Abstract P2-05-02: Functional genomic screening identifies ubiquitin-specific protease 11 (USP11) as a novel regulator of ER-alpha transcription in breast cancer

Abstract

Abstract 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. Further validation was carried out in a HEK293T USP11 knockout cell line, where reduced activity of an ERE-luciferase reporter was detected when compared to wild-type cells. This phenotype was rescued with a USP11 overexpression vector, both in the presence and absence of E2. 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, while RNA-seq revealed a downregulation of several putative ERα target genes and a downregulation of many cell cycle-associated proteins. To support the prognostic relevance of USP11, immunohistochemical staining of a breast cancer tissue microarray (103 ER+ patients available for final analysis) was performed. Kaplan-Meier analysis of this cohort revealed a highly significant association between high USP11 expression and poor overall (p=0.030) and breast cancer-specific survival (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 survival (HR 2, CI 1.37-2.91, p=0.00023). This correlation was also significant in ER-positive patients who had received tamoxifen only (HR 2.9, CI 1.63-5.15, p=0.00015). These results suggest a role for USP11 in driving cellular growth and identify USP11 as novel therapeutic target in breast cancer. Citation Format: Dwane L, Das S, Moran B, O'Connor AE, Mulrane L, Dirac AM, Jirstrom K, Crown JP, Bernards R, Gallagher WM, Ní Chonghaile T, O'Connor DP. Functional genomic screening identifies ubiquitin-specific protease 11 (USP11) as a novel regulator of ER-alpha transcription in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-02.