The two direct targets of mTORC1 are p70 S6 kinase and 4E BP1, which mediate its effect on protein translation. Activation of mTORC1, in response to nutrient availability and activation of the PI3K/AKT pathway, results in the hyperphosphorylation of 4E BP1 and the release of eIF4E, which, together with eIF4G, form a functional eIF4F mRNA cap binding complex and initiates translation. p70 S6 phosphorylates the 40S ribosomal subunit protein S6 and stimulates the translation of the 5 oligopyrimidine tract containing mRNAs. Several of these cell cycle regulators are dysregulated in BC, including eIF 4E, p27, D type cyclins, and c myc. Hence, mTORC1 may provide a novel target for the treatment of breast tumors that are endocrine resistant.

Evidence suggests that the mTORC1 inhibitor rapa mycin, and its derivatives, may have some antitumorogenic activity. Rapamycins/rapalogs are allosteric inhibitors that, when in complex with the immunophilin FKBP12, target the FRB domain adjacent to the catalytic site of mTORC1, leading to inactivation of p70 S6 kinase and activation of 4E BP1 as a repressor of cap dependent translation. resulting in the suppres sion of global protein synthesis. Until recently, rapalogs showed modest clinical activity in BC. Lately, however, two clinical studies reported substan tially greater activity of the rapalog everolimus in the metastatic setting, when administered after prior treatment with an AI. First, in the TAMRAD study, median time to tumor progression was 4. 5 months, 3. 7 to 8. 7 with tamoxifen and 8. 5 months with everolimus plus tamoxifen.

BOLERO 2 found that the median TTP with exemestane alone of 4. 1 months after failure of nonsteroidal AI was extended to 10. 6 months, a result so positive that it required early cessa tion of the trial. We report here that in isogenic derivatives of MCF7 cells, the activity of everolimus is enhanced after acqui sition of resistance to E deprivation, together with mechanistic data that improve understanding of this enhanced activity. We also report xenograft studies of the combination of everolimus with the AI letrozole and parallel studies in the ER BT474 cell line, whose endocrine resistance depends on HER2 amplifica tion that is associated with response to rapalogs.

The results provide mechanistic support for recent posi tive clinical data on the combination of RAD001 and endocrine therapy, as well as data on potential routes of escape, via enhanced HER2/3 signaling, that merit inves tigation for further improvements in treatment efficacy. Methods Antibodies These companies provided the following substances Cell Signaling Dacomitinib Technology, New England Biolabs, Hert forshire, UK . Millipore . Sigma, Poole, Dorset UK . Santa Cruz Biotech nology, Santa Cruz, USA . Novacastra Labora tories, Newcastle upon Tyne, UK. HRP conjugated secondary antibodies were obtained from Amersham Pharmacia, Amersham UK.