Variant HNF1 modulates epithelial plasticity of normal and transformed ovary cells.

Ovarian carcinoma arises from the ovarian surface epithelium, which undergoes phenotypic changes characteristic of müllerian epithelium during the first stages of tumorigenesis. The variant isoform of the hepatocyte nuclear factor 1 (vHNF1) is a transcription factor involved in the development of tissues derived from the müllerian duct. Here, we show that vHNF1 knockdown in two ovarian carcinoma cell lines, SKOV3 and IGROV1, leads to reduced E-cadherin (E-cadh) expression and decreased proliferation rate. Accordingly, SKOV3 cells ectopically expressing a dominant-negative (DN) vHNF1 mutant undergo an epithelial-mesenchymal-like transition, acquiring a spindle-like morphology, loss of E-cadh, and disrupted cell-cell contacts. Gene expression profiling of DNvHNF1 cells on the basis of a newly compiled list of epithelial-mesenchymal transition-related genes revealed a correlation between vHNF1 loss-of-function and acquisition of the mesenchymal phenotype. Indeed, phenotypic changes were associated with increased Slug transcription and functionality. Accordingly, vHNF1-transfected immortalized ovarian surface epithelial cells showed down-regulation of Snail and Slug transcripts. In DNvHNF1-transfected SKOV3 cells, growth rate decreased, and in vHNF1-transfected immortalized ovarian surface epithelial cells, growth rate increased. By immunohistochemistry, we found a strong association of vHNF1 with E-cadh in clear cell and in a subset of serous carcinomas, data that could potentially contribute in distinguishing different types of ovarian tumors. Our results may help in understanding the biology of ovarian carcinoma, identifying early detection markers, and opening potential avenues for therapeutic intervention.

Binding of nuclear caveolin-1 to promoter elements of growth-associated genes in ovarian carcinoma cells.

Caveolin-1 (cav-1), a member of a protein family associated mainly with cell membrane microdomains in many cell types, acts as a tumor suppressor in ovarian carcinoma cells. Biochemical analyses demonstrated that cav-1 was also localized in the nuclei of ovarian carcinoma cells, endogenously (SKOV3) or ectopically (IGtC3) expressing cav-1. By confocal analyses, the same cell lines as well as IGROV1 and SKOV3 cells transiently transfected with green fluorescent protein-cav-1 fusion protein showed nuclear punctate speckled pattern. Subnuclear distribution analysis revealed cav-1 mainly associated with the nuclear matrix, but also slightly with chromatin. Cav-1 was found in nuclear high-molecular weight complexes and by confocal analysis was found to co-localized with the inner nuclear membrane protein emerin. Cyclin D1 and folate receptor promoters were modulated by cav-1 in SKOV3 cells as demonstrated by transient transfection with or silencing of cav-1. Chromatin immunoprecipitation and supershift assays indicated that nuclear cav-1 can bind in vitro and in vivo to promoter sequences of both cyclin D1 and folate receptor genes. These data suggest that in ovarian carcinoma cells cav-1, localized in transcriptionally inactive chromatin, exerts a functional activity mediated, at least in part, by directly binding to sequences of genes involved in proliferation.

The variant hepatocyte nuclear factor 1 activates the P1 promoter of the human alpha-folate receptor gene in ovarian carcinoma.

The alpha folate receptor (alpha FR) is a membrane glycoprotein that binds folates, and mediates their uptake and that of antifolate drugs. alpha FR is absent on ovarian surface epithelium (OSE) but is detectable during early transforming events in this epithelium, with increasing expression levels in association with tumor progression. Analysis of transcriptional regulation of the alpha FR gene have revealed two promoter regions, P1 and P4, flanking exons 1 and 4, respectively, and a requirement for three SP1 sites and an INR element for optimal P4 activity. Here, we focused on the P1 transcription regulation in ovarian carcinoma cells. RNase protection assay indicated that the 5'-untranslated region is heterogeneous because of different start sites and alternative splicing of exon 3. A core region of the P1 promoter was sufficient for maximal promoter activity in ovarian carcinoma cell lines but not in OSE cells or in alpha FR-nonexpressing cell lines. Deletion and mutation analysis of this core promoter identified a cis-regulatory element at position +27 to +33 of the untranslated exon 1, which is responsible for maximum P1 activity. This element formed an abundant DNA-protein complex with nuclear proteins from ovarian cancer cells but not from other cell lines or OSE cells. Competition experiments and supershift assays demonstrated binding of the P1 cis-regulatory element by a transcription factor involved in embryonic development, the variant hepatocyte nuclear factor-1 (vHNF1). Analysis of RNA from various cell lines and surgical specimens confirmed that vHNF1 is expressed in ovarian carcinomas. Thus, vHNF1 regulates tissue-specific transcription in ovarian carcinoma.