| Triple negative breast cancer(TNBC)is characterized by the absence of estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor 2(HER2),accounting for 12–17% of the total breast cancer(BC)cases.Generally,TNBC are found in young patients and are associated with high histologic grade,visceral metastasis and distant recurrence.Because of the lack of ER,PR,and HER2,chemotherapy is the only treatment for TNBC at present;however,patients with TNBC usually have poor clinical outcomes.Previous studies have shown that expression of activator protein 1(AP-1)family is significantly elevated in TNBC,compared with that in other BC subtypes.The upregulation of AP-1 family member Fra-1 is associated with the aggressive phenotype and poor prognosis of TNBC patients.Knockdown of c-Jun or Fra-1 significantly inhibited proliferation and invasion of TNBC cells.T5224,an inhibitor of c-Fos/AP-1,specifically inhibits the DNA binding activity of c-Fos/c-Jun.At present,T5224 has been used for treatment of patients with arthritis in human clinical trials;however,the anti-tumor effect of T5224 on TNBC is not clear.Objective:To investigate the inhibition effect of T5224 on AP-1 expression and to investigate the anti-tumor effect and mechanism of T5224 on TNBC.Methods:Online databases Kaplan-Meier plotter and UCSC were used to investigate the expression level of Fra-1 in BC patients defined by differential expression of 50 genes(PAM50)and to investigate the relation between the expression level of Fra-1and the prognosis of BC patients.Real-time PCR and Western blot were used to confirm the expression level of Fra-1 and c-Jun in TNBC cells and non-TNBC cells both in m RNA level and protein level.WST-1 cell proliferation assay was used to determine the optimal dose of T5224.Realtime PCR and Western-blot were used to investigate the inhibition effect of T5224 on AP-1 expression.Wound-healing assay,cell transwell assay,and flow cytometry assay were used to investigate the effects of T5224 on the migration,invasion,and apoptosis of TNBC cells.In order to investigate the effect of T5224 on gene expression in TNBC cells,Illumina Hiseq Xten platform was used to perform whole-genome sequencing.R 4.0.3was used to analyze the differentially expressed genes(DEGs)and to perform Gene Ontology(GO)analysis and Pathway Enrichment analysis [Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis].Volcano plot and heatmap were performed using ggplot2 package and Pheatmap package,respectively.GSEA 4.1.0 was used to perform Gene Set Enrichment Analysis(GSEA).To determine the target genes of AP-1 transcription factor,the genome-wide DEGs after c-Jun knockdown,Fra-1 knockdown,and T5224 treatment in the BT549 cells were overlapped.Online databases(Kaplan-Meier plotter and Cistrome Data Browser)and Ch IP-qPCR were used to further determine the target genes of AP-1 transcription factor.WST-1 cell proliferation assay,wound-healing assay,cell transwell assay,and flow cytometry assay were used to investigate the biological function of target gene in TNBC cells.Results:1.Expression level of Fra-1 in BC patients and relation between Fra-1 and prognosis of BC patients.The Kaplan-Meier plotter database and UCSC database showed that compared with other subtypes of BC,Fra-1 was evaluated in Basal-like BC.We then investigated the Kaplan-Meier plotter database,revealing that BC patients or luminal BC patients with low Fra-1 expression had better recurrence-free survival outcomes and distance metastasis free survival outcomes than those with high Fra-1 expression [BC: HR =1.24(1.03,1.50),P = 0.024;HR = 1.7(1.27,2.26),P < 0.001;luminal: HR =1.30(1.03,1.64),P = 0.028;HR = 1.85(1.30,2.64),P = 0.001].HER2-positive patients with low Fra-1 expression had better distance metastasis free survival outcomes but worse overall survival than those with high Fra-1 expression [HR: 2.61(1.21,5.67),P = 0.031;HR: 0.35(0.12,1.00),P = 0.022].UCSC database also showed that BC patients or luminal BC patients with low Fra-1 expression had better distance metastasis free survival outcomes than those with high Fra-1 expression [BC: HR = 1.42(1.08,1.86),P = 0.008;luminal: HR = 1.61(1.10,2.35),P = 0.007].2.Effect of T5224 on TNBC cells1)Effect of T5224 on the expression of AP-1Using real-time PCR and Western-blot,we confirmed that the expression level of Fra-1 and c-Jun was higher in TNBC cells than those in non-TNBC cells both in m RNA level and protein level.WST-1 cell proliferation assay showed that the inhibition rate of 15?M T5224 on proliferation of BT549 cells was 43.56% and the inhibition rate of40?M T5224 on proliferation of HS578 T cells was 48.42% after 48 h treatment.We further analyzed the expression of Fra-1 and c-Jun in BT549 and HS578 T cells following T5224 treatment.We found that both Fra-1 and c-Jun were significantly downregulated at m RNA level and protein level in BT549 cells.In HS578 T cells,T5224 treatment significantly reduced c-Jun m RNA expression at 48 h and c-Jun protein expression at 72 h.Similarly,both Fra-1 m RNA and protein expressions were significantly reduced following 72 h of T5224 treatment.2)Effect of T5224 on the function of TNBC cellsWST-1 cell proliferation assay showed that BT549 cells were sensitive to T5224 at15?M and HS578 T cells were sensitive to T5224 at 40?M after 48 h treatment.Flow cytometry assay showed that the number of apoptotic cells in T5224 group was as 1.79 times as that in control group in BT549 cells and as 1.29 times as that in control group in HS578 T cells with a statistical significance(P < 0.01).Cell scratch assay showed that after 48 h,the free area in control group was significantly smaller than that in T5224group(P < 0.01).Cell transwell assay showed that the number of invaded cells in control group was statistically higher than that in T5224 group(P < 0.01).3)Effect of T5224 on gene expression in TNBC cellsGO analysis showed that the 5136 DEGs were involved in blood vessel morphogenesis,cell-substrate adhesion,response to topologically incorrect protein,type I interferon signaling pathway,focal adhesion,proteinaceous extracellular matrix,cell-cell adherens junction,cell adhesion molecule binding,cadherin binding,and growth factor binding.KEGG analysis showed that these DEGs were associated with PI3K-Akt signaling pathway,human papillomavirus infection,focal adhesion,and calcium signaling pathway.Moreover,GSEA analysis showed that after treatment with T5224,pathways in cancer,such as calcium signaling pathway,ERBB signaling pathway,JAK-STAT signaling pathway,and RIG-1 like receptor signaling pathway were down-regulated.Biological process and cellular component,such as de novo protein folding,response to topologically incorrect protein,and protein folding chaperone were upregulated;however,innate immune response,response to type I interferon,vasculature development,extracellular matrix binding,and growth factor binding were downregulated.3.Screening of target gene of AP-1 transcription factor and function of target gene in TNBC cells1)Screening of target gene of AP-1 transcription factorOLFML2A expression was correlated with c-Jun expression.Basal-like BC patients with low OLFML2 A had better distance metastasis free survival outcomes than those with high OLFML2 A expression [HR: 3.04(1.40,6.61),P = 0.029].We further found that c-Jun and Fra-1 bound to OLFML2 A from the Ch IP-Seq data in the Cistrome Data Browser database.Relation between OLFML2 A and AP-1 was validated using Ch IP-qPCR assay.2)Effect of OLFML2A-konckdown on the function of TNBC cellsWST-1 cell proliferation assay showed that the average value of survival TNBC cells in OLFML2A-konckdown group was lower than that in control group(P < 0.01).Flow cytometry assay showed that the number of apoptotic cells in OLFML2 Akonckdown group was as 1.53 times as that in control group in BT549 cells and as 1.15 times as that in control group in HS578 T cells with a statistical significance(P < 0.01).Cell scratch assay showed that after 48 h,the free area in control group was significantly smaller than that in OLFML2A-konckdown group(P < 0.01).Cell transwell assay showed that the number of invaded cells in control group was statistically higher than that in OLFML2A-konckdown group(P < 0.01).Conclusion:1.Fra-1 is overexpressed in TNBC patients and TNBC cells,while c-Jun is overexpressed in TNBC cells.2.BC patients or luminal BC patients with low Fra-1 expression had better recurrence-free survival outcomes and distance metastasis free survival outcomes than those with high Fra-1 expression3.T5224 downregulates the expression of c-Jun and Fra-1 in TNBC cells.4.T5224 has anti-tumour effect on TNBC cells.T5224 inhibits the proliferation,migration,and invasion of TNBC cells,but induces apoptosis of TNBC cells.5.Basal-like BC patients with low OLFML2 A expression had better distance metastasis free survival outcomes than those with high OLFML2 A expression.6.OLFML2 A knockdown inhibits proliferation,migration,and invasion of TNBC cells,but induces apoptosis of TNBC cells.7.AP1-overexpressing TNBC dependent on OLFML2 A,and targeting both AP-1 and OLFML2 A through T5224 may be a synergistic therapeutic strategy for this clinically challenging subset of BC. |
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