The Effects And Mechanisms Of Marine-derived Compound Asperolide A Aganist Triple Negative Breast Cancer Induced Bone Destruction

1.BackgroundsBreast cancer is a common malignant tumor that is accompanied by significant incidence(24.2%)and mortality(15%)in the world.Triple negative breast cancer(TNBC)is a molecular subtype of clinical breast cancer with negative expression of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2),accounting for about 15-20% of all breast cancer cases.Compared with hormone receptor-positive breast cancers,TNBC conveys more aggressive invasiveness and more likely to cause metastasis,resulting in less survival time of patients.Bone is reported as the most common distant metastasis site in TNBC,especially osteolytic bone metastases.Hence,inhibition of osteolytic lesions and regulation crosstalk between metastatic TNBC cells and bone microenvironment are the keys to treat advanced TNBC.Bisphosphonates is the recommended drug for cancer with bone metastasis,which could inhibit osteoclast activity,and protect against tumor metastases.However,it could not promote bone repair and inhibit TNBC progression.Therefore,screening novel compounds is very important.Screening lead compounds against tumor from natural products is an important way for drug development.The sources of natural products are diverse.Marine is called “blue drug bank”.The living environment of marine organisms is special and diverse(for example: low temperature,high salt,low oxygen,and high pressure,etc.),so their metabolites have diverse structures and special activities,providing abundant lead compounds for drug research and development.Seaweed is an important part of marine biological resources.Seaweed and its epiphyte extracts contain a large number of bioactive substances.According to the theory of traditional chinese medicine,seaweed has the effect of regulating qi and dispersing knots,which can be used to treat breast hyperplasia,fibroadenoma and other diseases,and also has a potential role in the prevention and treatment of osteoporosis.Wentilactone A,Wentilactone B,Botryosphaerin B,LL-Z1271-?,Asperolide A,Asperolide B,Asperolide C are a series of tetranorditerpene dilactone derivatives isolated from the secondary metabolites of marine microorganisms.The preliminary screening results showed that these compounds have anti-tumor and anti-inflammatory activities.Thus,our study was to investigate the effects of Asperolide A against anti-TNBC bone metastasis.2.ObjectsTo demonstrate the inhibitory effects of Asperolide A against the malignant phenotype of cancer such as proliferation,migration,invasion and metastasis.To clarify the molecular mechanism of Asperolide A against TNBC.To confirm the effects and mechanisms of Asperolide A against osteoclast differentiation,osteolysis and bone destruction in advanced TNBC.To explore the clinical significance of Asperolide A's target in TNBC.All in all,to lay the foundations for the development of new drugs for TNBC.3.Research methodsFirstly,we cultured human breast cancer cell lines MDA-MB-231,MDA-MB-463,and normal breast cell line MCF-10 A,then tested the cytotoxicity of Asperolide A.Wound healing test,transwell chamber shuttle invasive test,cell counting kit-8(CCK-8)experiment and flow cytometry test were performed to verify the effects of Asperolide A on the malignant phenotypes of TNBC cells.A metastatic model of TNBC in nude mice was performed to evaluate the in vivo effect of Asperolide A.In order to search the potential targets and confirm the mechanisms,we used Swissdock server to predict the protein targets of Asperolide A,modeling binding mode between Asperolide A and target proteins.Surface plasmon resonance(SPR)technology was used to analyze the kinetics of the reaction between Asperolide A and target proteins.Fluorescent probe labeling technology was used to show the binding site of Asperolide A in living TNBC cells.RNA interference technology,real-time quantitative polymerase chain reaction(q RTPCR),western blot,enzyme linked immunosorbent assay(Elisa)and immunofluorescence(IF)technology were used to test the changes of target genes and proteins.Oncomine,UALCAN databases and tumor tissue microarray chip were used to analyse the expression of target protein in TNBC patients.Then we cultured bone marrow-derived macrophages cells(BMMs),and performed tartrate resistant acid phosphatase(TRAP)staining,F-actin staining,hydroxyapatite test and reactive oxygen species(ROS)assay.At the same time,we cultured bone marrow stromal cells(BMSCs),and then alkaline phosphatase(ALP)staining and alizarin red(AR)staining were performed.4.Research resultsOur results showed that Asperolide A could suppress the phenotypes of TNBC cell lines.Besides,Asperolide A could inhibit receptor activator for nuclear factor-?b ligand(RANKL)secretion in TNBC cells.Bioinformatics analysis predicted that c-KIT was the potential target of Asperolide A,and the binding sites were tyrosine residues 568 and 570.Binding score was-6.93 kcal/mol.Besides,Asperolide A labeled with 5-carboxyfluorescein was clustered on the membrane of TNBC cells.After knockdown of c-KIT,the inhibitory effect of Asperolide A on TNBC cells was reduced.Asperolide A inhibited phosphorylation of phosphoinositide 3-kinase(PI3K),protein kinase b(PKB/AKT),mammalian target of rapamycin(m TOR)proteins.Compared with non-TNBC,expression of c-KIT was significantly upregulated in TNBC.The expression of c-KIT was associated with the stage of breast cancer.Besides,western blot results demonstrated that Asperolide A could inhibit the activation,differentiation,and function of osteoclasts via nuclear factor kappa B(NF?-B),c-Jun N-terminal kinase(JNK),and PI3K/AKT/m TOR signaling pathways.Animal model also confirmed that Asperolide A could inhibit TNBC induced bone destruction.5.ConclusionThough our research,we have demonstrated that Asperolide A,a small molecule compound derived from marine microorganisms,could inhibit activation of cKIT/PI3K/AKT/m TOR signaling pathway,could suppress the phenotypes and RNAKL secretion of TNBC cell lines,and could inhibit the activation and differention of osteoclasts via NF?-B and JNK pathway in vitro and bone destruction induced by TNBC in vivo.Asperolide A has potential to become a new anti-TNBC and secondary bone destruction drug.