Loss of the SxxSS Motif in a Human T-Cell Factor-4 Isoform Confers Hypoxia Resistance to Liver Cancer: An Oncogenic Switch in Wnt Signaling

Liver Research Center, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, Rhode Island, United States of America.
PLoS ONE (Impact Factor: 3.23). 06/2012; 7(6):e39981. DOI: 10.1371/journal.pone.0039981
Source: PubMed


Aberrantly activated Wnt/β-catenin signaling is important in hepatocellular carcinoma (HCC) development. Downstream gene expressions involving the Wnt/β-catenin cascade occur through T-cell factor (TCF) proteins. Here, we show the oncogenic potential of human TCF-4 isoforms based on the expression of a single conserved SxxSS motif.
We investigated the TCF-4J and K isoform pair characterized by the presence (K) or absence (J) of the SxxSS motif. The mRNA expression profiles were examined in 47 pairs of human HCCs and adjacent non-cancerous liver tissues by RT-PCR. Proliferation, sphere assays and immunoblot analysis were performed under normoxia and hypoxia conditions. The ability of HCC cells overexpressing TCF-4J (J cells) and K (K cells) to grow as solid tumors in nude mice was explored.
TCF-4J expression was significantly upregulated in HCC tumors compared to corresponding peritumor and normal liver and was preferentially expressed in poorly differentiated HCCs. In contrast, TCF-4K was downregulated in those same HCC tumors. TCF-4J-overexpressing HCC cells (J cells) revealed a survival advantage under hypoxic conditions, high proliferation rate and formation of aggregates/spheres compared to overexpression of TCF-4K (K cells). The hypoxic J cells had high expression levels of HIF-2α and EGFR as possible mechanisms to promote tumorigenesis. Increased stability of HIF-2α under hypoxia in J cells was associated with a decreased level of von Hippel-Lindau (VHL) protein, a known E3 ligase for HIF-αs. In a xenograft model, the J cells rapidly developed tumors compared to K cells. Tumor tissues derived from J cells exhibited high expression levels of HIF-2α and EGFR compared to the slow developing and small K cell derived tumors.
Our results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions.

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Available from: Hironori Koga
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    • "It has been recently demonst rated that TCF-4J expressing HCC cells develop tumors more rapidly in a xenograft model compare d to TCF-4K expressing cells. More important, TCF-4J is significantly upregulated in human HCC tumors compare d to corresponding peritumor tissue including normal liver [12]. These observations led us to test the hypothesis that a significant difference in generation of a malignant phenotyp e could be related, in part, to different ial TCF transcrip tional activity. "
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    ABSTRACT: BACKGROUND: T-cell factor (TCF) proteins represent key transcription factors that activate Wnt/β-catenin signalling. We have reported that a pair of TCF-4 isoforms (TCF-4C and TCF-4D) exhibit differential TCF transcriptional activity in hepatocellular carcinoma (HCC) cells, although their structure differs by only the presence (TCF-4D) or absence (TCF-4C) of exon 4. AIM: To demonstrate a regulatory role of exon 4 in HCC development. METHODS: TCF-4C and TCF-4D expression profiles were examined in 27 pairs of human HCC and adjacent liver tissues. The functional role of the TCF-4 isoforms was evaluated in OUMS-29 (an immortalized hepatocyte-derived) and HAK-1A (a well-differentiated HCC) cell lines using stable clones overexpressing the TCF-4 isoforms. RESULTS: TCF-4C was significantly upregulated in HCC tissues compared with corresponding peritumour and normal liver tissues; in contrast, there was no difference in TCF-4D expression. TCF-4C clones derived from both cell lines exhibited increased TCF activity, Wnt-responsive target genes, cell proliferation, cell cycle progression and resistance to chemotherapeutic drugs compared with TCF-4D clones. Capability of cell migration and colony formation was significantly higher in TCF-4C than TCF-4D clones. In a nude mice xenograft model, the HAK-1A-derived TCF-4C clone rapidly developed tumours compared with the TCF-4D clone. TCF-4C clone-derived tumours exhibited upregulation of Wnt-responsive target genes compared with the slow developing and small TCF-4D-derived tumours. CONCLUSION: These results demonstrate that the TCF-4C isoform lacking exon 4 is associated with a malignant phenotype compared with the exon 4-harbouring TCF-4D isoform, indicating that exon 4 of TCF-4 plays a prominent role in HCC development.
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