Serum response factor is alternatively spliced in human colon cancer

Abstract

Background

Serum response factor (SRF) is a transcription factor that plays an important role in cellular differentiation and cell cycle regulation. SRF function is regulated in part by alternative splicing. Little is known about the expression or role of these alternatively spliced isoforms during tumorigenesis. We hypothesized that there is a change in the splice variants during intestinal tumorigenesis and that this change promotes the tumor phenotype.

Materials and methods

SRF expression was determined by Western blotting of benign intestinal cells and human colon cancer cell lines. To determine the effect of alternative splicing of SRF on intestinal growth and proliferation, the predominant alternatively spliced isoform of SRF that we identified in colon cancer cells, SRFΔ5, was transfected into IEC-6 cells. IEC-6 and IEC-6SRFΔ5 cells were plated and cell numbers were determined at four time points.

Results

Western blotting demonstrates that full-length SRF is the predominant form of SRF in rat IEC-6 cells, normal human colonic mucosa, and HT-29 cells, derived from a well-differentiated human colonic adenocarcinoma. In the colon cancer cell lines derived from poorly differentiated tumors (WiDr, HCT 116, LoVo, and SW480), SRFΔ5 is the predominant isoform expressed. There was a significant increase in cell survival in IEC-6 cells transfected with SRFΔ5 compared to parental cells.

Conclusion

Our data demonstrate that an alternatively spliced isoform of SRF, SRFΔ5, is expressed in human colon cancer cell lines. Additionally, these data demonstrate that expression of SRFΔ5 may contribute to the tumor phenotype by affecting cell survival. This is the first study to document a change in expression of the alternatively spliced isoform of SRF in human malignancy.

Introduction

Serum response factor (SRF) is a 67-kDa protein that was initially identified as an activator of c-fos 1, 2. It is a member of the MADS (MCM1, Agamous, Deficiens, and SRF) box superfamily of transcription factors. SRF is encoded by seven exons, with the highly conserved DNA binding site and dimerization domains encoded by the first two exons and the transactivation region encoded by exons four through seven [3]. Earlier studies have shown that SRF binds as a dimer to the CArG box (CC(A/T)₆GG), which is the central element of the SRF binding site, termed the serum response element (SRE) 4, 5.

SRF is known to regulate the activity of ubiquitously expressed immediate early genes (IEGs), such as c-fos, Egr-1, and several muscle restricted genes, by binding to the SREs found on their promoters [6]. Through these downstream targets, SRF participates in many cellular processes, including cell cycle regulation, apoptosis, cell growth and differentiation, and cell-specific gene regulation 7, 8, 9.

The diversity of stimuli that activate SRF-dependent gene expression indicates that SRF is likely a common target of multiple pathways. One of the well studied pathways that targets SRF is the mitogen-activated protein kinase pathway (MAPK) that acts through the ternary complex factors (TCFs). SRF also activates downstream targets through non-TCF-dependent mechanisms through the activation of the small GTPase RhoA [10].

More recently, there has been increasing evidence that SRF plays a role in cellular transformation. Phiel et al. have shown that the nuclear accessibility of SRF is different in smooth muscle versus smooth muscle cells undergoing neoplastic transformation. Altered binding was also demonstrated in smooth muscle cells undergoing neoplastic transformation when compared to normal tissues [11]. Pschiari et al. have shown that SRF was both up-regulated and highly active in squamous epithelial tumor cells that had undergone mesenchymal transition [12]. It appears that SRF can support or hinder transformation depending on the cell type and other available cofactors.

The roles of SRF and the alternatively spliced forms of SRF in the homeostasis of the gastrointestinal tract in general, and the colon specifically, have not been reported. Several studies have documented the presence of SRF in endoderm during embryonic development 8, 13. However, since SRF knockout mice die early during early embryonic development the role of SRF in the adult gastrointestinal tract is not known. We have demonstrated the presence of nuclear SRF by immunohistochemical staining of the human and murine adult small intestine and colon (data not shown). Furthermore, we have demonstrated an increased staining for SRF as cells migrate out of the regenerating crypts (data not shown). Therefore, we sought to determine what, if any, role SRF plays in colon tumorigenesis.

Alternative splicing is a molecular mechanism that creates diversity from a single gene. Primary mRNA transcripts can be alternatively spliced to contain or delete specific exons. This process has been shown to affect the transactivation, binding specificity, localization, and regulation of the gene being spliced [14]. This alternative splicing, if containing the DNA binding site but lacking the transactivation site, can also give rise to a dominant negative isoform [15].

Alternatively spliced isoforms have been shown to play a role in several disease processes 15, 16, 17, 18, 19. Four isoforms of SRF were shown to alter smooth muscle promoter activity depending upon the type of muscle tissue in which they were expressed. Davis et al. showed that an alternatively spliced isoform of SRF lacking exons 4 and 5, SRFΔ4,5, was increased in failing hearts. Overexpression of the SRFΔ4,5 isoform resulted in inhibition of the expression of known SRF-dependent cardiac muscle genes [16]. Alternative splicing was also demonstrated in lung smooth muscle cells when the isoform of SRF lacking exon 5, SRFΔ5, was synthesized in human hypoplastic lungs but was suppressed during normal bronchial myogenesis [17]. All of these SRF isoforms lack regions of the C-terminus but have an intact N-terminus and therefore an intact DNA binding site.

In this study we sought to determine whether SRF was alternatively spliced in colonic epithelium and to characterize the relationship of the alternatively spliced isoform with the process of cellular transformation. We have found that SRF is alternatively spliced in human colonic mucosa and colon cancer cell lines, with the SRFΔ5 isoform being the predominant isoform in cell lines derived from poorly differentiated tumors. In a rat model of enteric epithelial cell transformation, the levels of SRFΔ5 correlated with the expression level of activated ras, an oncogene that is mutated in the majority of colon cancers. In addition, we also show that the overexpression of SRFΔ5 in benign, immortal rat intestinal epithelial cells leads to altered cell survival and dysregulation of apoptosis-related genes such as caspase 3 and fas.