The mechanistic target for rapamycin pathway is related to the phosphorylation score for estrogen receptor-α in human breast tumors in vivo

Abstract

Introduction: A phosphorylation score for estrogen receptor-alpha (ERα), called P7 score, was shown previously to be an independent prognostic factor in breast cancer patients treated with tamoxifen. Since mechanistic target of rapamycin (mTOR) activation is implicated in resistance to endocrine therapy in breast cancer we determined whether mechanistic target of rapamycin complex 1 (mTORC1) activation, measured by phosphorylation on S2448 (p-mTOR), was associated with the P7-score and/or clinical outcome in the same cohort.

Methods: mTOR phosphorylation status was determined at S2448 residue in vivo by immunohistochemistry in a cohort of more than 400 well-characterized ERα positive breast tumors. MCF7 cells were treated with estrogen and activation of mTOR pathway was determined by Western blotting.

Results: Contrary to earlier reports, p-mTOR expression, measured by immunohistochemistry, was negatively associated with size and nodal status. Additionally, p-S2448 mTOR expression was positively correlated with p-S118- ERα, p-S167-ERα and p-S282-ERα but negatively correlated with p-T311- ERα. Consistent with these, p-S2448 mTOR was negatively associated with P7-score and was significantly associated with overall survival (OS) (hazard ratio (HR) = 0.61, P = 0.028, 95% confidence interval (CI) 0.39 to 0.95, n = 337) and relapse-free survival (HR = 0.58, P = 0.0032, 95% CI 0.41 to 0.83, n = 337) following univariate but not multivariate analysis. Furthermore, we show that estrogen can regulate phosphorylation of mTOR and its down stream target p70S6 kinase. Additionally, recombinant mTOR can phosphorylate ERα in vitro.

Conclusions: These data suggest that in breast tumors where there is intact estrogen regulated signaling, mTOR is regulated by estrogen and therefore associated with an increased likelihood of responsiveness to endocrine therapy.

Figure 1

Kaplan-Meier estimates of relapse free survival from breast cancer recurrence (A, C) or breast cancer specific death (B, D) with respect to expression of total mTOR (A, B) and p-mTOR (C, D). P value represents the significance of a simple survival analysis without the proportional hazard assumption that was applied in the analyses presented in Tables 1 and 2. mTOR, mechanistic target of rapamycin.

Figure 2

p-S2448-mTOR expression as determined by immunohistochemistry is inversely related to P7-score in ER + primary breast cancer. Tumors were dichotomized into high P7-score (>3) and low P7-score (<3). The histograms show mean ± SEM for the two groups. The median H -scores were significantly different between the two groups for p-S2448-mTOR but not total mTOR using a Mann–Whitney two tailed statistical analysis. ER, estrogen receptor; mTOR, mechanistic target of rapamycin; p-S2448-mTOR, mTOR phosphorylated on serine 2448; SEM, standard error of the mean.

Figure 3

Effect of estrogen treatment on p-S2448-mTOR and p-p70S6Kinase in MCF7 human breast cancer cells. MCF7 were serum starved and estrogen depleted and then treated with and without E2 (10 nM) for the indicated time periods. Cell extracts were prepared and analyzed as described in the Methods section: 100 ug of extr0061ct was subject to Western blotting for p-mTOR, total mTOR, p-p70S6K and total p70S6K. Results represent one of four to seven independent experiments. Upper panel: for p-S2448-mTOR, E2 treatment for three hours showed a 1.5 ± 0.2 fold, n = 7 (mean ± SD) and at six hours 1.4 ± 0.3 fold, n = 5 (one way ANOVA P = 0.0006) increase. Middle panel: for p-T389-p70S6K, E2 treatment for three hours showed a 1.9 ± 0.5 fold, n = 10 (mean ± SD) and at six hours 1.6 ± 0.3 fold, n = 10 (one way ANOVA P < 0.0001) increase. ANOVA, analysis of variance; E2, estradiol; mTOR, mechanistic target of rapamycin; p-S2448 mTOR, mTOR phosphorylated on serine 2448; SD, standard deviation.

Figure 4

mTOR can phosphorylate ERα in vitro. A) 300 ng of rh-ERα alone (full-length human ERα), or in the presence of 100 ng of mTOR (mTOR/Raptor/MLST8 complex, catalytic domain) or 100 ng of p70S6K was subjected to in vitro kinase assays in the presence of 0.2 mM ATP for 30 minutess at 30°C. Reactions were pre-incubated for 15 minutess with DMSO (vehicle control) or with selective kinase inhibitors (100 nM mTOR inhibitor AZ8055 or 10 μM p70S6K inhibitor PF-4708671). Following incubation, an aliquot of the reaction mix was subjected to Western blotting and visualized with an antibody specific for phospho-serine residues. B) 100 ng of recombinant human p70S6K was incubated with 100 ng of rh-mTOR (catalytic domain) with and without ATP as described in the Methods section. At the end of the incubation an aliquot of each reaction was subjected to Western blotting and visualized with an antibody specific for p-T389-p70S6K. C) 300 ng of rh-ERα and 100 ng of recombinant full-length p70S6K were incubated together in the presence or absence of ATP as described in the Methods section. At the end of the incubation an aliquot of each reaction was subjected to Western blotting and visualized with an antibody specific for pS167-ERα. M = molecular mass marker. DMSO, dimethyl sulfoxide; E2, estradiol; mTOR, mechanistic target of rapamycin; rh, recombinant human.

Figure 5

ERα is co-immunoprecipitated with mTOR from MCF7 cells. MCF7 cells were grown in 5% CM (lane 1) or MCF7 were serum starved and estrogen depleted and then treated with (lane 3) and without E2 (lane 2) for 60 minutess. Cells were harvested, crosslinked with DSP and immunoprecipitated with an isotype-matched but irrelevant antibody (IgG) or an antibody specific for mTOR (sc-1549-R). Aliquots of immunoprecipitated complexes were subjected to Western blot analysis with antibodies to ERα or mTOR, as shown. Levels of ERα and mTOR in input lysates are shown in the two lower panels. A representative experiment is shown from a total of three independent experiments. CM, complete medium; DSP, dithiobis(succinimidylpropionate); E2, estradiol; ERα, estrogen receptor α; IgG, immunoglobulin G; mTOR, mechanistic target of rapamycin,