Effect And Mechanism Of Metformin On Breast Density In Overweigt/obese Premenopausal Women And The Breast Tumor-associated Adipose Microenvironment

Overweight/obesity,as an independent risk factor for breast cancer,increased the prevalence of breast cancer by 30-50%,which could cause multiple metabolic disorders,including insulin resistance,changes in expression and secretion of fat factors caused by chronic inflammation..At present,it is generally accepted that overweight/obese postmenopausal women have an increased risk of breast cancer.However,it is still controversial about the risk of breast cancer in overweight/obese premenopausal women.Therefore,we further explored the relationship between overweight/obesity and breast cancer risk in Chinese overweight/obese premenopausal women.There exists a certain correlation between obesity and breast density,which are both independent risk factors for breast cancer.Metformin(MET),a hypoglycemic drug,has attracted widespread attention in recent years for its anti-tumor effect.Substantial studies have showed that metformin had a certain adjuvant effect on prevention of breast cancer,but specific mechanism is not completely clear.Whether metformin can affect breast density to reduce the risk of breast cancer is still unknown.Addtionally,obese individuals diagnosed with breast cancer have higher risks of distant metastasis.Since obesity was closely related to cancer,and adipocytes were considered to be an important part of the tumor microenvironment(TME).The crosstalk between adipocyte and tumor cell continuously resmodel the TME,gradually forming a unique tumor-associated adipose microenvironment(TAME).Research showed that metformin can achieve anti-tumor effects by affecting the tumor microenvironment,such as inhibiting angiogenesis signals in the tumor microenvironment,affecting the formation of tumor-related fibroblasts,affecting tumor-related macrophages,inhibiting immune response,etc..However,the effect of metformin on tumor-related adipocytes(CAAs)in the microenvironment of breast cancer is still unclear.This study intends to discuss the aforementioned issues from both clinical and basic medicinal aspects.Experimental methods and results1.Study on the effect and mechanism of metformin on breast density in premenopausal obese women.ObjectiveTo explore the effect of metformin on breast density in premenopausaloverweight/obese women and its possible mechanism.MethodsSocial overweight/obese menopausal women recruited from April 1,2020 to June 31,2020 were randomly divided into metformin group(experimental group)and control group(placebo group),with diet and exercise guidance and follow-up for 12 months.During the trial,the changes in breast density and pathological biopsy positive rate of the population were mainly observed,while the secondary observation data included weight,body mass index(BMI),waist circumference,hip circumference,fasting blood glucose(FPG),fasting insulin(FIN),insulin resistance index(HOMA-IR),and low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),triglycerides(TG),total cholesterol(TC),systolic blood pressure(SBP),diastolic blood pressure(DBP),follicle stimulating hormone(FSH),estrogen(E),adipondecin(APN).ResultsA total of 120 overweight/obese women completed a one-year trial.In the control group,there were 7 cases of BI-RADS grade greater than level 4,of which 5 cases were positive for biopsy;in the metformin group,2 cases of cases of BI-RADS grade was greater than level 4,of which 1 case was positive for biopsy.Compared with the control group,the weight(61.8±4.3 VS 58.3±5.3,P<0.001),BMI(25.8±2.0 VS 23.1±1.5,P<0.001),waist circumference(87.5±6.51 VS 81.4±4.21,P<0.00),FPG(5.5±0.5 VS 5.0±0.6,P<0.001),FIN(19.6±4.5 VS 7.9±1.8,P<0.001),HOMA-IR(4.8±1.2 VS 1.7±0.4,P<0.001)and BI-RADS grading of the metformin group were improved,the APN significantly increased(3.4±0.9 VS 4.6±1.0,P<0.001),and the positive rate of pathological biopsy decreased(8.33±0.28 VS 1.67±0.13,P=0.01).Relevance analysis showed that BI-RADS grading was related to weight(P<0.001),BMI(P<0.001),FIN(P=0.039),HOMA-IR(P=0.044),APN(P<0.001)and the positive rate of pathological biopsy was related to weight(P=0.033),BMI(P<0.001),APN(P=0.001)and BI-RADS grading(P<0.001).Logistic regression analysis showed that family history of breast cancer(P=0.001),FPG(P=0.048),HOMA-IR(P=0.001),LDL-C(P=0.023)and APN(P=0.005)were risk factors for BI-RADS;family history of breast cancer(P=0.007),age(P=0.011),APN(P=0.004)and BI-RADS grading(P=0.023)were risk factors for biopsy positive rate.Conclusions1.Metformin combined with lifestyle intervention improved the body weight,BMI,waist circumference,FIN,insulin resistance,APN levels and breast density,and reduced the pathological biopsy positive rate of breast cancer in premenopausal overweight/obese women.2.Breast cancer family history,fasting blood glucose,HOMA-IR,LDL-C and APN were risk factors for BI-RADS classification.3.Breast cancer family history,age,APN and BI-RADS classification were biopsy risk factors of positive rate.2.The regulation of metformin based on cytokines affects the function of breast cancer cells onadipocytes.ObjectiveTo construct the breast cancer tumor-associated adipose microenvironment and investigate the effects of metformin on the formation of tumor-associated adipocytes formation and cytokine production.MethodsThe 4T1 breast cancer cells and mature adipocytes induced by 3T3-L1 pre-adipocytes was co-cultivated in transwell system,triglyceride content of adipocytes was detected,which was further verified by oil red O.Inflammatory cytokines IL-1β,IL-6 and TNF-α,and adipocytokines APN,leptin,resistin and visceral adipotin were detected by ELISA and RT-qPCR to observe whether 4T1 breast cancer cells promoted the transformation of mature adipocytes into CAAs.At the same time,metformin was given to intervene mature adipocytes,and the above cytokines were detected by ELISA,RT-qPCR and Western Blotting.The apoptosis of adipocytes was detected by flow cytometry,to observe whether the effect of 4T1 on the transformation of mature adipocytes into CAAs changed after metformin intervention.ResultsThe 3T3-L1 mature adipocytes were co-cultured with of 4T1 breast cancer cells,the adipocytes became smaller,the fat droplets decreased,and the triglyceride content reduced(2.75±0.18 VS 1.13±0.07,P<0.001).In the supernatant,the content of inflammatory factors IL-1β(498.03±62.65 VS 1104.47±112.04,P=0.001),IL-6(127.03±28.19 VS 451.97±111.47,P=0.008)and TNF-α(565.87±57.32 VS 1409.27±125.13,P<0.001)increased,the content of adipocytokines ANP(2692.63±173.60 VS 1116.87±116.66,P<0.001),leptin(1017.40±139.60 VS 682.27±91.23,P=0.025)and resistin(490.03±26.34 VS 222.13±31.88,P<0.001)decreased significantly.The mRNA and protein expressions of inflammatory cytokines IL-1 β,IL-6 and TNF-α increased in adipocytes,while the mRNA and protein expressions of adiponectin,leptin,resistin and visfatin decreased in adipocytes,suggesting that 4T1 breast cancer cells promoted the transformation of mature adipocytes into tumor-associated adipocytes.After the intervention of metformin,the levels of IL-1β,IL-6 and TNF-α decreased,the APN level increased,the level of leptin further lower,resistin and visfatin did not change significantly,the apoptosis of adipocytes increased。ConclusionsMetformin can inhibit the formation of CAAs in tumor-associated adipose microenvironment and suppress the production of tumor-associated adipocytes inflammatory factors,improving adiponectin and leptin levels and promoting apoptosis of CAAs.3.Metformin inhibits the development of breast cancer by regulating the interaction between adipocytes and breast cancer cells.ObjectiveTo observe the effects of metformin on proliferation,migration and invasion of breast cancer cells after treated with adipocytes.MethodsTranswell system co-culture and adipocytes supernatant culture were adopted.In the methods of culturing 4T1 breast cancer cells with adipocytes supernatant,adipocytes conditioned medium(Ad-CM)and tumor-associated adipocytes conditioned medium(CAA-CM)were collected to culture 4T1 breast cancer cells,and metformin intervention was given or not,to detect 4T1 breast cancer cell proliferation.The effect of mature adipocytes on migration and invasion of 4T1 breast cancer cells was observed by co-culture using the Transwell system.After metformin intervention,the effect of metformin on migration and invasion of breast cancer cells was also observed.ResultsAfter intervention with metformin,the proliferative ability of 4T1 breast cancer cells cultured in three kinds of medium decreased(P<0.01).There was no significant change in the proliferative ability of 4T1 breast cancer cells cultured with Ad-CM and CAA-CM.The migration and invasion of 4T1 breast cancer cells were enhanced after co-cultured with mature adipocytes or cultured in CAA-CM(P<0.01).There was no significant change in the migration and invasion ability of 4T1 breast cancer cells in Ad-CM culture.After metformin intervention,the migration and invasion ability of 4T1 cells decreased,while metformin had stronger inhibition on the migration and invasion of 4T1 breast cancer cells co-cultured with mature adipocytes.ConclusionsMetformin can inhibit the proliferation,migration and invasion of breast cancer cells by regulating the interaction between adipocytes and breast cancer cells in TAME.