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.
Highlights
From the ‡Department of Molecular Medicine, Aarhus University Hospital, Skejby, DK-8200 Aarhus N, Denmark, §Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel, ¶Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, ʈDepartment of Surgery P, Aarhus University Hospital, Aarhus Hospital, DK-8000 Aarhus C, Denmark
Most colorectal cancers (CRC)1 are believed to arise from adenomas, and a hallmark of colorectal tumor initiation is the constitutive activation of the Wnt pathway, which is observed in ϳ85% of colorectal tumors [1]
Several SR proteins are activated by phosphorylation of their serines by SRSF protein kinase 1 (SRPK1), which is up-regulated in colon, breast, and pancreatic cancer [13]
Summary
The Wnt pathway plays a significant role in other cancer types, such as lung, gastric, thyroid, and prostate cancer [3,4,5], and it has been shown to be involved in the regulation of RNA splicing as well [6]. The best described SRPK1 target is serine/arginine-rich splicing factor 1 (SRSF1, formerly known as SF2/ASF) [14, 15], an essential splicing factor that participates in both constitutive and AS, and which was recently shown to be a proto-oncogene [12] supporting the involvement of AS in cancer development. The aim of the study was to discover AS in colorectal adenoma and cancer samples, and we identified tumor-specific splicing of solute carrier family 39 (zinc transporter), member 14 (SLC39A14), a metal ion transporter with the capacity to transport divalent cations, such as zinc, iron, and cadmium. We show that knockdown of SRSF1 affects the splicing of SLC39A14
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