SMURF1-mediated ubiquitination of ARHGAP26 promotes ovarian cancer cell invasion and migration

Abstract

Rho GTPase-activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small GTP-binding protein RhoA (GTP-RhoA) to its inactive GDP-bound form and is a putative tumor suppressor gene associated with cell growth and migration. Here, the involvement of ARHGAP26 in ovarian cancer cell proliferation and migration was investigated. In this study, low ARHGAP26 expression was observed in ovarian cancer tissues and was associated with a poor overall survival and higher β-catenin expression in patients with ovarian cancer. A2780 and HEY cells with ARHGAP26 upregulation showed decreased cell proliferation, migration, and invasion, along with decreased GTP-RhoA, β-catenin, VEGF, MMP2, and MMP7 expression. ARHGAP26 upregulation in A2780 cells also inhibited lung metastasis in vivo. SKOV3 cells with ARHGAP26 downregulation demonstrated an inverse effect, which was inhibited by ARHGAP26 overexpression or DKK1, an antagonist of the β-catenin pathway. SMURF1, an E3 ubiquitin ligase, interacted with and induced ubiquitination of ARHGAP26. ARHGAP26 upregulation in SKOV3 cells significantly inhibited SMURF1 upregulation-induced cell migration and invasion. Overall, SMURF1-mediated ubiquitination of ARHGAP26 may promote invasion and migration of ovarian cancer cells via the β-catenin pathway.

Fig. 1. ARHGAP26 expression was decreased in ovarian cancer tissues.

ARHGAP26 mRNA expression in ovarian cancer tissues from TCGA (a) and our independent hospital (b) cohorts was detected. ARHGAP26 protein expression in our independent hospital (c) cohorts was detected via immunohistochemistry (IHC). Scale bars: 100 μm. d Overall survival of 85 patients with ovarian cancer from the hospital cohort was detected. **P < 0.01 and ***P < 0.001 compared with normal tissues. OC, ovarian cancer; normal, adjacent noncancerous tissues

Fig. 2. ARHGAP26 upregulation inhibited the proliferation of A2780 and HEY cells.

a, b ARHGAP26 expression in five ovarian cancer cell lines and the nonmalignant human ovarian surface epithelial cell line IOSE80 was detected. A2780 and HEY cells were transduced with ARHGAP26 expression lentivirus or control lentivirus (blank vector). ARHGAP26 expression (c, d) and cell proliferation (e, f) was detected via real-time PCR, western blotting, and CCK-8 assays. *P < 0.05 and ***P < 0.001 compared with IOSE80 or control cells

Fig. 3. ARHGAP26 upregulation inhibited the ovarian cancer cell invasion and migration.

A2780 and HEY cells were transduced with recombined ARHGAP26 expression lentivirus or control lentivirus (blank vector). Cell migration and invasion of A2780 (a, b) and HEY cells (c, d) were detected via Transwell analysis, and the expression of GTP-RhoA, total RhoA, β-catenin, VEGF, MMP2, and MMP7 in A2780 and HEY cells was detected via western blotting (e–g). Scale bars: 100 μm. h Histology of lungs metastases from mice intravenously injected with A2780 or HEY cells stably expressing ARHGAP26 or blank vector. Scale bars: 200 μm. ***P < 0.001 compared with control

Fig. 4. ARHGAP26 downregulation increased the proliferation, invasion, and migration of SKOV3 cells.

a, b SKOV3 cells were transfected with three siRNAs targeting AGRHAP26 or with control siRNA (siNC), and AGRHAP26 expression was detected via real-time PCR and western blotting. SKOV3 cells were transfected with siRNA-2 and siRNA-3. SKOV3 cell proliferation (c), migration, and invasion (d, e) were detected via CCK-8 and Transwell assays, and the expression of GTP-RhoA, total RhoA, β-catenin, VEGF, MMP2, and MMP7 (f, g) was detected via western blotting. Scale bars: 100 μm. *P < 0.05 and ***P < 0.001 compared with control

Fig. 5. DKK1 treatment inhibited ARHGAP26 downregulation-induced invasion and migration of SKOV3 cells.

SKOV3 cells transfected with AGRHAP26 siRNA-3 or siNC were treated with DKK1 (200 ng/mL) or transduced with recombined ARHGAP26 expression lentivirus, and expression of β-catenin, MMP2, MMP9, and VEGF (a, b), and SKOV3 cell migration and invasion (c, d) were detected via western blotting and Transwell analysis. Scale bars: 100 μm. ***P < 0.001 compared with siNC. ^###P < 0.001 compared with siRNA-3

Fig. 6. SMURF1 promotes ovarian cancer cell migration and invasion by inhibiting ARHGAP26.

a, b SMURF1 expression in five ovarian cancer cell lines and the nonmalignant human ovarian surface epithelial cell line IOSE80 was detected. c SKOV3 cell lysates were subjected to immunoprecipitation with control IgG, or anti-SMURF1 or anti-ARHGAP26 antibody. The immunoprecipitates were then blotted with anti-SMURF1 or anti-ARHGAP26 antibody. d SKOV3 cells transduced with recombined pLVX-Puro-SMURF1 or control lentivirus (blank vector) were treated with MG132 (50 μM) for 4 h, and the expression of ARHGAP26 was determined by western blotting. e SKOV3 cells were transduced with recombined pLVX-Puro-SMURF1 or control lentivirus (blank vector). After 48 h, the cells were collected and AGRHAP26 was immunoprecipitated and immunoblotted with control IgG, or anti-ARHGAP26 or anti-ubiquitin antibody. SKOV3 cells were transduced with recombined pLVX-Puro-SMURF1, pLVX-Puro-ARHGAP26, or control lentivirus (blank vector), and cell migration, invasion (f, g), and expression of β-catenin, VEGF, MMP2, and MMP7 (h, i) were detected using Transwell and western blotting assays. Scale bars: 100 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with IOSE80 cells or cells transduced with blank vector. ^###P < 0.001 compared with SMURF1. ^ΔP < 0.05 compared with blank vector plus MG132

Fig. 7. Expression of β-catenin and SMURF1 and correlation analysis in ovarian cancer tissues.

a, b β-Catenin and SMURF1 mRNA expression in ovarian cancer tissues and adjacent normal ovarian tissues from our independent hospital database was detected using real-time PCR. c, d Pearson’s correlation scatter plots for ovarian cancer tissues (n = 28). ***P < 0.001 compared with normal. OC, ovarian cancer; normal, adjacent noncancerous tissues