This investigation of the prognostic value of RBM3 in EOC reveals that RBM3 is an independent prognostic marker at both mRNA and protein levels. Gene expression analysis in a cohort of 267 EOC cases showed that high RBM3 mRNA expression was an independent predictor of a significantly improved RFS and OS. Immunohistochemical analysis in an independent cohort of 154 EOC cases demonstrated that RBM3 protein expression was associated with a significantly improved OS in univariate and multivariate analysis. This is in line with previous findings from two independent breast cancer cohorts where RBM3 was associated with more favourable clinicopathological parameters and a significantly improved survival, irrespective of adjuvant treatment [16
]. However, since platinum-based chemotherapy is a fundamental aspect of current EOC treatment regimens, we hypothesized that RBM3 might enhance platinum-sensitivity in vitro
. We initially confirmed lower RBM3 protein levels in the cisplatin-resistant ovarian cancer cell line A2780-Cp70 compared to their parental cisplatin-sensitive A2780 cells and using RNAi techniques, we demonstrated that silencing of RBM3 led to a decreased cisplatin response in ovarian cancer cells.
Taken together, these data demonstrate that RBM3 is a marker of good prognosis in EOC and a predictor of response to platinum-based chemotherapy, most likely a combination of both, particularly in the light of the previously demonstrated good prognosis associated with RBM3 expression in breast cancer patients, where the vast majority of patients received no adjuvant systemic chemotherapy [16
]. While future in-depth studies are warranted to further elucidate the functional mechanisms underlying RBM3's role in cisplatin-mediated cell death, the in vitro
data presented here provide sufficient evidence to support the hypothesis that, in addition to being a beneficial prognostic biomarker, RBM3 might also predict cisplatin response in EOC. Further studies are required to evaluate the role of RBM3 in predicting response to other platinum based agents, particularly in the setting of a prospective randomised control trial whereby stratification according to different treatment regimens can be performed.
Some aspects on the results presented here merit further attention. The reduced cytotoxic effect of cisplatin in siRBM3 transfected cells was to a large extent reflected by cell cycle alterations, e.g. a lower percentage of cells arrested in G2/M phase rather than by a decreased percentage of apoptotic cells. Cisplatin treatment is known to induce both cell cycle arrest and apoptosis (reviewed in [7
]), which was confirmed here in A2780 cells. However, while cisplatin treatment resulted in a significantly decreased cell cycle arrest in siRBM3 transfected A2780 cells, the percentage of apoptotic cells was not significantly reduced. Notably, despite being one of the most cisplatin sensitive ovarian cancer cell lines, A2780 cells have been shown to have a relatively low percentage of apoptotic cells following cisplatin treatment [30
]. As there was no evident difference in cell cycle characteristics between siRBM3-transfected and control-transfected A2780 cells without cisplatin treatment, but yet a significantly reduced proportion of siRBM3 treated cells in G2/M-phase arrest following cisplatin treatment, the main effects of RBM3 on cisplatin sensitivity might be reflected in cell cycle distribution rather than apoptosis. Although RBM3 mRNA expression has previously been associated with the pro-apoptotic Bax gene in breast cancer [29
], we were unable to demonstrate a down-regulation of Bax protein in siRBM3 transfected A2780 cells, further supporting the theory that RBM3 promotes cisplatin sensitivity through cell-cycle regulation.
It is evident that RBM3 is up-regulated in response to various conditions causing cellular stress, i.e. hypothermia [13
] hypoxia [26
], serum starvation [28
] and exposure to microgravity [33
]. We did not see an up-regulation of RBM3 upon cisplatin-induced stress, however, it would still be of interest to investigate the potential role of RBM3 in DNA repair response given the observed decrease of RBM3-silenced cells arrested in G2/M upon cisplatin treatment.
Apart from our previous study in breast cancer [16
], there are to our knowledge no other published data on the prognostic impact of tumor-specific RBM3 expression in human cancer. However, RBM3 has been identified as one of five down-regulated genes in an in vitro
model of melanoma progression [34
], implying a beneficial impact on prognosis also in this cancer form. Contrasting in vitro
data have also been published, whereby RBM3 has been proposed to be a proto-oncogene stabilizing COX-2 mRNA levels and protecting against mitotic catastrophe in colorectal cancer cell lines [27
A common observation in human tissue is that RBM3 is up-regulated in cancer [27
] and in proliferating non-malignant cells [28
], compared to normal cells. Quite in line with these findings, we found that siRNA mediated knockdown of RBM3 resulted in reduced proliferation in A2780 cells, which might in part explain the reduced cisplatin sensitivity and also the relatively modest reduction in apoptosis upon treatment in siRBM3 transfected A2780 cells. Yet, as a consequence of the observation that RBM3 seems to be necessary for the maintenance of cellular integrity during various stress conditions, it has been hypothesized that targeting RBM3 could prove to be an efficient novel therapeutic strategy against cancer [27
]. This viewpoint is challenged by the results presented here, but, evidently, drug induced effects by RBM3 modulation seem to differ between different cell line models, as down-regulation of RBM3 has been associated with enhanced response to adriamycin and cisplatin in androgen dependent but not androgen-independent prostate cancer cells [35
]. To what extent this variation is true in human tumors remains to be elucidated but to our knowledge, in contrast to ovarian cancer, prostate cancer is not routinely treated with platinum-based chemotherapeutic agents. It should also be emphasized that, in a translational context, the proposal that RBM3 is a proto-oncogene activated in response to adverse cellular conditions [27
] does not contradict the findings that its presence in an established tumor is associated with a favourable patient outcome as such findings would not have taken patient treatment into account.
The coincidence of a beneficial prognostic impact of RBM3 expression in EOC both at the mRNA and protein level demonstrated here is particularly relevant from a translational perspective as it would justify using IHC, which is a simpler, faster and less costly method than RT-PCR in the clinical setting. In our previous study in breast cancer [16
], the favourable prognostic impact of RBM3 was assessed by IHC in two independent patients cohorts using a polyclonal, monospecific antibody, initially developed within the HPA programme [23
]. In the present study, the favourable prognostic impact of RBM3 expression in EOC was demonstrated at both the gene expression and protein levels in two relatively large independent cohorts. RBM3 protein expression was assessed using a monoclonal antibody which displayed a single band of the expected size on Western blot and further validation showed a decreased RBM3 expression in siRNA transfected A2780 cells compared to controls, both as assessed by IHC and Western blotting. Notably, the previously used polyclonal antibody has also been validated in the A2780 cells with similar results to the monoclonal antibody; e.g. differential expression in A2780 and A2780-Cp70 cells and decreased expression in siRNA transfected cells (data not shown). Furthermore, analysis of the tumor specimens in Cohort II using the antibody that was used in the breast cancer study [16
] yielded concordant results regarding the prognostic impact of tumor-specific RBM3 expression (data not shown).
In breast cancer, nuclear RBM3 expression was associated with favourable clinicopathological parameters, including hormone receptor status [16
]. In this study, we found no association between RBM3 and ER or PR expression in EOC as assessed by IHC. This observation indicates that RBM3 might have different functions in the context of estrogen-related signalling in breast cancer and ovarian cancer. The potential clinical relevance of this is however less evident as the beneficial effect of high RBM3 expression in breast cancer was independent of tamoxifen treatment.