DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway

Abstract

Dickkopf-3 (DKK3), a tumor suppressor, is frequently downregulated in various cancers. However, the role of DKK3 in ovarian cancer has not been evaluated. This study aimed to assess aberrant DKK3 expression and its role in epithelial ovarian carcinoma. DKK3 expression was assessed using immunohistochemistry with tissue blocks from 82 patients with invasive carcinoma, and 15 normal, 19 benign, and 10 borderline tumors as controls. Survival data were analyzed using Kaplan-Meier and Cox regression analysis. Paclitaxel-resistant cells were established using TOV-21G and OV-90 cell lines. Protein expression was assessed using Western blotting and immunofluorescence analysis. Cell viability was assessed using the MT assay and 3D-spheroid assay. Cell migration was determined using a migration assay. DKK3 was significantly downregulated in invasive carcinoma compared to that in normal, benign, and borderline tumors. DKK3 loss occurred in 56.1% invasive carcinomas and was significantly associated with disease-free survival and chemoresistance in serous adenocarcinoma. DKK3 was lost in paclitaxel-resistant cells, while β-catenin and P-glycoprotein were upregulated. Exogenous secreted DKK3, incorporated by cells, enhanced anti-tumoral effect and paclitaxel susceptibility in paclitaxel-resistant cells, and reduced the levels of active β-catenin and its downstream P-glycoprotein, suggesting that DKK3 can be used as a therapeutic for targeting paclitaxel-resistant cancer.

Keywords: DKK3; P-glycoprotein; ovarian cancer; paclitaxel resistance; β-catenin.

Figure 1

Immunoreactivity of DKK3 and survival curves for patients with invasive serous ovarian cancer. (a) DKK3 immunoreactivity in normal epithelium and serous tumors. (b) Immunoreactivity of each score in invasive serous carcinoma. (c) Kaplan–Meier curves for disease-free survival. Magnification 200×.

Figure 2

Characteristics of paclitaxel-resistant cells. (a,b) Cells were seeded in triplicates in 96-well plates in 0.1 mL culture medium at a density of 1 × 10⁴ cells/well. After 24 h, the cells were treated with paclitaxel. MTT assay performed 48 h after treatment showed that the paclitaxel-resistant cells (TOV-21G/PTX and OV-90/PTX) had higher IC₅₀ than the parental cells. (c,d) Paclitaxel treatment reduced the viability of parental cells, while the paclitaxel-resistant cells continued to grow. (e) Western blot analysis revealed that DKK3 was lost and that β-catenin and P-glycoprotein were upregulated in paclitaxel-resistant cells (Res) compared to that in their parent cells (Con). β-Actin was used as the loading control. * p < 0.01.

Figure 3

Anti-proliferative effect of the secreted DKK3 on paclitaxel-resistant cells. (a) Western blotting results show DKK3 protein levels in the control and DKK3 conditioned medium (CM). MTT assay performed after 24 h of incubation with CM (*** p < 0.001). (b) The viability of TOV-21G and TOV-21G/PTX cells after treatment with DKK3 CM diluted to 0%, 50%, and 90%, respectively, with control CM (** p < 0.01). (c) The OV-90 and OV-90/PTX cells were incubated with CM with or without DKK3. Western blotting results show DKK3 protein levels in the control and DKK3 CM. MTT assay after 24 h of treatment (*** p < 0.001). (d) Western blotting reveals recombinant human DKK3 levels. Two concentrations of the protein (10 μg/mL and 50 μg/mL) were used to treat cells for 72 h (* p < 0.05). (e,f) TOV-21G/PTX and OV-90/PTX cells were treated with CM with or without paclitaxel (100 ng/mL and 200 ng/mL, respectively) (** p < 0.01). (g) 3D spheroids of OV-90 and OV-90/PTX cells were generated in a microwell array and the images were captured after incubation for 6 d (magnification, 40×; scale bar 300 μm). (h) Spheroid viabilities and diameters were compared. The asterisks on the graph denote statistically significant differences (p < 0.01) between the DKK3 CM group and the control CM group. The diameter of spheroids in the DKK3 CM group was compared to that in the DKK3 CM with paclitaxel group (#, p < 0.01). (i,j) OV-90/PTX and TOV-21G/PTX cells were treated as mentioned above and migration rates were evaluated using the migration assay. (k,l) The paclitaxel-resistant cells were incubated with control and DKK3 CMs. At the indicated time points, the cells were harvested and subjected to Western blotting. The graphs were plotted based on the band densities measured using the ImageJ software. * p < 0.05.

Figure 4

Secreted DKK3 enhanced paclitaxel susceptibility via inhibition of the β-catenin-P-glycoprotein signaling pathway. (a,b) The cells were incubated with control and DKK3 CM. Immunofluorescence analysis after 24 h showed that FLAG-DKK3 was present in the perinuclear area, attenuating TR-non-phospho-β-catenin signaling. Scale bar, 50 μm. (c,d) The cells were incubated with control and DKK3 CM and subjected to Western blotting. The graphs were plotted based on the band densities (* p < 0.01). (e,f) To activate endogenous β-catenin, cells were treated with LiCl for 24 h and subjected to Western blot analysis. β-Actin was used as the loading control. The intensities of the Western blot bands were normalized to that of β-actin for comparison (* p < 0.01).