Alternative splicing of SLC39A14 in colorectal cancer is regulated by the Wnt pathway

Abstract

Alternative splicing is a crucial step in the generation of protein diversity and its misregulation is observed in many human cancer types. By analyzing 143 colorectal samples using exon arrays, SLC39A14, a divalent cation transporter, was identified as being aberrantly spliced in tumor samples. SLC39A14 contains two mutually exclusive exons 4A and 4B and the exon 4A/4B ratio was significantly altered in adenomas (p = 3.6 × 10(-10)) and cancers (p = 9.4 × 10(-11)), independent of microsatellite stability status. The findings were validated in independent exon array data sets and by quantitative real-time reverse-transcription PCR (qRT-PCR). Aberrant Wnt signaling is a hallmark of colorectal tumorigenesis and is characterized by nuclear β-catenin. Experimental inactivation of Wnt signaling in DLD1 and Ls174T cells by knockdown of β-catenin or overexpression of dominant negative TCFs (TCF1 and TCF4) altered the 4A/4B ratio, indicating that SLC39A14 splicing is regulated by the Wnt pathway. An altered 4A/4B ratio was also observed in gastric and lung cancer where Wnt signaling is also known to be aberrantly activated. The splicing factor SRSF1 and its regulator, the kinase SRPK1, were found to be deregulated upon Wnt inactivation in colorectal carcinoma cells. SRPK1 was also found up-regulated in both adenoma samples (p = 1.5 × 10(-5)) and cancer samples (p = 5 × 10(-4)). In silico splicing factor binding analysis predicted SRSF1 to bind predominantly to the cancer associated exon 4B, hence, it was hypothesized that SRPK1 activates SRSF1 through phosphorylation, followed by SRSF1 binding to exon 4B and regulation of SLC39A14 splicing. Indeed, siRNA-mediated knockdown of SRPK1 and SRSF1 in DLD1 and SW480 colorectal cancer cells led to a change in the 4A/4B isoform ratio, supporting a role of these factors in the regulation of SLC39A14 splicing. In conclusion, alternative splicing of SLC39A14 was identified in colorectal tumors and found to be regulated by the Wnt pathway, most likely through regulation of SRPK1 and SRSF1.

Fig. 1.

SLC39A14 alternative splicing is dysregulated in colorectal neoplasia. A, Exon structure of SLC39A14 isoforms 1 and 2. The isoforms differ in exon 4 usage. Exons 4A and 4B are mutually exclusive. B, Expression levels of the individual exons of SLC39A14 measured in normal mucosa (n = 24) and tumor (n = 84) samples using exon arrays. Error bars represent S.E. C, SLC39A14 exon 4A/4B ratio in normal mucosa samples, adenomas, and carcinomas, same data as in B. D, Technical validation of SLC39A14 exon 4A/4B ratio by qRT-PCR. E, The total transcript level of SLC39A14 measured by qRT-PCR on the same samples as in D. F, Total protein level of SLC39A14 detected by immunohistochemistry in normal epithelial cells and carcinoma cells, respectively.

Fig. 2.

SLC39A14 exon 4A/4B ratio is regulated by Wnt signaling in colorectal carcinoma cells. A, dnTCF1 or dnTCF4 expression was induced in stably transfected DLD1 or Ls174T cells. The 4A/4B ratio in dnTCF1 and dnTCF4 Wnt-model systems was measured by qRT-PCR. B, qRT-PCR of β-catenin mRNA from DLD1 cells transfected with 20 nm control or β-catenin siRNA. C, 4A/4B ratio in the siRNA transfected cells described in B. Error bars represent standard deviation of three (A) or two (B and C) replicas. * = p < 0.05.

Fig. 3.

Expression of the splicing regulators SRPK1 and SRSF1 is regulated by Wnt signaling. SRPK1 and SRSF1 expression was quantified by qRT-PCR in the Wnt-model systems described in Fig. 2. A, Expression of SRPK1 in the dnTCF1, dnTCF4 (left panel) and β-catenin knockdown models (right panel). B, Expression of SRSF1 in the same models as in A. * = p < 0.05.

Fig. 4.

SRPK1 and SRSF1 expression in normal mucosa and tumor samples. SRPK1 and SRSF1 expression was quantified by qRT-PCR in colorectal tissue samples. A, Expression of SRPK1 and B, Expression of SRSF1.

Fig. 5.

Prediction of SRSF1 binding sites in exon 4A and 4B and surrounding intronic sequences. A, Combined binding site scores for SRSF1 binding motifs crsmsgw and ugrwgvh. The scores summarize the clustering potential and evolutionary conservation of the candidate binding sites. B, Exon 4A and 4B specific hits. Δscores represent the difference between 4A and 4B scores in every sequence position of a pairwise alignment between both exons. Highly positive Δscore values indicates 4A specific motifs. Conversely, highly negative Δscore account for 4B specific motifs.

Fig. 6.

Knockdown of SRPK1 and SRSF1 in DLD1 cells and the effect on 4A/4B ratio. A, SRPK1 and SRSF1 qRT-PCR expression in DLD1 cells treated with either control, SRPK1, or SRSF1 siRNA (50 nm or 100 nm). B, 4A/4B ratio following knockdown of SRPK1 or SRSF1 as in A. C, SRPK1 and SRSF1 qRT-PCR expression following their simultaneous knockdown in DLD1 cells treated with 100 nm siRNA in total. D, 4A/4B ratio following simultaneous knockdown of SRPK1 and SRSF1 as in C.