Progesterone receptor membrane component 1 as a potential prognostic biomarker for hepatocellular carcinoma

Abstract

Aim: To investigate the clinicopathological significance of progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 in hepatocellular carcinoma (HCC).

Methods: We performed immunohistochemical staining to evaluate the estrogen receptor (ER), progesterone receptor (PR), PGRMC1, and PGRMC2 in a clinical cohort consisting of 89 paired HCC and non-tumor liver samples. We also analyzed HCC data (n = 373) from The Cancer Genome Atlas (TCGA). We correlated the expression status of PGRMC1 and PGRMC2 with clinicopathological indicators and the clinical outcomes of the HCC patients. We knocked down or overexpressed PGRMC1 in HCC cell lines to evaluate its biological significance in HCC cell proliferation, differentiation, migration, and invasion.

Results: We found that few HCC cases expressed ER (5.6%) and PR (4.5%). In contrast, most HCC cases expressed PGRMC1 (89.9%) and PGRMC2 (100%). PGRMC1 and PGRMC2 exhibited significantly lower expression in tumor tissue than in non-tumor tissue (P < 0.001). Lower PGRMC1 expression in HCC was significantly associated with higher serum alpha-fetoprotein expression (P = 0.004), poorer tumor differentiation (P = 0.045) and liver capsule penetration (P = 0.038). Low PGRMC1 expression was an independent predictor for worse disease-free survival (P = 0.002, HR = 2.384, CI: 1.377-4.128) in our cases, as well as in the TCGA cohort (P < 0.001, HR = 2.857, CI: 1.781-4.584). The expression of PGRMC2 did not relate to patient outcome. PGRMC1 knockdown promoted a poorly differentiated phenotype and proliferation of HCC cells in vitro, while PGRMC1 overexpression caused the opposite effects.

Conclusion: PGRMC1 is a non-classical hormonal receptor that negatively regulates hepatocarcinogenesis. PGRMC1 down-regulation is associated with progression of HCC and is a poor prognostic indicator.

Keywords: Hepatocellular carcinoma; Hormonal receptor; Progesterone receptor membrane component 1; Prognosis; Proliferation.

Figure 1

Western blot analysis of PGRMC1 and PGRMC2 expression in 10 paired hepatocellular carcinoma tumor (T)/non-tumor liver (NT) samples (A). B: Progesterone level in HCC tissue samples and non-tumor liver tissue; C: A comparison of progesterone levels in HCC (T) and non-tumor liver (NT) tissue samples; D: Serum progesterone level in the corresponding HCC patients. ^bP < 0.01.

Figure 2

PGRMC1 and PGRMC2 expression levels, expressed as positive immunohistochemical staining percentages in the clinical cohort (A and B) and as normalized mRNA expression in the TCGA cohort (C-F). A: A comparison of PGRMC1 and PGRMC2 staining in HCC (T), non-tumor liver (NT) and normal liver (N) tissue samples; B: A comparison of PGRMC1 staining in HCC samples with different degrees of tumor differentiation; C: A comparison of PGRMC1 and PGRMC2 mRNA expression levels in HCC (T) and non-tumor liver (NT) samples; D: A comparison of PGRMC1 mRNA expression levels in HCC samples with different tumor grades; E: A comparison of PGRMC1 mRNA expression levels in HCC samples with different tumor stages; F: A comparison of PGRMC2 mRNA expression levels in HCC samples with different tumor grades. ^aP < 0.05, ^bP < 0.01, ^eP < 0.001.

Figure 3

Representative images of PGRMC1 immunohistochemical staining in hepatocellular carcinoma. A-C: Well-differentiated HCC; D-F: Moderately differentiated HCC; G-I: Poorly differentiated HCC. Note that higher PGRMC1 expression was observed in non-tumor liver tissue samples (NT) as compared to HCC tissue samples (T) (A, D and G) and a proportion of HCC cells showed loss of PGRMC1 staining in (E) (A, D and G: 40 ×; B, C, E, F, H, and I: 100 ×).

Figure 4

Kaplan-Meier analysis of the relationships of PGRMC1 and PGRMC2 expression with disease-free survival (DFS) in the clinical cohort (A-B) and TCGA cohort (C-D). ^aP < 0.05, ^eP < 0.001.

Figure 5

Alpha-fetoprotein and glypican-3 expression in PGRMC1-knockdown HepG2 and Hep3B cells and PGRMC1-overxpressing PLC/PRF/5 and Huh7 cells (A). B: The correlation of PGRMC1 with AFP and GPC3 in HCC tissue samples in the TCGA cohort; C: Cell proliferation assay (XTT) of PGRMC1-knockdown HepG2 and Hep3B cells and PGRMC1-overxpressing PLC/PRF/5 and Huh7 cells. The experiment was performed in triplicate. ^aP < 0.05, ^bP < 0.01, ^eP < 0.001. AFP: Alpha-fetoprotein; GPC3: Glypican-3.