PO-345 The role of the deubiquitinase USP11 in endocrine-driven breast cancer

Abstract

IntroductionApproximately 80% of breast cancers overexpress the oestrogen receptor α (ERα) and depend on this key transcriptional regulator for growth. The discovery of novel mechanisms controlling ERα function represent major advances in our understanding of breast cancer progression and potentially offer new therapeutic opportunities. Here, we investigated the role of deubiquitinating enzymes (DUBs), which remove ubiquitin moieties from proteins, in regulating ERα activity in breast cancer.Material and methodsTo identify DUBs involved in ERα transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all 108 known or putative human DUB genes. We found that suppression of the BRCA-associated DUB, USP11, downregulated ERα transcriptional activity in ZR-75–1 cells. Dual luciferase reporter assays and qRT-PCR were used to determine ERα activity in stable ZR-75–1 USP11 knockdown cell lines. PTMScan technology, which allows for enrichment of ubiquitinated proteins and quantitative profiling by mass spectrometry, was used to reveal the ubiquitinome in these cells and results are pending. RNA sequencing (RNA-seq) technology was carried out in estrogen-independent LCC1 USP11 knockdown cells, were USP11 was hypothesised to play a vital role. Finally, immunohistochemistry (IHC) was used to investigate the prognostic relevance of USP11 in breast cancer patients.Results and discussionsKnockdown of USP11 in ZR-75–1 cells decreased ERα transcriptional activity and mRNA expression of ERα target genes. These results were further validated in a HEK293T USP11 CRISPR knockout model with ectopic ERα expression. Interestingly, USP11 expression was upregulated in LCC1 cells, an isogenic, estrogen-independent model derived from MCF-7 cells. Knockdown of USP11 in LCC1 cells decreased the expression of multiple ERα target genes and cell cycle-associated genes, as determined by RNA-seq. IHC staining of a breast cancer tissue microarray (103 ER +patients) and subsequent Kaplan-Meier analysis of this cohort revealed a 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 expression and poor prognosis.ConclusionThese results suggest a role for USP11 in ERα transcriptional activity and reveal a novel mechanism as to how this receptor is regulated in breast cancer.