Here we sought to determine the relationship between STAT3 activity and Galectin‐3 (Gal‐3) and to investigate the cytotoxic effect of PectaSol‐C Modified Citrus Pectin (Pect‐MCP) as a specific competitive inhibitor of Galectin‐3 (Gal‐3) in combination with Paclitaxel (PTX) to kill the ovarian cancer cell SKOV‐3 multicellular tumor spheroid (MCTS). To this order, SKOV‐3 cells in 2D and 3D cultures were treated with exogenous Gal‐3 for the assessment of STAT3 activity. Two‐way ANOVA main effect and IC 50 of each drug Paclitaxel (PTX) and Pect‐MCP or in combination were obtained from MTT assay results. The phosphorylated STAT3 levels, migration, invasion, integrin mRNA and p‐AKTser 473 levels were assessed in the absence or presence of each drug alone or in combination. Gal‐3 expression levels were assessed in human serous ovarian cancer (SOC) specimens and its correlation with different integrin mRNA levels was further assessed. Our results showed that Gal‐3 expression level was significantly increased in MCTS compared to monolayer SKOV‐3 cells which triggered STAT3 phosphorylation. Moreover, Pect‐MCP synergized with PTX to kill SKOV3 MCTS through abrogation of STAT3 activity and reduced expression of its downstream target HIF‐1α, reduced integrin mRNA levels, and subsequently decreased AKT activity. There were higher expression levels of Gal‐3 in human high‐grade SOC specimens compared to the normal ovary and borderline SOC which positively and significantly correlated with α5, β2 and β6 integrin mRNA levels. Together, these results revealed for the first time that Pect‐MCP could be considered as a potential drug to enhance the PTX effect on ovarian cancer cells MCTS through inhibition of STAT3 activity.

Wnt5A signals through various receptors that confer versatile biological functions. Here, we used Wnt5A overexpressing human ovarian SKOV-3 and OVCAR-3 stable clones for assessing integrin expression, cell proliferation, migration, invasion, and the ability of multicellular aggregates (MCAs) formation. We found here, that Wnt5A regulates differently the expression of its receptors in the stable Wnt5A overexpressing clones. The expression levels of Frizzled (FZD)-2 and -5, were increased in different clones. However ROR-1, -2 expression levels were differently regulated in clones. Wnt5A overexpressing clones showed increased cell proliferation, migration, and clonogenicity. Moreover, Wnt5A overexpressing SKOV-3 clone showed increased MCAs formation ability. Cell invasion had been increased in OVCAR-3-derived clones, while this was decreased in SKOV-3-derived clone. Importantly, αv integrin expression levels were increased in all assessed clones, accompanied by increased cell attachment to fibronectin and focal adhesion kinase activity. Moreover, the treatment of clones with Box5 as a Wnt5A/FZD5 antagonist abrogates ITGAV increase, cell proliferation, migration, and their attachment to fibronectin. Accordingly, we observed significantly higher expression levels of ITGAV and ITGB3 in human high-grade serous ovarian cancer specimens and ITGAV correlated positively with Wnt5A in metastatic serous type ovarian cancer. In summary, we hypothesize here, that Wnt5A/FZD-5 signaling modulate αv integrin expression levels that could be associated with ovarian cancer cell proliferation, migration, and fibronectin attachment.

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Wnt5A and receptor tyrosine kinase-like orphan receptor 2 (ROR2) proteins both regulate developmental processes, cell movement, and cell polarity. The purpose of this study was to evaluate a possible regulatory role of Wnt5A on ROR2 expression in human ovarian cancer cell lines. Moreover, the expression of Wnt5A and ROR2 mRNA and protein levels were assessed in human epithelial serous ovarian cancer (HSOC) specimens. ROR2 was strongly decreased in cells treated with siRNA against Wnt5A compared with scramble-treated or lipofectamine-treated cells (P < 0.001). There was 34% decreased cell invasion (P < 0.01) in Wnt5A knock-down cells compared with lipofectamine-treated and scramble-treated cells; however, cell invasion remained unchanged upon addition of anti-ROR2 antibody to the culture media of these cells. In contrast, addition of anti-ROR2 antibody to the culture media for lipofectamine-treated and scramble-treated cells led to 32% decreased cell invasion (P < 0.01). Normal ovarian specimens were negative, and variable immunostaining was observed in HSOC for Wnt5A and ROR2 immunostaining. Furthermore, there was a positive correlation between Wnt5A and ROR2 expression in high-grade SOC samples at the mRNA level (P < 0.05; r = 0.38). This is the first report to show the regulatory role of Wnt5A on ROR2 expression in ovarian cancer.

In this paper, we investigate whether Wnt5A is associated with the TGF-β1/Smad2/3 and Hippo-YAP1/TAZ-TEAD pathways, implicated in epithelial to mesenchymal transition (EMT) in epithelial ovarian cancer. We used 3D and 2D cultures of human epithelial ovarian cancer cell lines SKOV-3, OVCAR-3, CAOV-4, and different subtypes of human serous ovarian cancer compared to normal ovary specimens. Wnt5A showed a positive correlation with TAZ and TGFβ1 in high- and low-grade serous ovarian cancer specimens compared to borderline serous and normal ovaries. Silencing Wnt5A by siRNAs significantly decreased Smad2/3 activation and YAP1 expression and nuclear shuttling in ovarian cancer (OvCa) cells. Furthermore, Wnt5A was required for TGFβ1-induced cell migration and invasion. In addition, inhibition of YAP1 transcriptional activity by Verteporfin (VP) altered OvCa cell migration and invasion through decreased Wnt5A expression and inhibition of Smad2/3 activation, which was reverted in the presence of exogenous Wnt5A. We found that the activation of TGFβ1 and YAP1 nuclear shuttling was promoted by Wnt5A-induced integrin alpha v. Lastly, Wnt5A was implicated in activating human primary omental mesothelial cells and subsequent invasion of ovarian cancer cells. Together, we propose that Wnt5A could be a critical mediator of EMT-associated pathways.