The Effects And Mechanism Of TAM Treated Huc-MSC On Endocrine Resistance In ER~+ Breast Cancer

Objective:Breast cancer is the most common malignant tumor in women,and its incidence and mortality are highest in female tumors.The incidence of breast cancer has increased.Breast cancer can be divided into different subtypes,including Luminal A,Luminal B,HER2+(HR+),HER2+(HR-),and triple-negative breast cancer.Among them,the Luminal A type and Luminal B type are ER~+breast cancer,accounting for more than 70%of the number of all breast cancer patients.In the treatment,ER~+breast cancer is mostly used in endocrine treatment.Tamoxifen is one of the most common endocrine drugs for ER~+breast cancer treatment.However,TAM drug resistance also caused researchers’attention.Huc-MSC is a stem cell with multi-directional differentiation potential.In breast cancer,studies have revealed that Huc-MSC appears to promote the progression of tumors via direct effects or acting on a tumor microenvironment.Huc-MSC may promote growth and metastasis of breast cancer via a variety of molecular mechanisms,but the role and mechanisms of Huc-MSC play in the TAM drug resistance have not been demonstrated.We are intended to study the interaction between Huc-MSC and breast cancer cells in breast cancer,and further clarify the role and mechanism of Huc-MSC in TAM resistance.Methods:We first determined the median lethal concentration of TAM to ER~+breast cancer cells by CCK8 assay.To verify the effects of TAM treated Huc-MSC on the drug resistance of breast cancer cells,we divided cells into five groups:Control group is a negative control group that does not add any intervention;TAM group,5.0ug/m L of TAM was added into the cell culture medium;MSC-CM+TAM group,50%of the untreated Huc-MSC medium was added into the cell culture medium;DMSO+MSC-CM+TAM group,50%DMSO treated Huc-MSC supernatant was added into the cell culture medium;TAM-MSC-CM+TAM group,50%TAM treated Huc-MSC supernatant was added into the cell culture medium.The survival rate of the cells of different treatment groups was determined via CCK8 assay,and the apoptosis proportion of different groups was verified by flow cytometry.We further verify the results of the cell assays via the nude mouse subcutaneous tumor model.In terms of mechanism,to determine the effect of TAM-treated Huc-MSC supernatant on breast cancer cell stemness,we use the Western blotting assay to detect the expression of stemness genes in different cell groups.To further verify the effect of the TAM-treated Huc-MSC supernatant on the proliferation ability of breast cancer cells,we conducted the cell monoclonal formation assay.To explore the media molecules of Huc-MSC secrete,we conducted liquid chip sequencing which including 48 different cell regulating factors.We analyzed the result via the LIMMA package in R language.To further clarify the function of the TAM secreted IL-6 on the breast cancer cells,we conducted o transcription sequencing for different treatment breast cancer cells,and the result was analyzed via the DESeq2 package of R language.The results were verified again via the WB assays.So far,we have found the"intermediary molecule"of Huc-MSC regulating the TAM resistance of breast cancer cells.Finally,we neutralized the supernatant of Huc-MSC via neutralizing the antibody of this molecule,observed the changes of cell stemness,apoptosis and proliferation before and after neutralization,and verified the expression changes of key molecules in the downstream signal pathway by Western blotting.Results:we determined that the median lethal concentration of TAM to breast cancer cells was 5ug/m L by the CCK8 assay.Furthermore,the CCK8 assay was also employed to determine that the supernatant of TAM-treated MSC cell culture medium(TAM-MSC-CM)could make ER~+breast cancer cells have obvious drug resistance to TAM.The apoptosis assay revealed that the supernatant of TAM-treated Huc-MSC cell culture medium could significantly inhibit the apoptosis of ER~+breast cancer cells.As for in vivo experiment,we constructed 6different groups of subcutaneously transplanted tumor models in nude mice,and confirmed in vivo that TAM-treated Huc-MSC supernatant(TAM-MSC-CM)could make ER~+breast cancer resistant to TAM therapy.In terms of mechanism,Western blotting,sphere formation and clone formation assays determined that the Huc-MSC supernatant treated with TAM could significantly enhance the stemness of breast cancer cells,significantly enhance the sphere-forming and clone-forming ability of breast cancer cells.Further bio-plex demonstrated that TAM-treated Huc-MSC might enhance the stemness and malignant phenotype of breast cancer cells and induce drug resistance of TAM by secreting IL-6.Transcriptome sequencing and Western blotting showed that breast cancer cells enhanced the stemness of breast cancer cells and promoted TAM resistance via activating the WNT/β-catenin signal pathway.Finally,we used the IL-6 antibody to neutralize IL-6 in the supernatant of Huc-MSC.The results showed that after neutralizing IL-6,the WNT pathway in breast cancer cells was significantly inhibited.It is proved that Huc-MSC treated with TAM activates WNT/β-catenin signal pathway in breast cancer cells via secreting IL-6,and then enhances tumor stemness and TAM resistance.Conclusion:Our study confirmed the reciprocal relationship between TAM-treated Huc-MSCs and tumor parenchymal cells.TAM-treated Huc-MSCs act on ER~+breast cancer cells by secreting IL-6,which promotes tumor stemness and endocrine resistance by activating the WNT/β-catenin signaling pathway.Our study elucidates the mechanism of endocrine resistance in ER~+breast cancer,thus providing a new idea for endocrine therapy of ER~+breast cancer.