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Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8− tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up‐regulation of E‐cadherin and down‐regulation of Twist, p120‐catenin, and β‐catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal–epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell–cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several‐fold increase in EV number in cell culture and the circulation of tumour‐bearing animals. We observed increased protein levels of E‐cadherin and p120‐catenin in these EVs; furthermore, Tspan8 and p120‐catenin were co‐immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The purpose of this study was three fold: (1) to reveal the implications of Wnt5A for cytokine and chemokine production by human ovarian cancer cell line SKOV-3 cells, (2) to determine the influence of Wnt5A on chemotactic SKOV-3 cell migration, and (3) to assess the effect of inflammatory mediators on Wnt5A expression levels and to describe its underlying molecular mechanisms. A cytokine array was performed using a conditioned medium harvested from SKOV-3 cells transfected with specific siRNAs against Wnt5A or with scrambled siRNA and a transfection reagent alone as negative controls for 48 h. Chemotactic cell migration was performed using transwells. Inflammation-induced Wnt5A expression was determined by treating cells with recombinant human (rh) IL-1β, IFNβ, or TNFα alone or in combination with STAT3 and NF-κB inhibitors for different time durations. The cytokine array showed the suppression of GCSF, GM-CSF, IL-1α, IL-2, IL-13, and MCP-3 production, whereas cell RANTES and IL-7 showed increased levels in Wnt5A knock-down cells compared with those in controls. Chemotactic migration decreased significantly when the conditioned medium from Wnt5A knock-down cells was applied to the upper chamber of the transwell. Compared with the control, there were 30-fold and five-fold increases in Wnt5A mRNA levels in cells treated, with rhIL-1β and rhIFNβ, respectively after 8 h (P < 0.001). Compared with the control, TNF-α had a 1.8-fold increased levels of Wnt5A mRNA after 4 h (P < 0.01). Both NF-κB and STAT3 inhibitors decreased inflammation-induced Wnt5A expression. This study revealed a previously unrecognized immunomodulatory role of endogenous Wnt5A in ovarian cancer cells, which could further influence chemotactic cell migration.

BackgroundWnt5A, which is a member of the non-transforming Wnt protein family, is implicated in inflammatory processes. It is also highly expressed by ovarian cancer cells. ROR2, which is a member of the Ror-family of receptor tyrosine kinases, acts as a receptor or co-receptor for Wnt5A. The Wnt5A–ROR2 signaling pathway plays essential roles in the migration and invasion of several types of tumor cell and influences their cell polarity. We investigated the modulation of Wnt5A–ROR2 by inflammatory mediators and its involvement in the migration of the human ovarian cancer cell line SKOV-3.MethodsSKOV-3 cells were treated with LPS (lipopolysaccharide), LTA (lipoteichoic acid) and recombinant human IL-6 alone or in combination with STAT3 inhibitor (S1155S31-201) or NF-kB inhibitor (BAY11-7082) for 4, 8, 12, 24 and 48 h. The Wnt5A and ROR2 expression levels were determined at the gene and protein levels. Cells were transfected with specific siRNA against Wnt5A in the absence or presence of human anti-ROR2 antibody and cell migration was assessed using transwells.ResultsThere was a strong downregulation of Wnt5A expression in the presence of STAT3 or NF-kB inhibitors. Cell stimulation with LTA or IL-6 for 8 h led to significantly increased levels of Wnt5A (5- and 3-fold higher, respectively). LPS, LTA or IL-6 treatment significantly increased ROR2 expression (2-fold after 48 h). LPS- or LTA-induced Wnt5A or ROR2 expression was abrogated in the presence of STAT3 inhibitor (p < 0.001). IL-6-induced Wnt5A expression was abrogated by both STAT3 and NF-kB inhibitors (p < 0.001). Although not significant, IL-6-induced ROR2 expression showed a modest decrease when STAT3 inhibitor was used. Moreover, cell migration was decreased by 80 % in siRNA Wnt5A-transfected cells in the presence of anti-human ROR2 antibody (p < 0.001).ConclusionsThis study revealed for the first time that inflammatory mediators modulate Wnt5A and ROR2 through NF-kB and STAT3 transcription factors and this may play a role in ovarian cancer cell migration. The results described here provide new insight into the role of the Wnt5A–ROR2 complex in ovarian cancer progression in relation to inflammation.

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.

BACKGROUND AND OBJECTIVES:The prediction of in vitro fertilization (IVF) outcomes by anti-Müllerian hormone (AMH) measurement is getting increasing attention from clinicians. This study compares the relationship between serum or intrafollicular AMH levels and IVF outcomes in women with and without polycystic ovary syndrome (PCOS).METHODS:This prospective study was carried out in two university-based fertility clinics. Serum samples were collected on cycle day 3 and follicular fluid (FF) was collected on the day of oocyte retrieval from 26 women with PCOS and 42 normo-ovulatory controls. AMH levels were measured in the samples using immunoenzymatic assay. The relationship between serum or FF AMH levels and IVF outcomes, including number of oocytes retrieved, oocyte maturation rate, fertilization rate, implantation rate, high quality grade embryo rate, and biochemical and clinical pregnancy rates were further assessed.RESULTS:Median serum basal AMH and FF AMH levels were significantly higher in the PCOS group as compared to controls, the values being 14.2 ng/mL vs. 3.2 ng/mL (P<.001) and 8.2 ng/g protein vs. 4.7 ng/g protein (P<.01), respectively. In both groups, serum basal AMH levels showed a positive correlation with number of oocytes retrieved (r=0.323; P=.037 in control vs. r=0.529; P=.005 in PCOS). In the control group, there was a positive relationship between serum basal AMH levels and percentage of matured oocytes (r = 0.331; P=.032) and implantation rate (r=0.305; P=.05).CONCLUSION:Serum basal, and not intrafollicular, AMH levels may be a good predictive factor for quantitative and qualitative IVF outcomes in normo-ovulatory, but not in PCOS patients.

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.

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