Clinicopathological and Functional Evaluation Reveal NBS1 as a Predictor of Platinum Resistance in Epithelial Ovarian Cancers

Abstract

Platinum resistance seriously impacts on the survival outcomes of patients with ovarian cancers. Platinum-induced DNA damage is processed through DNA repair. NBS1 is a key DNA repair protein. Here, we evaluated the role of NBS1 in ovarian cancers. NBS1 expression was investigated in clinical cohorts (protein level (n = 331) and at the transcriptomic level (n = 1259)). Pre-clinically, sub-cellular localization of NBS1 at baseline and following cisplatin therapy was tested in platinum resistant (A2780cis, PEO4) and sensitive (A2780, PEO1) ovarian cancer cells. NBS1 was depleted and cisplatin sensitivity was investigated in A2780cis and PEO4 cells. Nuclear NBS1 overexpression was associated with platinum resistance (p = 0.0001). In univariate and multivariate analysis, nuclear NBS1 overexpression was associated with progression free survival (PFS) (p-values = 0.003 and 0.017, respectively) and overall survival (OS) (p-values = 0.035 and 0.009, respectively). NBS1 mRNA overexpression was linked with poor PFS (p = 0.011). Pre-clinically, following cisplatin treatment, we observed nuclear localization of NBS1 in A2780cis and PEO4 compared to A2780 and PEO1 cells. NBS1 depletion increased cisplatin cytotoxicity, which was associated with accumulation of double strand breaks (DSBs), S-phase cell cycle arrest, and increased apoptosis. NBS1 is a predictor of platinum sensitivity and could aid stratification of ovarian cancer therapy.

Keywords: NBS1; biomarker; ovarian cancer; platinum sensitization.

Figure 1

NBS1 and epithelial ovarian cancers. (A) Immunohistochemical staining of NBS1 in ovarian cancers. (B) Kaplan-Meier curve for NBS1 nuclear protein expression and progression free survival (PFS) in ovarian cancer. (C) Kaplan-Meier curve for NBS1 nuclear protein expression and overall survival (OS) in ovarian cancer. (D) Kaplan-Meier curve for NBS1 cytoplasmic protein expression and PFS in ovarian cancer. (E) Kaplan-Meier curve for NBS1 cytoplasmic protein expression and OS in ovarian cancer. (F) Kaplan-Meier curve for NBS1 mRNA expression and PFS in ovarian cancer. (G) Kaplan-Meier curve for NBS1 mRNA expression and OS in ovarian cancer. Scale bar = 50 µM, red line= high expression, black line = low expression.

Figure 2

Sub-cellular localization of NBS1 following cisplatin in ovarian cancer cells. (A) Clonogenic assay showing Cisplatin sensitivity in A2780, PEO1 compared to A2780cis and PEO4 cells. (B) NBS1 level nuclear and cytoplasmic extracts in platinum resistant (A2780cis, PEO4) and platinum sensitive (A2780, PEO1) cells treated with 5µM cisplatin. Lysates collected 48 h post-treatment. (C) NBS1 nuclear level quantification A2780, A2780cis, PEO1, and PEO4 cells. (D) NBS1 cytoplasmic level quantification A2780, A2780cis, PEO1, and PEO4 cells. UN = untreated, T = treated. ‘**’ = p value < 0.001.

Figure 3

NBS1 depletion and platinum sensitivity in A2780cis cells. (A) NBS1_KD in A2780cis cells using siRNA construct 1. (B) NBS1 protein quantification in scrambled control and siRNA construct 1 in A2780cis cells. (C) Cisplatin sensitivity in A2780cis control and A2780cis_RAD50_KD cells using siRNA construct 1. (D) NBS1_KD in A2780cis cells using siRNA construct 2. (E) NBS1 protein quantification in scrambled control and siRNA construct 2 in A2780cis cells. (F) Cisplatin sensitivity in A2780cis control and A2780cis_RAD50_KD cells using siRNA construct 2. (G) Representative data showing percentage of γH2AX positive cells by flow cytometry in control and NBS1_KD A2780cis cells untreated or treated with cisplatin (G1) and quantification of % γH2Ax positive cells by flowcytometry (G2) (FL1 Log: FACS fluorescence channel used to measure yH2AX staining). (H) Representative data showing cell cycle progression by flow cytometry in control and NBS1_KD in A2780cis cells untreated or treated with cisplatin (H1) and quantification of cell cycle analysis by flow cytometry (H2) (FL3 Lin: FACS fluorescence channel used for measuring PI staining). (I) Representative data showing apoptotic cells accumulation by flow cytometry in control and NBS1_KD in A2780cis cells untreated or treated with cisplatin (I1) and quantification of apoptotic cells by flow cytometry (I2) (FL1 Log: FACS fluorescence channel used to measure Annexin V staining (horizontal) and FL3 log to measure PI staining (vertical). Early apoptosis rate is calculated from FL1+/PI−, while late apoptosis rate is from FL1+/PI+). UN = untreated, T = treated. ‘*’ = p < 0.01, ‘**’ = p < 0.001.

Figure 4

(A) NBS1_KD in PEO4. (B) NBS1 protein quantification in scrambled control and siRNA construct 1 in PEO4 cells. (C) Cisplatin sensitivity in PEO4 control and PEO4_NBS1_KD cells. (D) The percentage of γH2Ax positive cells by flowcytometry. (E) Cell cycle analysis by flow cytometry (F) annexin V apoptosis analysis by flow cytometry. UN = untreated, T = treated. ‘**’ = p < 0.001, ‘***’ = p < 0.0001.