The Study On Efficacy And Mechanism Of Anlotinib In The Treatment Of Metastatic Breast Cancer

Background:Breast cancer is the malignant tumor with the highest incidence in the world,and the median time of survival in metastatic breast cancer is about 2-3 years.HER2-negative breast cancer lacks specific targeted therapy.So it has been a hot and difficult issue in the current research field to find other targeted therapy drugs that can improve the therapeutic effect while alone or combined with chemotherapy.Tumor angiogenesis plays an important role in tumor growth and invasion.Anlotinib is a new type of anti-angiogenesis small molecule tyrosinase inhibitor,which targets the vascular endothelial growth factor receptor(VEGFR)-1(Fltl),VEGFR-2,VEGFR-3(Flt4),platelet-derived growth factor receptors(PDGFRs)and c-KIT,inhibiting tumor angiogenesis and tumor cells proliferation,it is effective in advanced non-small cell,soft tissue sarcoma and so on.This study aims to explore the efficacy,safety and biomarkers of anlotinib in the treatment of HER2-negative metastatic breast cancer.Methods:Recruitment criteria are patients aged between 18-75 years old with HER2-negative metastatic breast cancer who have received at least first-line chemotherapy and endocrine therapy for hormone receptors-positive breast cancer patients after metastasis,and have received anthracyclines or taxel in neoadjuvant,adjuvant,or post-metastasis therapy.During the research,anlotinib was taken 12 mg daily for 14 consecutive days and 21 days as a cycle.The patients received CT or MRI at baseline and every 2 cycles during treatment for efficacy evaluation until the disease progressed or unacceptable toxicity occurred.The primary research endpoint is the objective response rate(ORR),and the secondary research endpoints include disease control rate(DCR),progression free survival(PFS),and overall survival(OS),safety and biomarkers.At the same time,10 mL of venous blood was collected every 2 treatment cycles for circulating tumor DNA(ctDNA)detection.Hybrid capture high-throughput sequencing is used to detect mutations(Mutant),copy number variants(CNV),single nucleotide variants(SNV),structure variation(SV),exploring the relationship between ctDNA variation and efficacy or adverse events.Results:A total of 26 patients were enrolled in this study,with a median age of 56(30-75).The median follow-up time was 10.5 months.Among them,4 patients achieved partial response,ORR was 15.4%,17 patients achieved stable disease,DCR was 80.8%,and median PFS was 5.22 months(95%confidence interval 2.86-6.24).During the follow-up period,2 patients died without reaching the median OS,and 14 patients(53.8%)survived for more than 10 months.The most common treatment-related adverse events were hypertension(57.7%),elevated thyroid-stimulating hormone(34.6%)and hand-foot syndrome(23.1%).The median PFS of patients with hand-foot syndrome was longer than that of those without(median PFS was 5.22 vs.2.86,P=0.62).Peripheral venous blood was collected from 17 patients for ctDNA testing.Among them,17 patients were collected baseline blood samples,and 11 patients were collected peripheral blood samples at baseline and at least one efficacy evaluation point.The main types of gene changes are copy number variants(CNV),point mutations and structure variation(SV).The most common mutant genes are TP53(11/17,64.7%),PIK3CA(7/17,41.2%),BRCA1(3/16,17.6%)and ESR1(3/16,17.6%).The median PFS of patients with TP53 mutation(P=0.057)or PIK3CA variant allele frequency(VAF)greater than 1%(P<0.01)was significantly shortened.The change trend of ctDNA VAF measured in 81.7%of patients was consistent with the change in tumor burden detected by imaging.Conclusions:(1)Anlotinib has potential curative effects and the adverse events is tolerable in the second-line and further HER2-negative metastatic breast cancer;(2)The appearance of hand-foot syndrome during treatment may be a predictor of efficacy.(3)The presence of TP53 mutation and VAF of PIK3CA greater than 1%may be a predictive biomarker of poor efficacy;(4)The dynamic changes of ctDNA variant alleles may predict tumor burden.Background:Triple negative breast cancer(TNBC)refers to breast cancer in which the expression of estrogen receptor,progesterone receptor and human epidermal growth factor receptor-2 are all negative,accounting for about 20%of the total.TNBC has a low age of onset,strong invasiveness,easy recurrence and metastasis,and lack of effective therapeutic targets.Anlotinib is a class 1.1 anti-angiogenic small molecule tyrosine kinase inhibitor independently developed by my country,and it has shown potential efficacy in metastatic TNBC breast cancer.However,there are still some patients who are not sensitive to anlotinib.Exploring the factors that affect the sensitivity of Anlotinib is a key issue that needs to be resolved urgently.Methods:(1)In this experiment,MDA-MB-231 and HCC38 were selected as the experimental objects,and the cell viability was detected by CCK-8 experiment by culturing the above-mentioned cells and treating them with anlotinib;(2)Treating MDA-MA-231 cells with anlotinib and performing transcriptome sequencing to explore the targets of anlotinib;(3)MDA-MA-231 and HCC38 were treated with anlotinib of 0,2.5,5,10,20,40uM and 24,48,72 hours respectively.Western Blot was used to detect the expression of Smurf2 and related autophagy proteins;(4)Transfect Flag-Smurf2 into MDA-MA-231 by plasmid transfection method,and then give them anlotinib.Western Blot were used to detect the expression of Smurf2 and related autophagy proteins;(5)Detect the interaction between Smurf2 and ATG7 by immunoprecipitation;overexpression of Flag-Smurf2 with 0,1,2,4ug and use western Blot to detect the expression of ATG7;(6)Construct an MDA-MB-231 nude mouse xenograft tumor model,and treated with anlotinib,then compare the tumor volume change within two groups.Western Blot were used to detect the expression of Smurf2,ATG7 and related autophagy proteins in tumor tissue,and use immunohistochemistry to detect the expression of Smurf2 in tumor tissue.Results:(1)Anlotinib can inhibit the viability of TNBC cells;(2)Anlotinib targets include PI3K-AKT pathway,MAPK pathway,Rapl signal transduction pathway,etc.,which are related to cell autophagy;Anlotinib inhibits the transcription of E3 ubiquitin ligase Smurf2;(3)As the concentration of Anlotinib increases,the expression of Smurf2 in the cell gradually decreases,and the level of autophagy gradually increases;(4)Overexpression of Flag-Smurf2 can inhibit autophagy induced by Anlotinib;(5)Smurf2 and ATG7 can interact with each other;(6)Gradient overexpression of Flag-Smurf2 can reduce the expression of ATG7;(7)Anlotinib can induce autophagy in vivo,inhibit the expression of Smurf2,and increase the expression of ATG7Conclusions:Anlotinib inhibits protein degradation mediated by the ubiquitin-proteasome pathway by down-regulating Smurf2,causing the accumulation of ATG7 and then inducing autophagy in TNBC cells,which ultimately leads to a decrease in drug sensitivity.