Abstract 4163: Expression of estrogen response element binding proteins in human breast carcinoma improves prediction of clinical outcome

Abstract

Abstract Purpose: Unique ERE-binding proteins (ERE-BP) were detected in cytosolic extracts of breast cancer while investigating estrogen receptor-α (hERα) recognition of ERE sequences. Our goal is to compare properties of these novel ERE-BP with those of hERα and hERβ, and determine their clinical relevance. Procedures: Using de-identified tissue biopsies, cytosols were prepared in 40 mM Tris buffer, pH 7.4 with 1.5 mM EDTA, 10 mM Na2MoO4, 10% glycerol, 1 mM PMSF and 10 μM monothioglycerol while nuclear pellets were extracted in the same buffer with 0.4M NaCl and without 10 mM Na2MoO4. Extracts were incubated 16 hr, 4°C with [32P]ERE and separated by electrophoretic mobility shift (EMSA) and super-shift assays (SSA) with ERE sequences present in 5’-flanking regions of estrogen responsive genes. ERE-BP level was estimated from band intensity of an EMSA profile. ERE-BP was correlated with cancer features (e.g. pathology, stage, nodal status), patient characteristics (e.g., family history, age, race) and clinical outcome to determine ERE-BP status for predicting clinical behavior of breast cancer. Results: Both recombinant hERα and ERE-BP reference specimens were developed as assay controls. Compared to cytosolic levels, ERE-BP exhibited higher expression in nuclear extracts. When either disease-free (DFS) or overall survival (OS) was analyzed as a function of above or below median ERE-BP expression using Kaplan-Meier plots, patients with biopsies in the above median group exhibited decreased DFS and OS compared to women in the below median group. Overall 5-year survival was 68% for the above median group and 85% for those in the below median group. No correlation of ERE-BP level was observed with patient age, race, stage, nodal status or tumor marker levels; however ERE-BP over-expression correlated with increased tumor grade. There was no difference in survival between patients with ERα positive cancer in either the above median ERE-BP or below median ERE-BP groups. However, patients with ERα negative cancers expressing high ERE-BP exhibited the poorest OS of any group. Five year OS for patients in the ERα negative/above median ERE-BP group was only 55% compared to 78% in the ERα negative/below median ERE-BP group suggesting clinically relevant subtypes of ER negative carcinomas. Conclusions: Results support our hypothesis that presence of these novel ERE-binding proteins in a breast carcinoma is an indicator of clinical behavior. ERE-BP expression in combination with ERα negative status in breast cancer appears to provide an improved means of identifying patients at greatest risk of recurrence and decreased survival. Supported in part by NIH/NCI 1R43CA106059-01A1, Phi Beta Psi Charity Trust, a UofL Research on Women Grant and a CTSB grant from the Commonwealth of Kentucky. TLK is a recipient of a DOD BCRP Pre-doctoral Traineeship Award. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4163. doi:10.1158/1538-7445.AM2011-4163