Session: MON 355-388-Sex Hormone Receptor Action & Reaction
Poster Board MON-373
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.
Nothing to Disclose: LCM, CB, TB, KU, AS
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