Abstract P3-04-11: ER is required for mTORC1 inhibitor-induced feedback activation of PI3K/AKT in ER+ breast cancer cells and patients' tumors
Authors: Yang W , Chen VS , Schwartz GN , Marotti JD , Rosenkranz KM , Gui J , Miller TW 
Published: March 2017

The mTORC1 inhibitor everolimus (afinitor) is approved for the treatment of patients with advanced/metastatic ER+/HER2- breast cancer in combination with the steroidal aromatase inhibitor exemestane following progression on a non-steroidal aromatase inhibitor. The BOLERO-2 and TAMRAD studies demonstrated that combined everolimus/anti-estrogen therapy provided longer PFS compared to anti-estrogen alone. However, it has not been clarified whether continued treatment with an anti-estrogen backbone is beneficial in the setting of mTORC1 inhibition.

Upon activation by mTORC1, p70S6K phosphorylates the insulin-like growth factor-1 receptor (IGF-1R)/insulin receptor (InsR) effector IRS-1 to promote IRS-1 degradation, which in turn decreases activation of phosphatidylinositol 3-kinase (PI3K), AKT, and mTORC1. IGF1R, IRS1, and IRS2 are ER-inducible genes, and crosstalk between the ER and IGF-1R pathways has been described. We hypothesized that mTORC1 inhibition with everolimus will upregulate IGF-1R/InsR/IRS-1/2 signaling to activate PI3K/AKT and promote cancer cell survival, while combined inhibition of ER and mTORC1 will block PI3K/AKT activation by decreasing IGF-1R and IRS-1/2, providing rationale for combined targeting of ER and mTORC1.

In 3 ER+ breast cancer cell lines, everolimus treatment increased phospho-AKT levels. ER inhibition with fulvestrant suppressed the induction of P-AKT by everolimus. IGF-1R/InsR inhibition with OSI-906, and RNAi-mediated knockdown of IGF-1R, InsR, or IRS-1/2, decreased everolimus-induced P-AKT. Everolimus sensitized IGF-1R/InsR to IGF-1 that was suppressed by fulvestrant but enhanced by 17b-estradiol. Although fulvestrant decreased IGF-1R and InsR protein levels, phospho-receptor tyrosine kinase profiling showed that fulvestrant increased P-IGF-1R and P-InsR. Acting downstream of IGF-1R/InsR, fulvestrant prevented everolimus-induced PI3K/AKT activation by blocking binding between the p85 regulatory subunit of PI3K and IRS-1, possibly by decreasing IRS-1/2 levels. In summary, everolimus-induced activation of PI3K/AKT requires IGF-1R/InsR/IRS-1/2 signaling facilitated by ER. Combined treatment with fulvestrant and everolimus synergistically inhibited growth in 4 ER+ cell lines.

To determine whether ER promotes PI3K/AKT activation induced by mTORC1 inhibition in patients' tumors without exposing patients to everolimus, we analyzed live tumor tissues from post-menopausal patients with ER+/HER2- breast cancer treated +/- letrozole for 10-21 d before surgical tumor resection. Tumor cores (1 mm diameter) were used for ex vivo culture in DMEM +/- everolimus +/- OSI-906 for 1 h, and lysates were analyzed by immunoblot. Everolimus significantly increased P-AKT in tumors from untreated patients (n=10). OSI-906 did not affect P-AKT, but OSI-906 suppressed everolimus-induced P-AKT. In tumors from letrozole-treated patients (n=7), neither everolimus nor OSI-906 affected P-AKT. These data collectively suggest that ER activation is required for activation of PI3K/AKT induced by mTORC1 inhibition, and provide rationale for therapeutic combinations of anti-estrogens and mTORC1 inhibitors.

Citation Format: Yang W, Chen VS, Schwartz GN, Marotti JD, Rosenkranz KM, Gui J, Miller TW. ER is required for mTORC1 inhibitor-induced feedback activation of PI3K/AKT in ER+ breast cancer cells and patients' tumors [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-11.