considered a useful therapeutic target in hepatocellular carcinoma (HCC). Here, we investigated whether Pgrmc1 is involved in HCC progression. In clinical datasets, PGRMC1 transcription level was positively correlated with EGFR levels; importantly, PGRMC1 level was inversely correlated with the survival duration of HCC patients. In a diethylnitrosamine (DEN)-induced murine model of HCC, the global ablation of Pgrmc1 suppressed the development of HCC and prolonged the survival of HCC-bearing mice. We further found that increases in hepatocyte death and suppression of compensatory proliferation in the livers of DEN-injured Pgrmc1-null mice were concomitant with decreases in nuclear factor κB (NF-κB)-dependent production of interleukin-6 (IL-6). Indeed, silencing of Pgrmc1 in murine macrophages led to reductions in NF-κB activity and IL-6 production. We found that the anti-proinflammatory effect of Pgrmc1 loss was mediated by reductions in EGFR level and its effect was not observed after exposure of the EGFR inhibitor erlotinib. This study reveals a novel cooperative role of Pgrmc1 in supporting the EGFR-mediated development of hepatocellular carcinoma, implying that pharmacological suppression of Pgrmc1 may be a useful strategy in HCC treatment.

Keywords: EGFR; HCC; Pgrmc1; inflammation; liver cancer.

Figure 1

Correlation of PGRMC1 and EGFR with survival duration in patients with hepatocellular carcinoma (HCC). (A,B) Kaplan–Meier curves for the relationships of the mRNA levels of (A) PGRMC1 and (B) EGFR with the overall survival duration of patients with hepatocellular carcinoma in two independent cohorts from the GEO database, GSE76427 (left) and GSE20140 (right). (C) EGFR mRNA expression levels in HCC patients with high or low levels of PGRMC1 in the GSE76427 (left) and GSE20140 (right) cohorts.

Figure 2

Loss of Pgrmc1 extends the survival duration and suppresses the development of mice bearing hepatocellular carcinoma (HCC). (A) Kaplan–Meier curve of wild-type (WT) and Pgrmc1 knockout (KO) mice bearing HCC. For the induction of HCC, mice were injected with diethylnitrosamine (DEN) (25 mg/kg, i.p.) at 2-week-old of age, and their survival rate was monitored until 104 weeks. (B) Gross images of the livers of WT and Pgrmc1-null mice with DEN-induced HCC. Mice (2-weeks-old) were injected with DEN (25 mg/kg/week, i.p.) once a week for 4 weeks and sacrificed at 50 weeks for tumor assessment. Scale bar, 0.5 cm. (C) Tumor incidence at 50-weeks-old WT (n = 11) and Pgrmc1 KO (n = 12) mice with DEN-induced HCC. (D) Plasma ALT level in 50-weeks-old WT and Pgrmc1 KO mice with DEN-induced HCC. * p < 0.05 in Student’s t-test. (E) Tumor number, (F) individual tumor size, and (G) average tumor size in the livers of 50-week-old WT and Pgrmc1 KO mice with DEN-induced HCC. * p < 0.05 in Student’s t-test.

Figure 3

Loss of Pgrmc1 aggravates liver injury while suppressing compensatory proliferation. (A) WT and Pgrmc1-null mice were injected with high-dose DEN (200 mg/kg, i.p.), and their plasma and livers were collected after 48 h. (B) Plasma level of alanine aminotransferase (ALT). (C) Expression of high mobility group box 1 (HMGB1) in the plasma. The expression level was normalized to that of plasma albumin and expressed relative to the WT group. (D) TUNEL staining in the livers of WT and Pgrmc1-null mice. TUNEL-positive cells were counted and expressed relative to the WT group. Scale bar, 100 μm. (E) Ki67 immunostaining in the livers of WT and Pgrmc1-null mice. Ki67-positive cells were counted and expressed relative to the WT group. Scale bar, 100 μm. (F) mRNA expression levels of C-Myc, Cyclin D, and Hgf in the livers of WT and Pgrmc1-null mice. The expression level was normalized to that of Rplp0 and expressed relative to the WT group. * p < 0.05 in Student’s t-test. All full blot images were provided in Supplementary File (Figure S3).

Figure 4

Loss of Pgrmc1 suppresses EGFR activation. (A) PGRMC1, EGFR, and pEGFR expression levels in DEN-injected livers of WT and Pgrmc1-null mice. The expression levels were normalized to that of β-actin and expressed relative to the WT group. (B) mRNA expression of Egfr in DEN-injected livers of WT and Pgrmc1-null mice. The expression levels were normalized to that of Rplp0 and expressed relative to the WT group. * p < 0.05 in Student’s t-test. (C,D) PGRMC1, EGFR, and pEGFR expression levels in primary hepatocytes (C) and primary non-parenchymal cells (D) from WT and Pgrmc1-null mice. The expression levels were normalized to that of β-actin and expressed relative to the WT group. * p < 0.05 in Student’s t-test. All full blot images were provided in Supplementary File (Figure S3).

Figure 5

Loss of Pgrmc1 suppresses IL-6 production in macrophages. (A,B) Plasma level of IL-6 (A) and hepatic mRNA levels of IL-1α, IL-1β, and Tnf (B) in DEN-injected WT and Pgrmc1-null mice. The mRNA expression levels were normalized to that of Rplp0 and expressed relative to the WT group. * p < 0.05 in Student’s t-test. (C,D) Culture supernatant level of IL-6 (C) and mRNA expression levels of IL-1α, IL-1β, and Tnf (D) in Raw 264.7 cells transfected with Control (Ctrl) or Pgrmc1-siRNA (KD) and treated with or without necrotic debris for 3 h. The mRNA expression levels were normalized to that of Rplp0 and expressed relative to the Ctrl group. * p < 0.05 in one-way ANOVA followed by Tukey’s post hoc test. (E,F) Control (Ctrl) or Pgrmc1-siRNA (KD)-transfected cells were treated with erlotinib (50 μM) and necrotic debris for 8 h. Level of IL-6 (E) and mRNA expression levels of IL-1α, IL-1β, and Tnf (F) in the supernatant of erlotinib-treated Raw 264.7 cells. The mRNA expression levels were normalized to that of Rplp0 and expressed relative to the Ctrl group. * p < 0.05 in one-way ANOVA followed by Tukey’s post hoc test.

Figure 6

EGFR inhibition by erlotinib nullifies the anti-proinflammatory role of Pgrmc1 depletion. (A) Western blot analysis of PGRMC1, EGFR, pEGFR, IκBα, pIκBα, NF-κB (p65), and pNF-κB (p65) and (B) mRNA expression levels of Egfr in Raw 264.7 cells transfected with Control (Ctrl) or Pgrmc1-siRNA (KD) and treated necrotic debris for 3 h. The protein expression levels were normalized to that of β-actin and expressed relative to the Ctrl group. The mRNA expression levels were normalized to that of Rplp0 and expressed relative to the Ctrl group. * p < 0.05 in Student’s t-test. (C,D) Control (Ctrl) or Pgrmc1-siRNA (KD)-transfected cells were treated with erlotinib (50 μM) and necrotic debris for 8 h. (C) Western blot analysis of PGRMC1, EGFR, pEGFR, IκBα, pIκBα, NF-κB (p65), and pNF-κB (p65) and (D) mRNA expression levels of Egfr in erlotinib-treated Raw 264.7 cells. The protein expression levels were normalized to that of β-actin and expressed relative to the Ctrl group. The mRNA expression levels were normalized to that of Rplp0 and expressed relative to the Ctrl group. * p < 0.05 in Student’s t-test. All full blot images were provided in Supplementary File (Figure S3).