Selective activation of tumor growth-promoting Ca2+ channel MS4A12 in colon cancer by caudal type homeobox transcription factor CDX2
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Colon cancer-associated MS4A12 is a novel colon-specific component of store-operated Ca2+ (SOC) entry sensitizing cells for epidermal growth factor (EGF)-mediated effects on proliferation and chemotaxis. In the present study, we investigated regulation of the MS4A12 promoter to understand the mechanisms responsible for strict transcriptional restriction of this gene to the colonic epithelial cell lineage. DNA-binding assays and luciferase reporter assays showed that MS4A12 promoter activity is governed by a single CDX homeobox transcription factor binding element. RNA interference (RNAi)-mediated silencing of intestine-specific transcription factors CDX1 and CDX2 and chromatin immunoprecipitation (ChIP) in LoVo and SW48 colon cancer cells revealed that MS4A12 transcript and protein expression is essentially dependent on the presence of endogenous CDX2. In summary, our findings provide a rationale for colon-specific expression of MS4A12. Moreover, this is the first report establishing CDX2 as transactivator of tumor growth-promoting gene expression in colon cancer, adding to untangle the complex and conflicting biological functions of CDX2 in colon cancer and supporting MS4A12 as important factor for normal colonic development as well as for the biology and treatment of colon cancer.
KeywordsSW48 Cell Colonic Epithelial Cell Supervisory Board Colonic Epithelium siRNA Duplex
Membrane-spanning 4-domains subfamily A (MS4A) is an evolving family of structurally related cell surface proteins. Prominent members of this group are B lymphocyte differentiation antigen CD20 (MS4A1), the high-affinity IgE receptor β chain (FcεRIβ; MS4A2), and hematopoietic cell cycle regulator HTm4 (MS4A3) [1, 2, 3]. As reported recently, a genome-wide in silico search for differentiation genes of colonic epithelial cells led us to MS4A12, a poorly characterized member of this family . In contrast to other MS4A proteins, which are known to be restricted to cells of hematopoietic or lymphatic lineages, MS4A12 is not expressed in such cell types [2, 3]. Instead, we showed that expression of MS4A12 is strictly confined to the apical region of normal colonic epithelial cells. In the course of malignant transformation expression of MS4A12 is frequently and strongly maintained in colon cancers. Functional studies revealed that MS4A12 is a SOC channel modulating growth factor-mediated capacitative Ca2+ entry, which is a convergent point of many signal transduction pathways controlling important cellular functions. In line with these findings, silencing of MS4A12 expression in colon cancer cells by RNAi results in attenuation of EGF-dependent effects. In particular, proliferation, motility, and chemotactic invasion of cells are significantly impaired. Cancer cells expressing MS4A12, in contrast, are sensitized to EGF and respond to low concentrations of this growth factor .
The molecular mechanisms regulating tissue-specific expression of MS4A12 have not been specified so far, however, identification of these mechanisms is likely to further shed light on the biological functions of MS4A12, its role in colon cancer progression, and its suitability as therapeutic molecular target. Therefore, we now aimed to identify the cis-elements and trans-factors that regulate MS4A12 promoter activity.
Next, to determine whether CDX1 and CDX2 were able to bind to this functional CDX element, an ELISA-based transcription factor binding assay was performed. Incubation of nuclear cell extract from LoVo cells as source of CDX transcription factors with a biotinylated oligonucleotide capture probe representing the putative CDX binding sequence resulted in detection of specific CDX1 and CDX2 protein-DNA complexes (Fig. 1c), albeit signals were much stronger for CDX2 compared to CDX1. Mutation of the biotinylated oligonucleotide probe suppressed binding of both transcription factors. Complex formation was also inhibited by excess amounts of the non-biotinylated capture probe as competitor, but not by a competitor probe with a mutated binding site, confirming specificity of these interactions. Due to the highly conserved homeodomain, CDX1 and CDX2 typically bind and transactivate many of the same DNA elements, but there are reports of gene promoters in which selective binding of CDX2 was observed [6, 7, 8, 9]. In accordance with such reports, our data imply preferential binding of CDX2 to the functional element.
Our findings are of particular interest for several reasons. First, they provide a rationale for the strict transcriptional restriction of MS4A12 to the colonic epithelium and its absence from other normal cell types. In the adult intestine CDX2 expression increases progressively from the duodenum to the distal intestine, and is at its greatest levels in the proximal colonic epithelium [10, 11]. CDX2 target genes are expressed in a number of different patterns in the small intestine and colonic epithelium. These include genes like mucin 2 (MUC2), Kruppel-like factor 4 (KLF4), and guanylcyclase 2C (GUCY2C), which are expressed throughout the intestine [9, 12, 13]. The observation that MS4A12 expression is strictly confined to the colonic epithelial cells is concordant with other CDX2 target genes showing colon-restricted expression, like colon-specific carbonic anhydrase I (CA1) . Precise temporal and spatial regulation of gene expression within the intestinal and colonic epithelium are known to depend upon specific interactions between CDX2 and other transcription factors [15, 16, 17, 18], as well as the phosphorylation state of CDX2 [19, 20], which will be mapped for MS4A12 in future studies. In colon cancer cells CDX2 expression was initially reported to be reduced compared to normal colonic mucosa with an inverse relationship between CDX2 expression and advanced stages of cancers [21, 22, 23, 24]. These data, however, have been disproved by recent studies showing strong and robust expression of CDX2 in >80% of colon cancers [25, 26, 27], complying with the high and frequent expression of MS4A12 in colon cancer.
Second, our data add to untangle the complex and conflicting biological functions of CDX2 in colon cancer. CDX2 has been reported to possess growth-inhibitory as well as growth-promoting functions in cancer cells. The suppressive function of CDX2 on tumor cell proliferation observed in several studies [28, 29, 30] has been explained by CDX2-mediated activation of negative cell cycle regulators, such as cyclin-dependent kinase inhibitor 1A (CDKN1A), a known inhibitor of G1 cyclin/cyclin-dependent kinase complexes . On the other hand, it was shown that CDX2 enhances proliferation and has tumorigenic potential in the human colon cancer cell lines LoVo and SW48  used in this study. Until now the molecular basis for the growth-promoting functions of CDX2 in these cells has not been elucidated. Together these conflicting findings point to a complex role for CDX2 in the regulation of cell proliferation. It suggests that CDX2 has different functions depending on the cellular and tissue context it is expressed in.
Our findings provide first evidence for the capability of this caudal-related protein to induce growth-promoting genes in colon cancer. Moreover, they link CDX2 to EGF-mediated cancer cell proliferation, migration and invasion, and provide a basis to further dissect the role of CDX2 in colon cancer progression.
Conflicts of interests
MK is scientific advisor, ÖT is CEO/CSO, CH is member of the supervisory board, and US is CMO of Ganymed Parmaceuticals AG, a company holding patent applications on MS4A12 as therapeutic target for monoclonal antibody therapy of cancer.
This work was supported by the GO-Bio funding of the Federal Ministry of Education and Research (BMBF).
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