S1: Image of full size blot for figure 1A; S2: Image of full size blot for figure 1B; S3: SRC3-pS543 and SRC3 chromatin binding patterns in MCF7 cells; S4: Venn diagram of SRC1, SRC2 and SRC3-pS543 enriched binding sites; S5: A. ChIP-QPCR validation of differential enriched chromatin binding sites for total SRC3 and SRC3-pS543; S6: Hormone dependence and phospho-specificity of SRC3-pS543 at sites either or not shared with total SRC3; S7: Overlap of ERïƒ¡ï€ and SRC3 with other publically available datasets; S8: Fulvestrant decreases SRC3-pS543/chromatin interactions at sites shared with ER; S9: Venn diagram, showing the overlap of GO pathways enriched for genes proximal to chromatin binding sites for SRC3-pS543 unique, SRC3 unique or shared regions; S10: 2D graph of normalized ChIP-seq data, illustrating signal for ER, total SRC3, H3K4me1, H3K4me2, H3K4me3 and H3K27Ac binding events at genomic regions occupied by SRC3-pS543; S11: Histological analysis of SRC3 and SRC3-pS543 expression in representative tumour sections; S12: Scatterplot analysis for total SRC3 versus SRC3-pS543 signal in the Nottingham Tenovus Primary Breast Carcinoma Series; S13: Disease Free Survival and breast cancer specific survival in breast cancer patients based on SRC3 protein expression; S14: Disease Free Survival and breast cancer specific survival in patients with HER2 positive and SRC3 high breast cancers compared with all other patients.
ARTICLE ABSTRACT
Purpose: The steroid receptor coactivator SRC3 is essential for the transcriptional activity of estrogen receptor α (ERα). SRC3 is sufficient to cause mammary tumorigenesis, and has also been implicated in endocrine resistance. SRC3 is posttranslationally modified by phosphorylation, but these events have not been investigated with regard to functionality or disease association. Here, we investigate the spatial selectivity of SRC3-pS543/DNA binding over the human genome and its expression in primary human breast cancer in relation with outcome.Experimental Design: Chromatin immunoprecipitation, coupled with sequencing, was used to determine the chromatin binding patterns of SRC3-pS543 in the breast cancer cell line MCF7 and two untreated primary breast cancers. IHC was used to assess the expression of SRC3 and SRC3-pS543 in 1,650 primary breast cancers. The relationship between the expression of SRC3 and SRC3-pS543, disease-free survival (DFS), and breast cancer specific survival (BCSS) was assessed.Results: Although total SRC3 is selectively found at enhancer regions, SRC3-pS543 is recruited to promoters of ERα responsive genes, both in the MCF7 cell line and primary breast tumor specimens. SRC3-pS543 was associated with both improved DFS (P = 0.003) and BCSS (P = 0.001) in tamoxifen untreated high-risk patients, such a correlation was not seen in tamoxifen-treated cases, the interaction was statistically significant (P = 0.001). Multivariate analysis showed SRC3-pS543 to be an independent prognostic factor.Conclusions: Phosphorylation of SRC3 at S543 affects its genomic interactions on a genome-wide level, where SRC3-pS543 is selectively recruited to promoters of ERα-responsive genes. SRC3-pS543 is a prognostic marker, and a predictive marker of response to endocrine therapy. Clin Cancer Res; 22(2); 479–91. ©2015 AACR.
