Background: Crosstalk between estrogen receptor (ER) and growth factor receptor (GFR) downstream signaling pathways [PI3K/AKT/mTOR and MEK\p42\44MAPK (MAPK)] has been associated with endocrine resistance. Single downstream inhibitors like everolimus partially reverse endocrine resistance. However, more than one downstream escape pathway may contribute to endocrine resistance. Furthermore, disruption by a single downstream inhibitor of the negative feedback loops that balance the amplified signals from GFRs may result in compensatory pathway activation. Therefore we investigated whether dual downstream signaling inhibitions are required to more effectively overcome tamoxifen-resistant growth, using in vitro and in vivo models.
Materials and Methods: The effects of different kinase inhibitors (i) AZD2014 (mTORi), AZD5363 (AKTi), and AZD6244 (MEKi) on endocrine therapy [tamoxifen (Tam) and fulvestrant (Fulv)] were tested in ER+ MCF7 and T47D Tam-resistant derivatives (TamR). In vitro growth and apoptosis were assessed using methylene blue and c-PARP, respectively. Western blot analysis was used to analyze the effect of each inhibitor or combination on their respective pathway substrates. Nude mice with transplantable MCF7 TamR xenografts at a size of 200 mm3 were randomized to continued Tam, continued Tam + (mTORi, AKTi, MEKi, mTORi+MEKi, AKTi+MEKi) or Fulv ± the inhibitor combinations.
Results: We found that in two ER+ models MCF7 TamR and T47D TamR in vitro, both mTORi and AKTi were effective in restoring growth inhibition, and effective in inhibiting their respective pathways. Interestingly, inhibition of mTOR and AKT resulted in upregulation of pMAPK. However, while MEKi did inhibit its pathway; it did not restore growth inhibition by the antiestrogens. On the other hand, dual inhibition, adding the MEKi to either mTORi or AKTi, resulted in a more potent reduction of cell growth as well as of downstream signaling. In the TamR derivative of MCF7, ER is still maintained and plays a role in resistance, unlike the T47D model, where there is little to no ER expression after TamR develops. Thus, as might be expected, combining downstream inhibitors with potent ER blockade by Fulv enhances the effect of single and dual downstream signaling inhibitors mainly in the MCF7 TamR model. Finally, Fulv in addition to dual downstream inhibitions (mTORi+MEKi or AKTi+MEKi) delayed MCF7 TamR xenograft growth significantly more than Fulv with single downstream inhibitors.
Conclusion: This study provides evidence that dual inhibition of GFR downstream pathways is needed to overcome activation of escape mechanisms that are up-regulated with acquired endocrine resistance and after resistance to single pathway inhibitors. Although the downstream inhibitors alone significantly inhibit TamR growth, combination with Fulv robustly slowed growth of TamR tumors in vivo. Based on these results further studies combining MEK inhibition with inhibitors of the PI3K pathway and ER downregulators are warranted.
Citation Format: Agostina Nardone, Gladys Morrison, Xiaoyong Fu, Martin Shea, Tamika Mitchell, Teresa Klinowska, C Kent Osborne, Rachel Schiff. Reversal of endocrine therapy resistance with inhibitors of AKT, mTOR, or MEK as single agents or in combination [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-03.