Effect Of CXCR4 On Angiogenesis Of Breast Cancer Under Hypoxia And Its Mechanism

Background and Purpose:Breast cancer is the most common malignancy in women worldwide,accounting for about 11.7 percent of new cancer cases diagnosed in 2020.In China,the incidence and mortality rate of breast cancer also rank first.Current treatments such as surgery,radiation and chemotherapy have greatly improved the survival rate of breast cancer patients,but as the disease progresses,30%of breast cancers may metastasize.Some breast cancer patients have metastases even at the diagnosis stage of the disease,and this type of tumor can develop drug resistance,adversely affecting the quality of life of breast cancer patients.Therefore,it is very important to understand the specific pathogenesis of breast cancer and find a new treatment to improve the survival of patients.Hypoxia is one of the main features in the development of breast cancer,which usually promotes the survival,invasion and metastasis of breast cancer cells.The transcription factor that plays a major role in anoxic environment is hypoxia-inducing factor-1（Hypoxia-inducible factor,HIF-1）,which is composed of functional subunit HIF-αand structural subunit HIF-β.When there is insufficient oxygen in the environment,the degradation of HIF-1αwill be inhibited,resulting in increased expression of hypoxia-inducing factor-1α（HIF-1α）,so that HIF-1αcan promote metastasis and angiogenesis of breast cancer cells by inducing transcription of downstream pro-angiogenic factors,so as to maintain tumor survival and growth.Angiogenesis is the process of making new blood vessels from existing blood vessels.In the early stages of growth,due to insufficient blood supply,tumor cells will lie dormant,with a volume of no more than 2mm³,keeping themselves alive with minimal consumption.As the disease progresses,tumor cells secrete a lot of angiogenic cytokines,which promote the formation of new blood vessels to get the nutrients and oxygen they need to grow.The tumor cells that restored the blood supply began to proliferate rapidly.Blood vessels are also pathways for breast cancer metastasis.Tumor cells enter circulation through blood vessels to survive,and then colonize other tissues and organs through exosmosis.Therefore,angiogenesis is the key to tumor development.However,the angiogenesis of neoplasms is abnormal,and the blood vessels function with an abnormal vascular network of disorder,infantilism,and high permeability,which often leads to poor perfusion of the tumor.However,poorly perfused tumors will further create a hypoxic environment,making the tumors more aggressive and reducing the spread of chemotherapy drugs and the efficiency of radiotherapy.The steps of angiogenesis include the degradation of the basement membrane,migration and proliferation of endothelial cells,and finally the synthesis of a new basement membrane.The migration of endothelial cells is influenced by chemokines.Chemokines are chemotactic cytokines with small molecular weight that regulate various cellular functions such as cell activation differentiation and transport.According to the arrangement of two cysteine residues at the amino terminal,chemokines can be divided into four subtypes:XC,CC,CXC and CX3C.According to the classification of chemokine ligands,chemokine receptors can be divided into four subtypes:XCR,CCR,CXCR and CX3CR,all of which are G-protein-coupled receptors.Chemokines bind to their receptors to play a biological role.Stromal cell derived factor-1（SDF-1,CXCL12）is a homeostatic chemokine that controls angiogenesis,hematopoiesis,and embryogenesis.However,more and more studies have shown that it mediates the growth,metastasis and angiogenesis of primary tumors.CXCR4,which binds only to its unique ligand CXCL12,is a highly conserved 7-transmembrane G-protein-coupled receptor that is strongly expressed in many types of cancer,including breast cancer.In breast tumors,endothelial cells with high levels of CXCR4 expression migrate along CXCL12 gradients.CXCR4+angiogenic cells include immature and mature hematopoietic cells,endothelial progenitors,and smooth muscle cell progenitors,which have direct or indirect angiogenic properties.CXCL12 plays a role in the mobilization and recruitment of these cells,supporting revascularization and tumor growth in cells and tissues lacking blood supply.It has also been found that the accumulation of HIF-1αin the state of hypoxia can also promote the expression of CXCR4.Therefore,finding the association between hypoxia,chemokines and angiogenesis is crucial for the treatment of breast cancer angiogenesis.This study will discuss the effect and mechanism of CXCR4 on breast cancer angiogenesis under hypoxia conditions.Method:1.CoCl₂ was used as the inducer of hypoxia to simulate the cell hypoxia of breast cancer cells under the normal oxygen state.2.Western blot was used to detect the main hypoxia regulatory factors,CXCR4,the expression of cell proliferation and angiogenesis related proteins（HIF-1α,CXCR4,PCNA,VEGF-A,CD31）and the expression of related signaling pathway proteins Akt/mTOR.3.The expression of VEGF-A was detected by ELISA.4.Angiogenesis assay to detect the ability of cell angiogenesis.Experimental results:1.CoCl₂ simulated the hypoxia of breast cancer cells and detected the expression of HIF-1αin hypoxia cell lines.It was found that the expression of HIF-1αwas increased in the hypoxia of MCF-7 and MDA-MB-231 breast cancer cell lines,and the proliferation and angiogenesis of the cells were enhanced.The results showed that hypoxia can promote the proliferation and angiogenesis of breast cancer cells.2.Transient transfection of CXCR4-c DNA and CXCR4-si RNA showed that CXCR4expression was also increased in anoxic cell lines.Cell proliferation and angiogenic protein expression were enhanced in the hypoxia group,in the CXCR4 overexpression group,and in the hypoxia group combined with CXCR4 overexpression group,while cell proliferation and angiogenic protein expression were decreased in the CXCR4 down-regulated group.The results indicated that hypoxia can increase the expression of CXCR4,which can promote the proliferation and angiogenesis of breast cancer cells,and hypoxia and CXCR4 have a synergistic effect.3.Same as the above experimental treatment,culture medium of breast cancer cells was collected for ELISA and angiogenesis experiments respectively.In the angiogenesis experiment,HUVEC cells were cultured using breast cancer cell culture medium as conditioned medium.We found that in the hypoxia group,CXCR4 overexpression group and hypoxia combined with CXCR4 overexpression group,VEGF-A and angiogenesis ability were enhanced,while in the CXCR4 down-regulated group,VEGF-A and angiogenesis ability were decreased.4.Western blot assay detected the expression of Akt/mTOR in related signaling pathways,and the results showed that the expressions of p-Akt and p-mTOR were enhanced in hypoxia group,CXCR4 overexpression group and hypoxia combined with CXCR4 overexpression group,while in CXCR4 down-regulated group,the expression of p-Akt and p-mTOR was enhanced.The expression of p-Akt and p-mTOR was decreased.In addition,we used PI3K/mTOR pathway inhibitor PF-04691502,and Western Blot results showed that down-regulated CXCR4 and administration of the pathway inhibitor resulted in the lowest protein expression of p-Akt and p-mTOR.In conclusion,these results suggest that hypoxia may induce the expression of CXCR4 and VEGF through HIF-1α,and CXCR4 may also promote the expression of VEGF,thereby regulating cell proliferation and angiogenesis through the Akt/mTOR signaling pathway.This study will verify the mechanism of breast cancer angiogenesis and provide theoretical basis for the treatment of breast cancer.