RBM3-regulated genes promote DNA integrity and affect clinical outcome in epithelial ovarian cancer

Abstract

The RNA-binding motif protein 3 (RBM3) was initially discovered as a putative cancer biomarker based on its differential expression in various cancer forms in the Human Protein Atlas (HPA). We previously reported an association between high expression of RBM3 and prolonged survival in breast and epithelial ovarian cancer (EOC). Because the function of RBM3 has not been fully elucidated, the aim of this study was to use gene set enrichment analysis to identify the underlying biologic processes associated with RBM3 expression in a previously analyzed EOC cohort (cohort 1, n = 267). This revealed an association between RBM3 expression and several cellular processes involved in the maintenance of DNA integrity. RBM3-regulated genes were subsequently screened in the HPA to select for putative prognostic markers, and candidate proteins were analyzed in the ovarian cancer cell line A2780, whereby an up-regulation of Chk1, Chk2, and MCM3 was demonstrated in siRBM3-treated cells compared to controls. The prognostic value of these markers was assessed at the messenger RNA level in cohort 1 and the protein level in an independent EOC cohort (cohort 2, n = 154). High expression levels of Chk1, Chk2, and MCM3 were associated with a significantly shorter survival in both cohorts, and phosphorylated Chk2 was an adverse prognostic marker in cohort 2. These results uncover a putative role for RBM3 in DNA damage response, which might, in part, explain its cisplatin-sensitizing properties and good prognostic value in EOC. Furthermore, it is demonstrated that Chk1, Chk2, and MCM3 are poor prognostic markers in EOC.

Figure 1

Identification of cellular processes associated with RBM3 expression in EOC. (A) Flowchart illustrating a novel approach to biomarker discovery whereby transcriptomic and proteomic data can be integrated to identify new biomarkers. (B) GSEA demonstrated that increased RBM3 expression was associated with a number of processes including DNA-dependent replication P < .01, chromatin remodeling P < .05, DNA replication P < .01, DNA integrity checkpoint P < .05, and DNA damage checkpoint P < .05.

Figure 2

Downregulation of RBM3 affects the expression of MCM3, Chk1, and Chk2. The expression of MCM3, Chk1, Chk2, and RBM3 were examined by (A) Western blot analysis and (B) reverse transcription-PCR in A2780 cells 48 hours after transfection of cells with three different siRNAs targeting RBM3 (nos. 58, 59, and 60). Data shown are mean ± SEM of four, for siRBM3 nos. 58 and 59, and three for siRBM3 no. 60, independent experiments performed in triplicate. (C) Immunohistochemical staining of Chk1, Chk2, and MCM3 in EOC tumors denoted as negative, intermediate, and strong.

Figure 3

Increased mRNA (cohort 1) and protein expression (cohort 2) of MCM3, Chk1, and Chk2 are associated with an impaired survival. Kaplan-Meier analysis of RFS and OS according to (A) MCM3, (B) CHK1, and (C) CHK2 mRNA levels in cohort 1. Kaplan-Meier analysis of OS according to immunohistochemical (A) MCM3, (B) Chk1, and (C) Chk2 staining in cohort 2.

Figure 4

The impact of RBM3 on phosphorylation of Chk1 and Chk2 and the association of pSer345-Chk1 and pT68-Chk2 with OS in EOC. The impact of RBM3 on checkpoint response was examined in siRBM3-transfected cells by Western blot analysis using antibodies against (A) pSer345-Chk1 and pT68-Chk2. (B) Immunohistochemical staining of pSer345-Chk1 and pT68-Chk2 in EOC tumors. (C) Kaplan Meier analysis of OS according to immunohistochemical staining of pS345-Chk1 and pT68-Chk2 in cohort 2 in strata defined as high versus low expression.