BML-111 Inhibits EMT,Migration And Metastasis Of TAMs-stimulated Triple-negative Breast Cancer Via ILK Pathway

Background and purpose: Breast cancer(BC)is the most common cancer with the highest mortality among women in the world.Triple-negative Breast Carcinomas(TNBC)lacks the expression of ER,PR,HER2 and other common target receptors.It has higher invasiveness and obvious heterogeneity of tumor microenvironment,Compared with other types of BC,TNBC has higher recurrence rate and metastasis rate.In addition to conventional surgery and chemotherapy,there is no more effective treatment.Most of the immune cells in tumor microenvironment are tumor associated macrophages(TAMs),and type M2 TAMs are closely related to tumor growth,metastasis and prognosis.Lipoxin A4(LXA4)plays an important role in the development of many kinds of tumors.BML-111 is the most common analogue.Up to now,the treatment and effect of LXA4 and its analogues on TNBC have not been elucidated.This experiment is to study the effect of BML-111 on EMT,migration and metastasis of TNBC tumor cells induced by TAMs.We speculated that BML-111 could inhibit EMT,migration and metastasis of TNBC tumor cells.Methods:1.We collected the information and samples of triple negative breast cancer patients in the Second Affiliated Hospital of Nanchang University from 2017 to 2019,detected and compared the expression rate of E-cadherin,Ki67 index and the number of TAMs in the primary sites of breast cancer patients with or without metastasis.2.Cell experiment group:Guoup 1: TNBC cells(MDA-MB-231,4T1)were cultured in complete medium;Guoup 2: TNBC cells + BML-111 were cultured in complete medium;Guoup3: TNBC cells + BML-111 + BOC-2(lipoxin receptor antagonist)were cultured in complete medium;Guoup 4: TNBC cells were cultured in conditioned medium;Guoup 5: TNBC cells +CM+ BML-111;Guoup 6: TNBC cells + CM+BML-111 + BOC-23.The effects of BML-111 on the activity and increment of MDA-MB-231 and 4T1 cells induced by TAMs were detected;4.The effects of BML-111 on the migration and metastasis of MDA-MB-231 and 4T1 cells induced by TAMs were detected;5.The effects of BML-111 on the EMT of MDA-MB-231 and 4T1 cells induced by TAMs were detected.6.The effect of BML-111 on the expression of ILK,Akt,GSK3 β,phophorylated Akt(p-Akt),phophorylated GSK3 β(p-gsk3 β)in TAMs induced three negative breast cancer cells was detected by Western blot.7.To construct and study the effect of BML-111 on tumor growth and metastasis in animal model.Resμlts:1.Clinical specimens: the expression of E-cadherin was higher in cancer tissues of TNBC non metastasis group,and the expression of CD68 in the center of TNBC was significantly lower than that in the non metastasis group,while the expression of CD68 in the periphery of TNBC was significantly higher than that in the metastasis group.In addition,the positive rate of E-cadherin was negatively correlated with metastasis and clinical stage,but not with age,tumor size,pathological grade and Ki-67 index.The positive expression of CD68 in the central area of TNBC was positively correlated with metastasis and pathological grade.The positive expression of CD68 in the peripheral area of TNBC was higher correlated with metastasis and clinical stage.2.BML-111 can inhibit the survival rate of TAMs induced MDA-MB-231 and 4T1 cells,and BML-111 can inhibit the formation of three-dimensional cell clusters of TAMs induced MDA-MB-231 and 4T1 cells,which has a long-term effect on TNBC cells.3.Scratch test and Transwell test showed that BML-111 could inhibit the migration of TNBC cells,especially the cells induced by TAMs.4.The results of immunofluorescence showed that BML-111 could increase the expression of E-cadherin in MDA-MB-231 and 4T1 cells,especially in TAMs induced cells,but the effect of BML-111 could be blocked by BOC-2.After TAMs induction,the expression of vimentin in MDA-MB-231 and 4T1 cells increased significantly,but BML-111 inhibited the expression of vimentin.5.WB test showed that BML-111 had significant inhibition on the expression of ILK protein in TAMs induced MDA-MB-231 and 4T1 cells,but had no effect on the expression of Akt and GSK3 β protein,but BML-111 could inhibit the expression of p-Akt and p-gsk3 β protein in MDA-MB-231 and 4T1 cells,and had more obvious inhibition on TAMs induced cells.6.4T1-MIND model: BML-111 treatment group mice tumor size is about 50% of the blank control group.7.The number and relative area of lung metastasis in BML-111 group were significantly lower than those in the control group,and the Ki-67 index and F4 / 80 expression in BML-111 group were lower than those in the control group.Conclusion:1.BML-111 inhibited the proliferation,migration and EMT of TNBC cells induced by TAMs.2.BML-111 regulates the ILK/Akt/GSK3β signaling pathway in TAMs induced TNBC cells.3.BML-111 could restrain tumor growth and lung metastasis in 4T1-MIND mice.