XIAP gene expression and function is regulated by autocrine and paracrine TGF-β signaling

Background: X-linked inhibitor of apoptosis protein (XIAP) is often overexpressed in cancer cells, where it plays a key role in survival and also promotes invasiveness. To date however, the extracellular signals and intracellular pathways regulating its expression and activity remain incompletely understood. We have previously showed that exposure to each of the three TGF-beta (transforming growth factor beta) isoforms upregulates XIAP protein content in endometrial carcinoma cells in vitro. In the present study, we have investigated the clinical relevance of TGF-b isoforms in endometrial tumours and the mechanisms through which TGF-b isoforms regulate XIAP content in uterine cancer cells. Methods: TGF-b isoforms immunoreactivity in clinical samples from endometrial tumours was assessed using immunofluorescence. Two model cancer cell lines (KLE endometrial carcinoma cells and HeLa cervical cancer cells) and pharmacological inhibitors were used to investigate the signalling pathways regulating XIAP expression and activity in response to autocrine and paracrine TGF-b in cancer cell. Results: We have found immunoreactivity for each TGF-b isoform in clinical samples from endometrial tumours, localizing to both stromal and epithelial/cancer cells. Blockade of autocrine TGF-b signaling in KLE endometrial carcinoma cells and HeLa cervical cancer cells reduced endogenous XIAP mRNA and protein levels. In addition, each TGF-b isoform upregulated XIAP gene expression when given exogenously, in a Smad/NF-kappa B dependent manner. This resulted in increased polyubiquitination of PTEN (phosphatase and tensin homolog on chromosome ten), a newly identified substrate for XIAP E3 ligase activity, and in a XIAP-dependent decrease of PTEN protein levels. Although each TGF-b isoform decreased PTEN content in a XIAP-and a Smad-dependent manner, decrease of PTEN levels in response to only one isoform, TGF-beta 3, was blocked by PI3-K inhibitor LY294002. Conclusions: XIAP gene expression and function is positively regulated by exposure to the three TGF-b isoforms in a Smad-dependent manner, similar to constitutive XIAP gene expression which depends on autocrine TGF-beta/Smad signalling.