Verification of predicted alternatively spliced Wnt genes reveals two new splice variants (CTNNB1 and LRP5) and altered Axin-1 expression during tumour progression

Abstract

Background: Splicing processes might play a major role in carcinogenesis and tumour progression. The Wnt pathway is of crucial relevance for cancer progression. Therefore we focussed on the Wnt/beta-catenin signalling pathway in order to validate the expression of sequences predicted as alternatively spliced by bioinformatic methods. Splice variants of its key molecules were selected, which may be critical components for the understanding of colorectal tumour progression and may have the potential to act as biological markers. For some of the Wnt pathway genes the existence of splice variants was either proposed (e.g. beta-Catenin and CTNNB1) or described only in non-colon tissues (e.g. GSK3beta) or hitherto not published (e.g. LRP5).

Results: Both splice variants--normal and alternative form--of all selected Wnt pathway components were found to be expressed in cell lines as well as in samples derived from tumour, normal and healthy tissues. All splice positions corresponded totally with the bioinformatical prediction as shown by sequencing. Two hitherto not described alternative splice forms (CTNNB1 and LRP5) were detected. Although the underlying EST data used for the bioinformatic analysis suggested a tumour-specific expression neither a qualitative nor a significant quantitative difference between the expression in tumour and healthy tissues was detected. Axin-1 expression was reduced in later stages and in samples from carcinomas forming distant metastases.

Conclusion: We were first to describe that splice forms of crucial genes of the Wnt-pathway are expressed in human colorectal tissue. Newly described splicefoms were found for beta-Catenin, LRP5, GSK3beta, Axin-1 and CtBP1. However, the predicted cancer specificity suggested by the origin of the underlying ESTs was neither qualitatively nor significant quantitatively confirmed. That let us to conclude that EST sequence data can give adequate hints for the existence of alternative splicing in tumour tissues. That no difference in the expression of these splice forms between cancerous tissues and normal mucosa was found, may indicate that the existence of different splice forms is of less significance for cancer formation as suggested by the available EST data. The currently available EST source is still insufficient to clearly deduce colon cancer specificity. More EST data from colon (tumour and healthy) is required to make reliable predictions.

Figure 1

The Wnt-pathway. Picture taken from KEGG database . The selected genes involved in the analysis presented here are marked by red rectangles.

Figure 2

Alternative splice forms of selected Wnt-components. At top genomic DNA is depicted with all exons. In the middle the cDNA-structure of the normal form is given, the coding region is indicated in yellow, at the bottom the alternative isoform is shown; skips are indicated in red, inserts in blue. On the right side the length in bp is given of the pertinent sequence. For all cases, only the single splice event is shown. Potential other splice sites are neglected. A: CTNNB1(a), B: CTNNB1(b), C: CtBP1, D: GSK-3β, E: Axin-1, F: LRP5

Figure 3

Determination of optimal RT-PCR conditions for detection of both GSK-3β splice forms. On the left (A) GSK 3β expression is shown in dependence of various annealing temperatures. On the right (B) side GSK 3β expression is shown in dependence of the number of cycles. In figure C both annealing and extension times are varied. Abbreviations: H₂O, negative control; normal, normal GSK 3β isoform; alt, alternative splicing product.

Figure 4

Expression of CTNNB1 and beta-Actin. Expression of CTNNB1 and beta-Actin in LS174T, SW-480 cell lines and tissue cDNA from colorectal mucosa and cancer. Abbreviations: H₂O, negative control; normal, normal CTNNB1 isoform; alt.1 and alt.2, alternative splicing products.

Figure 5

GSK 3β expression. GSK 3β expression in LS 174T cell line and tissue cDNA from colorectal mucosa and cancer. Abbreviations: H₂O, negative control; normal, normal GSK 3β isoform; alt, alternative splicing product.

Figure 6

Axin-1 expression. Axin-1 expression in LS 174T cell line and tissue cDNA from colorectal mucosa and cancer. In the left part of figure PCR product with primer A and in the right part PCR product with primer B. Abbreviations: H₂O, negative control; normal, normal Axin-1 isoform; alt, alternative splicing product.

Figure 7

LRP 5 expression. LRP 5 expression in LS 174T, SW 480 cell lines and tissue cDNA from colorectal mucosa and cancer. Abbreviations: H₂O, negative control; normal, normal LRP5 isoform; alt, alternative splicing product.

Figure 8

Axin-1, primer location for real-time amplicons and electrophoresis after real-time PCR The upper part shows the primer location for real-time amplicons. T1: 104 bp amplicon (normal isoform), T2: 94 bp amplicon (alternative isoform). The lower part gives the electrophoresis result after real-time PCR. Line 1: LS 174S T1 primer product, line 2: negative control (H₂O) for T1, line 3: marker, line 4: LS 174T, T2 primer product, line 5: negative control (H₂O) for T2

Figure 9

Expression of normal and alternative axin splice forms. Box-plots of constitutive normal (con) and alternative (alt) forms in carcinoma (T) and mucosa (M) (cases N = 14).

Figure 10

Axin-expression in correlation with tumour invasion. Box-plots of expression values of constitutive normal (con) and alternative (alt) forms in correlation with stage T (tumour invasion). T: carcinoma, M: mucosa; t = 2: Tumour invades muscularis propria, t = 3: Tumour invades through the muscularis propria into the subserosa, or into nonperitonealized pericolic or perirectal tissues, t = 4 : Tumour directly invades other organs or structures, and/or perforates visceral peritoneum (cases N = 14).

Figure 11

Axin-expression in correlation with stage distant metastasis. Box-plots of expression values of constitutive normal (con) and alternative (alt) forms of Axin in cancer tissue in correlation with stage distant metastasis. M = 0: no tumour metastasis, M = 1: distant metastasis at the time point of surgery (cases N = 14).