Mechanism Of Fatostatin Inducing Tamoxifen Sensitivity In Breast Cancer

BackgroundBreast cancer is the major cause of cancer death among women and is responsible for 14%of all cancer-related deaths worldwide.Approximately 65-75%of breast cancer patients express estrogen receptors(ERs)or progesterone receptors(PRs)[1].And activation of the estrogen receptor(ER)signaling pathway affects over 70%of breast cancers patients[2]Tamoxifen,a selective estrogen receptor modulator(SERM),has been devoted in breast cancer for more than 40 years[3].In ER-positive breast cancer,5 years of Tamoxifen utilization substantially impedes the annual recurrence rate and reduces the breast cancer mortality rate by one-third in both pre-and postmenopausal women[4].But unfortunately,Tamoxifen resistance remains an obstacle in the treatment of hormone-dependent breast cancer[5].While up to one third of patients are resistant to Tamoxifen at the beginning of treatment,the majority of patients who initially respond to Tamoxifen will later also become resistant[6].Fatostatin(formerly called 125B11)is a diarylthiazole compound that blocks ER-to-Golgi transport of SCAP[7].Mechanistic studies showed that Fatostatin binds SCAP directly and blocks Golgi-specific glycosylation on SCAP,suggesting that Fatostatin inhibits ER-to-Golgi transport of SCAP(8).Fatostatin was originally developed to inhibit insulin-induced adipogenesis and decreased the amounts of fatty acid,triglyceride and low-density lipoprotein and reduce body weight by perturbing the nuclear translocation of SREBP in obese mice with low cytotoxicity[9].Fatostatin has been shown to have the potential efficacy in glioma cells[9]It reported that Fatostatin suppresses cell proliferation,tumorigenesis and progression and induces apoptotic death in prostate cancer cells in vitro and in vivo by blocking SREBP activation and inhibiting fatty acid and cholesterol biosynthesis as well as AR signaling.So how does a lipostatin work in an estrogen-receptor-positive breast cancer cell line?Does its combination with Tamoxifen increase the sensitivity of estrogen-receptor-positive breast cancer cells to Tamoxifen?Its molecular mechanism deserves further exploration.In this study,the molecular mechanism of adipostatin on estrogen receptor-positive breast cancer cells and the effect of Tamoxifen on estrogen receptor-positive breast cancer were discussed through the basic research of molecular biology,providing an important theoretical basis for the combined drug use of breast cancer patients.ObjectiveTo clarify the role of Fatostatin in estrogen receptor breast cancer progression,we construct xenograft tumorigenicity BALB/c nu/nu female mice model with treatment of Tamoxifen and Fatostatin,and then examined the expression of estrogen receptor,apoptosis relative protein marker,cell cycle relative protein marker,autophagy relative protein marker,ki67 in tumor tissure by immunohistochemical(IHC)staining and western blotting and analyzed the effect of combination of Fatostatin and Tamoxifen for estrogen receptor positive breast cancer.In addition,we determined the role of combination of Tamoxifen and Fatostatin in regulating estrogen receptor positive breast cancer cell proliferation,migration and invasion in vitro,and clarified the potential mechanisms underlying tumor-suppressor activity by Fatostatin in estrogen receptor positive breast cancer.Our study demonstrated for the first time that Fatostatin and Tamoxifen could induce autophagy and apoptosis in ER-positive breast cancer cells via suppress PI3K/Akt/mTOR pathway,and the combination treatment of Fatostatin and Tamoxifen play a crucial role in inhibiting tumor development.Methods1.We used Immunohistochemistry to detect the expression level of estrogen receptor apoptosis relative protein marker,cell cycle relative protein marker,autophagy relative protein marker,ki67 in tumor from xenograft mouse model with treatment of Tamoxifen and Fatostatin2.We tested the expression levels of estrogen receptor by Western blot,apoptosis relative protein marker,cell cycle relative protein marker,autophagy relative protein marker,ki67 in human estrogen receptor breast cancer cell lines(MCF-7 and T47D)with treatment of Tamoxifen and Fatostatin3.MTT and colony formation assays were used to measure the influence of Fatostatin and Tamoxifen on cell proliferation in vitro;Cell-cycle analysis by flow cytometry were used to confirm the influence of Fatostatin and tamoxfein on cell cycle in vitro.4.TUNEL assays and apoptosis by PI-Annexin-V staining were used to determine the effect of Tamoxifen and Fatostatin on cell apoptosis in vitro?western blot assays were used to examine the expression levels of apoptosis-related proteins(Bcl-2?Bax?PARP?cleaved PARP?Caspase3?cleaved Caspase3?Caspase8?cleaved Caspase8?Caspase9?cleaved Caspase9)after treatment of Tamoxifen and Fatostatin.MDC and acidic vesicular organelles staining by fluorescence microscope were used to confirm the effect of Tamoxifen and Fatostatin on cell autophagy in vitro.Western blot assays were used to examine the expression levels of autophagy-related proteins(mTOR,p-mTOR,Atg12,Atg13.Atg7,P62,LC3B)after treatment of Tamoxifen and Fatostatin?5.Transwell assays and wound healing assays were performed to investigate Tamoxifen and Fatostatin effect on the migration and invasion of cancer cells in vitro.6.Western blot was used to detect the estrogen receptor by treatment with CHX,MG132 and Chloroquine combining with Fatostatin?And then confirm that Fatostatin degraded estrogen receptor by ubiquitination pathway by using CO-IP assay.ResultsThe combination of fatostain and TAM reduced the viability and motility of ER-positive breast cancer cells MTT assay was used to measure cell viability.As shown in figure combined therapy with Fatostatin and TAM significantly reduced the viability of both MCF-7 and T47D cells in a time-and dose-dependent manner.Cell viability decreased sharply after administration of 40uM Fatostatin with 20?M TAM,independent of the treatment time?A colony formation assay revealed that combined treatment decreased the proliferation rate of both MCF-7 and T47D cells Migration and invasion assays were carried out to measure cell motility.The combination of 10uM Fatostatin and 10 ?M TAM inhibited migration and invasion in MCF-7 cells and T47D more than single drug treatments.Combination of Fatostatin and Tamoxifen to induce autophagic cell death in ER-positive breast cancer cellsTo quantify autophagic cell death in cells treated with Fatostatin,TAM,or both,we usedflow cytometry analysis and an AVO staining assay,both Fatostatin and TAM induced autophagic cell death.Combining the two treatments induced the formation of more autophagosomes than either of the drugs aloneFatostatin with Tamoxifen to induce apoptosis in ER-positive breast cancer cells.We used the TUNEL assay to detect the modes of cell death induced by Fatostatin and TAM.Fatostatin also synergized with Tamoxifen to induce autophagy and apoptosis in ER-positive breast cancer cell.Additionally,intact cells,early apoptotic cells,and late apoptotic or dead cells can be identified using PI-annexin-V double staining.This method showed that after combined drug treatment,late apoptosis or cell death rates and early apoptosis rates increased in a dose-dependent manner in both MCF-7 and T47D cells.Fatostatin with Tamoxifen to induce cell-cycle arrest in ER-positive breast cancer cellsCell-cycle distribution was analyzed by flow cytometry to determine the inhibitory effect of Fatostatin and TAM.MCF7 and T47D cells were exposed to Fatostatin,TAM,or both for a total of 48 h.G0/G1 arrest increased in cells exposed to these drugs compared to untreated cells.All treatments also concomitantly decreased the proportion of cells in the S phase.These results revealed that both Fatostatin slow MCF-7 Figure and T47D cell proliferation via cell-cycle arrest at the G2 phase,and TAM slowed MCF-7 Figure and T47D Figure cell proliferation via cellcycle arrest at the G0/G1 phase,which is consistent with previous studies.Furthermore,after combined drug treatment,the percentage of cells in the S phase decreased dramatically in a dose-dependent manner in both MCF-7 and T47D cells.TAM and Fatostatin target AKT in ER-positive breast cancer cellsMCF-7 and T47D cells were treated with Fatostatin(0,5,or 10uM),TAM(0,5,or 10?M),or both Figure for 48 hours.As shown in Figure levels of proteins involved in the AKT/mTOR pathway(AKT,p-AKT,mTOR,p-mTOR,p70S6K,p-p70S6K),cell autophagy(p-S6,P62,Atg7,Beclinl,Lc3b),and apoptosis(Cleaved-caspase-3,Cleaved-caspase-9,Cleaved-PARP,Bcl-2,Bax)were all altered by treatment.Expression values were normalized to controls.Because AKT induces changes in cell cycle distribution,we measured the effects of treatment on the levels of select cell cycle regulatory proteins.Cyclin D1 stimulates the G1-S checkpoints,and the role of?-GSK3? as an upstream regulator of Cyclin D1 was consistent with the flow cytometry analysis.Combined TAM and Fatostatin treatment inhibited the gro.wth of subcutaneous tumorsIn order to demine whether the curative effect of combination treatment was greater than either monotherapy in vivo,MCF-7 breast cancer cells were subcutaneously injected into the right flank of BALB/c nu/nu mice.As shown in Figure xenograft tumor growth was reduced in the combined treatment group as compared to the control group and both monotherapy groups.On day 40,tumors in the combined treatment group(mm3)were smaller than those in the Fatostatin treated(mm3),TAM treated(mm3),and untreated control(mm3)groups.In order to examine the mechanism underlying the inhibition of tumor growth by Fatostatin and TAM in vivo,we measured Lc3b,Bcl-2,and Ki67 protein levels using immunohistochemical staining,Bcl-2 and Ki67 levels decreased and Lc3b levels increased in the combined treatment group compared to the control group and monotherapy groups.Furthermore,combined treatment increased Bax,Beclin-1,and Lc3b levels and decreased Bcl-2 and Cyclin D1 levels compared to the control and monotherapy groups in vivo.These results suggest that Fatostatin with Tamoxifen to induce autophagy and apoptosis in ER-positive breast cancer cells in vivo.Conclusion.1.For the first time,our studies found that Fatostatin degraded estrogen receptor protein by ubiquitination pathway of K48.2.fatoatatin could increase the sensitive to Tamoxifen in estrogen receptor breast cancer.The combination of Fatostatin and Tamoxifen could inhibited estrogen receptor positive breast cancer proliferation,migration and invasion and induce apoptosis.Meanwhile the combination of Fatostatin and Tamoxifen promoted autophagy process by inhibiting PI3K/AKT/mTOR pathway.Fatostatin might be a new therapeutic treatment for estrogen receptor positive breast cancer.