Estrogens play an important role in development and progression of breast cancer. Aromatase inhibitors (AIs) such as letrozole, that block the synthesis of estrogen are proving to be superior to antiestrogens (AEs) and may replace tamoxifen as first line treatment for postmenopausal ER positive breast cancer patients. However, resistance to letrozole therapy remains a major concern. To study the mechanisms of this resisitance we have employed a model system designated as Long Term Letrozole Treated (LTLT-Ca). Human MCF-7 breast cancer cells transfected with aromatase were grown as tumors in female ovariectomized nude mice and then treated with letrozole. The tumors regressed initially but later on began to grow. After 56 weeks of treatment the tumors were removed. The cells were isolated from the tumors and the LTLTCa cell line was developed. LTLT-Ca cells were then routinely cultured in the presence of 1μM of letrozole. These cells were completely cross-resistant to other AIs and AEs. Compared to the parental MCF-7Ca cells, up-regulation of growth factor receptors Her-2 (4 fold) and IGF-I β (3 fold) and activation of down-stream targets such as mTOR (3 fold) was observed using western immunoblotting. The overexpression of mTOR was associated with activation of the downstream effectors p70S6K (2 fold) and S6 ribosomal protein (3 Fold). LTLT-Ca cells responded to growth inhibitory effects of rapamycin (IC50= 55.6 nM) and dual EGFR/Her-2 tyrosine kinase inhibitor AEE788 (IC50= 1.5 nM) These cells showed a partial restoration of growth inhibitory effects of letrozole, anastrozole, tamoxifen and fulvestrant after concommitant treatment with AEE788. The cell viability decreased from 83.28% with AEE788 (1nM) alone to 58.21% with AEE (1nM) plus letrozole (1μM). However, rapamycin was unable to restore the responsiveness of these cells to letrozole suggesting that letrozole resistance and mTOR activation may not be connected. Activation of mTOR is usually associated with aberrent activity of Akt. However, LTLT-Ca cells exhibited activation of mTOR inspite of low levels of Akt kinase activity of 0.9 fold compared to MCF-7Ca cells. This suggests that activation of mTOR is independent of Akt activation. It has been hypothesized that when all the erbB activity is blocked, the cells become very sensitive to changes in the mTOR dependent translation. Hence, inhibition of both Her-2 mediated tyrosine kinase signaling and mTOR dependent translation could provide better inhibition of proliferation of breast cancer cells that are refractory to aromatase inhibitor letrozole and may also restore responsiveness to letrozole.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]