Antimicrobial Peptides LL-37 Induces Proliferation,Migration And Invasion Of Breast Cancer Cells Through Activation Of Wnt Signal Pathway

Objective: To investigate whether the antimicrobial peptide LL-37/CRAMP enhances the proliferation,invasion and migration of breast cancer cells through activating the Wnt/β-catenin signaling pathway,and triggers myeloid macrophages’ differentiation into M2 type in tumor immune microenvironment.Background: Breast cancer is now the most common malignancy in the world,and it has been threatening women’s health increasingly.Antimicrobial peptide LL-37 is a small molecule polypeptide that is widely present in host cells and organisms and has a variety of biological activities.In addition to antibacterial activity,it plays a vital role in anti-infection,anti-tumor,participation in immune regulation,angiogenesis,and damage repair.The latest researches revealed that LL-37 has different effects on the occurrence and development of various tumors.The data in The Cancer Genome Atlas(TCGA)database shows that LL-37 ascends in primary breast cancer compared to that in normal breast tissue,which suggests it may contribute to giving vantage to the breast cancer progression.Studies have reported that LL-37 activated PI3K/Akt signaling pathway is responsible for the migration of breast cancer cell lines.The activated Akt phosphorylates a variety of substrates including GSK3β,thereby inhibiting cell apoptosis and promoting survival.Additionally,GSK3β,as one of serine/threonine family kinase,is a key factor in the Wnt signaling pathway that negatively regulates β-catenin.However,in breast cancer,the relationship between LL-37/CRAMP’s promotion function and Wnt/β-catenin signaling pathway has not been elucidated.There are a large number of immune cells in the tumor microenvironment,such as tumor-associated macrophages(TAMs),dendritic cells(DCs),myeloid-derived suppressor cells(MDSCs),lymphocytes,etc.Tumor cells remodeled tumor microenvironment on the contrary affects the behavior and status of tumor cells.Based on the linkage between LL-37 and Akt signal in previous research,we speculate that LL-37 could promote the proliferation,invasion and migration of breast cancer cells by further activating Wnt/β-catenin signals and changes in the polarization phenotype of macrophages to exert carcinogenic effects.Methods:1.Firstly,the expression level of LL-37 in primary breast cancer and normal breast tissue was validated with application of The Cancer Genome Atlas(TCGA)database.In vitro experiments,MCF-10A(human normal breast epithelial cell line)as control subjects,real-time PCR was used to detect the transcription level of the antimicrobial peptide CAMP gene in a variety of human breast cancer cell lines MCF-7,MDA-MB-231,and BT549.In order to further explore the role of LL-37/CRAMP,the endogenous gene Cramp in PY8119 was knocked down for a certain period of time.Transcription levels of indicators related to proliferation and migration in cells were tested by use of real-time PCR.2.Human breast cancer cell line MCF-7 and murine breast cancer cell line PY8119,4T1 were treated with recombinant polypeptide LL-37/CRAMP for a certain period of time.Colony formation experiment,CCK8,wound healing experiment and Transwell asssay were employed to detect the proliferation,invasion and migration capabilities to clarify the prominent role of LL-37.The total RNA and total protein in breast cancer cells were collected,and the instructive markers of cell proliferation and migration,the important signal molecules in PI3K/Akt and Wnt/β-catenin signaling pathways such as β-catenin,GSK3β,AXIN2,LEF1,MMP-9 and the activation degree of internal related receptors such as LRP5/6,FZD1,EGFR,FPR2,and TLR4 were examined by means of real-time PCR and Western Blot.Immunofluorescence technique was conducted to explore the localization of β-catenin in cells and the expression intensity of p-β-catenin after LL-37 treatment in breast cancer cells,which further exposits the activation of Wnt/β-catenin signaling pathway.3.The exogenous β-catenin specific inhibitor XAV939 was applied in the experiments of breast cancer cells MCF-7 and PY8119.The expression changes of p-β-catenin/β-catenin and p-GSK3β/GSK3β in experimental groups with employment of XAV939 were detected by real-time PCR and Western Blot.4.The murine breast cancer cell lines PY8119 and 4T1 were pretreated with recombinant polypeptide CRAMP in vitro.In the cancer cells-macrophages co-culture system,the inflammatory factor CXCL1,M1 macrophage polarization markers NOS2 and M2 markers ARG1 were detected to verify that LL-37 is an aid to shape the macrophage immunophenotype.Results:1.The TCGA human cancer database revealed that compared with normal tissues,LL-37 in primary breast cancer is significantly up-regulated,in the company of high expression of mRNA level of CAMP in MCF-7compared to MCF-10 A.Moreover,knockdown of endogenous Cramp in PY8119 attenuated c-myc,PCNA,MMP-9,and fibronectin transcriptional levels,indicating that proliferation and migration abilities of breast cancer cells were weakened.2.At the cellular level,the observation manifested that LL-37/CRAMP clearly improves the proliferation,lateral and longitudinal migration and invasion capabilities of MCF-7 and PY8119 cells.Quantitative results showed that LL-37/CRAMP caused an increase of Cyclin D1 and MMP-9,the markers of proliferation and migration.3.After exogenous addition of CRAMP,the mRNA level of Akt in PY8119 cells was evidently higher than that in the untreated group,and the Wnt-responsive genes,such as AXIN2,β-catenin,LEF1,and fibronectin,increased in a time-dependent manner.Furthermore,the fold changes of the transcriptional levels of AXIN2,β-catenin,and LEF1 in PY8119,4T1 and MCF-7 showed an accretion after integration.At the protein level,due to the regulation by LL-37/CRAMP,p-β-catenin/β-catenin was decreased and p-GSK3β/GSK3β was increased in three breast cancer cell lines.The green fluorescence of p-β-catenin severely faded,and the red fluorescence ofβ-catenin overlaps with the blue fluorescence of the DAPI,turning to pink,which indicates its translocation into the nucleus under the confocal immunofluorescence microscopy.Besides,the mRNA levels of Wnt signaling pathway receptors LRP5/6 and FZD1 were increased in PY8119 cells after being exposed to CRAMP,as was EGFR.4.After combination treatment of LL-37/CRAMP and XAV939,the protein expression of p-β-catenin/β-catenin and p-GSK3β/GSK3β in MCF-7 and PY8119 showed opposite trends from before,p-β-catenin/β-catenin was up-regulated and p-GSK3β/GSK3β was down-regulated.5.In the breast cancer cells-macrophages co-culture system,CRAMP pretreated PY8119 and 4T1 cells leaded to the repression of M1 macrophage polarization marker NOS2,accompanied by the elevation of M2 macrophage polarization marker ARG1,revealing a M2 polarization trend of macrophages in co-culture system.Conclusions: This study confirmed that the antimicrobial peptide LL-37/CRAMP ascends in breast cancer and markedly enhances the proliferation,invasion and migration capabilities of breast cancer cells by activating Wnt/β-catenin signaling and promotes macrophages in co-culture system to polarize to M2 immunophenotype,exhibiting tumor-promoting effects in vitro.Accordingly,we believe that LL-37/CRAMP is a vital regulator of breast cancer occurrence and development.Our findings provide new experimental research basis about the mechanism of LL-37 in the progression of breast cancer.It is contributed to a kind of probability that LL-37-Wnt/β-catenin could be a dormant target spot for the clinical diagnosis and therapy of breast cancer via offering new theoretical principle for controlling breast cancer progression.