Abstract 3040: Functional genomic screening identifies USP11 as a novel regulator of ERα transcription in breast cancer

Approximately 70% of breast cancers overexpress the estrogen receptor α (ERα) and depend on this key transcriptional regulator for growth and differentiation. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer attractive new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which act to remove ubiquitin moieties from proteins, in regulating transcriptional activity of ERα in breast cancer. To identify DUBs involved in the regulation of ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all human DUB genes. The DUB library consisted of pools of four non-overlapping shRNAs targeting all 108 known or putative DUBs (432 shRNAs in total). We found that suppression of a number of DUBs markedly repressed or enhanced the activity of an estrogen-response-element (ERE) luciferase reporter following estradiol (E2) stimulation. Of particular interest, suppression of the BRCA2-associated DUB, USP11, was found to down-regulate ERα transcriptional activity. Subsequent validation using two individual siRNAs targeted to USP11 revealed a notable reduction in expression of endogenous ERα target genes in the ZR-75-1 cell line, as quantified using qRT-PCR. Immunoprecipitation of ERα revealed no physical interaction with USP11, however E2 stimulation resulted in translocation of USP11 to the nucleus, suggesting a potential role in E2-induced transcription. Furthermore, USP11 expression was found to be upregulated in the estrogen-independent cell line LCC1 when compared to their parental MCF7 cells. Knockdown of USP11 in LCC1 cells resulted in decreased mRNA expression of a panel of ERα target genes, suggesting a role for USP11 in an estrogen independent setting. To support the prognostic relevance of USP11, immunohistochemical staining of a breast cancer tissue microarray (n=144) was performed. Kaplan-Meier analysis of this cohort revealed a highly significant association between poor overall survival (OS) (p=0.030) and breast cancer-specific survival (BCSS) (p=0.041). In silico analysis of publically available breast cancer gene expression datasets further supported an association between high USP11 mRNA levels and poor prognosis. We observed a significant correlation between high expression of USP11 mRNA in ER-positive patients and poor distant metastasis-free sur