Abstract S3-7: Kinome Screening for Regulators of Estrogen Receptor Identifies a Novel Kinase as a New Therapeutic Target in Breast Cancer
Authors: Giamas G , Filipovic A , Jacob J , Shifa BA , Green AR , Ellis IO , Lenz H-J , Stebbing J. 
Published: April 2011

Abstract
Background: Estrogen Receptor (ER) positive breast cancer is usually treated with hormone reduction or anti-estrogens. Still, only half the ER+ breast tumors respond to tamoxifen. Mutations in ER are rarely found; instead other mechanisms have been associated with resistance, namely phosphorylation of ERα. Since phosphorylation has been shown to be important for the stimulation of its transcriptional activity, focusing on the role of kinases and the influence of site specific phosphorylation in modulating activity may reveal fundamental insights and derive new druggable targets for exploitation in the clinic.
Materials and Methods: A systematic high-throughput RNAi screening using a large Human Kinase siRNA Library was carried out to identify novel kinases able to phosphorylate and/or modulate ERα activity; quantitative real-time RT-PCR analysis of pS2, PR and GREB1, three well characterized ERα-regulated genes, was performed after silencing each kinase individually. We then examined the effects of each newly identified kinase on various ERα features/functions including: i) ERα stability (Western blot), ii) ERα sub-cellular localization (confocal microscopy) and iii) ERα interactions with major components of the transcriptional machinery (co-immunoprecipitation). In addition, we examined the effects of these kinases on i) cell-cycle progression (FACS), ii) proliferation rate (MTT) and, iii) apoptosis levels (Annexin V, Caspase assays). We assessed clinical significance of our observations, by analyzing the expression of the novel kinases in BC tissue microarrays (TMAs, n=614) by immunohistochemistry.
Results: We have categorized the newly identified kinases in two groups depending on their effects on ERα transcriptional activity (30% up-regulation - 70% down-regulation). Among them, we discovered a novel receptor tyrosine kinase (RTK) whose role and/or involvement in ERα regulation has not been described thus far. We demonstrated that silencing of this kinase resulted in a great down-regulation (&gt;70-80%) of all 3 ERα-regulated genes tested (pS2, GREB1, PGR). In addition, upon kinase-silencing, we observed a large decrease in ERα protein levels, suggesting an involvement of this kinase in ERαα stability. Moreover, silencing of this kinase resulted in a &gt;50% decrease of breast cancer cells viability (P&lt;0.001). Analysis of the BC TMAs, the kinase of interest was expressed in weak (27%), moderate (35%) and high levels (38%). High expression correlated significantly with high tumor grade (P&lt;0.001) and predicted for worse BC specific overall survival (p=0.03).
Discussion: As RTKs are considered the primary mediators of the signalling network that transmit extracellular signals into the cell, they represent the first ‘line’ of drug target development in cancer treatment. We propose that our newly identified kinase could be a successful and life-saving drug target for the progression of various types of cancer.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S3-7.