Crucial Roles of Sp1 and Epigenetic Modifications in the Regulation of the CLDN4 Promoter in Ovarian Cancer Cells

Abstract

Claudins form a large family of tight junction proteins that have essential roles in the control of paracellular ion flux and the maintenance of cell polarity. Many studies have shown that several claudin family members are abnormally expressed in various cancers. In particular, CLDN4 (encoding claudin-4) is overexpressed in ovarian cancer. However, although CLDN4 overexpression is well established, the mechanisms responsible for this abnormal regulation remain unknown. In the present study, we delineate a small region of the CLDN4 promoter critical for its expression. This region contains two Sp1 sites, both of which are required for promoter activity. However, because of the ubiquitous expression of Sp1, these sites, although necessary, are not sufficient to explain the patterns of gene expression of CLDN4 in various ovarian tissues. We show that the CLDN4 promoter is further controlled by epigenetic modifications of the Sp1-containing critical promoter region. Cells that overexpress CLDN4 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN4 promoter region, and the reverse is observed in cells that do not express CLDN4. Moreover, the CLDN4-negative cells can be induced to express CLDN4 through treatment with demethylating and/or acetylating agents. Because CLDN4 is elevated in a large fraction of ovarian cancer, the mechanism leading to deregulation may represent a general pathway in ovarian tumorigenesis and may lead to novel strategies for therapy and an overall better understanding of the biology of this disease.Claudins are major components of tight junctions, which are essential in the control of paracellular ion flux and the maintenance of cell polarity. The presence of phosphorylation sites and PDZ-binding motifs in claudins suggest a role in signal transduction for these proteins (1-3). So far, 23 members of the claudin family have been identified, and the expression of the various claudins has been shown to vary among or even within tissues, probably depending on the exact physiological requirements (1). The gene for claudin-4, CLDN4, is normally expressed in the epithelial cells of colon mucosa, mammary gland, renal tubules, and thyroid gland.Recently, several claudin genes have been shown to be abnormally expressed in human malignancies (4). In particular, CLDN4 has now been shown to be significantly elevated in several malignancies such as breast, pancreas, prostate, and ovary (4). In ovarian cancer, we and several other groups have shown that the genes for CLDN3 and CLDN4, as well as their corresponding proteins (claudin-3 and -4) are frequently up-regulated (5-10). Because many CA125-negative ovarian cancers overexpress these claudins, they may represent diagnostically useful biomarkers for this disease (11). Furthermore claudin-3 and claudin-4 are receptors for the cytotoxic Clostridium perfringens enterotoxin (12), and intra-abdominal administration of Clostridium perfringens enterotoxin might represent an interesting new avenue for ovarian cancer therapy (13). Alt...

Expression of tight-junction-associated proteins in human gastric cancer: downregulation of claudin-4 correlates with tumor aggressiveness and survival

“…Therefore, disruption of the tight junction can cause the loss of cell polarity, resulting in an abnormal infl ux of growth factors, which could provide autocrine and paracrine stimulation to tumorigenic epithelial cells [5]. However, the exact roles of tight-junction-associated proteins in gastric cancer remain unclear [18].…”

Claudin-4 overexpression is associated with epigenetic derepression in gastric carcinoma

“…30 Similarly, alteration of claudin-4 expression in bladder carcinoma was found to be associated with DNA methylation, 21 and epigenetic modifications including histone acetylation and DNA demethylation have been reported to be involved in the increase of CLDN4 expression in ovarian cancer cells. 32,33 Interestingly, our recent study 33 revealed that claudin-4 overexpression during ovarian tumorigenesis is associated with loss of repressive histone methylations, suggesting that epigenetic derepression also contributes to tumorigenesis by activation of cancer-promoting genes. However, the molecular mechanism responsible for the alteration of claudin-4 expression during gastric tumorigenesis is unclear.…”

Epigenetic Up-Regulation of Leukemia Inhibitory Factor (LIF) Gene During the Progression to Breast Cancer

“…Accumulating evidence supports the role of such epigenetic silencing mechanisms as methylation and inactive histone modifications in the transcriptional inactivation of tumor suppressor genes during cancer developmental processes (Ballestar and Esteller, 2008;Baylin and Jones, 2007;Robertson, 2005). In contrast, tumor progression may involve the epigenetic activation of oncogene-like genes, as evidenced by the upregulation of galectin-7, CLDN4, uPA, and MMP-2 genes in several cancer cells via DNA hypomethylation in their promoter regions (Chernov et al, 2009;Demers et al, 2009;Guo et al, 2002;Honda et al, 2006;Pakneshan et al, 2004;Shvachko, 2009). This epigenetic activation involves the reversal of inactive epigenetic marks including histone deacetylation by histone acetylation and DNA methylation by DNA hypomethylation.…”

“…Methylation of CpG dinucleotides located within DNA recognition elements may interfere with the binding of transcription factors, which inhibits transcription (Siegfried et al, 1999). Methylated CpG dinucleotides interfere with SP1 binding and further the reduced binding of Sp1 may be involved in suppression of transcriptional activity of the genes such as AQP5, and CLDN4 (Clark et al, 1997;Honda et al, 2006;Kudo, 1998;Mancini et al, 1999;Motegi et al, 2005;Zhu et al, 2003). In agreement with these findings, we observed that the normal breast epithelial cells MCF-10A had a heavily methylated potential SP1-binding site, the ninth CG of the LIF promoter, while benign and carcinoma MCF10 variants did not.…”

“…Certain cancer cell types methylate the BubR1, 7-dehydrocholesterol reductase (Dhcr7), aquaporin-5 (AQP5), RUNX3, lysophosphatidic acid receptor-1 (lpa1), galectin-3, CDX1, MUC5B, PDZ-LIM domain-containing protein 2 (PDLIM2), LOT1 (PLAGL1/ZAC1), TNFSF7 (CD70) genes, leading to their transcriptional suppression DNA hypomethylation at CpG islands within the promoters (Abdollahi et al, 2003;Ahmed et al, 2007;Kim et al, 2005;Motegi et al, 2005;Park et al, 2005;Perrais et al, 2001;Qu et al, 2010;Suh et al, 2002;Tsujiuchi et al, 2006;Yu et al, 2010). The upregulation of galectin-7, CDX4, and urokinase-type plasminogen activator (uPA), and MMP-2 genes in several cancer types including breast cancer, ovarian cancer, and lymphoma cells is mediated by DNA hypomethylation (Chernov et al, 2009;Demers et al, 2009;Guo et al, 2002;Honda et al, 2006;Pakneshan et al, 2004). For transcription factors such as STAT1, NFAT and Oct-1, methylation at specific CpGs has been shown to directly inhibit protein binding and thus inhibit transcription in colon carcinoma cells and human T cells (McGough et al, 2008;Murayama et al, 2006).…”