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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    • "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: T-cell factor (TCF) proteins represent key transcription factors in Wnt signaling. We show that the SxxSS motif in TCF-4 regulates transcriptional activity in HCC cells. TCF-4K mutants increased transcriptional activity compared to TCF-4K (bearing the SxxSS); the binding pattern of co-factors in TCF-4K mutants was similar to that in TCF-4J (lacking the SxxSS). TCF activity in TCF-4K cells was suppressed by homeodomain-interacting protein kinase 2 (HIPK2), but not in TCF-4J cells. Together, our data indicates that the SxxSS motif in TCF-4K regulates transcriptional activity by modifying co-factors in the β-catenin/TCF-4 transcriptional complex and these events may be mediated through HIPK2.
    Cancer letters 04/2013; 336(2). DOI:10.1016/j.canlet.2013.03.031 · 5.62 Impact Factor
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    ABSTRACT: The Wnt/β-catenin signalling pathway regulates genes involved in cell proliferation, survival, migration and invasion through regulation by T-cell factor (TCF)-4 transcription factor proteins. However, the role of TCF-4 isoforms generated by alternative splicing events in hepatocellular carcinoma (HCC) is unknown. Here, we investigated TCF-4 isoforms (TCF-4J and K)-responsive target genes that are important in hepatic oncogenesis and tumour development. Gene expression microarray was performed on HCC cells overexpressing TCF-4J and K isoforms. Expression level of selected target genes was evaluated and correlations were made between their expression level and that of TCF-4 isoform in 47 pairs of human HCC tumours. Comparison by gene expression microarray revealed that 447 genes were upregulated and 343 downregulated more than 2.0-fold in TCF-4J compared with TCF-4K expressing cells. We validated expression of 18 selected target genes involved in Wnt/β-catenin, insulin/IGF-1/IRS1 and Notch signalling pathways in 47 pairs of human HCCs and adjacent uninvolved liver tissues. It was observed that 13 genes (CLDN2, STK17B, SPP1, AXIN2, WISP2, MMP7, IRS1, ANXA1, CAMK2N1, ASPH, GPR56, CD24 and JAG1) activated by TCF-4J isoform in HCC cells, were also upregulated in HCC tumours compared with adjacent peritumour tissue; more importantly, 10 genes exhibited a significant correlation with the TCF-4J expression level in tumour. TCF-4 isoforms (TCF-4J and K) activated different downstream target genes in HCC. The biological consequence of TCF-4J isoform expression was upregulation of genes associated with tripartite Wnt/β-catenin, insulin/IGF-1/IRS1 and Notch signal transduction pathway activation, which contribute to the pathogenesis of HCC.
    Liver international: official journal of the International Association for the Study of the Liver 04/2013; 33(7). DOI:10.1111/liv.12188 · 4.85 Impact Factor
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    Liver international: official journal of the International Association for the Study of the Liver 05/2013; 33(10). DOI:10.1111/liv.12189 · 4.85 Impact Factor
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