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Title: Identification and characterisation of transcriptional regulatory proteins that modulate estrogen receptor α activity
Author: Ahamed, Eliyaz
ISNI:       0000 0004 2686 9847
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2009
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Breast cancer is the most common malignancy in women in the Western world, accounting for 18% of all female cancers. Two-thirds of breast cancers express Estrogen receptor-α (ERα) and ERα is a predictive and positive prognostic marker. Estrogens stimulate breast cancer growth, primarily through activation of ERα and hormonal therapies used in breast cancer treatment either inhibit estrogen synthesis to prevent ERα activation or bind to ERα to inhibit its activation. Unfortunately resistance to these drugs is a common clinical problem. Altered phosphorylation of ERα has been proposed as one of the mechanisms of resistance to endocrine therapy. Phosphorylation of ERα can result in its activation, both in the presence and absence of ligand. The key phosphorylation sites have been mapped to transcription activation function AF1. In particular, phosphorylation of serine residues at amino acid positions 104, 106 and 118 in AF1 results in stimulation of ERα activity. A wealth of evidence demonstrates an important role for the ERK1/2 MAPK signal transduction pathway in non-responsiveness to endocrine therapies. However, it remains unclear as to how phosphorylation potentiates the activity of AF1. It is likely that phosphorylation of AF1 stimulates the recruitment of transcriptional coactivators that mediate the transcriptional activity of AF1. Identification and characterisation of such coactivators would further our understanding of the role of phosphorylation in the molecular mechanism of transcriptional regulation by ER α Two approaches were used to identify coactivators that may mediate the effects of ERα phosphorylation on its activity. In the first approach, bacterial two-hybrid system was used to screen a breast cDNA expression library. In the second, p68 and p72 RNA helicases, two known transcriptional coregulators, previously reported to interact with AF1 of ERα, were characterised as coactivators of ERα. Both proteins stimulated activity of ERα and show synergism with the well-characterised nuclear receptor coactivator, SRC-1. Surprisingly, reporter gene assays showed that phosphorylation of ERα is not essential for the coactivator function of p68. Importantly, RNA interference studies revealed complex effects of p68 and p72 on ERα activity and consequently on estrogen-regulated gene expression. p72 knockdown reduced the expression of the estrogen-regulated genes, pS2, Cathepsin D and GREB1, and inhibited the growth of the ERα-dependent MCF-7 breast cancer cell line. Surprisingly, p68 knockdown did not affect ER signalling. Data are also presented to suggest that the balance and interplay between p68 and p72 may be responsible for the previously described regulation of ERα activity by p68. Collectively, the data presented show that p72 may be more important than p68 in modulating transcriptional responses of ERα in breast cancer cells.
Supervisor: Ali, Simak ; Coombes, Charles Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral