PI3K/Akt/mTOR Inhibitors Effect On The Response Of Endocrine-and Targeted-therapeutic Drugs In Breast Cancer Stem Cells

Objective:To inhibit PI3K in PI3K/Akt/mTOR signaling pathways and study its effects on breast cancer stem cells (BCSCs) and evaluate its response to endocrine drug. To inhibit mTOR in PI3K/Akt/mTOR signal pathway to study its effects on trastuzumab-resistant breast cancer cells and BCSCs, and evaluate its influence on trastuzumab resistance.Methods:1. Using culture method of balloon cells in serum-free medium suspension, to enrich breast cancer cell line MCF-7-BCSCs. Using flow cytometry to elect ESA+CD44+CD24-/low subpopulation and clonal culture. Construction of eukaryotic expression vector of aromatase CYP19(pH β-Aro-CYP19), transfected in MCF-7BCSCs and non-stem cells, thus obtaining high aromatase expression BCSCs (BCSCs-CYP19) and non-stem cells (MCF-7-CYP19) subcloning. Detecting cell proliferation by MTT, evaluating cell migration by scratch experiment, detecting cell cycle and apoptosis by flow cytometry, evaluating colony formation ability by soft agar experiment, to compare inbitition effects of BKM120and letrozole on BCSCs-CYP19and MCF-7-CYP19inhibition. Using Western blot to study protein expression of AKT1, PI3K, and S6in PI3K/Akt/mTOR pathway through BKM120or combined letrozole.2. By adding different concentrations of trastuzumab into BT474for6months to get trastuzumab-resistant BT474R cells, and confirmed BT474R successfully constructed by MTT assay. Using culture method of balloon cells in serum-free medium suspension to enrich BT474-BCSCs and BT474R-BCSCs. Using flow cytometry to elected ESA+CD44+CD24-/low subpopulation and clonal culture. Using Western blot to detect protein expression of PI3K, AKT1, S6, mTOR in PI3K/Akt/mTOR pathway, and evaluate effects of this signal pathway on trastuzumab resistance. To silence mTOR expresion of BT474R-BCSCs, and evaluate colony formationg by colony assaycell colony formation; Using Western Blot to detect PI3K, AKT1, S6, mTOR expression when mTOR low-expression of BT474R-BCSCs and to evaluate its inhibition after downgrade mTOR expression. Results:1. Effects of BKM120on BCSCs-CYP19:Compared with control group, BKM120could inhibit BCSCs-CYP19growth by dose-dependence. BKM120could reduce migration and colony formation of BCSCs-CYP19, and significantly reduced expression of PI3K, AKT1, S6and mTOR.2. Effects of BKM120combined letrozole on BCSCs-CYP19:Combined BKM120and letrozole can inhibit BCSCs-CYP19growth and proliferatio. Compared with control group, BCSCs-CYP19stayed in G0-G1phase increased significantly and induced early apoptosis; Drugs combination can inhibit clonogenic capacity of BCSCs-CYP19and down-regulate expression of PI3K, AKT1and S6.3. PI3K/Akt/mTOR pathway kept active in trastuzumab-resistance. Through silencing mTOR of BCSCs-BT474R, found that resistance to trastuzumab reduced, expression levels of upstream protein of mTOR did not change significantly, but downstream protein such as S6phosphorylation significantly inhibited(P<0.001).4. mTOR inhibitor everolimus can effectively inhibit clonogenic capacity of BCSCs-BT474R in vitro and growth of BT474R-BCSCs.Conclusions:1. BKM120can inhibit BCSCs-CYP19proliferation. Inhibition effects of BKM120combined letrozole is more effective than BKM120, which inducing early apoptosis, down-regulating expression of important downstream products in PI3K/Akt/mTOR pathway, to provide a theoretical basis for reversal of endocrine-resistance.2. PI3K/Akt/mTOR pathway is active in trastuzumab-resistant process and mTOR plays an important role. mTOR inhibitor everolimus can effectively inhibit BCSCs-BT474R and may provide a new regimen for reverse trastuzumab-resistance.