BACKGROUND It was demonstrated previously that the Snail family of transcription factors and Smad‐interacting protein 1 (Sip1) regulate E‐cadherin and matrix metalloproteinase 2 (MMP‐2) expression, cellular morphology, and invasion in carcinoma. For the current study, the authors analyzed the relation between the expression of Snail, Slug, and Sip1; the expression of MMP‐2 and E‐cadherin; and clinical parameters in patients with metastatic ovarian and breast carcinoma. METHODS One hundred one fresh‐frozen, malignant effusions from patients who were diagnosed with gynecologic carcinomas (78 ovarian carcinomas and 23 breast carcinomas) were studied for mRNA expression of Snail, Slug, Sip1, MMP‐2, and E‐cadherin using reverse transcriptase‐polymerase chain reaction analysis. Snail mRNA and E‐cadherin protein expression levels also were studied in ovarian carcinoma effusions using in situ hybridization and immunocytochemistry. The results were analyzed for possible correlation with clinicopathologic parameters in both tumor types. RESULTS E‐cadherin mRNA expression was lower in breast carcinoma (P = 0.001), whereas Snail expression was higher (P = 0.003). The Snail/E‐cadherin ratio (P < 0.001) and the Sip1/E‐cadherin ratio (P = 0.002) were higher in breast carcinomas. Sip1 mRNA expression (P < 0.001) and Slug mRNA expression (P < 0.001) were correlated with the expression of MMP‐2 in ovarian carcinomas. The Sip1/E‐cadherin ratio was higher in primary ovarian carcinomas at the time of diagnosis compared with postchemotherapy ovarian carcinoma effusions (P = 0.003), higher in Stage IV tumors compared with Stage III tumors (P = 0.049), and higher in pleural effusions compared with peritoneal effusions (P = 0.044). In a univariate survival analysis of patients with ovarian carcinoma, a high Sip1/E‐cadherin ratio predicted poor overall survival (P = 0.018). High E‐cadherin mRNA expression predicted better disease‐free survival (P = 0.023), with a similar trend for a low Slug/E‐cadherin ratio (P = 0.07). High Snail mRNA expression predicted shorter effusion‐free survival (P = 0.008), disease‐free survival (P = 0.03), and overall survival (P = 0.008) in patients with breast carcinoma. CONCLUSIONS Transcription factors that regulate E‐cadherin were expressed differentially in metastatic ovarian and breast carcinoma. Snail may predict a poor outcome in patients who have breast carcinoma metastatic to effusions. E‐cadherin expression generally was conserved in effusions from patients with ovarian carcinoma, but the subset of patients with postulated Sip1‐induced repression of this adhesion molecule had a significantly worse outcome. This finding was in agreement with the stronger suppression of E‐cadherin by Snail and Sip1 in breast carcinoma effusions, a clinical condition associated with extremely poor survival. Cancer 2005. © 2005 American Cancer Society.

Unlike most epithelial cancers, E-cadherin expression is upregulated in ovarian carcinoma effusions compared with corresponding primary tumors. In the present study, we analyzed the anatomic site-specific expression of transcription factors that negatively regulate E-cadherin in ovarian carcinoma. Using reverse-transcription polymerase chain reaction, mRNA in situ hybridization, and Western blotting, we analyzed the expression and localization of the Snail, Slug, and SIP1 transcription factors and E-cadherin in 78 effusions, 41 primary carcinomas, and 15 solid metastases. Slug mRNA and protein expression was highest in metastases (p=0.042 and p<0.001, respectively). Snail mRNA was comparable at all anatomic sites, but higher protein expression was found in primary tumors and solid metastases compared with effusions (p<0.001). SIP1 mRNA expression was higher in effusions (p<0.001) compared to other sites. Confocal microscopy analysis of fresh and cultured cells from effusion specimens revealed cytoplasmic localization of the Snail protein in primary tumor cells, with a nuclear shift following culturing of these cells. In conclusion, E-cadherin and its negative regulators show site-dependent expression in ovarian carcinoma. In solid tumors, E-cadherin is negatively regulated by Snail and Slug. In effusions, SIP1 may be the main regulator of E-cadherin, but with a lesser level of suppression compared with primary tumors and solid metastases.

The PI 3-K and Akt signaling pathway regulates all phenotypes that contribute to progression of human cancers, including breast cancer. Akt mediates signal relay by phosphorylating numerous substrates, which are causally implicated in responses such as cell growth, survival, metabolic reprograming and migration and invasion. Here we identify a new Akt substrate, the adherens junction protein Afadin, that is phosphorylated by Akt at Ser1718. We show that under conditions of physiological IGF-1 signaling and oncogenic PI 3-K and Akt, Afadin is phosphorylated by all Akt isoforms, and that this phosphorylation elicits a relocalization of Afadin from adherens junctions to the nucleus. Phosphorylation of Afadin also results in a marked increase in breast cancer cell migration that is dependent on Ser1718 phosphorylation. We also observe nuclear localization in breast cancer tissues, indicating that regulation of Afadin by the PI 3-K and Akt pathway has pathophysiological significance. Implications Phosphorylation of the adhesion protein Afadin by Akt downstream of the PI 3-K pathway, leads to re-distribution of Afadin and controls cancer cell migration.

The present study investigated the intracellular regulation of E-cadherin in ovarian carcinoma. E-cadherin expression and regulation by Snail and Pak1 were studied in ES-2 and OVCAR-3 ovarian cancer cells in vitro. Twist1, Zeb1 and Vimentin mRNA expression and HIF-1alpha protein expression were analyzed in 80 and 189 clinical specimens, respectively. OVCAR-3 cells incubated with an anti-E-cadherin antibody formed smaller and looser spheroids compared to controls. Snail silencing using Small Hairpin RNA in ES-2 cells reduced invasion and MMP-2 activity, with unaltered cellular morphology. Using dominant negative (DN) and constitutively active (CA) Pak1 constructs, we found that DN Pak1 ES-2 and OVCAR-3 clones had reduced attachment to matrix proteins, invasion and MMP-2 activity compared to CA and wild-type cells. DN Pak1 ES-2 cells also bound less to LP9 mesothelial cells. DN Pak1 OVCAR-3 cells had lower Vimentin levels. Snail expression was lower in cultured effusions compared to primary carcinomas, and was cytoplasmic rather than nuclear. Twist1 (P < 0.001), Zeb1 (P = 0.003) and Vimentin (P = 0.03) mRNA expression was significantly higher in solid metastases compared to primary carcinomas and effusions. HIF-1alpha protein expression was lower in effusions compared to primary carcinomas and solid metastases (P = 0.033). Our data suggest that the previously reported E-cadherin re-expression in ovarian carcinoma effusions is regulated by Pak1. The transient nature of E-cadherin expression during ovarian carcinoma progression is probably the result of partial epithelial-to-mesenchymal transition (EMT) and the reverse process of mesenchymal-to-epithelial-like transition (MET). Expression of the EMT-related molecules Twist, Zeb1, Vimentin and HIF-1alpha is anatomic site-dependent in ovarian carcinoma.