The Effects And Mechanisms Of Breast Cancer Stem Cells Resistance Mediated By HIF-2α

Objective:Breast cancer is one of the most common malignant tumors in the world.With the development of research,CSCs(cancer stem cells)theory provides a new idea for the treatment of breast cancer.CSCs is a small group of cells with the ability of unlimited proliferation and differentiation charactered by strong ability of selfrenew,high expression of stemness markers,antiapoptosis,high resistance protein.At present,CSCs has been found in many solid tumors such as breast cancer,colorectal cancer,liver cancer,brain cancer and other cancers.Targeting breast cancer stem cells(BCSCs)is of great significance for the clinical design of a more effective and reasonable chemotherapy regimen.In solid tumors,CSCs often stay in hypoxia microenvironment niches which induce hypoxia inducible factors’(Hypoxia inducible,factor,HIFs)accumulation.HIFs is a transcription factor composed of basic helix-loop-helix-Per-ARNT-Sim,contains a oxygen content sensitive subunit isoforms(HIF-1?,HIF-2?,HIF-3?)ligand and a beta subunit(ARNT).Although HIF-1?and HIF-2?is highly homologous in structure,but they often function in different mechanisms.In recent years,studies have found that,in cancer stem cells,HIF-1?and HIF-2?are involved in regulating of stemness,especially the function of HIF-2?has attracted much attention,but how HIF-2?play the function to mediate resistance has not been reported.Endoplasmic reticulum stress(ERS)is a subcellular pathological process of endoplasmic reticulum physiological function disorder,through activating the unfolded protein response(UPR)mediated intracellular signal transduction pathways.Activation of the UPR pathway enhances drug resistance had been confirmed in a variety of tumor cells.Recent studies have reported that HIF-2?and UPR are closely related to the drug resistance.But in breast cancer stem cells how HIF-2?mediated UPR pathway in regulation of resistance to conventional chemotherapy has not been reported.(-)-Epigallocatechin-3-gallate(EGCG)was monomers isolated from tea catechins,is a major biological activity component of the tea polyphenols,having the antioxidation and anti-aging effect.A lot of studies have found that in different animal models and tumor cell lines,EGCG as antioxidants can inhibit the proliferation and induce apoptosis,reduce the chemoresistance.However,whether EGCG regulates breast cancer stem cells drug resistance through targeting HIF-2?drug resistance has not been reported yet.Wnt,Nothch signaling pathway are two highly conserved and stem cell self-renewal closely related pathways.Currently the mechanism how HIF-2?interact with Wnt/beta-catenin and Notch pathway regulating the tumorigenesis is still controversial.And the relationship of HIF-2?with BCSCs stemness and drug sensitivity has not been reported.Therefore,this study is aiming to research how HIF-2?regulate stemness and drug sensitivity of breast cancer,and endoplasmic reticulum stress UPR pathway and Wnt pathway and Notch pathway,by used non-adherent suspension culturing MCF-7 and T47D in breast cancer cells without serum,successfully enriched stem-like MCF-7 MS and T47D MS mamosphere,which will provide theoretical and experimental basis for clinical treatment.Methods:In serum-free non-adherent suspension culture,MCF-7 and T47D cells enriched T47D MS and MCF-7 MS mamosphere.Flow cytometrydry detectsCD44~+CD24~-cell ratio which is breast cancer stem cells marker.Western blot detects stem cell marker OCT4 and Nanog protein expression levels.Soft agar colony forming assays self-renewal capacity.Transwell migration experiment detects cell migration and invasion ability,restore serum culture observed MCF-7 MS,T47D MS mamospheres cells differentiation potential,nude mice experiment in vivo detected the tumorigenicity ofMCF-7 MS and T47D MS and identified characteristics of the self-renewal,differentiation,invasion and tumorigenicity and stemness.Lentivirus vector transfected and selected HIF-2?overexpressing MCF-7 and T47D stably transfected breast cancer cells,and HIF-2?scilence MCF-7 MS and T47D MS stabletransfection cell lines,qRT-PCR and western detect the expression of HIF-2?and downstream target genes HES-1,VEGFA.After scilencing HIF-2?(11)the CCK-8 assay was used to detecte the drug sensitivity of paclitaxel;soft agar colony formation to detect self-renew ability;serum recovery experiment to detect differentiation ability,western blot detects the expression change of OCT4,Nanog,CK14.To investigate the role of UPR pathway in HIF-2?regulating breast cancer stem cells drug resistance,after silecing HIF-2?(11)western blot detected the expression of related endoplasmic reticulum stress protein GRP78,IRE1,PERK,XBP1s,p-IRE1,p-PERK,P-GP;LC-MS/MS detected the concentration of PTX in cells;mitoSOX detected intracellular level of mtROS;qRT-PCR detected the expression of SOD2.On the nude mice,we constructed the HIF-2?silencing xenograft model,and recorded the growth of tumor in nude mice and the change of drug sensitivity to PTX.By immunohistochemical staining we detected the expression of HIF-2?,P-GP,GRP78 in tumor tissue;western blot detected UPR pathway related protein,of LC-MS/MS detected PTX concentration in tumor tissue.EGCG combined with PTX was used to treat MCF-7 MS cells,and MTT assay was used to detect the changes of drug sensitivity to PTX.Western blot detected the expression of GRP78 and HIF-2?.To obseve how EGCG impact HIF-2?to regulate the chemosensitivity of breast cancer stem cells in vivo,nude mice inoculated with MCF-7MS cells were used to construct nude mice xenograft model,and EGCG and PTX were injected intraperitoneally to investigate the effect of EGCG combined with PTX on the growth of transplanted tumor and the drug sensitivity of PTX in nude mice.The expression of HIF-2?and GRP78 was detected by immunohistochemical staining.To observed the EGCG and HIF-2?affinity in vitro and specific binding sites,MOE was used to predicte the combination of EGCG and HIF-2?(11)OWLS was used to detect the affinity in vitro.In MCF-7 and MDA-MB-231 HIF-2?overexpression cells,western blot detected the Wnt,Notch signaling pathway related protein expression.After treated with Wnt pathway inhibitor DKK-1 or Notch pathway inhibitor L685,458,MTT assay was used to detect cell survival rate;the change of cell apoptosis was detected by flow cytometry,the soft agar colony formation assay was used to detect the change of cell clone forming ability.In MCF-7 and MDA-MB-231 cells,western blot was used to detect the change of Wnt and Notch pathway after treated with DKK-1 and L685,458.Results:1.MCF-7 and T47D breast cancer cells in serum-free conditions can be enriched to form MCF-7 MS and T47D MS;compared with the parental MCF-7 and T47D cells,MCF-7MS and T47D MS cells have a high proportion of CD44~+CD24~-cells rate and high expression of stem cell markers OCT4 and Nanog,with the ability of forming cloney,migration and invasion,differentiation and in vivo tumorigenicity.2.MCF-7 MS and T47D MS cells were resistant to PTX,and highly expressed of HIF-1?/HIF-2?,especially HIF-2?.Lentiviral transfect MCF-7 MS and T47D MS with shRNA-HIF-2?plasmid could silence the HIF-2?expression,increase sensitivity to PTX,the increase of intracellular PTX,reduce the MCF-7 MS and T47D MS cell clone forming ability and the expression of OCT4 and Nanog;lentiviral transfect MCF-7 and MDA-MB-231 cells with HIF-2 alpha-cDNA plasmid,could reduce sensitivity to PTX,enhance the MCF-7 and MDA-MB-231 cell clone forming ability and expression of stem cell markers OCT4 and Naong.3.Silencing of HIF-2?significantly reduced the expression of UPR pathway related protein and P-GP;decreased SOD2 mRNA expression,increased mtROS expression.After treating with mitoTMPOL,the mtROS inhibitor,the drug sensitivity of HIF-2?silencing MCF-7 MS cells reverse,the level of mtROS decrease,and decreased the level of UPR pathway to be reversed.4.silent HIF-2?MCF-7 MS cells in nude mice bearing the tumor volume and tumor weight were reduced and enhanced the drug sensitivity to PTX tumor bearing mice;the expression of HIF-2?and GRP78,UPR pathway related protein decreased in tumor tissue,P-GP protein expression decreased,PTX increased savings within the organization.5.EGCG combined with PTX treatment of MCF-7 MS cells can enhance the drug sensitivity of MCF-7 cells to PTX,inhibit the ability of self-renewal,and inhibit the expression of HIF-2?and GRP78.EGCG can bind to HIF-2?PAS region and inhibit the transcriptional activity of HIF-2?.6.The overexpression of HIF-2?in MCF-7 and MDA-MB-231 cells could active Wnt pathway and Notch pathway;Wnt pathway inhibitor DKK-1 and Notch pathway inhibitor L685,458 reversed the resistance of MCF-7 and MDA-MB-231 in breast cancer cells induced by HIF-2?to PTX;colony formingn ability decreased;the proportion of apoptotic cells increased significantly.Conclusions:1.By serum free suspension culture MCF-7 and T47D breast cancer cells can successfully enrich MCF-7 MS and T47D MS breast cancer cell with strong self-renewal ability,migration and invasion,differentiation and in vivo tumorigenicity.2.Silencing HIF-2?can inhibit drug resistance of breast cancer stem cells to PTX.3.Effects of HIF-2?mediated endoplasmic reticulum stress UPR pathway by regulating mtROS to impact the resistance of breast cancer stem cells to PTX.4.EGCG by targeting HIF-2?PAS region,inhibited HIF-2?transcriptional activity,enhance the killing effect of PTX on MCF-7 MS cells.5.HIF-2?enhances the stemness of breast cancer cells and induces resistance to PTX by activating Wnt and Notch pathway.