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ABSTRACT: Invasive migration of carcinoma cells is a prerequisite for the metastatic dissemination of solid tumours. Numerous mechanisms control the ability of cancer cells to acquire a motile and invasive phenotype, and subsequently degrade and invade the basement membrane. Several genes that are up-regulated in breast carcinoma are responsible for mediating the metastatic cascade. Recent studies have revealed that the NFAT (nuclear factor of activated T-cells) is a transcription factor that is highly expressed in aggressive breast cancer cells and tissues, and mediates invasion through transcriptional induction of pro-invasion and migration genes. In the present paper we demonstrate that NFAT promotes breast carcinoma invasion through induction of GPC (glypican) 6, a cell-surface glycoprotein. NFAT transcriptionally regulates GPC6 induction in breast cancer cells and binds to three regulatory elements in the GPC6 proximal promoter. Expression of GPC6 in response to NFAT signalling promotes invasive migration, whereas GPC6 silencing with shRNA (small-hairpin RNA) potently blocks this phenotype. The mechanism by which GPC6 promotes invasive migration involves inhibition of canonical β-catenin and Wnt signalling, and up-regulation of non-canonical Wnt5A signalling leading to the activation of JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase). Thus GPC6 is a novel NFAT target gene in breast cancer cells that promotes invasive migration through Wnt5A signalling.
Biochemical Journal 08/2011; 440(1):157-66. · 4.90 Impact Factor
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ABSTRACT: The NFAT (nuclear factor of activated T cells) family of transcription factors plays a fundamental role in the transcriptional regulation of the immune response. However, NFATs are ubiquitously expressed, and recent evidence points to their important functions in human epithelial cells and carcinomas. Specifically, NFAT has been shown to be active in human breast and colon carcinoma cells and to promote their invasion through Matrigel. The mechanisms by which NFAT promotes invasion have not been defined. To identify NFAT target genes that induce carcinoma invasion, we have established stable breast cancer cell lines that inducibly express transcriptionally active NFAT. Gene expression profiling by cDNA microarray of cells induced to express NFAT revealed up-regulation of cyclooxygenase-2 (COX-2). Increased NFAT expression and activity induced COX-2 expression as well as prostaglandin E2 synthesis. This induction was more prominent when NFAT was activated by phorbol 12-myristate 13-acetate and calcium ionophore ionomycin and was blocked by the NFAT antagonist cyclosporin A. Breast cancer cells with elevated COX-2 expression showed increased invasion through Matrigel, and this was reduced in cells treated with COX-2 inhibitors. Conversely, loss of NFAT1 protein expression using small interfering RNA led to a reduction in COX-2 transcription and reduced invasion. Similarly, Matrigel invasion was reduced in cells in which COX-2 expression was reduced using specific siRNA. These findings demonstrate that NFAT promotes breast cancer cell invasion through the induction of COX-2 and the synthesis of prostaglandins.
Journal of Biological Chemistry 06/2006; 281(18):12210-7. · 4.77 Impact Factor
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ABSTRACT: The phosphoinositide 3-kinase (PI 3-K) signaling axis is intimately associated with deregulated cancer cell growth, primarily by promoting increased survival through Akt/PKB (protein kinase B). However, there is relatively little information on the role of Akt in cancer cell motility, a key phenotype of invasive carcinomas. Here we report that activation of Akt inhibits carcinoma migration and invasion of breast cancer cells. Conversely, downregulation of Akt using RNA interference increased migration and invasion. Akt blunts invasion by inhibiting the transcriptional activity of NFAT (nuclear factor of activated T cells). Specifically, signaling through Akt reduces NFAT expression levels due to ubiquitination and proteasomal degradation, mediated by the E3 ubiquitin ligase HDM2. These results indicate that while Akt can promote tumor progression through increased cell survival mechanisms, it can block breast cancer cell motility and invasion by a mechanism that depends, at least in part, on the NFAT transcription factor.
Molecular Cell 12/2005; 20(4):539-50. · 14.18 Impact Factor
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ABSTRACT: Reduced expression of topoisomerase II is one of the mechanisms observed in cell lines and clinical samples that are resistant to topoisomerase II-targeting agents. The Chinese hamster lung cell line DC-3F/9-OH-E made resistant to 9-OH ellipticine and cross-resistant to other topoisomerase II inhibitors has previously been shown to express lower level of topoisomerase IIalpha isoform, than the parental DC-3F cell line. We have shown here that topoisomerase IIalpha promoter activity is lower in the resistant cell line. The promoter sequence responsible for the differential expression of Chinese hamster topoisomerase IIalpha gene was localized in a small promoter region, which harbors three inverted CAAT elements (ICEs) that bind transcription factor NF-Y, two GC boxes that bind Sp1 and a TATA-like element that binds unknown factors. Immunoblot analysis of cell lysates showed that the resistant line expressed reduced levels of NF-Y subunits and attenuated level of p53. Although p53 has been reported being involved in the regulation of topoisomerase II expression, it is not responsible for the reduced topoisomerase IIalpha expression in the drug resistant line. Mutational analysis of individual elements suggested that the resistant cell line has relaxed responses to ICE mutations, and the TATA-like element plays a predominant role in the regulation of topoisomerase IIalpha. Furthermore, gel mobility shift assays showed that the resistant line has a differential binding to the novel TATA-like element, which may be responsible for the down-regulation of topoisomerase IIalpha gene.
Gene 12/2004; 342(1):145-55. · 2.34 Impact Factor
