The Function And Mechanism Of Dopamine Receptor DRD2 In Breast Cancer

ObjectiveAccording to the latest Cancer Statistics,breast cancer is the most common cancer and the second leading cause of cancer-related death in female.The 5-year relative survival rate has increased with the development of mammography screening,but there is still a lack of effective treatment for patients with recurrent and metastatic breast cancer.Tumor suppression genes(TSGs)play an important role in the development of breast cancer.Identifying new TSGs and revealing their molecular mechanisms will help to provide new biomarkers for prognosis prediction and comprehensive treatment.TSGs are often located on regions of loss of heterozygosity(LOH),and their expression is often reduced or even silenced due to promoter methylation.To identify new potential TSGs in breast cancer,we have obtained breast cancer tissues and normal breast tissues and then performed RNA sequencing(RNA-seq)assay.And a potential TSG was selected.The mRNA expression and promoter methylation status were analyzed in multiple online databases.Then its mRNA expression,promoter methylation status,biological functions and possible molecular mechanisms were further determined in breast cancer.This study would help to reveal the possible molecular mechanisms of the selected TSG in breast cancer,which might provide experimental basis of a potential new biomarker in breast cancer.Methods1.The obtained breast cancer tissues and normal breast tissues were applied to RNA-seq for differences in gene expression at the transcriptional level.Among the top 30 genes with significant differences,Dopamine Receptor D2(DRD2)was selected for further study.The online database Meth HC database was used to analyze the mRNA expression and promoter methylation status of DRD2 in breast cancer tissues and normal breast tissues.The online database TCGA was also used to observe the mRNA expression and promoter methylation status of DRD2 in normal breast tissues and breast cancer tissues.The Immunohistochemistry(IHC)was used to detect protein expression level of DRD2 in normal and breast cancer tissues.The kmplot database was used to explore the effect of DRD2 expression in breast cancer tissues on the survival time of patients with different phenotypes of breast cancer.Furthermore,TCGA was further analyzed to explore the correlation between DRD2 mRNA expression level or promoter methylation status and clinical characteristics of breast cancer patients.2.The mRNA expression levels of DRD2 were detected in human breast cancer cell lines and normal immortalized epithelial cell lines using Reverse transcription polymerase chain reaction(RT-PCR),and Methylation specific polymerase chain reaction(MSP)was further used to determine DRD2 promoter methylation status in human breast cancer cell lines and normal immortalized epithelial cell lines.To further confirm whether the loss or downregualted DRD2 expression is regulated by promoter methylation,the demethylation agent was used in DRD2expression-silenced MDA-MB231 and BT549 cells,and q RT-PCR was used to further examine DRD2 expression.3.Cell lines stably expressing DRD2 were constructed in human breast cancer cell lines MDA-MB21 and BT549.The effects of DRD2 on the biological functions of breast cancer cells in vitro were examined using the following experiments: Cell Counting Kit-8(CCK8),colony formation assay,and soft agar formation assay were used to detect the effect of ectopic overexpressed DRD2 on breast cancer cell growth.Flow cytometer(FCM)and Acridine orange/Ethidium bromide(AO/EB)assay were further applied to investigate the effects of overexpressed DRD2 on the cell cycle and apoptosis respectively.Would healing assay,migration assay and invasion assay were applied to determine the effect of overexpressed DRD2 on the metastatic ability of breast cancer cells.The murine breast cancer cell line 4T1 stably expressing DRD2 was used to construct the subcutaneous tumorigenic model to investigate the effect of DRD2 on the development of breast cancer in vivo.And a co-cultured system of breast cancer cells and macrophages was constructed by using Transwell? to study the effects of DRD2-expressing breasrt cancer cells on breast cancer-associated macrophages.And the effects of effect of breast cancer-associated macrophages on DRD2-expressing breast cancer cells were also investigated4.Western Blot(WB),Co-immunoprecipitation(Co-IP),the Antibody Array,Mass Spectrometry,real-time fluorescence quantitative polymerase chain reaction(q RT-PCR),Hematoxylin and Eosin(HE)staining,immunofluorescence(IF)and IHC were further used to investigate the molecular mechanism of DRD2 on breast cancer development.Results1.mRNA and protein levels of DRD2 were lower in breast cancer tissues compared to normal tissues.Meanwhile,the promoter of DRD2 was in a hypermethylated state in breast cancer.DRD2 mRNA expression was restored after using the demethylation agent 5-aza-2-deoxycytidine(Aza)in DRD2-silenced breast cancer cell lines.2.In vitro,ectopic overexpressed DRD2 inhibited breast cancer cells growth and metastasis ability of breast cancer cells.DRD2 also blocked the cell cycle at G2/M phase.DRD2 significantly inhibited epithelial mesenchymal transition(EMT).Ectopic expression of DRD2 promoted apoptosis in vitro and in vivo.DRD2 also promoted necroptosis of breast cancer.In vivo,ectopic expression of DRD2 inhibited growth of breast cancer.3.During the crosstalk between breast cancer cells and macrophages,macrophages induced pyroptosis of breast cancer cells,which process was executed by GSDME in a DRD2-dependemt manner.Meanwhile,DRD2-expressing breast cancer cells reprogrammed breast cancer-associated macrophages to M1 phenotype.4.Overexpression of DRD2 was more common in HER2-patients,and DRD2 overexpression prolonged survival times of HER2+ patients.mRNA expression levels of EGFR and HER2 in breast cancer cells were suppressed by DRD2 overexpression,and DRD2 protein binds to EGFR.5.DRD2 could be induced endocytosis upon stimulation by non-selective ligands LPS or macrophages.Upon internalization,DRD2 binded and activated β-arrestin2.Activated β-arrestin2 and TAK1 competitively combined TAB1,which finally blocked the activation TAK1,the upstream of NF-κB signaling.And DRD2 also inhibited phosphorylation of p65 and the nuclear entry of p65.In resting status,DRD2 downregulated the expression of DDX5 and eEF1A2 by protein-protein interaction.And the downregulation would inhibit the proliferation and migration of breast cancer cells,as well as antagonize the activation of NF-κB signaling pathway.ConclusionIn breast cancer cells and breast cancer tissues,DRD2 expression is silenced or dowregualted due to promoter methylation.In breast cancer,DRD2 functioned as a TSG and reprogrammed the polarization of breast cancer-associated macrophages.On the one hand,DRD2 restricted the activation of NF-κB signaling pathway by interacting with β-arrestin2,DDX5 and eEF1A2;on the other hand,the GSDME-executed pyroptosis of breast cancer cells was induced by macrophages in a DRD2-dependent manner.