Date of Award

2011

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Dr Leonie Young

Keywords

Breast Neoplasms, Aromatase.

Abstract

Aromatase inhibitors (Al) are the newest form of endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer and have demonstrated excellent efficacy in clinical trials; however, a proportion of patients treated with Als will relapse with resistant disease. Resistance to endocrine therapy is characterised by cross-talk between ERa and membrane growth factor receptors, such as the epidermal growth factor receptor (EGFR) family, resulting in increased signalling. In Al-resistant molecular models this results in proliferation in the absence of estrogen. The steroid receptor coactivator AIBI increases transcriptional activity of ERa. The aim of this study was to elucidate the role of AlBl as an ERa coactivator in development of resistance to Al treatment, and the consequent effects on estrogenregulated genes. An aromatase-overexpressing breast cancer cell line was established by transfecting the aromatase gene into ER-positive breast cancer cells. This cell line was treated with the Al, letrozole, over an extended period until cells became resistant to the drug. Proliferation assays demonstrated that growth of the Alresistant cell line was independent of estrogen and therefore was not inhibited by letrozole. Co-immunoprecipitation showed that interaction of ERa with AlBl was intensified in Al-resistant cells compared with Al-sensitive cells, in a ligandindependent manner. Treatment with epidermal growth factor (EG F) increased the association of ERa with AlBl in Al-resistant cells. EGF also stimulated proliferation of these cells, indicating an increased dependence of these cells on growth factormediated signalling. In these Al-resistant cells, recruitment of ERa and AlBl to the promoters of estrogen-responsive genes, pS2 and c-myc was demonstrated in the presence of letrozole, but recruitment to the cyclinDl promoter remained sensitive to estrogen treatment. These patterns of recruitment were matched by mRNA and protein expression levels of the three target genes. Inhibition of c-Jun N-terminal kinase (JNK) reduced the protein expression of cyclinD1. It was proposed that this differential gene regulation was due to indirect binding of ER to the cyclinDl promoter, in contrast to pS2 and c-myc, where ER binds directly to estrogen response elements (ERE). Fluorescent coassociation of ERa with AlBl demonstrated increased colocalisation of these proteins in Al-resistant cells compared with Al-sensitive cells. In primary breast cancer cultures from ER-positive patients who had not received endocrine therapy, coassociations of ER with AlBl were increased by androstenedione and disrupted by letrozole. In a tissue microarray of 447 breast cancer patients, expression of AlBl was demonstrated to predict disease recurrence in patients who were treated with an Al. This research has demonstrated that epidermal growth factor signalling is involved in activating increased interactions between ERa and AlBl in the development of resistance to Al treatment, resulting in ligand-independent proliferation of Al-resistant cells. AlBl is emerging as an important marker of response to Al therapy in breast cancer.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

File Size

12 MB

Comments

A thesis submitted to the Royal College of Surgeons in Ireland for the degree of Doctor of Philosophy from the National University of Ireland in 2011

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