E-cadherin (cadh), a member of a family of integral membrane glycoproteins that represent the major component of adherens junctions (AJs), mediates cell-cell adhesion through the calcium-dependent homophilic interaction of its extracellular domain. Metastatic human carcinomas frequently lose E-cadh expression, whereas epithelial ovarian cancer (EOCs) maintain properties characteristic of Mu¨llerian epithelium during tumor progression, including E-cadh expression. Here, we examined the potential role of cell-cell contacts in EOCs through E-cadh homophilic interactions in PI3K/AKT activation whose altered signaling has been implicated in EOC pathogenesis. We show that E-cadh is predominantly expressed at cell-cell contacts and its functionality is necessary and sufficient for the activation of the PI3K/ AKT pathway. E-cadh knockdown and phosphoinositide-3-kinase (PI3K) inhibition complement each other in impairing cell-cycle progression and proliferation of ovarian carcinoma cells. E-cadh is stably bound to the PI3K complex, and the de novo formation of E-cadh/bcatenin complexes following calcium deprivation and subsequent calcium restoration recruits the PI3K p85 subunit to the site of the cell-cell contacts. The finding that E-cadh-mediated AJ formation contributes to PI3K/AKT activation in EOC cells by a mechanism that appears to be restricted to these cells provides the underpinning for therapeutic strategies that exploit PI3K inhibition to halt EOCs.

No abstract

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.

The human folate receptor (FR) is overexpressed in ovarian carcinoma. FR transcripts are heterogeneous due to the use of two promoters, P1 and P4, and alternative splicing of exon 3. RNase protection assay and RT-PCR revealed higher levels of the transcripts that include exon 3 in lines and specimens from ovarian carcinoma. A P1^chloramphenicol acetyltransferase (CAT) construct containing exon 3 demonstrated efficient reporter expression only in ovarian carcinoma. 5P P and 3P P deleted variants of the P1^CAT construct were analyzed by RT-PCR of the exogenous transcripts and reporter activity. A 5P P splice site and 35 bp downstream intronic region of exon 3 appeared to regulate enhanced FR expression in ovarian carcinoma. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

The alpha-folate receptor (alpha FR) is overexpressed in 90% of nonmucinous ovarian carcinomas. In addition to the known role of alpha FR binding and mediating the internalization of folates, functional interaction of alpha FR with signaling molecules was recently shown. To identify a model to study the role of alpha FR in ovarian carcinoma, we characterized the alpha FR gene in the ovarian carcinoma cell line CABA I in comparison to a reference line, IGROV1. In CABA I cells, Northern blot analysis revealed an alpha FR transcript of the expected length and FACS analysis indicated receptor expression on the cell membrane; however, RNase protection assay revealed no specific signals. Southern blot and genomic PCR analysis suggested the presence of a rearrangement(s) involving the 5' region of the gene in CABA I cells as compared to IGROV1 cells. Cloning and sequencing of CABA I alpha FR cDNA revealed several point mutations. The partitioning of alpha FR in membrane microdomains from CABA I cells and its association with regulatory molecules was comparable to that of IGROV1 cells. By contrast, the alpha FR expressed on the CABA I cell membrane bound folic acid with lower affinity, and ectopic expression of the corresponding cDNA in CHO cells confirmed impaired folic acid binding. Thus, CABA I cells may provide a tool to delineate functional domains of the alpha FR.