Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma

Abstract

Background: Similarly to other tumor types, an imbalance between unrestrained cell proliferation and impaired apoptosis appears to be a major unfavorable feature of hepatocellular carcinoma (HCC). The members of IAP family are key regulators of apoptosis, cytokinesis and signal transduction. IAP survival action is antagonized by specific binding of Smac/DIABLO and XAF1. This study aimed to investigate the gene and protein expression pattern of IAP family members and their antagonists in a series of human HCCs and to assess their clinical significance.

Methods: Relative quantification of IAPs and their antagonist genes was assessed by quantitative Real Time RT-PCR (qPCR) in 80 patients who underwent surgical resection for HCC. The expression ratios of XIAP/XAF1 and of XIAP/Smac were also evaluated. Survivin, XIAP and XAF1 protein expression were investigated by immunohistochemistry. Correlations between mRNA levels, protein expression and clinicopathological features were assessed. Follow-up data were available for 69 HCC patients. The overall survival analysis was estimated according to the Kaplan-Meier method.

Results: Survivin and Livin/ML-IAP mRNAs were significantly over-expressed in cancer tissues compared to non-neoplastic counterparts. Although Survivin immunoreactivity did not correlate with qPCR data, a significant relation was found between higher Survivin mRNA level and tumor stage, tumor grade and vascular invasion.The mRNA ratio XIAP/XAF1 was significantly higher in HCCs than in cirrhotic tissues. Moreover, high XIAP/XAF1 ratio was an indicator of poor prognosis when overall survival was estimated and elevated XIAP immunoreactivity was significantly associated with shorter survival.

Conclusion: Our study demonstrates that alterations in the expression of IAP family members, including Survivin and Livin/ML-IAP, are frequent in HCCs. Of interest, we could determine that an imbalance in XIAP/XAF1 mRNA expression levels correlated to overall patient survival, and that high XIAP immunoreactivity was a poor prognostic factor.

Figure 1

High XIAP/XAF1 ratio is a poor prognostic factor in HCC patients. Kaplan-Meier survival curves of HCC patients characterized by XIAP/XAF1 mRNA ratio > 2 (high, solid line) or ≤ 2 (low, dashed line) when compared by log-rank test. Overall survival was significantly different among the two subgroups of patients (p = 0.03).

Figure 2

Protein expression pattern of Survivin, XIAP and XAF1 in HCC tissues and non-neoplastic liver parenchyma. IAP members immunoreactivity was estimated by tissue microarray in a subset of HCC patients (n = 40). A-D, Representative Survivin cytoplasmatic immunostaining in a tumor core (A), in a tumor proximal to cirrhosis (C, N: cirrhosis, K: HCC), and in adjacent and long-distance non-neoplastic parenchyma (B and D, respectively). XIAP marked (score 12) and moderate (score 8) immunoreactivity is shown for HCC (E and I, respectively) as well as for cirrhosis (F and L, respectively). G and H, Nuclear XAF1 staining is shown for tumor and non-neoplastic liver whereas XAF1 cytoplasmatic expression in HCC and cirrhosis is shown in panels M and N, respectively. Original magnification ×50 and ×250, for tissue cores and insets, respectively.

Figure 3

High XIAP expression is a poor prognostic factor in HCC patients. Kaplan-Meier survival curves of HCC patients characterized by high XIAP expression (solid line) or low (dashed line) when compared by log-rank test. Overall survival was significantly different among the two subgroups of patients (p = 0.03).