Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma

Abstract

Objectives: Hepatocellular carcinoma (HCC) is a leading cancer-related cause of death. Unfortunately, recurrence is common even after curative treatment of early-stage patients, and no adjuvant treatment has yet been established. Aberrant expression of OLFM4 in human cancers has been reported; yet, its specific function during tumor development remains poorly understood, and its role in HCC is unknown. The purpose of this study is to examine the prognostic significance of OLFM4 and its functional relevance in determining recurrence in patients with early-stage HCC.

Methods: Immunohistochemical staining to assess expression, cellular distribution, and prognostic significance of OLFM4 was performed in a tissue microarray comprising 157 HCC tissues and matched nontumor tissues. In addition, expression of OLFM4-coding mRNA was assessed in a separate patients' cohort. The findings were validated by in vitro functional studies using siRNA directed against OLFM4 to assess its effect on cell motility and proliferation.

Results: The fraction of HCC samples exhibiting positive OLFM4 staining was higher in comparison with that observed in hepatocytes from matched nontumor tissue (61% vs 39%). However, cytoplasmic-only staining for OLFM4 was associated with vascular invasion (P = 0.048), MMP-7 expression (P = 0.002), and poorer survival (P = 0.008). A multivariate analysis confirmed the independent significance of OLFM4 in determining patients' outcome (5-year survival [58.3% vs 17.3%; HR: 2.135 {95% confidence interval: 1.135-4.015}; P = 0.019]). Correspondingly, inhibition of OLFM4 by siRNA modulated the expression of MMP-7 and E-cadherin, causing inhibition of cell proliferation, motility, and migration.

Discussion: To the best of our knowledge, we provide the first report on the prognostic significance of OLFM4 in HCC and identify its mechanistic role as crucial mediator of MMP family protein and E-Cadherin in determining cell invasion and metastasis formation.

Figure 1.

Expression and cellular distribution of OLFM4 in tumor and nontumor tissue samples. (a, b). Staining of OLFM4 in HCC cells and in nontumor cells: proportion of samples showing no staining (none), cytoplasmic staining (cytoplasm only), membrane staining (membrane only), or both (m + c). Representative cytoplasm staining pattern in HCC samples: (c) score 0, negative; (d) score 1, weak; (e) score 2, strong. Representative typical positive membrane staining in HCC tissues: (f) score 0, negative; (g) score 1, less than 30% of cells; (h) 30%–70% of cells; (i) over 70% of cells. Magnification, ×40. HCC, hepatocellular carcinoma.

Figure 2.

Assessment of the correlations between OLFM4 and E-cadherin (a) or MMP-7 (b) mRNA expression levels according to the analysis of an independent HCC cohort. HCC, hepatocellular carcinoma.

Figure 3.

Prognostic significance of OLFM4 according to cellular distribution in patients undergoing hepatectomy. (a) Overall survival (OS) of the entire patients' collective as determined by the Kaplan-Meier method. (b) OS according to evidence of vessel invasion. OS according to cytoplasm staining for OLFM4 (positive or negative (c)) or according to semiquantitative assessment of staining intensity (d). OS according to membrane staining for OLFM4 (positive or negative (e)) or according to semiquantitative assessment of staining intensity (f). (g) OS according to OLFM4 staining in HCC tissues from LMU Munich. (h) Kaplan-Meier survival curves according to OLFM4 mRNA expression levels in a second independent cohort of patients with HCC from TCGA. +Censored cases. HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas.

Figure 4.

Overall survival according to OLFM4 staining in non-HCC tissues from LMU Munich cohort of hepatectomy. +Censored cases. HCC, hepatocellular carcinoma.

Figure 5.

Overall survival (a) and prognostic significance of multifocal HCC (b) in liver transplant recipients from LMU Munich cohort. +Censored cases. HCC, hepatocellular carcinoma.

Figure 6.

In vitro assessment of the effect of OLFM4 silencing on MMP-7 and E-cadherin. (a) Effect of transfection of Huh7 and PLCPRF5 cells with siRNA specifically targeting OLFM4 or non-coding siRNA (Beta-gal, 50 nM) for 24 hours on the expression of the indicated molecules after immunoblotting. (b) Typical pattern of OLFM4 expression at immunofluorescence after transfection with control-targeting or OLFM4-targeting siRNA. Green: DAPI staining of the nuclei; Red: immunofluorescence of OLFM4.

Figure 7.

Effect of OLFM4 silencing on cell migration and invasion. (a, b) Assessment of cell motility by wound healing assay in Huh7 or PLCPRF5 cells transfected with siRNA-OLFM4 or control-si-RNA. (c) Quantitative analysis of healing area in percentages. (d, e) The migration and invasion of Huh7 and PLCPRF5 cells transfected with siRNA-OLFM4 compared with the controls by transwell assay after 24 hours. (f, g) Quantitative analysis of migration and invasion cells. (h) The E-cadherin expression of Huh7 and PLCPRF5 cells transfected with siRNA-OLFM4 compared with the controls by immunofluorescence after 24 hours. (i–j) Effects of OLFM4 silencing by siRNA on sub-G1 events, cell cycle distribution, and cell viability in Huh7 (i) and PLCPRF5 (j) cell lines. Values represented mean ± SD from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, Student t-test. Blue: DAPI staining of nuclei; green, immunofluorescence of E-cadherin.

Figure 8.

Schematic diagram describing the hypothesized effect of OLFM4, MMP family members and on invasion and metastasis formation in HCC. HCC, hepatocellular carcinoma.