Abstract
BackgroundApproximately half of all human genes use alternative transcription start sites (TSSs) to control mRNA levels and broaden the transcriptional output in healthy tissues. Aberrant expression patterns promoting carcinogenesis, however, may arise from alternative promoter usage.ResultsBy profiling 108 colorectal samples using exon arrays, we identified nine genes (TCF12, OSBPL1A, TRAK1, ANK3, CHEK1, UGP2, LMO7, ACSL5, and SCIN) showing tumor-specific alternative TSS usage in both adenoma and cancer samples relative to normal mucosa. Analysis of independent exon array data sets corroborated these findings. Additionally, we confirmed the observed patterns for selected mRNAs using quantitative real-time reverse-transcription PCR. Interestingly, for some of the genes, the tumor-specific TSS usage was not restricted to colorectal cancer. A comprehensive survey of the nine genes in lung, bladder, liver, prostate, gastric, and brain cancer revealed significantly altered mRNA isoform ratios for CHEK1, OSBPL1A, and TCF12 in a subset of these cancer types.To identify the mechanism responsible for the shift in alternative TSS usage, we antagonized the Wnt-signaling pathway in DLD1 and Ls174T colorectal cancer cell lines, which remarkably led to a shift in the preferred TSS for both OSBPL1A and TRAK1. This indicated a regulatory role of the Wnt pathway in selecting TSS, possibly also involving TP53 and SOX9, as their transcription binding sites were enriched in the promoters of the tumor preferred isoforms together with their mRNA levels being increased in tumor samples.Finally, to evaluate the prognostic impact of the altered TSS usage, immunohistochemistry was used to show deregulation of the total protein levels of both TCF12 and OSBPL1A, corresponding to the mRNA levels observed. Furthermore, the level of nuclear TCF12 had a significant correlation to progression free survival in a cohort of 248 stage II colorectal cancer samples.ConclusionsAlternative TSS usage in colorectal adenoma and cancer samples has been shown for nine genes, and OSBPL1A and TRAK1 were found to be regulated in vitro by Wnt signaling. TCF12 protein expression was upregulated in cancer samples and correlated with progression free survival.
Highlights
Half of all human genes use alternative transcription start sites (TSSs) to control mRNA levels and broaden the transcriptional output in healthy tissues
A very good inter-observer agreement was seen for all tested IHCs, and we found that Transcription factor 12 (TCF12) staining intensity was significantly associated with progression-free survival when data was dichotomized with the respect to strong intensity samples versus negative/weak/moderate intensity samples
transcription factors (TFs) binding analysis suggested that p53 and SOX9 might be responsible for altering the TSS use as they are both upregulated in cancer and have enriched binding sites in the promoters associated with the TSS preferred by the neoplastic cells
Summary
Half of all human genes use alternative transcription start sites (TSSs) to control mRNA levels and broaden the transcriptional output in healthy tissues. CRC can be divided into two major subgroups: microsatellite stable (MSS) and microsatellite unstable cancers (MSI), the latter being characterized by a TCF4 (gene symbol TCF7L2) leading to expression of target genes including MYC, SOX9 and Cyclin D1. The generation of proteome diversity from a rather limited number of genes is primarily due to alternative splicing and alternative promoter usage, the latter leading to multiple transcripts from the same gene with different transcription start sites (TSSs). Genome-wide predictive analysis has shown that alternative promoters are overrepresented in genes involved in development and transcriptional control, whereas genes with only a single promoter are more frequently involved in general cellular processes [9]. While the long isoform of TCF1 interacts with b-catenin and stimulates transcription of Wnt-target genes, the short isoform, unable to bind b-catenin but with the DNA binding domain intact, acts as a dominant negative regulator of b-catenin mediated Wnt signaling [13]
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