Preclinical and clinical evidence links tamoxifen (TAM)-resistant estrogen receptor (ER)-positive breast cancers with overexpressed receptor tyrosine kinases (RTKs) like HER2 and/or constitutive downstream PI3K/AKT signaling that activates the mammalian target of rapamycin, mTOR. Activated mTOR signaling is known to be associated with estrogen (E2) induced breast cancer cell growth; and while inhibitors of mTOR like RAD001 (everolimus) or CCI-779 (temsirolimus) show preclinical ability to enhance TAM sensitivity, phase II/III clinical trials are evaluating their ability to improve ER-positive breast cancer responsiveness under E2 deprivation. We employed TAM-sensitive (MCF7) and TAM-resistant (MCF7/HER2, BT474) ER-positive breast cancer models to evaluate the antitumor activity of RAD001 alone, with or without E2 deprivation, and in combination with TAM (100 nM). Despite HER2 induced increases in phospho-AKT, phospho-ERK1/2, phospho-p70S6K and phospho-4E-BP1, TAM-resistant MCF7/HER2 cells showed no significant difference in RAD001 sensitivity when compared to TAM-sensitive MCF7 cells (24-120 h dose response to 0-100 nM RAD001 by MTT assay). In the presence of E2, all three cell lines exhibited comparable antiproliferative responses to RAD001 with IC50 values of ~0.2 nM (120 h). Within 24 h of treatment all cells showed RAD001 inhibition of E2 induced cyclin D1 and the downstream mTOR substrates, phospho-p70S6k and phospho-4E-BP1, without any apoptotic increase in p85 PARP. For MCF7 cells in particular growth inhibition was E2 dependent, since with E2 deprivation 100 nM RAD001 inhibited MCF7 cell proliferation <20%. Despite modest RAD001 response differences between TAM-sensitive and TAM-resistant cells, mTOR inhibition did not increase TAM sensitivity in any of the ER-positive breast cancer cells. Under maximal RAD001 growth inhibitory conditions (+E2), all cell lines showed a paradoxical 2-to-4 fold increase in phospho-AKT(Ser473) within 24 h. Moreover, this paradoxical RAD001 induction of phospho-AKT was suppressed by the HER2 RTK inhibitor, AG825 (25 μM), which further enhanced the antiproliferative activity of RAD001 in all three cell lines. In summary, growth inhibition by the mTOR inhibitor RAD001 appears HER2 independent and E2 dependent in both TAM-sensitive and TAM-resistant ER-positive breast cancer models, where it fails to enhance TAM responsiveness. This failure of RAD001 to sensitize ER-positive breast cancer cells to TAM may result from rapid and paradoxical RAD001 induction of phospho-AKT which can be prevented by RTK inhibitors like AG825. Thus, optimal use of RAD001 to treat ER-positive breast cancers with or without endocrine therapy may be in combination with RTK inhibitors that prevent phospho-AKT induction.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA