4338954792152307 revised-2 Inhibitors of Apoptosis Proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma Authors:

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 its 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. Background Hepatocellular carcinoma (HCC), one of the most common malignant tumors worldwide, can be managed with surgical resection or transplantation in selected cases, whereas advanced tumors responds poorly to currently available medical therapies [1]. The understanding of the molecular pathways leading to the development of HCC may provide important data to develop new therapies. Similarly to other tumor types, an imbalance between unrestrained cell proliferation and impaired apoptosis appears to be a major unfavorable feature of HCC [2]. Recent studies have documented the over-expression of anti-apoptotic factors such as the inhibitors of apoptosis proteins (IAPs) in a variety of solid tumors and cancer cell lines [3]. Eight human IAPs have been identified so far: NAIP (BIRC1), c-IAP1 (BIRC2), c-IAP2 (BIRC3), X-linked IAP (XIAP, BIRC4), Survivin (BIRC5), Apollon (BRUCE, BIRC6), Livin/ML-IAP (BIRC7) and IAP-like protein 2 (BIRC8) [4]. The members of IAP family, defined by the presence of a baculovirus IAP repeat (BIR) protein domain, are key regulators of apoptosis, cytokinesis and signal transduction [3]. In addiction to BIR domains, some members of this family as XIAP, c-IAP1, c-IAP2 and Livin also have a RING domain that allows these proteins to act as E3 ubiquitin ligases [5]. The E3 ubiquitin ligase activity of the IAPs is capable of promoting ubiquitination and proteasomal degradation of caspases, TRAF2 and several other partners [4]. XIAP is unique among IAP proteins, because of its ability to inhibit and directly bind to activated caspases. Through its BIR2 domain with its N-terminal linker, XIAP binds to the active site of effectors caspase-3 or -7 and prevents substrate binding and subsequent catalysis [5]. On the other hand the BIR3 domain sequestrates active caspase-9. Furthermore, XIAP has been shown to promote nuclear factor -B (NF-B) activation by enhancing the translocation of NF-B from the cytoplasm into the nucleus, by increasing the degradation of inhibitor B protein and through its association with TAK1 kinase and its cofactor TAB1 [6]. The two main antagonists of IAP proteins are Smac/DIABLO and XAF1, involved in the balance and regulation of apoptotic stimuli. Smac/DIABLO is released from mitochondria together with cytochrome c after initiation of intrinsic apoptotic cascade. Smac appears to function as a general IAP inhibitor in that it is shown to bind to XIAP, cIAP1, cIAP2, Survivin, Livin and BRUCE [3]. Conversely, the nuclear protein XAF1 exclusively interacts with XIAP, restraining this IAP antiapoptotic action even in healthy cells. The mechanism by which XAF1 antagonizes XIAP is not completely explained, although recent studies showed that XAF1 is able to sequestrate XIAP from the cytoplasm into the nucleus. Moreover, XAF1 expression is low or absent in several tumor cell lines [7]. Since XIAP, Smac/DIABLO and XAF1 are antagonistic regulators, it is reasonable to assume that their relative expression ratios, rather than the expression of a single regulator, determine susceptibility for apoptosis [8]. This study aimed to investigate the gene and protein expression pattern of IAP family members and its antagonists in a series of human HCCs and to assess their clinical and prognostic significance.