The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer

Abstract

Paclitaxel is widely used as a first-line chemotherapeutic drug for patients with ovarian cancer and other solid cancers, but drug resistance occurs frequently, resulting in ovarian cancer still presenting as the highest lethality among all gynecological tumors. Here, using DIGE quantitative proteomics, we identified UBC13 as down-regulated in paclitaxel-resistant ovarian cancer cells, and it was further revealed by immunohistochemical staining that UBC13 low-expression was associated with poorer prognosis and shorter survival of the patients. Through gene function experiments, we found that paclitaxel exposure induced UBC13 down-regulation, and the enforced change in UBC13 expression altered the sensitivity to paclitaxel. Meanwhile, the reduction of UBC13 increased DNMT1 levels by attenuating its ubiquitination, and the up-regulated DNMT1 enhanced the CHFR promoter DNA methylation levels, leading to a reduction of CHFR expression, and an increased in the levels of Aurora A. Our findings revealed a novel function for UBC13 in regulating paclitaxel sensitivity through a DNMT1-CHFR-Aurora A pathway in ovarian cancer cells. UBC13 could potentially be employed as a therapeutic molecular drug for reversing paclitaxel resistance in ovarian cancer patients.

Fig. 1. Differential expressions of proteins between paclitaxel sensitive and resistant ovarian cancer cells by proteomic analysis

a Analysis of SKOV3 and SKOV3-TR30 samples by DIGE. Fluorescent (left) and monochrome (right) images of the DIGE Gel. Proteins extracted from SKOV3 (SK) and SKOV3-TR30 (ST) were labeled with Cy3 and Cy5, respectively. The labeled samples were initially separated in the first dimension (x-axis) on a non-linear gradient pH 3-10, then separated in the second dimension (y-axis) on a 12% polyacrylamide gel. The circled spots were identified by MALDI-TOF/TOF MS/MS analysis shown in the supplementary material Table S1–S3. b Three-dimensional volumetric models of UBC13 in SKOV3 (top) and SKOV3-TR30 (bottom) for DIGE spots. c The expression levels of UBC13 shown on DIGE were calculated by DeCyder analysis and presented as standardized log abundance. SK presents SKOV3 and ST presents SKOV3-TR30. d Western blotting of UBC13 in A2780 vs. A2780-TR and SKOV3 vs. SKOV3-TR30 cells, respectively. One of three representative results is shown in (d)

Fig. 2. Analysis of the association of immunohistochemistry of UBC13 expression with the survival of patients

a Representative UBC13 staining from ovarian cancer tissues. Scale bars represent approximately 500 μm (top) and 200 μm (bottom). b Kaplan-Meier survival curves for PFS and OS in ovarian cancer patients with low-expression and high-expression UBC13 protein by log-rank test

Fig. 3. Paclitaxel induces UBC13 down-regulation, and UBC13 modulates the paclitaxel sensitivity through the DNMT1, CHFR, and Aurora A pathway

a A2780 and b SKOV3 cells were treated with 5, 10, 20, and 30 nM paclitaxel for 24 h. Western blotting was performed with the indicated antibodies. Cell viability assays in A2780 and SKOV3 cells with c UBC13-knockdown and d UBC13-overexpression that were treated with paclitaxel at the indicated concentrations. Results are shown as means ± SEM for at least three separate experiments (* P < 0.05, ** P < 0.01, *** P < 0.001 for siRNA1 or plasmid; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 for siRNA2). Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with e UBC13-knockdown and f UBC13-overexpression. One of three representative results is shown in (a, b, e, and f)

Fig. 4. UBC13 controls DNMT1 stability via ubiquitination and DNMT1 participates in UBC13 regulation of the paclitaxel sensitivity

DNMT1 ubiquitination in a A2780 and b SKOV3 cells with UBC13-overexpression or c A2780 and d SKOV3 with UBC13-knockdown without or with HA-ubiquitin. Cells were treated with MG-132 (20 μM, 8 h) prior to preparation of lysates and then subjected to IP followed by western blot with the indicated antibodies. e Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. f Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were treated with paclitaxel at the indicated concentrations. h Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown. Results are shown as means ± SEM for at least three separate experiments in (e and g) (*P < 0.05, **P < 0.01, ***P < 0.001 for siRNA1; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 for siRNA2). One of three representative results is shown in (a–d, f, and h)

Fig. 5. DNMT1 maintains CHFR gene expression via promoter DNA methylation, and CHFR participates in UBC13 regulating paclitaxel sensitivity

a Detection of methylation status at the promoter region of the CHFR gene in A2780, SKOV3, and 3AO cells with DNMT1-knockdown by Bisulfite sequencing. Ten lines with circles represent the same sequence of ten clones from one sample. CpG sites are shown as filled circles (methylated) or unfilled circles (unmethylated). The lower panel shows the summary data. *P < 0.05, ***P < 0.001, compared with NC siRNA group (chi-square test). b mRNA expression of CHFR in A2780, SKOV3, and 3AO cells with DNMT1-knockdown. c Cell viability assays in CHFR-overexpression A2780 and SKOV3 cells, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. d Western blotting of the indicated proteins with CHFR overexpression, which were transfected in advance with UBC13 specific shRNA and selected with G418 (400 μg/mL) for 14 days. e Cell viability assays in A2780 and SKOV3 cells with CHFR-knockdown, which were transfected in advance with pEGFP-UBC13 plasmid and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at indicated concentrations. f Western blotting of the indicated proteins with CHFR knockdown, which were transfected in advance with pEGFP-UBC13 plasmid and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with CHFR-knockdown treated with paclitaxel at the indicated concentrations. h Western blotting of the indicated proteins with CHFR-knockdown. i Cell viability assays in A2780 and SKOV3 cells with CHFR-overexpression, which were treated with paclitaxel at the indicated concentrations. j Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with CHFR-overexpression. Results are shown as means ± SEM for at least three separate experiments in (b, c, e, g, and i) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 in (a, c, and i); *P < 0.05, **P < 0.01, ***P < 0.001 for siRNA1, ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 for siRNA2 in (e and g). One of three representative results is shown in (d, f, h, and j)

Fig. 6. Paclitaxel exposure reduces UBC13 and CHFR, increases DNMT1 and Aurora A

Western blotting of the indicated proteins in a A2780 and b SKOV3 cells treated with 2, 5, and 10 nM paclitaxel for the indicated times. Cells without paclitaxel treatment were assigned to be the blank control (0 h). One of three representative results is shown. c Working model: UBC13 regulates paclitaxel resistance via DNMT1/CHFR/Aurora A in ovarian cancer cells. UBC13 reduces DNMT1 through ubiquitination, results in DNA hypomethylation of the CHFR promoter, and decreases Aurora A. Reduction of UBC13 increases stability of DNMT1, leads to hypermethylation of the CHFR promoter, elevates Aurora A levels, and prompts paclitaxel resistance in ovarian cancer cells. Ub: ubiquitin