Estrogen Suppresses Anoikis Via Extrcellular Signal-regualted Kinase(ERK)-calpain-focal Adhesion Kinase(FAK)signaling In Breast Cancer Cells

Objective: This study was performed to investigate the effects of estrogen(E2)on the resistance to anoikis and a possible role of calpain-focal adhesion kinse(FAK)signaling underlying the estrogen action in breast cancer cells,for the purpose of searching a high-efficiency therapy.Methods: Human breast cancer cell line MCF-7(ER+)was employed as a model system and poly-Hema-coated culture was used to induce anoikis and where required,estrogen-sensitive MCF-10 A and estrogen insensitive-MDA-MB-231 cells were used.Cells were treated with E2and/or pretreated with MEK or FAK inhibitors where needed.Western blotting was used to assess the phosphorylation of ERK and FAK,MTT assay and trypan blue staining/ cell counting were employed to evaluate cell viability,and Hoechst staining was used to verify apoptosis.Results: 1)E2 enhanced anoikis resistance and increased phosphorylation of ERK and FAK in MCF-7 breast cancer cells.Compared to anchorage-dependent culture,suspension culture with poly-hema profoundly reduced survival;Treatment of model cells in suspension culture dramatically decreased cell survival and apoptosis.Additionally,E2 treatment led to increased phosphorylation of ERK and FAK.2)Inhibitors for ERK or calpain greatly reversed phosphorylation of FAK and anti-anoikis effect induced by E2 stimulation.Treatment of MCF-7 cells with E2 for 12 h significantly enhanced phosphorylation of ERK and FAK,while MEK inhibitor U0126 treatment suppressed the phosphorylation of ERK or FAK,as well as anoikis resistance.In addition,calpain inhibitor calpeptin inhibited ERK phosphorylation and resistance to anoikis.3)Calpain activity in breast cancer cells is much higher than that in normal breast cells.Calpain in MCF-7 cells is highly sensitive to stimulation with E2 or epithelial growth factor(EGF),which waseffectively suppressed by calpeptin.Calpain in MBA-MD-231 cells was also highly active to EGF exposure,while calpain remained intact in exposure to E2 or EGF in MCF-10 A breast cells.4)FAK inhibitor dramatically reduced E2-induced FAK phosphorylation and anoikis effect.Treatment of model cells with FAK inhibitor(FI)suppressed E2-induced phosphorylation of both FAK and ERK.FI also suppressed E2-sitmulated anoikis resistance and reduced cell survival significantly.Conclusions:1)E2 contributes to enhanced cell viability and increased resistance to anoikis in MCF-7 breast cancer cells;2)ERK-CANP/FAK signaling may be involved in the E2-stimulated survival during suspension culture;3)A positive feedback loop may exist between calpain/FAK and ERK.Higher activity of calpain and a positive feedback may strengthen MCF-7 cells' responsiveness to estrogen sitmulation.