Screening Of Breast Cancer Angiogenesis Related Genes And Expression And Clinical Significance Of ACKR1 In Breast Cancer

Background: Breast cancer is the most common malignancy in women and the leading cause of cancer-related deaths in women worldwide.Despite significant advances in early diagnosis,surgical intervention,and local and systemic adjuvant therapy,mortality from breast cancer remains high.In recent years,molecular targeted therapies have played an important role in the individualized treatment of breast cancer.For example,trastuzumab,an anti-HER2 monoclonal antibody,has been shown to improve survival in breast cancer patients;however,it targets only some molecular subtypes of breast cancer and the prognosis remains suboptimal.Angiogenesis is a key factor in the development and metastasis of many cancers and also plays a critical role in the development and progression of breast cancer.Therefore,effective therapeutic targets and prognostic biomarkers are needed to improve the prognosis of breast cancer patients.Objective: To explore the angiogenic subtypes of breast cancer and reveal the etiology and molecular characteristics of breast cancer.To screen out the genes associated with breast cancer angiogenesis.The molecular characteristics of ACKR1,a gene related to breast cancer angiogenesis,and its relationship with breast cancer angiogenesis were described,and ACKR1 was experimentally demonstrated as a possible biomarker for breast cancer diagnosis and prognosis.The relationship between ACKR1 and clinicopathological features was analyzed in the context of clinicopathological indicators of breast cancer patients,and the significance of ACKR1 in the prognostic assessment of breast cancer patients was discussed.Methods: 1.Based on the angiogenesis gene set derived from Ami GO2 and the breast cancer data in the Cancer Genome Atlas(TCGA),establish a new angiogenesis subtype model through consensus clustering;2.Analyze the genes and mi RNAs regulation of this subtype,And screen out the breast cancer angiogenesis-related gene ACKR1;3.Explore the immune landscape,molecular and clinical features and enrichment methods of breast cancer angiogenesis subtypes;4.Collect cancer and normal tissue samples from breast cancer patients,and pass Western Blot,q RT-PCR,and immunofluorescence techniques were used to detect ACKR1 m RNA and protein expression levels in cancer and normal tissues.Results:1.A total of 1094 breast cancer patients and 474 angiogenic genes were obtained in this study,and two angiogenic subtypes were established by consensus clustering,with subtype 1 including 275 patients and subtype 2including 813 patients.2.A total of 643 differentially expressed genes(top ten up-regulated genes(ADH1B,SCGB2A2,ADIPOQ,CILP,TFF1,C7,OGN,FABP4,ABCA8,and ACKR1)and down-regulated genes(PRAME,CXorf61,MAST1,ART3,RCOR2,ROPN1,A2ML1,HORMAD1,MSLN and CA9))and 109 mi RNAs.3.53 GO(Gene Ontology)and 20 KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichments were revealed,including extracellular matrix structural components and protein digestion and uptake,and ECM-receptor(extracellular matrix-receptor)interactions.In addition,GSEA(Gene Set Enrichment Analysis)showed that the enriched marker pathways in subtype 2were associated with cancer progression,such as TGF beta signaling,KRAS signaling upstream,P53 pathway,inflammatory response,and IL2/STAT5 signaling.Notably,four pathways closely associated with breast cancer progression were enriched in subtype2,including estrogen response early estrogen response late,epithelial-mesenchymal transition(EMT),and especially angiogenesis.In addition,the mutation site tumor heterogeneity and tumor mutation burden were significantly higher in the non-angiogenic subtypes than in the angiogenic subtypes.In addition,stromal score,immune score and ESTIMATE score were significantly higher in the angiogenic subtype than in the angiogenic subtype,whereas tumor purity was greatly reduced in the angiogenic subtype.Finally,14 of the 15 immune checkpoints were highly expressed in the angiogenic subtype,indicating that the immunosuppression in these patients was caused by breast cancer.4.The results of Western Blot,q RT-PCR,and immunofluorescence techniques showed that the relative expression of ACKR1 protein in cancer and normal tissues were 0.67±0.45 and 1.42±0.98,respectively,and the expression of ACKR’s protein in cancer tissues was significantly higher than that in normal tissues,and the difference was significant(P<0.05);ACKR1 m RNA The relative expression in cancer and normal tissues were 0.19±0.08 and 0.32±0.09,respectively,and the expression of ACKR1 m RNA in normal tissues was significantly higher than that in cancer tissues,and the difference was significant(P<0.05);in addition,the immunofluorescence technique again demonstrated that the expression content of ACKR1 protein in normal tissues was higher than that in cancer tissues.Combined with the clinicopathological data of patients,we found that the ACKR1 m RNA expression level was correlated with lymph node metastasis and clinical stage of patients(P < 0.05),while it was not correlated with patients’ age,menopause,tumor size,histological grade and ki-67 and P53 proteins(P > 0.05).Conclusions:1.This study established a novel model of breast cancer angiogenic subtype by histological analysis and characterized the immune microenvironment and genomic alterations in breast cancer,which may provide new evidence for inhibiting breast cancer progression and immunotherapeutic response.2.Biological analysis revealed that ADH1 B,SCGB2A2,ADIPOQ,CILP,TFF1,C7,OGN,FABP4,ABCA8 and ACKR1 may be favorable prognostic factors for breast cancer,while PRAME,CXorf61,MAST1,ART3,RCOR2,ROPN1,A2ML1,HORMAD1,MSLN and CA9 may be unfavorable prognostic factors for breast cancer.3.ACKR1 m RNA and protein expression levels were significantly higher in normal breast tissues than in breast cancer tissues,and ACKR1 may serve as a new biomarker for breast cancer diagnosis and prognosis,and possibly as a new target for breast cancer biotherapy.