Abstract
PurposeAberrantly 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.MethodsWe 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.ResultsTCF-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.ConclusionsOur results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions.
Highlights
The Wnt/b-catenin signaling pathway plays a crucial role in cell fate determination and stem cell renewal in adult tissue [1,2]
We determined if three pairs of isoforms (TCF-4A and B; T-cell factor (TCF)-4G and H; and TCF-4J and K) exhibited different expression profiles that may suggest SxxSS-dependent regulation in human Hepatocellular carcinoma (HCC) tumors
A similar expression profile of these Tcell factor-4 (TCF-4) isoforms was observed in a second cohort of HCC-adjacent uninvolved tissue pairs from individuals where 85% (17/20) of the tumors were related to chronic hepatitis C virus (HCV) infection (Fig. 1B, Table 2)
Summary
The Wnt/b-catenin signaling pathway plays a crucial role in cell fate determination and stem cell renewal in adult tissue [1,2]. The alternative splicing sites in the central domain of TCF-4 can generate isoforms with or without the conserved LVPQ and SxxSS motifs located at the end of exon 7 and the beginning of exon 9, respectively [11,12]. We identified and characterized 14 (12 of which are unique) TCF-4 isoforms derived from human HCC cell lines [13]. Among such isoforms, three structurally identical pairs (TCF-4J and K; TCF-4A and B; TCF4G and H) were observed that differed only by the presence (K, A, and H) or absence (J, B, and G) of a SxxSS motif. Previous studies suggest that the SxxSS motif may modulate transcriptional activity of TCF-4 [12]; both the cell-based and in vivo functional consequences of the SxxSS motif expression as well as subsequent gene regulatory activity have not been determined
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