The Role And Mechanism Of FAM64A In Endocrine Resistance Of Breast Cancer

BackgroudThe prognosis of hormone receptor-positive breast cancer patients can be improved significantly with endocrine therapy.However,about 50% patients will develop to resistance of endocrine therapy eventually.The mechanism of endocrine resistance of breast cancer is still unclear.Studies have showed that the expression of FAM64A(Family with Sequence Similarity 64)was up-regulated in a variety of tumor tissues compared with normal tissues.FAM64 A could act as an indicator of poor prognosis of cancer patients.The Gene Expression Profilling Interactive Analysis(GEPIA)database in The Cancer Genome Atlas(TCGA)database showed that there were differences in the expression of FAM64 A in different types of breast Cancer,and the expression of FAM64 A was negatively correlated with the expression of estrogen receptor 1(ESR1)which suggestes that FAM64 A might be related to endocrine resistance of breast cancer.ObjectiveWe firstly detected the expression of FAM64 A in different types of breast cancer tissues to investigate the role of FAM64 A in the breast cancer genesis and development.We used cytology tests and mouse experiments to study the effects of FAM64 A in the development and progression of breast cancer cells,we also detected the sensitivity of TAM treatment of breast cancer in different expression level of FAM64 A.We also investigated the molecular mechanism of endocrine resistance induced by FAM64 A.MethodsThe expression level of FAM64 A in different types of breast cancer tissues was predicted and analyzed through online database,and the influence of FAM64 A on the prognosis of different breast cancer tissues was analyzed.We conducted Western Blotting(WB)to detected the expression level of FAM64 A in different breast cancer cell lines,and we also analyzed the relationship between the expression of FAM64 A and ESR1.We transfected FAM64 A gene to overexpress the expession level of FAM64 A in breast cancer cell lines,and through transfection of siRNA and viral packaging shRNA of FAM64 A we silenced the endogenous expression level of FAM64 A in breast cancer cell lines.We used MTT,clonal formation and cell cycle assays to observe the effect of FAM64 A on the proliferation of breast cancer cells.We performed Transwell assays,Wound Healing assays and F-actin assays to observe the effect of FAM64 A on the migration ability of breast cancer cells.IHC staining was used to compare the effect of FAM64 A on EMT and the relationship between the expression level of FAM64 A ER.Then we detected the sensitivity of ER positive breast cancer cells to TAM treatment after overexpression of FAM64 A by MTT assay,cloning assay and animal experiments.After that we used Co-IP assays to explore the interaction between FAM64 A and TWIST1 and further more to discover the binding sites.Finally,we conducted ChIP assays and luciferase reporter assays to detecte the binding effect of between FAM64 A and the intron 7 and promoter of ER.At the sametime,we researched the binding interaction effect of between FAM64 A and Mi2/NuRD through IP and WB assays.ResultsFirstly,we found that FAM64 A is highly expressed in hormone receptor negative breast cancer tissues and cell lines.In addition,FAM64 A is positively correlated with the Basal markers such as Epidermal Growth Factor Receptor(EGFR),while FAM64 A is negatively correlated with Luminal markers such as ESR1 and FOXA1(P < 0.01).We also found that FAM64 A could significantly affect the DFS and OS of ER positive breast cancer patients,while FAM64 A had no significant effect on DFS and OS in ER negative patients.We also found that FAM64 A was overexpressed in MDA-MB-231,MDA-MB-468 and BT549 cells which had a high degree of malignancy,and FAM64 A being low-expressed in MCF7 and T47 D which had a low degree of malignancy.Overexpression of FAM64 A in MDA-MB-231、SK-BR-3 cells can promote the proliferation and invasion of breast cancer cells.Then we found that FAM64 A can promote the growth of breast cancer cells in the mice tumor tissues.The results of IHC showed that the expression levels of Ki-67,Vimentin and TWIST1 were up-regulated,while the expression levels of E-cadherin and ER were down-regulated,and overexpression of FAM64 A could upregulate the expression level of EGFR but down-regulate the expression levels of ER and FOXA1 proteins in MCF7 cells.Through collection of breast cancer patients’ clinical samples,we found that the expression level of FAM64 A was negatively correlated with the expression level of ER.In 293 FT cells,IP experiments showed that the expresstion of FAM64 A has a correlation with TWIST1.The ChIP assays and luciferase reporter assays also detected that FAM64 A gene can bind with the intron 7 and promoter of ER.At the same time,the IP assays explored the interaction between FAM64 A and Mi2/NuRD.ConclusionThere was a negative correlation between the expression of FAM64 A and ESR1.The upregulation of FAM64 A predicted a poor prognosis of breast cancer patients.The sensitivity of endocrine therapy decreased after upregulation the expression of FAM64 A.TWIST1 is a direct bind target of FAM64 A.FAM64A inhibited the transcription of ER by recruiting Mi2/NuRD complex,thus we concluded that the expression of FAM64 A may contribute to the resistance of breast cancer endocrine therapy.