The Role And Molecular Mechanism Of CXCLs/ERK/LIF Signaling Pathway In Cancer-associated Adipocyte-mediated Breast Cancer Cell Migration And Invasion

The invasion and metastasis are the leading causes of death in breast cancer patients.The occurrence of a tumor is closely related to its microenvironment.Adipocytes are rich in the microenvironment of breast cancer.In the early stage of breast cancer,local cancer cells invade adipose tissue and induce adipocytes to transform into cancer-associated adipocytes(CAA).CAA exhibits altered phenotype and secrets many cytokines and chemokines,which promote the invasion and metastasis of adjacent breast cancer cells.However,its role in the breast cancer microenvironment and related molecular mechanisms is still unclear.Objective:To study how CAA in the breast cancer microenvironment affects the progression of breast cancer and the interaction between adipocytes and breast cancer cells,focusing on the role and molecular mechanism of leukemia inhibitory factor(LIF).Method:1.The preadipocytes were cultured and induced into mature adipocytes.We established a co-culture system of adipocytes and breast cancer cells with transwell chambers in vitro.Wound healing,transwell migration,and matrix gel invasion assays were used to evaluate migration and invasion ability of breast cancer cells after co-cultured with adipocytes.The expression of LIF in adipocytes and LIF content in the CAA culture medium(CAA-CM)after co-cultured with breast cancer were analyzed by Q-PCR and ELISA.Western blot was used to detect the activation of LIF downstream signals in breast cancer cells.Si RNA was used to knock down the expression of Stat3 in breast cancer cells,and the effects of CAA and LIF on migration and invasion of breast cancer cells were detected.Then,Hematoxylin-eosin(H&E)staining and immunohistochemistry were used to analyze the expression of LIF and p-STAT3 in tissue sections of breast cancer patients.2.The selective pathway inhibitors were used to explore the upstream pathways of LIF and Q-PCR was used to detect the upstream signaling pathways that regulate LIF expression in CAA.Western blot was used to analyse the regulatory relationship of the upstream signal pathway of LIF in CAA.The expression and localization of LIF upstream signal in CAA were analyzed by immunofluorescence.3.The differentially expressed genes between control and co-cultured breast cancer cells were analyzed by RNA-seq,and genes of the essential secreted proteins regulating LIF were screened and verified by Q-PCR.Q-PCR,ELISA and Western blot were used to analyze the effects of the receptor inhibitors of ELR+CXC chemokines and recombinant proteins CXCL3 and CXCL8 regulation on expression and secretion of LIF and its upstream signal pathway.The effects of recombinant CXCL3 and CXCL8 on the upstream transcription factor localization of LIF were analyzed by immunofluorescence.Q-PCR and Western blot were used to analyze the effects of CXCL3 neutralizing antibody on the expression,secretion,and activation of the upstream signaling pathway of CXCLs-induced LIF in breast cancer.The expressions of CXCL1-3 and CXCL8 in breast cancer and adjacent adipose tissue were detected by Q-PCR.Result:1.The primary human preadipocytes were induced into mature adipocytes.The adipocytes were co-cultured with breast cancer cells in the transwell chamber for 36 hours,Q-PCR results showed that the expressions of PPAR-γ and C/EBP-α,markers of adipocyte differentiation,were significantly decreased in adipocytes,while the expressions of HSL and pro-inflammatory cytokines IL-1β,IL-6,and CCL2 were significantly increased.Oil red O staining showed that lipid droplets in adipocytes co-cultured with breast cancer cells were smaller than those in adipocytes cultured alone,indicating that adipocytes transformed into CAA after co-cultured with breast cancer cells.2.After co-cultured with breast cancer cells,the level of LIF m RNA in adipocytes was significantly increased,and the content of LIF in the culture medium of adipocytes increased significantly.The results of H&E staining and immunohistochemistry showed that compared with adipocytes in normal breast tissue,adipocytes adjacent to breast cancer tissue expressed high levels of LIF.3.Both recombinant human LIF(rh LIF)and CAA-CM can significantly promote the migration and invasion of breast cancer MDA-MB-231 and BT549 cells,and also the phosphorylation of Stat3.LIF neutralizing antibodies can reverse the migration and invasion,and the phosphorylation of Stat3 of MDA-MB-231 and BT549 cells promoted by CAA-CM.4.Stattic,a specific inhibitor of Stat3,can reverse the migration and invasion of MDA-MB-231 and BT549 cells and intracellular Stat3 phosphorylation promoted by CAA-CM or rh LIF.Knockdown of Stat3 expression in MDA-MB-231 cells by si RNA significantly inhibited the migration and invasion of MDA-MB-231 cells induced by CAA-CM.The results of immunohistochemistry on clinical breast cancer tissue specimens showed that the expression of LIF in breast cancer adjacent adipocytes are positively correlated with the phosphorylation of Stat3 in breast cancer cells.5.When LY3214996 and PD98059(both are ERK1/2 inhibitors),JSH-23,and BAY 11-7082(both are NF-κB inhibitors,and the latter can inhibit p65phosphorylation)and Stattic(Stat3 inhibitor)were added into the co-cultured system,respectively,LIF m RNA expression was significantly inhibited in adipocytes.In addition,after co-cultured with MDA-MB-231 cells,ERK1/2,NF-κB p65,and Stat3 phosphorylation levels in adipocytes were significantly enhanced.Treatment of the co-cultured system with LY3214996 significantly inhibited the phosphorylation of ERK1/2,Stat3 and p65 in adipocytes.Immunofluorescence results indicate that compared with the adipocytes cultured alone,Stat3 and p65 protein of CAA in the nucleus significantly increased after co-cultured with breast cancer cells.6.The differentially expressed secretory protein genes were screened by RNA-seq in breast cancer MDA-MB-231 cells co-cultured with adipocytes,among which CXCLs(CXCL1,CXCL2,CXCL3,and CXCL8),members of the ELR+CXC chemokine subfamily,were significantly differentially expressed.These differentially expressed secretory protein genes verified by Q-PCR were consistent with the results of RNA-seq screening.7.SB225002,a specific inhibitor of CXCR2,was able to inhibit the expression of LIF m RNA,the content of LIF in CAA-CM,and also ERK1/2,NF-κB,and Stat3 phosphorylation in adipocytes co-cultured with MDA-MB-231 cells.Both CXCL3 and CXCL8 could induce the phosphorylation of ERK1/2,NF-κB p65,and STAT3 and up-regulate the expression of LIF in adipocytes,which was significantly inhibited by SB225002.Moreover,the effect of the CXCL3 neutralizing antibody was consistent with that of SB225002 in the co-cultured system.8.The Q-PCR results showed that m RNA levels of CXCL1,CXCL2,CXCL3,and CXCL8,members of the ELR+CXC chemokine subfamily,were significantly up-regulated in breast cancer tissue and paracancerous adipose tissue compared with normal breast tissue.9.The Q-PCR results showed that LIF could up-regulate the expression of CXCL1,CXCL2,CXCL3,and CXCL8 in breast cancer cells.Therefore,LIF and CXCLs form a malignant positive feedback loop during the interaction between breast cancer cells and adipocytes.Conclusion:Breast cancer cell-derived and CAA-derived CXCLs activated the ERK1/2signaling pathway by binding to CXCR2 receptor of CAA,and subsequently activated transcription factors Stat3 and NF-κB p65,which were phosphorylated and entered the nucleus,then initiated LIF expression in CAA.Therefore,LIF secretion increased in CAA,which activated the Stat3 signal by paracrine to promote the migration and invasion of breast cancer cells.