Background: Approximately 80% of human breast carcinomas present as estrogen receptor α-positive (ER+ve) disease and ER status is a critical factor in treatment decision-making. However, ER+ve tumors are heterogeneous in both their molecular profiles and response to therapy. Up to 50% of patients derive little or no clinical benefit from treatment and many exhibit de novo or intrinsic resistance to hormonal therapy. Aim: To investigate factors associated with the level of expression of the ER gene that may be potential determinants of the biology of ER+ve tumors. Methods: Pre- and 2wk post-anastrozole tumor biopsies were obtained from 104 postmenopausal women with stage I to IIIB ER+ early breast cancer. RNA extracted from biopsies was analyzed on Illumina 48K microarrays and quantitative trait analysis by Spearman correlation was used to identify genes whose expression in tumors correlated significantly with ESR1. Results: Expression of three previously uncharacterized open reading frames (ORFs) located immediately upstream of ESR1 on chromosome 6q21, RMND1, C6ORF97 and C6ORF211, were highly correlated with ESR1 (Rs=0.672, 0.637 and 0.546 respectively, p<0.0001). Exploration of other publicly available datasets revealed that this relationship was present in other groups of ER+ve tumors. In our tumor set, change in expression of these genes upon estrogen deprivation remained tightly correlated with change in ESR1 expression. Array CGH of microdissected tumor samples on tiling path 32K BAC arrays revealed that DNA copy number changes did not account for the correlation. Analysis of expression of all four genes in cultured MCF7 and BT474 cells by quantitative RT-PCR over a timecourse showed that the correlation was maintained in vitro. Treatment with the ERα antagonist ICI 182,780 did not affect ORF expression or the correlation of the genes with ESR1, suggesting that their transcriptional co-activation is not directly mediated by ERα. siRNA inhibition of C6ORF211 suppressed proliferation in MCF7 cells (1.6-fold, p<0.0001). In pre-treatment tumor biopsies, C6ORF211 correlated with a published proliferation metagene (p=0.04), mirroring the effect of C6ORF211 knockdown on proliferation seen in vitro. C6ORF211 was also found to be expressed more highly in Luminal B versus Luminal A tumors (p=0.0005), despite ESR1 levels not being significantly different between these two molecular subgroups. Summary and conclusions: There is a previously unpublished association between ESR1 and three ORFs immediately adjacent to ESR1 on chromosome 6. Their co-expression is not a result of co-amplification or directly mediated by ERα transcriptional activity. Our data indicate that C6ORF211 influences proliferation in vitro and in tumors hence it remains possible that some of the biological effects previously attributed to ER could be mediated by these co-expressed genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2916.