Rapamycin effects on mTOR signaling in the MCF10AT series of human breast epithelial cells: a model for proliferative breast disease and carcinoma

3731 Human proliferative breast disease (PBD) refers to a sequence of progressive morphologic changes, including hyperplasia that occurs in the breast tissue prior to breast cancer development. The MCF10AT cell series of human breast epithelial cell includes the normal MCF10A (10A), premalignant MCF10AT (10AT) and MCF10ATG3B (10ATG3B), and fully malignant MCF10CA1a (10CA1a) cells and serves as a unique model system for development of PBD and carcinoma. The effects of rapamycin on mammalian target of rapamycin (mTOR) signaling in the MCF10AT cell lineage were examined. Rapamycin (100 nM) inhibited the proliferation of 10A, 10AT, and 10ATG3B cells by 48, 58 and 53%, respectively. In contrast, rapamycin inhibited 10CA1a tumor cell proliferation by only 26%. Treatment of cells with rapamycin (100 nM) for 48 h resulted in the percentage of cells in G 1 phase being increased by 16.6, 17.5, 9.2 and 4.9% in the 10A, 10AT, 10ATG3B, and 10CA1a cells, respectively. The presence of apoptotic cells, reflected by the subG 1 peak, was not detectable in MCF10AT cell lineage. Rapamycin inhibited significantly the phosphorylation/activation of p70S6K, 4E-BP1 and STAT3 in the MCF10AT cell lineage. The 4E-BP1:eIF4E ratio, an intrinsic marker for sensitivity to rapamycin, increased by 1.7- to 3.3-fold in MCF10AT cell lineage, but did not correlate well with either cell proliferation or cell cycle data. Rapamycin decreased phospho-S6RP levels by ~86%, ~67%, ~85% and ~22% in the 10A, 10AT, 10ATG3B and 10CA1a cells, respectively. These results correlated well with the decreased sensitivity of the 10CA1a cells to rapamycin-mediated inhibition of cell proliferation and G 1 phase arrest. Rb and pRb protein levels were significantly decreased after treatment of rapamycin (100 nM). The protein expression of cyclin D3 was significantly decreased in a time-dependent manner, with the levels at 36.0, 29.4, 21.7 and 37.4 % for 10A, 10AT, 10ATG3B and 10CA1a cells, respectively, relative to untreated controls. Cdk2 protein levels declined by ~45,~30, ~32, and ~36% in the 10A, 10AT, 10AT3B, and 10CA1a cells, respectively, relative to untreated controls. Collectively, these data show that rapamycin inhibited cell proliferation by G 1 cell cycle arrest in the 10A, 10AT and 10ATG3B cells, whereas the 10CA1a tumor cells were less sensitive to rapamycin effects. The effects of rapamycin on Rb, pRb, cyclin D3 and cdk2 levels contributed to G 1 cell cycle arrest in the 10A, 10AT and 10AT3B cells, with a lesser effect noted for the 10CA1a tumor cells. Supported by NIH grant ES10595 and EHS Center grant P30 ES06639. Keywords: MCF 10AT cells; mTOR, rapamycin, cyclin D3, cdk2