Identification of T-cell factor-4 isoforms that contribute to the malignant phenotype of hepatocellular carcinoma cells

Abstract

The Wnt/β-catenin signaling pathway is frequently activated in hepatocellular carcinoma (HCC). Downstream signaling events involving the Wnt/β-catenin cascade occur through T-cell factor (TCF) proteins. The human TCF-4 gene is composed of 17 exons with multiple alternative splicing sites. However, the role of different TCF-4 isoforms in the pathogenesis of HCC is unknown. The purpose of this study was to identify and characterize TCF-4 isoforms in HCC. We identified 14 novel TCF-4 isoforms from four HCC cell lines. Functional analysis following transfection and expression in HCC cells revealed distinct effects on the phenotype. The TCF-4J isoform expression produced striking features of malignant transformation characterized by high cell proliferation rate, migration and colony formation even though its transcriptional activity was low. In contrast, the TCF-4K isoform displayed low TCF transcriptional activity; cell proliferation rate and colony formation were reduced as well. Interestingly, TCF-4J and TCF-4K differed by only five amino acids (the SxxSS motif). Thus, these studies suggest that conserved splicing motifs may have a major influence on the transcriptional activity and functional properties of TCF-4 isoforms and alter the characteristics of the malignant phenotype.

Alternative splicing isoform of T cell factor 4K suppresses the proliferation and metastasis of non-small cell lung cancer cells

“…Therefore, we supposed that TCF-4K might regulate relevant target genes like C-myc to affect cell proliferation. In contrast to the conclusions of Tsedensodnom et al (2011) from their research on hepatoma carcinoma cells, we found that over-expression of TCF-4K could significantly inhibit the migration of A549 cells, which might be caused by the down-regulation of migration-relevant target genes. Therefore, our data indicate that TCF-4K might function as tumor suppressor in NSCLC by down-regulating the Wnt pathway.…”

“…Sequence analysis was performed using the NCBI nucleotide blast web service, and validated that the 1700 bp band that was missing in lung cancer cells represented an alternative splicing isoform of TCF-4. This isoform had also been previously identified as an inhibitory transcriptional factor of the Wnt pathway in hepatocellular carcinoma cell lines (Tsedensodnom et al, 2011). The TCF4 gene includes five essential domains for transcription function: an N-terminal β-catenin-binding domain (BCBD, exon 1), a Groucho-related-gene (Grg)/ transducin-like enhancer of split (TLE) transcriptional corepressor binding site (exons 9 and 10), a high mobility-group-box (HMG-box) DNA-binding domain (exons 10 and 11), an adjacent nuclear localization signal (NLS, exon 12), and a C-terminal binding protein binding site (CtBP, exon 17).…”

“…First-strand cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) according to manufacturer instruction, using 1 μg total RNA. For identifying total TCF-4 transcription products, polymerase chain reaction (PCR) was carried out with a primer pair 1F (forward, 5'-CCG CTC GAG CGG ATG CCG CAG CTG AAC GGC GG-3') and 17R (reverse, 5'-CGC GGA TCC GCG CTA TTC TAA AGA CTT GGT GAC GAG CGA CAG CGG CT-3') that spans from the beginning of exon 1 to the end of exon 17 (Tsedensodnom et al, 2011). The PCR product of approximately 1700 bp from the 16HBE cell line was cloned into the pEGFP-N1vector (Sangon, Shanghai, China) using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) and the individual clones were DNA sequenced at Sangon Biotech Co., Ltd. (Shanghai, China).…”

“…TCF4 isoforms have been cloned and sequenced in hepatocarcinoma cell (HCC) lines, and have revealed 14 different TCF-4 isoforms that have been named alphabetically. It has been validated that HCC cells expressed TCF-4K isoform displayed low TCF transcriptional activity; cell proliferation rate, and colony formation (Tsedensodnom et al, 2011).…”

TCF/LEFs and Wnt Signaling in the Nucleus

“…Original studies in Xenopus systems showed that forms with SxxSS behave like repressors, whereas forms missing this motif act like activators (Liu et al 2005). This was recently confirmed in a study of human hepatocellular carcinoma in which of the 14 alternatively spliced TCF4 isoforms expressed in liver cancer cells, those that contained the SxxSS motif were growth suppressive, whereas those lacking the element were Wnt activating and growth promoting (Tsedensodnom et al 2011). The fact that SxxSS forms of TCF4 are generated through alternative splicing means that the prevalence of these forms could differ among cell types and cell stages.…”