Wnt5A modulates integrin expression in a receptor-dependent manner in ovarian cancer cells

Abstract

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.

Figure 1

Wnt5A regulates its receptors and affects proliferation and clonogenicity of Wnt5A overexpressing clones. (A) Expression of Wnt5A in Clone 9 of SKOV-3 cells and Clone 3 and 2 of OVCAR-3 cells referred further as C9/SKOV-3 clone, C3/OVCAR-3 clone, and C2/OVCAR-3 clone was assessed by western blot (left and lower panel) compared to mock. The bands corresponding to GAPDH of the same blot was added separately. C9/SKOV-3, C3/OVCAR-3, and C2/OVCAR-3 clones were transfected with siRNA against Wnt5A showed a reduction of Wnt5A at protein levels (right and lower panels) compared to scramble (scr). (B,C) Wnt5A regulates its receptors as revealed by RT-qPCR analysis of frizzled-2 (FZD-2), frizzled-4 (FZD-4), frizzled-5 (FZD-5), ROR-1 and ROR-2 expression levels in C9/SKOV-3 and C3/OVCAR-3 clones in Wnt5A knock-down or Box5 treated clones relative to mock or scrambled (scr). GAPDH was used as an internal control. (D) Trypan blue exclusion test showed increased cell proliferation of C9/SKOV-3, and C3/OVCAR-3 clones compared to mock or Box5-treated cells. (E) The proportion of C9/SKOV-3, and C3/OVCAR-3 clones in the G2/M phase was increased compared to mock as revealed by cell cycle analysis. (F) Increased clonogenicity of C9/SKOV-3, and C3/OVCAR-3 clones compared to mock (left panel, scale bar: 100 µm). The right panel represents a quantitative assessment of colonies in the C9/SKOV-3 clone, C3/OVCAR-3 clone, and mock cells after 11 days. Results of RT-qPCR were normalized related to GAPDH used as an internal control. Mean ± SD from at least three independent experiments. a: compared to mock, b: compared to Wnt5A overexpressing clones *P < 0.05; **P < 0.01; ***P < 0.001 compared to mock.

Figure 2

Wnt5A overexpression modulates mesenchymal markers and reverts the mesenchymal morphology of SKOV-3 cells. (A) C9/SKOV-3 clone showed epithelial-like morphology compared to spindle morphology of non-transfected cells. Wnt5A knock-down or blocking with Box5 partially rescued morphology alteration of C9/SKOV-3 clone (arrows) (scale bar: 100 µm). (B,C) C9/SKOV-3 clone showed increased E-cadherin immunostaining and expression compared to mock that was reverted using siRNA Wnt5A or Box5 (scale bar: 50 µm). The bands corresponding to GAPDH of the same blot was added separately. (D) Decreased mRNA levels of the mesenchymal markers CDH-2, SNAIL, and FN1 in C9/SKOV-3 clone compared to the mock which was reversed with siRNA Wnt5A. (E) The increased expression level of mesenchymal markers in C3/OVCAR-3 clone compared to mock which was reverted in Wnt5A knock-down. Results of RT-qPCR were normalized related to GAPDH used as an internal control. Mean ± SD of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared to mock.

Figure 3

Wnt5A alters motility and invasiveness of Wnt5A overexpressing SKOV-3 and OVCAR-3 clones. (A) Increased migration of C9/SKOV-3 clone which was abrogated in the presence of Box5 (left panel, scale bar: 100 µm). The right panel shows the migration rate of cells that were assessed based on the distance of the selected wounded area at time intervals of 0, 3, 6, 12, and 24 h, and the percent of wound closure was determined for each time point. (n = 3, mean ± SD). (B) Wound-healing analysis of C3/OVCAR-3 clone during a 24-h time course in the presence or absence of Box5. Increased motility of C3/OVCAR-3 clone was abrogated in the presence of Box5 (250 µM) (left and right panel, scale bar: 100 µm). (C) C9/SKOV-3 clone showed decreased cell invasion compared to mock (upper panel, photos are representative of one of three performed experiments). The lower panel shows quantification of cell invasion by counting cells at ten random fields (n = 3, mean ± SD; scale bar: 100 µm). (D) C3/OVCAR-3 clone showed increased cell invasion compared to mock (upper panel, photos are representative of one of three performed experiments). The lower panel shows quantification of cell invasion by counting cells at ten random fields (n = 3, mean ± SD; scale bar: 100 µm). (E) Decreased and increased mRNA levels of MMP-13 in C9/SKOV-3, and C3/OVCAR-3 clones, respectively compared to mock. Results of RT-qPCR were normalized related to GAPDH used as an internal control. Mean ± SD of three independent experiments. a: compared to mock, and b: compared to Wnt5A overexpressing clones. *P < 0.05; **P < 0.01; ***P < 0.001 compared to mock.

Figure 4

Wnt5A increases MCAs formation and decreases the compactness of C9/SKOV-3 MCAs. (A) MCAs morphology during 3 and 6 days culture (left panel). MCAs formation rate of C9/SKOV-3 clone vs. mock cells (right panel) was quantitated after 6 days in 50 µl droplet under an inverted microscope. (B) C9/SKOV-3 clone was transfected with siRNA against Wnt5A or scramble siRNA (25 nM) and the formation of C9/SKOV-3 MCAs was followed over 7 days. Wnt5A knockdown reverted the loose structure of MCAs formed by the C9/SKOV-3 clone. Photos represent one of the three independently performed experiments (scale bare: 100 µm). *P < 0.05; **P < 0.01; ***P < 0.001 compared to mock.

Figure 5

Wnt5A modulates integrin expression in Wnt5A overexpressing clones in both 2D and 3D cell culture. (A) The integrin expression level was assessed in 3D and (B) 2D Wnt5A overexpressing C9/SKOV-3 clone and mock cells by using the Alpha/Beta Integrin-Mediated Cell Adhesion Array Combo Kit. Values were normalized related to mock from two independent experiments each performed in duplicate (mean + SD). (C) RT-qPCR analysis of integrin subunits in C9/SKOV-3 clone with or without siRNA Wnt5A or Box5 relative to mock or scrambled (scr). (D) RT-qPCR analysis of integrin subunits in C3/OVCAR-3 clone with or without siRNA Wnt5A or Box5 relative to mock or scrambled (scr). GAPDH was used as an internal control and the data represent mean ± SD (n = 3) *P < .05; **P < .01; ***P < .001 compared to mock.

Figure 6

fibronectin- and laminin-dependent adhesion of Wnt5A overexpressing clones and subsequent FAK activation. (A) C9/SKOV-3 clone or mock cells were seeded on fibronectin (FN)—or laminin (LN)-coated wells in the presence or absence of Box5 and the percent of adhered cells was assessed after 30 or 60 min. Left panel: Photos represent one of the three independent experiments after 60 min (scale bar: 100 µm). Right panel: Adhered cells were stained with crystal violet and quantitated. (B) C9/SKOV-3 clone or mock cells were seeded on collagen type-I or IV-coated wells and the percent of adhered cells was assessed after 30 or 60 min. Left panel: Photos represent one of the three independent experiments (scale bar: 100 µm). Right panel: Adhered cells were stained with crystal violet and quantitated. (C) Mock or C9/SKOV-3 clone with or without Box5 was seeded on FN- or LN-coated wells and immunostained with anti- pTyr³⁹⁷-FAK after 30 or 60 min. Photos are representative of three independent experiments (scale bar: 50 µm). (D) C9/SKOV-3 clone and Mock cells were mixed with 25 µg/ml of fibronectin (FN) and were cultured on agarose-coated 12 well plates for 24 h. Increased compaction of cells was shown in the presence of FN in Wnt5A overexpressed cells compared to mock. (E) C3/OVCAR-3 clone or mock cells were seeded on FN, LN-, Collagen- (I and IV types) coated wells in the presence or absence of Box5, and the percent of adhered cells was assessed after 30 or 60 min. Upper panel: Photos represent one of the three independent experiments after 60 min (scale bar: 100 µm). Lower panel: Adhered cells were stained with crystal violet and quantitated. The results are expressed as mean + SD relative to mock from three independent experiments each performed in triplicate. (Scale bar: 100 µm). *P < .05; **P < .01; ***P < .001 relative to mock.

Figure 7

Venn diagram of common GO (BP) and Protein–protein interaction network for Wnt5A, and integrins and their expression levels in human serous histological subtypes. (A) The heatmap shows the expression levels of integrins, Wnt5A, and cadherins obtained by hierarchical cluster analysis. Each column in the figure represents a sample, and each row represents a gene. The colors in the graph indicate the magnitude of gene expression in the sample. The black-red gradient indicates that the genes are medium–high expressed in the samples, and the blue indicates that the gene expression is low. (B) Common KEGG pathways analysis of ITGAV, ITGA5, and Wnt5A (C) All GO (BP) are grouped into three comparison groups represented by three circles. The overlapping portions of the different circles represent the number of GO (BP) common to these comparison groups. (D) Common GO (BP) for Wnt5A and ITGAVB3. (E) Common GO (BP) for Wnt5A and ITGA5B1. (F) Protein–protein interaction (PPI) network of Wnt5A and ITGAV, ITGB3, ITGA5, and ITGB1, the thickness of the edge indicates a strong interaction between the two proteins. N: normal; BL: Borderline; LGSOC: Low grade serous ovarian cancer; HGSOC: High grade serous ovarian cancer.

Figure 8

Inhibition of integrin αv decreases proliferation and migration of Wnt5A overexpressed clones. C3/OVCAR-3 clone or mock cells were treated with 25 μM CWHM- 12 as a specific inhibitor of αv integrin (A) Trypan blue exclusion (B) Transwell cell migration assay (scale bar: 100 μM). The results are expressed as mean + SD relative to untreated cells from three independent experiments each performed in triplicate. *P < .05; **P < .01.