Ghamartaj Hossein

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.

Steroid-associated osteonecrosis (SAON) is a chronic disease that leads to the destruction and collapse of bone near the joint that is subjected to weight bearing, ultimately resulting in a loss of hip and knee function. Zn²⁺ ions, as an essential trace element, have functional roles in improving the immunophysiological cellular environment, accelerating bone regeneration, and inhibiting biofilm formation. In this study, we reconstruct SAON lesions with a three-dimensional (3D)-a printed composite made of poly (epsilon-caprolactone) (PCL) and nanoparticulate Willemite (npW). Rabbit bone marrow stem cells were used to evaluate the cytocompatibility and osteogenic differentiation capability of the PCL/npW composite scaffolds. The 2-month bone regeneration was assessed by a Micro-computed tomography (micro-CT) scan and the expression of bone regeneration proteins by Western blot. Compared with the neat PCL group, PCL/npW scaffolds exhibited significantly increased cytocompatibility and osteogenic activity. This finding reveals a new concept for the design of a 3D-printed PCL/npW composite-based bone substitute for the early treatment of osteonecrosis defects.

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.

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 IC₅₀ 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‐AKTser⁴⁷³ 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 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.

It has been reported that secreted frizzled-related protein-4 known as an antagonist of Wnt signaling pathway plays a role in luteinization process of rodent granulosa cells. The purpose of this study was twofold: 1) to determine whether recombinant human secreted frizzled-related protein-4 (rhSFRP-4) could directly induce terminal differentiation of rat Granulosa Cells (GCs) and 2) to understand how the modulation of β-catenin and Protein Kinase B (PKB)/AKT activity by exogenous SFRP-4 could be involved in steroidogenesis. GCs were firstly stimulated with Follicle-Stimulating Hormone (FSH) named as FSH-primed cells then were treated with luteinizing hormone (LH). Then estradiol (E2) and progesterone (P4) production levels were assessed in the absence or presence of rhSFRP-4 treatment. The expression levels of activated β-catenin, pAKTser 473 , pGSK3βser 9 were assessed by western blot or immunofluoresence. In the presence of rhSFRP-4, there was 38% decreased E2 levels compared to untreated FSH-primed cells (p<0.05), and P4 production subsequently decreased. However, in GCs pre-treated with rhSFRP-4 prior to addition of FSH, P4 levels increased 2-fold compared with untreated cells (p<0.05). Unexpectedly, treatment with rhSFRP-4 prior to LH stimulation inhibited LH-induced P4 secretion. Treatment with low (0.5 ng/ml) but not high (50 ng/ml) concentrations of rhSFRP-4 led to significantly increased levels of pGSK3βser 9 (1.6-fold) and nuclear active β-catenin (2.8-fold) in GCs compared with untreated cells. Interestingly, pre-treating GCs with rhsFPR4 prior to LH stimulation resulted in a 38% decrease in pAKTser 473 levels compared with those in LH-treated cells (p<0.05). Taken together, our results showed that rhSFRP-4 could directly induce terminal differentiation in GCs via the modulation of β-catenin and PKB/AKT pathways and that it does so in a dose-dependent manner.

Wnt5A, 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. SKOV-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. There 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). This 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.

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.