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
