Date of Award

Winter 2018

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Darran O’Connor

Second Supervisor

Dr Tríona Ní Chonghaile

Funder/Sponsor

Irish Cancer Society

Keywords

Cancer, Breast Cancer, Deubiquitination, Molecular Biology

Abstract

Approximately 80% of breast cancers overexpress the estrogen 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.

To identify DUBs involved in ERα transcriptional activity, an RNAi loss-of-function screen using a library of shRNA vectors targeting all 108 known or putative human DUB genes was performed. Suppression of the BRCA-associated DUB, USP11 was found to downregulate ERα transcriptional activity in ZR-75-1 cells and was the sole focus of this study.

Knockdown of USP11 in ZR-75-1 cells decreased ERα transcriptional activity and mRNA expression of ERα target genes. Furthermore, estradiol (E2) stimulation of these cells resulted in upregulation of USP11 in the nucleus and the formation of USP11 nuclear foci. 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 publicly available breast cancer gene expression datasets further supported this observation. 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 sequencing (RNA-seq). Finally, knockdown of USP11 in ZR-75-1 cells induced significant changes in the proteome, as determined by liquid chromatography mass spectrometry (LC-MS).

This study, for the first time, determines a role for USP11 in ERα transcriptional activity. With further support, USP11 may represent a novel biomarker and therapeutic target in ERα-positive (ERα+) breast cancer.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

6.54 MB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2018.

Available for download on Sunday, November 15, 2020

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