SERPINB3 is associated with TGF-β1 and cytoplasmic β-catenin expression in hepatocellular carcinomas with poor prognosis

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most important sanitary problems for its prevalence and poor prognosis. To date, no information is available on the prognostic value of the ov-serpin SERPINB3, detected in primary liver cancer but not in normal liver. The aim of the study was to analyse SERPINB3 expression in liver cancer in relation with molecular signatures of poor prognosis and with clinical outcome.

Methods: Liver tumours of 97 patients were analysed in parallel for SERPINB3, TGF-β and β-catenin. In a subgroup of 67 patients with adequate clinical follow-up, the correlation of molecular findings with clinical outcome was also carried out.

Results: High SERPINB3 levels were detectable in 22% of the patients. A significant correlation of this serpin with TGF-β at transcription and protein level was observed, whereas for β-catenin a strong correlation was found only at post-transcription level. These findings were in agreement with transcriptome data meta-analysis, showing accumulation of SERPINB3 in the poor-prognosis subclass (S1). High levels of this serpin were significantly associated with early tumour recurrence and high SERPINB3 was the only variable significantly associated with time to recurrence at multivariate analysis.

Conclusions: SERPINB3 is overexpressed in the subset of the most aggressive HCCs.

Figure 1

SERPINB3, TGF-β1 and β-catenin mRNA in human HCCs. TGF-β1 (A) and β-catenin (B) mRNA were analysed by real-time PCR in relation to the presence (SERPINB3+ N=44) or absence (SERPINB3− N=53) of SERPINB3 mRNA. The y axis represents the relative mRNA amount of the normalised genes, calculated by dividing the non-normalised values by the housekeeping genes and expressed as arbitrary units. The analysis was performed with Mann–Whitney test. In the lower panels correlation of SERPINB3 with TGF-β1 (C) and β-catenin (D) mRNA in the same HCC sample are depicted.

Figure 2

Immunohistochemical analysis in human HCCs. Representative example of immunohistochemistry for SERPINB3, TGF-β1 and β-catenin, counterstained with hematoxylin, in serial sections of HCC specimens showing low SERPINB3 mRNA (A) or high SERPINB3 mRNA (B). Original magnification are reported. (C) Graphical representation of the quantitative analysis of each staining expressed as the mean of percentage of positive parenchyma staining per area in analysed tumour samples. Bars represent s.d. values. *P<0.0001 (HCCs with low SERPINB3 mRNA vs HCCs with high SERPINB3 mRNA).

Figure 3

SERPINB3 expression and human HCC molecular subclass. SERPINB3 expression level according to molecular subclass of HCC. SERPINB3 expression levels were extracted from publicly available transcriptome dataset (NCBI Gene Expression Omnibus accession number GSE10186) (n=97). Samples are ordered according to previously reported molecular subclasses of HCC determined by a meta-analysis of the following transcriptome datasets: S1 (activation of TGF-β/WNT pathways, EMT-like disseminative phenotype), S2 (MYC/AKT activation, AFP positivity) and S3 (preserved hepatocyte function) (Hoshida et al, 2009).

Figure 4

miRNA 146b-5p in relation to SERPINB3 expression. miR-146b-5p levels in control HepG2 (SERPINB3−) and in HepG2 cells expressing SERPINB3 (SERPINB3+) (A). Data are represented as mean of three independent experiments and bars represent s.e. miR-146b-5p values distribution in liver tumour specimens positive or negative for SERPINB3 (B). Central bars represent mean and external bars represent s.e.

Figure 5

Correlation of molecular variables with clinical outcome. Using 24 months as the cutoff value, all the recurrences were divided into early recurrences (⩽24 months) and late recurrences (>24 months). Distribution of mRNA values of SERPINB3 (A) and of the molecular variable TGF-β1 (B), which was found significantly correlated at transcription level with SERPINB3, is reported. Central bars represent mean and external bars represent s.e. Statistical analysis was performed using unpaired t-test with Welch's correction.

Figure 6

Kaplan–Meier curves of time to recurrence according to SERPINB3 expression. Patients with high (>median value) expression of SERPINB3 showed shorter recurrence-free survival, compared with patients with low or undetectable levels of SERPINB3. Statistical analysis was performed using log-rank (Mantel–Cox) test.