Phosphorylation of Estrogen Receptor alpha by mTOR

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 355-388-Sex Hormone Receptor Action & Reaction
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-373
Leigh Campbell Murphy*1, Chrissie Bruce2, Tarek Bader3, Kanyarat Ung3 and Anuraag Shrivastav4
1Univ of Manitoba, Winnipeg, MB, Canada, 2CancerCare Manitoba, Winnipeg, MB, Canada, 3University of Manitoba, Winnipeg, MB, Canada, 4Univ of Winnipeg, Winnipeg, MB, Canada
It was previously established that a phosphorylation score (P7-score) for estrogen receptor alpha (ERα), representing the presence or absence of up to seven different phosphorylated sites on ERα detected in any breast tumor, was an independent prognostic factor in breast cancer patients subsequently treated with tamoxifen (1).  More recently we found that detection of mTOR (mechanistic target of rapamycin) activation, (pS2448-mTOR), was associated with the P7-score and clinical outcome in the same cohort (2). Furthermore, using Kinexus kinase substrate motif prediction analysis ( we identified several serine residues in ERα that were potentially substrates for FRAP/mTOR. These data suggested the hypothesis that estrogen may regulate activation of mTOR pathways and that mTOR may directly phosphorylate ERα.

The ability of estrogen to regulate phosphorylation of mTOR in MCF7 ER+ human breast cancer cells was determined following estrogen-depletion for 3 days, and serum starvation overnight prior to treatment with estrogen (10nM) for various times. Estradiol (E2) treatment for 3-6 hours was associated with a small (mean ± SD; at 3 hours 1.5 ± 0.2 fold, n =7; at 6 hours 1.5 ± 0.3, fold, n = 4) but consistent induction of phosphorylation of mTOR at Ser 2448 as measured by western blotting. E2 treatment for six hours also resulted in phosphorylation of p70S6kinase, as previously shown (3). These data provide support for E2 regulated activation of mTOR and an mTOR downstream target, p70S6kinase in estrogen responsive MCF7 breast cancer cells.

To determine the potential of mTOR to directly phosphorylate ERα, an in vitro kinase assay was performed using recombinant- ERα(rh-ER) incubated with recombinant-mTOR (catalytic domain, rh-mTOR). Activity of rh-mTOR (catalytic domain) was shown by its ability to phosphorylate recombinant full-length human-p70S6kinase (rh-S6K) on T389 in an in vitro assay followed by western blotting with antibodies specific for pT389-p70S6kinase. The mTOR induced increase in pT389-rh-S6K was inhibited by pre-incubation with a selective mTOR inhibitor, AZD 8055. Incubation of rh-mTOR and rh-ER using in vitro kinase assays followed by western blotting with antibodies to phospho-serine, showed a 4.4  ± 1.7 fold (mean ± SD, n =3) increase in phosphorylated ERα at serine residues, which was inhibited by pre-incubation with a selective mTOR inhibitor, AZD 8055. This pattern was observed in three independent experiments. These data establish the potential for mTOR to phosphorylate ERα, although studies are required to determine if this occurs in intact cells in vivo.

1. Georgios Skliris,  Zoann Nugent, Brian Rowan, Carla Penner, Peter Watson, Leigh Murphy. (2010) A phosphorylation code for estrogen receptor alpha predicts clinical outcome to endocrine therapy in breast cancer. Endocrine-Related Cancer 17(3):589-972. Anuraag Shrivastav, Sri Seelkallu, Zoann Nugent, and Leigh Murphy. Mammalian Target of Rapamycin activation in Estrogen Receptor-a Positive Human Breast Tumors is a Predictor of Better Clinical Outcome. 2012 94th Endocrine Society meeting, Houston 23-26 June3. J Yu, EP Henske. (2006) Estrogen activation of mTOR is mediated via tuberin and the small GTPase Ras homologue enriched in brain. Cancer Res 66(19): 9461-6

Nothing to Disclose: LCM, CB, TB, KU, AS

*Please take note of The Endocrine Society's News Embargo Policy at

Sources of Research Support: This work was supported by the Canadian Institutes of Health Research (CIHR), the Canadian Cancer Society Research Institute (CCSRI) and the CancerCare Manitoba Foundation (CCMF). This study was also supported by the Manitoba Breast Tumour Bank, a member of the Canadian Tumour Repository Network and is funded in part by CCMF, CIHR and Canadian Breast Cancer Foundation (CBCF).