Cell cycle effects of RAD001 alone or in combination with endocrine agents Cells were seeded into 10 cm dishes. Monolayers were treated with the drug combinations indicated for 24 hours. Cells were pulse labeled with 10 uM bromodeoxyuridine for 2 hours and then fixed and stained with anti bromo deoxyuridine conjugated FITC and propidium iodide. Fluorescence activated cell signaling was used to analyze changes in the cell cycle. To assess the effect on cell cycle regulatory proteins, similarly treated cell monolayers were lysed and subjected to immu noblotting at the same time. Immunofluorescence Cells were prepared as described previously. After 24 hours of treatment with the drugs indicated, cells were fixed and incubated with a monoclonal anti human p27 antibody, as previously described.

Cells were then incubated in the pre sence of Alexa Fluor 488 conjugated goat anti mouse IgG secondary antibody, counter stained with TO PRO 3, and mounted onto glass slides by using Vectashield mounting medium. Images were collected sequen tially in two channels on a Leica TCS SP2 confocal microscope. The p27 positive nuclei were counted in each image by using the count tool in Adobe Photoshop CS6 Extended and were expressed as a percentage of total nuclei present. Values shown are mean percentages standard deviation. Human tumor xenografts Experiments were carried out in accordance with Home Office guidelines and approved by the Institute of Cancer Research Ethics Committee. Female Ncr Foxhead nude mice were kept under sterile conditions with free access to food and water.

Mice were ovariecto mized and then allowed to acclimatize for 7 to 14 days. MCF7 AROM 1 and BT474 AROM 3 xenografts were initiated by inoculation of 100 ul cell suspension contain ing 107 cells in basement membrane matrix into the right flank. Growth was main tained by androstenedione support through intradermal injection of androstenedione pellets. Tumors were grown to approximately 7 to 8 mm diameter and assigned to treatment groups with no statistically significant difference in mean volume before treatment. Mice were continued with androstenedione support and ran domized to receive daily doses of vehicle /90% polyethylene glycol, RAD001, tamoxifen, letrozole, or RAD001 in combination with tamoxifen or letrozole. All drugs were administered by oral gavage and were given daily for a total of 24 days.

Tumor growth was assessed twice weekly in Brefeldin_A all con trol and treatment arms by caliper measurements of the two largest diameters. Volumes were then calculated according to the formula a b2 ��/6, where a and b are orthogonal tumor diameters. Tumor volumes were then expressed as fold change in volume at the start of treatment. Overall statistical difference was calculated by using the Kruskal Wallace test, and the statistical differences between individual treatment arms was calculated by using the Mann Whitney test.