Abstract 2959: Characterization and function of unique T-cell factor-4 splicing isoforms that exhibit tumor repressive phenotypes in hepatocellular carcinoma

Abstract

Abstract Background and Aims: The canonical Wnt signaling is frequently activated in hepatocellular carcinoma (HCC). Although β-catenin is the principle effector protein of Wnt signaling, the success of relaying signals to the nucleus depends on the T-cell factor (TCF) family of transcription factors that bind to promoters of Wnt-responsive target genes. The human TCF-4 gene (TCF7L2) is composed of 17 exons with multiple alternative splicing sites. In spite of theoretical possibility of constructing more than 500 isoforms, only two TCF-4 splice variants have been identified so far. Therefore, the objective of this study is to identify different TCF-4 isoforms and explore their putative role in hepatic oncogenesis. Methods: The TCF-4 isoforms were detected by RT-PCR. Their expression was confirmed by using RT-PCR with splice site-specific primer pairs in HCC cell lines, HCC tumor and peritumor tissues, and normal liver. To examine their functional properties, mammalian expression plasmids encoding each isoform were prepared and protein products were verified by Western blotting. The TCF transcriptional activity, cell proliferation, migration and transformation were measured to evaluate their biological functions. Results: We identified and subsequently cloned 14 different TCF-4 splicing variants derived from HCC cell lines. Two previously known forms TCF-4B (short) and TCF-4E (long) were found among the 14 isoforms. The 12 new isotypes were named in alphabetical order according to increasing size of their protein products. Out of the 14 TCF-4 isoforms, we have detected 8 short forms (A-I except for E), 5 long forms (J-M plus E), and a novel isoform designated as TCF-4X. The structure of TCF-4X included an extra exon, labeled as exon 5a, between exons 5 and 6 indicating that TCF7L2 may have 18th exon in the genome. Functional analysis of these isotypes revealed several distinctive phenotypes. TCF-4B showed the highest TCF transcriptional activity and enhanced cell growth rate, but lower cell migration and transforming ability compared to control. TCF-4J exhibited lower transcriptional activity but the highest cell proliferation, migration and colony formation rate. In contrast, TCF-4K displayed the lowest TCF transcriptional activity, inhibited cell proliferation, and eliminated colony-forming ability. In addition, their expression profile by RT-PCR indicated that TCF-4J was highly expressed in HCC tumors compared to peritumor and normal liver, while TCF-4K was barely detected in HCC cell lines and tumor tissues. Conclusions: Our study demonstrates that at least 14 different TCF-4 isoforms are expressed in HCC and their function is distinguishable based on the structure of the splicing sites. Whereas the TCF-4J isoform produced the most characteristic features of the malignant phenotype, TCF-4K exhibited tumor repressive traits. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2959.