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. 2016 Jul 28:21:9.
doi: 10.1186/s11658-016-0003-3. eCollection 2016.

WNT5A-ROR2 is induced by inflammatory mediators and is involved in the migration of human ovarian cancer cell line SKOV-3

Somayeh Arabzadeh  1 , Ghamartaj Hossein  1 , Zahra Salehi-Dulabi  1 , Amir Hassan Zarnani  2   3
Affiliations

WNT5A-ROR2 is induced by inflammatory mediators and is involved in the migration of human ovarian cancer cell line SKOV-3

Somayeh Arabzadeh et al. Cell Mol Biol Lett. .

Abstract

Background: 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.

Methods: 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.

Results: 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).

Conclusions: 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.

Keywords: Inflammation; Migration; NF-kB/STAT3 signaling pathways; Ovarian cancer; ROR2; Wnt5A.

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Figures

Fig. 1
Fig. 1
Wnt5A expression induced by LPS, LTA and IL-6. SKOV-3 cells were treated with 1 μg/ml LPS (a); 30 μg/ml LTA (b) or 100 ng/ml IL-6 (c) for the indicated times. The left panels show normalized values (means ± SD) from three independent quantitative PCR analyses for Wnt5A expression. Data were normalized related to 18 s RNA as the internal control. The right panels show normalized values (means ± SD) from three independent western blots for Wnt5A. The western blots represent one of three independent experiments. GAPDH levels were used as the internal control. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 as compared with the control (Ctrl)
Fig. 2
Fig. 2
ROR2 expression induced by LPS, LTA and IL-6. Western blot analysis of ROR2 expression in SKOV-3 cells treated with 1 μg/ml LPS (a), 30 μg/ml LTA (b) and 100 ng/ml IL-6 (c) for the indicated times. Normalized values (means ± SD) from three independent western blots for ROR2. The western blots represent one of three independent experiments. GAPDH levels were used as the internal control. *p ≤ 0.05 as compared with the control (Ctrl)
Fig. 3
Fig. 3
NF-kB and STAT3 inhibitors alter SKOV-3 cell viability and affect Wnt5A and ROR2 expression. a – MTT assay of cell viability in the presence of BAY11-7082 as an NF-kB inhibitor (5, 10 μM) or S31-201 as a STAT3 inhibitor (25, 50 μM). b – Normalized values (means ± SD) from three independent western blots for Wnt5A in the presence of BAY11-7082 for the indicated times. c – Normalized values (means ± SD) from three independent western blots for Wnt5A in the presence of S31-201 for the indicated times. D – Western blot analysis of Wnt5A in cells treated with BAY11-7082 (5, 10 μM) or S31-201 (25, 50 μM) for the indicated times. The western blots represent one of three independent experiments. E – Normalized values (means ± SD) from three independent western blots for ROR2 in the presence of BAY11-7082 or S31-201 for 24 and 48 h. F- Western blot analysis of ROR2 in cells treated with BAY11-7082 (5 μM) or S31-201 (25 μM) for 24 and 48 h. The western blots represent one of three independent experiments. GAPDH levels were used as the internal control. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 as compared with the control (Ctrl)
Fig. 4
Fig. 4
Involvement of NF-kB and STAT3 signaling pathways in inflammation-induced Wnt5A expression. SKOV-3 cells treated with inflammatory mediators alone or in combination with BAY11-7082 as an NF-kB inhibitor or S31-201 as a STAT3 inhibitor for 24 h. LPS (a) and LTA (b). The upper panels show normalized values (means ± SD) from three independent western blots for Wnt5A expression for the indicated times. The lower panels represent one of three independent western blot analysis of Wnt5A. c – rhIL-6, with the left panel showing normalized values (means ± SD) from three independent western blots for Wnt5A expression for the indicated times and the right panel representing one of three independent western blot analyses of Wnt5A. GAPDH levels were used as the internal control. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 as compared with the control (Ctrl)
Fig. 5
Fig. 5
Involvement of NF-kB and STAT3 signaling pathways in inflammation-induced ROR2 expression. The left panels show normalized values (means ± SD) from three independent western blots for ROR2 expression for the indicated times and the right panels represent one of three independent western blot analyses of ROR2 in SKOV-3 cells treated with LPS (a); LTA (b) and rhIL-6 (c) alone or in combination with BAY11-7082 or S31-201 for 48 h. GAPDH levels were used as the internal control. ***p ≤ 0.001 as compared with the control (Ctrl)
Fig. 6
Fig. 6
Wnt5A is involved in rhIL-6-induced migration in SKOV-3 cells. a – Western blot quantification showed a 70 % reduction in Wnt5A expression in siRNA Wnt5A-transfected cells compared with the controls. The western blots represent one of three independent experiments. GAPDH levels were used as the internal control. b – In vitro cell migration assay of non-transfected or siRNA Wnt5A-transfected cells in the absence or presence of rhIL-6. The upper panel showed photos of transwells in the indicated conditions and the lower panel showed quantification of migrated cells in ten random fields. The percentage of migrated cells was expressed as: (number of migrated cells/number of seeded cells × 100). The migration index was expressed relative to control cells (set as 100 %). All of the experiments were carried out three times and the results are expressed as means ± SD. Magnification: 100x. *p ≤ 0.05; **p ≤ 0.0 as compared with the control (Ctrl)
Fig. 7
Fig. 7
Wnt5A–ROR2 is implicated in SKOV-3 cell migration. a – In vitro cell migration assay of non-transfected or siRNA Wnt5A-transfected cells in the absence or presence of anti-ROR2 antibody. The upper part or photos shows photos of transwells with the indicated conditions. The graph shows quantification of counted migrated cells in ten random fields. All of the experiments were carried out three times and the results are expressed as means ± SD. Magnification: 100x. **p ≤ 0.01 as compared with the control (Ctrl)
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