Abstract 4316: A dynamic interplay of KSR1, p53 and BRCA1 in breast cancer.

Abstract

Abstract Background: We have previously performed a human kinome screen and identified kinase suppressor of ras 1 (KSR1) as a potential regulator of estrogen receptor α (ERα) in breast cancer. Recent studies have suggested that KSR1, originally known as a positive modulator interacting with RAS and RAF in the MAPK pathway, may have dual functions as a scaffold protein and a kinase. Although KSR1 has been implicated in Ras-dependent cancers, its role in breast cancer has not been studied. Methods: The clinical significance of KSR1 levels and single nucleotide polymorphisms (SNPs) were assessed by analyzing its expression in tissue microarrays (n=1000) by immunohistochemistry in univariate and multivariate analyses. Expression of KSR1 in a panel of breast cancer cell lines were measured. A KSR1 stable over-expression breast cancer cell line (MCF7-KSR1) was generated to study its effects on growth and colony formation by 3D matrigel and soft agar assays. A xenograft mouse model was used to examine the effects of KSR1 on tumor development in vivo. Stable isotope labelling by amino acids in cell culture (SILAC) was used to analyse KSR1-regulated proteins and pathways. Additional molecular biology techniques (including immunoprecipitation, luciferase reporter and uibiquitination assay) were performed to study the action of KSR1 on p53 and BRCA1. Results: KSR1 was differentially expressed in breast cancer patients. High KSR1 expression correlated significantly with longer DFS (p=0.014) and OS (p=0.012) in > 20 yrs follow-up, independent of tumor stage, grade and size. KSR1 was associated positively with BRCA1 (p=0.002) and inversely with p53 levels (p=0.038). Two KSR1 SNPs’ were identified with no correlations with either DFS or OS. RT-qPCR and western blotting showed that KSR1 is ubiquitously expressed in breast cancer cell lines. In vitro 3D matrigel and soft agar assays revealed that MCF7-KSR1 had a significant decreased number of colonies and formed smaller size colonies compared to MCF7-parental cells. In vivo breast cancer mouse model showed tumor volumes of xenografts over-expressing KSR1 were significantly reduced compared to controls. SILAC analysis revealed various novel KSR1-regulated proteins and phospho-proteins in breast cancer. Our results showed over-expression of KSR1 decreases the transcriptional activity of p53 by reducing DBC1 phosphorylation, which leads to a reduced interaction of DBC1-SIRT1 and therefore allowing SIRT1 to deacetylate p53. Finally, KSR1 stabilizes BRCA1 by up-regulating BARD1 and promoting the interaction of BRCA1-BARD1. Conclusion: KSR1 is an independent prognostic factor in breast cancer; high KSR1 correlates with longer DFS and OS. KSR1 over-expression inhibits tumor formation and growth in vitro and in vivo. KSR1 is part of a dynamic interplay with p53 and BRCA1; it reduces p53 transcriptional activity by abolishing the interaction of SIRT1-DBC1, while it stabilizes BRCA1 by enhancing BRCA1-BARD1 interaction. Citation Format: Hua Zhang, Justin Stebbing, Aleksandra Filipovic, Ian O. Ellis, Heinz-Josef Lenz, Georgios Giamas. A dynamic interplay of KSR1, p53 and BRCA1 in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4316. doi:10.1158/1538-7445.AM2013-4316