The p63 isoform ΔNp63α inhibits epithelial-mesenchymal transition in human bladder cancer cells: Role of miR205.
ABSTRACT Epithelial-mesenchymal transition (EMT) is a physiological process that plays important roles in tumor metastasis, stemness, and drug resistance. EMT is typically characterized by the loss of the epithelial marker E-cadherin and increased expression of EMT-associated transcriptional repressors, including ZEB1 and ZEB2. The miR-200 family and miR-205 prevent EMT through suppression of ZEB1/2. p53 has been implicated in the regulation of miR-200c, but the mechanisms controlling miR-205 expression remain elusive. Here we report that the p53 family member and p63 isoform, Np63α, promotes miR-205 transcription and controls EMT in human bladder cancer cells. Np63α, E-cadherin and miR-205 were co-expressed in a panel of bladder cancer cell lines (n=28) and a cohort of primary bladder tumors (n=98). Stable knockdown (KD) of Np63α in the epithelial bladder cancer cell line UM-UC6 decreased the expression of miR-205 and induced the expression of ZEB1/2, effects that were reversed by expression of exogenous miR-205. Conversely, overexpression of Np63α in the mesenchymal bladder cancer cell line UM-UC3 induced miR-205 and suppressed ZEB1/2. Np63α KD reduced the expression of the primary and mature forms of miR-205 and the miR-205 host gene (miR-205HG) and decreased binding of RNA Pol II to the miR-205HG promoter, inhibiting miR-205HG transcription. Finally, high miR-205 expression was associated with adverse clinical outcomes in bladder cancer patients. Together, our data demonstrate that Np63α-mediated expression of miR-205 contributes to the regulation of EMT in bladder cancer cells and identify miR-205 as a molecular marker of the lethal subset of human bladder cancers.
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ABSTRACT: Trp63, founding member of the Trp53 family, contributes to epithelial differentiation and is expressed in breast neoplasia. Trp63 features two distinct promoters yielding specific mRNAs encoding two major TRP63 isoforms, a transactivating transcription factor and a dominant negative isoform. Specific TRP63 isoforms are linked to cell cycle arrest, apoptosis, survival and epithelial mesenchymal transition. Although TRP63 overexpression in cultured cells is used to elucidate functions, little is known about Trp63 regulation in normal and cancerous mammary tissue. This study used ChIP-seq to interrogate transcription factor binding and histone modifications of the Trp63 locus in mammary tissue and RNA-seq and immunohistochemistry to gauge gene expression. H3K4me2 and H3K4me3 marks coincided only with the proximal promoter, supporting RNA-seq data showing the predominance of the dominant negative isoform. STAT5 bound specifically to the Trp63 proximal promoter and Trp63 mRNA levels were elevated upon deleting STAT5 from mammary tissue, suggesting its role as a negative regulator. The dominant negative TRP63 isoform was localized to nuclei of basal mammary epithelial cells throughout reproductive cycles, and retained in a majority of the triple negative cancers generated from loss of full-length BRCA1. Increased expression of dominant negative isoforms was correlated with developmental windows of increased progesterone receptor binding to the proximal Trp63 promoter and decreased expression during lactation was correlated with STAT5 binding to the same region. TRP63 is present in the majority of triple negative cancers resulting from loss of BRCA1 but diminished in less differentiated cancer subtypes and in cancer cells undergoing epithelial mesenchymal transition.Endocrine Related Cancer 04/2014; DOI:10.1530/ERC-14-0032 · 4.91 Impact Factor
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ABSTRACT: The recent discovery of tiny microRNAs (miRNAs) has brought about awareness of a new class of regulators of diverse pathways in many physiological and pathological processes, such as tumorigenesis. They modulate gene expression by targeting plethora of mRNAs, mostly reducing the protein yield of a targeted mRNA. With accumulation of information on characteristics of miR-205, complex and in some cases converse roles of miR-205 in tumor initiation, progression and metastasis are emerging. miR-205 acts either as an oncogene via facilitating tumor initiation and proliferation, or in some cases as a tumor suppressor through inhibiting proliferation and invasion. The aim of this review is to discuss miR-205 roles in different types of cancers. Given the critical effects of deregulated miR-205 on processes involved in tumorigenesis, they hold potential as novel therapeutic targets and biomarkers.Asian Pacific journal of cancer prevention: APJCP 01/2014; 15(2):577-83. DOI:10.7314/APJCP.2014.15.2.577 · 1.50 Impact Factor
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ABSTRACT: AIM: To investigate role of putative mitogen-activated protein kinase activator with WD40 repeats (MAWD)/MAWD binding protein (MAWBP) in gastric cancer (GC). METHODS: MAWBP and MAWD mRNA expression level was examined by real-time reverse transcriptase-polymerase chain reaction and semi-quantitative polymerase chain reaction in six GC cell lines. Western blotting was used to examine the protein expression levels. We developed GC cells that stably overexpressed MAWBP and MAWD, and downregulated expression by RNA interference assay. Proliferation and migration of these GC cells were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), soft agar, tumorigenicity, migration and transwell assays. The effect of expression of MAWBP and MAWD on transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) was examined by transfection of MAWBP and MAWD into GC cells. We detected the levels of EMT markers E-cadherin, N-cadherin and Snail in GC cells overexpressing MAWBP and MAWD by Western blotting. The effect of MAWBP and MAWD on TGF-β signal was detected by analysis of phosphorylation level and nuclear translocation of Smad3 using Western blotting and immunofluorescence. RESULTS: Among the GC cell lines, expression of endogenous MAWBP and MAWD was lowest in SGC7901 cells and highest in BGC823 cells. MAWBP and MAWD were stably overexpressed in SGC7901 cells and knocked down in BGC823 cells. MAWBP and MAWD inhibited GC cell proliferation in vitro and in vivo. MTT assay showed that overexpression of MAWBP and MAWD suppressed growth of SGC7901 cells (P < 0.001), while knockdown of these genes promoted growth of BGC823 cells (P < 0.001). Soft agar colony formation experiments showed that overexpression of MAWBP and MAWD alone or together reduced colony formation compared with vector group in SGC7901 (86.25 ± 8.43, 12.75 ± 4.49, 30 ± 6.41 vs 336.75 ± 22.55, P < 0.001), and knocked-down MAWBP and MAWD demonstrated opposite effects (131.25 ± 16.54, 88.75 ± 11.12, 341.75 ± 22.23 vs 30.25 ± 8.07, P < 0.001). Tumorigenicity experiments revealed that overexpressed MAWBP and MAWD inhibited GC cell proliferation in vivo (P < 0.001). MAWBP and MAWD also inhibited GC cell invasion. Transwell assay showed that the number of traverse cells of MAWBP, MAWD and coexpression group were more than that in vector group (84 ± 16.57, 98.33 ± 9.8, 29 ± 16.39 vs 298 ± 11.86, P < 0.001). Coexpression of MAWBP and MAWD significantly decreased the cells traversing the matrix membrane. Conversely, knocked-down MAWBP and MAWD correspondingly promoted invasion of GC cells (100.67 ± 14.57, 72.66 ± 8.51, 330.67 ± 20.55 vs 27 ± 11.53, P < 0.001). More importantly, coexpression of MAWBP and MAWD promoted EMT. Cells that coexpressed MAWBP and MAWD displayed a pebble-like shape and tight cell-cell adhesion, while vector cells showed a classical mesenchymal phenotype. Western blotting showed that expression of E-cadherin was increased, and expression of N-cadherin and Snail was decreased when cells coexpressed MAWBP and MAWD and were treated with TGF-β1. Nuclear translocation of p-Smad3 was reduced by attenuating its phosphorylation. CONCLUSION: Coexpression of MAWBP and MAWD inhibited EMT, and EMT-aided malignant cell progression was suppressed.World Journal of Gastroenterology 05/2013; 19(18):2781-2792. DOI:10.3748/wjg.v19.i18.2781 · 2.43 Impact Factor