Abstract P1-04-04: KSR1 is involved in functional interaction between p53 and BRCA1 and is an independent predictor of overall survival in breast cancer

Abstract

Abstract Background: Our recent human kinome screening to identify new regulators of estrogen receptor α (ERα) suggested an important role of kinase suppressor of ras 1 (KSR1) in breast cancer. KSR1 was originally identified as a positive regulator interacting with both RAS and RAF in the mitogen-activated protein kinase (MAPK) pathway. Recent studies have suggested that KSR1 may have dual functions as a scaffolding protein and also a protein kinase. Although KSR1 has been implicated in Ras-dependent cancers, its clinical significance and function in breast cancer have not been elucidated. Methods: The clinical significance of KSR1 was assessed by analyzing its expression in breast cancer tissue microarrays (TMAs, n=1000) by immunohistochemistry. Pearson chi2 and Fisher's Exact Test were used to correlate the expression levels with various tumor biomarkers; Kaplan Meier analyses were used to investigate impact on disease-free (DFS) and overall survival (OS). Multivariate Cox-proportional hazards analysis was also performed to evaluate the significant of KSR1 as an independent factor in breast cancer-specific survival (BCSS) and disease-free interval (DFI). The expression levels of KSR1 in breast cancer cell lines were measured by RT-qPCR and western blotting. A KSR1 stable over-expression breast cancer cell line (MCF7-KSR1) was generated to study its effect on growth and colony formation by 3D matrigel assay and soft agar assay in vitro. Furthermore, MCF7-KSR1 stable cells were also used in nude mice xenograft model in vivo. Luciferase reporter assay was employed to examine the effect of KSR1 on p53 transcriptional activity. Further immunofluorescence staining, western blotting and reporter assays were performed to elucidate the interaction between KSR1, BRCA1 and p53. Results: KSR1 was expressed with low (60%) and high levels (40%) in breast cancer patients. High expression correlated significantly with longer DFS (p = 0.014) and longer OS (p = 0.012) in more than 20 yrs follow-up. Interestingly, KSR1 was positively associated with BRCA1 (p = 0.002) and reversely with p53 (p = 0.038). High KSR1 levels were a significant predictor of longer breast cancer-specific survival BCSS (p = 0.001) and longer disease free interval DFI (p = 0.002) independent of tumour stage, grade and size. RT-qPCR and western blotting demonstrated that KSR1 is ubiquitously expressed in breast cancer cell lines. In vitro 3D matrigel and soft agar assay showed that MCF7-KSR1 cells had a significant decreased number of colonies and formed smaller size colonies compared to MCF7-vector cells. Furthermore, over-expression of KSR1 (MCF7-KSR1) significantly suppressed the growth of breast cancer xenografts in nude mice. Finally, KSR1 was involved in the functional interaction between BRCA1 and p53 and elevated KSR1 potentially resulted in up-regulated BRCA1. KSR1 also inhibited p53-dependent transcriptional activity through suppression of p53 acetylation and promotion of p53 neddylation. Conclusion: KSR1 is an independent prognostic factor in breast cancer and high KSR1 expression correlates with longer DFS and OS. Furthermore, it is potential that KSR1 is important in tumour suppression by its involvement in functional feedback regulation of BRCA1 and p53. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-04.