Abstract 1933: Preferential ubiquitination of hypoxia-inducible factors depends on the SxxSS motif in human T-cell factor-4 isoforms derived from hepatocellular carcinoma

The T-cell factor (TCF)-4 is a key transcriptional protein activated by Wnt/β-catenin signaling. We previously cloned 14 TCF-4 isoforms derived from human hepatocellular carcinoma (HCC) cell lines. The TCF-4J and K pair were characterized based on the presence (K) or absence (J) of the functional motif SxxSS. Recently, we have demonstrated that loss of the SxxSS motif in the TCF-4 isoforms conferred robust tumorigenic potential to HCC, involving the hypoxia-inducible factor (HIF)-2α (AASLD Oct. 30, 2010; abstract #560). It is known that the cellular amount of HIF proteins is tightly regulated by ubiquitination and such proteins appear to be important in cancer stem cell biology. Thus, the AIM of the current study was to examine how HIF protein expression levels were regulated in the SxxSS motif-dependent manner. Methods: The human HCC cell line HAK-1A was used in this study. Hypoxia was induced with cobalt chloride (CoCl2) or 1% oxygen exposure. TCF-4K mutants were prepared with substitution of serine (S) to alanine (A) by site-directed mutagenesis. Cell clones overexpressing TCF-4J, K, and K mutants such as S269A, S272A, and S273A were established. Ubiquitination of HIF proteins were assessed by immunoprecipitation (IP)/Western blot analysis using the proteasome inhibitor MG-132. Results: Increased expression of both HIF-1α and HIF-2α was preferentially found in the TCF-4J-overexpressing cells (J cells) under severe hypoxia, which was in contrast to the levels in the TCF-4K-overexpressing cells (K cells) and the mock-transfected cells (mock cells). This increase in both HIF expressions in J cells was accompanied by downregulation of Von Hippel Lindau (VHL) protein, a component of E3 ligase for HIFs. When cells were incubated with 10 μM MG-132 for 4 h under severe hypoxia, the expression levels of HIF-1α and HIF-2α were highly increased in K cells, suggesting degradation of HIFs in an ubiquitin-dependent fashion. Indeed, both the robust poly-ubiquitination of HIF-2α and the interaction between VHL and HIF-2α were detected by the IP/Western analysis in K cells. To further elucidate the detailed mechanisms of the augmented HIF-2α ubiquitination through SxxSS, the possible impact derived from the K mutants on the ubiquitination process was investigated. As a result, the S269A mutant demonstrated predominant intracellular accumulation of HIF-2α in the treatment with MG-132 under severe hypoxia, suggesting that de-phosphorylation at serine 269 in SxxSS facilitated the proteasomal degradation of HIF-2α. Conclusion: This is an initial demonstration that the TCF-4 isoforms are involved in the proteolytic processing of HIFs in HCC. The findings from this study may provide insights into the understanding and possible role of the canonical Wnt/β-catenin/TCF-4 signaling pathway in the regulation of HIF-2α, which is a novel cancer stem cell marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1933. doi:10.1158/1538-7445.AM2011-1933