| Background:Among female cancer patients worldwide,the incidence and mortality of breast cancer are the first,and the metastasis and recurrence of breast cancer are the main factors leading to the death of patients.Triple negative breast cancer（TNBC）,namely estrogen receptor（ER）,progesterone receptor（PR）,human epidermal growth factor receptor 2（human epidermal growth factor receptor,HER-2）were all negative.This subtype of patients accounts for about 20%-25%of the total breast cancer patients.Its clinical characteristics are:young patients,prone to recurrence and metastasis,and poor prognosis.Due to the lack of effective therapeutic targets,the treatment of triple negative breast cancer is still a clinical problem.Therefore,there is an urgent need to explore new target molecules and targeted drugs.Existing studies have shown that the expression level of acetaldehyde dehydrogenase 1A3 in metastatic lesions of TNBC patients is significantly higher than that of primary tumors.Meanwhile,the expression of ALDH1A3 was significantly positively co rrelated with the Aldefluor activity of breast cancer cell lines（R=0.91）,and compared with Aldefluor-/CD44-TNBC cells,Aldefluor+/CD44+TNBC cells could significantly promote lung metastasis in transplanted tumor mice（P<0.05）,suggesting that ALDH1A3 may be related to TNBC metastasis.However,the prognostic significance of ALDH1A3 expression and the therapeutic significance of targeting ALDH1A3 to inhibit TNBC metastasis need further study.Therefore,this study intends to use bioinformatics methods to analyze the correlation between ALDH1A3 expression and prognosis of TNBC patients and enrichment of metastasis-related signaling pathways,and further use the targeted ALDH1A3 small molecule inhibitor YD1701 to explore its effect on TNBC cell migration,invasion and self-renewal ability.Purposes:1.To explore the correlation between the expression of ALDH1A3 and the prognosis of TNBC patients.2.To explore genes enrichment which related to biological processes and signaling pathway of metastasis in TNBC patients with ALDH1A3 high expression.3.To explore the ability of ALDH1A3 targeting small molecule inhibitor YD1701 to bind to ALDH1A3 protein in TNBC cells and its ability to inhibit Aldeflour activity.4.To explore the effects of ALDH1A3 targeting small molecule inhibitor YD1701 on the invasion,migration and tumorsphering ability of TNBC cells.Methods:1.Search the GEO database for datasets（GSE10893,GSE25065,GSE2603）that contain relevant prognostic information and patient gene expression information such as the survival and metastasis status,overall survival,and metastasis-free survival of TNBC patients.Group patients according to survival and metastasis status,and draw receiver receiver operating characteristic curve（ROC）.Patients were grouped by the ALDH1A3 expression value with the highest Jordon index,which was the Cutoff value of the ALDH1A3 expression group.The contingency table analysis in SPSS（V25.0）software was used to analyze the correlation between ALDH1A3 expression and the clinical pathological parameters of TNBC patients,such as age of onset and tumor grade.The column analysis in Graphpad（V6.01）software was used to compare the expression levels of ALDH1A3 in patients with different metastatic states,and Kaplan-Meier survival analysis function was used to analyze the overall survival（OS）and metastasis-free survive（MFS）.The ALDH1A3 expression grouping and clinical prognosis parameters of patientswere imported into SPSS（V25.0）software,and univariate and multifactorial COX regression survival analysis was performed to evaluate the significance of ALDH 1A3 expression and the prognosis of OS and MFS in TNBC patients.2.Gene set enrichment analysis（GSEA）（V3.0）software was used for GSEA analysis of the GSE10893,GSE25065,GSE7390,GSE11121 datasets,and the R2genome analysis and visualization platform（hgserver1.amc.nl/cgi-bin/r2/main.cgi）,performing a Kyoto Encyclopedia of Genes and Genomes（KEGG）signal enrichment analysis on GSE25065,GSE7390,GSE11121 datasets and a dataset containing TNBC patient gene expression information.Assess genes enrichment which related to biological processes and signaling pathway of metastasis in TNBC patients with ALDH1A3 high expression.SiRNA knocked down the expression of ALDH1A3 in MDA-468 cells,and explored its effect on migration ability in scratch experiments.3.Western-blot and Aldeflour analysis were used to detect the expression of ALDH1A3,Aldeflour activity in TNBC cell lines（HCC38,MDA-231,MDA-468）,and the change of Aldeflour activity of TNBC cells after treatment with ALDH1A3targeting small molecule inhibitor YD1701.4.Cellular thermal shift assay（CETSA）was used to detect the effect of YD1701treatment on the thermal stability of ALDH1A3 in MDA-468 cells,and draw a binding curve to test the binding ability of YD1701 to ALDH1A3 in TNBC cells.5.Scratch and Transwell invasion experiments were performed to detect the migration and invasion ability of TNBC cells after YD1701 treatment.Stem cell enrichment cultureexperiments were performed to detect the effects of YD1701treatment on the tumorsphering ability of Aldefluor+MDA-468 cells.Results:1.The results of contingency table analysis of the GSE10893 data set show that ALDH1A3 expression in TNBC patients is significantly related to distant metastasis（P=0.005）,the analysis results of the GSE25065 and GSE2603 data sets also show that ALDH1A3 expression is significantly related to distant metastasis（P=0.031,P=0.001）.The comparison of ALDH1A3 expression in patients with metastasis and non-metastasis in the above three datasets shows that ALDH1A3 expression in patients with TNBC metastasis is higher than that in patients without metastasis(P=4.450×10-2;P=3.740×10-2;P=1.260×10-2).Kaplan-Meier survival analysis showed that in the GSE10893 dataset,compared with lower ALDH1A3 group（n=150）,OS and MFS in patients with high ALDH1A3 group（n=49）were significantly shortened(median overall survival time:715 days vs 915 days,P=7.000×10-4;median non-metastatic survival time:630 days vs 780 days,P=1.200×10-3);In the GSE25065 dataset,MFS in ALDH1A3 patients with high expression of TNBC（n=66）was significantly shortened compared with patients with lower expression（n=132）(median non-metastatic survival time:1131 days vs 1165 days,P=4.220×10-2).Analysis of the GSE2603dataset also showed that MFS in patients with high expression of ALDH1A3 was significantly shortened(median non-metastatic survival time:1246 days vs 2051 days,P=1.000×10-4).Further analysis of the above dataset by multi-factor COX regression model analysis showed that in the GSE10893 data set,the expression of ALDH1A3was significantly correlated with the patient’s OS(P=1.000×10-3)and MFS(P=1.000×10-3).The results of data analysis of the GSE25065 data set showed that tumor grade(P=4.700×10-2),INSS(P=7.000×10-3)staging,and ALDH1A3 expression(P=4.800×10-2)were significantly correlated with MFS in TNBC patients.Analysis of the GSE2603 data set showed that ALDH1A3 expression(P=1.000×10-3)was significantly correlated with MFS.2.The GSEA analysis of the GSE10893,GSE25065,GSE7390 and GSE11121datasets showed that the genes related to the EMT pathway(P=4.300×10-2,P=0.000,P=0.000,P=0.000)of patients with high expression of TNBC in ALDH1A3 were significantly enriched.Results of KEGG analysis of the public dataset of the Tumor breast（TNBC）and GSE25065,GSE7390,and GSE11121 datasets on the R2 website showed that genes of actin cytoskeleton regulation(P=2.400×10-6,P=3.992×10-3,P=0.000,P=5.474×10-3),extracellular matrix receptor interaction(P=5.000×10-6,P=3.651×10-2,P=0.000,P=0.000),transendothelial migration(P=2.700×10-5,P=2.439×10-2,P=2.053×10-2,P=1.873×10-3)were significantly enriched,and significant activation of EMT-related signaling pathway genes such as Ras(P=2.900×10-5,P=1.855×10-3,P=1.799×10-2,P=0.000)and NF-κB(P=3.200×10-4,P=8.214×10-3,P=3.357×10-2,P=3.150×10-2)in TNBC patients with ALDH1A3 highly expressed.3.The results of WB and Aldefluor analysis showed that the expression of ALDH1A3 is higher in MDA-468 cells than MDA-231 and HCC38 cells,and The positive rate of Aldefluor（44.11%）was higher in MDA-468 cells than that of MDA-231（4.28%）and HCC38（1.77%）cells.After YD1701 treatment,the Aldefluor positive rate of MDA-468 cells decreased（27.89%vs 0.75%）.4.CETSA results showed that YD1701 can improve the spatial structure stability of ALDH1A3 expressed in MDA-468 cells under high temperature environment and can be specifically recognized by the ALDH1A3 monoclonal antibody,indicating that YD1701 can bind to ALDH1A3 in TNBC cells.MDA-468 cells were treated with different concentrations YD1701 and subjected to CETSA.The results showed that the half-binding concentration of YD1701 and ALDH1A3 in MDA-468 cells was10.55μg/mL.5.The scratch test results show that YD1701 can significantly inhibit the migration ability of the TNBC cell line and is concentration-dependent(HCC38:0 vs 30μg/mL,P=1.070×10-9;0 vs 60μg/m L,P=3.300×10-10;30μg/mL vs 60μg/mL,P=1.079×10-2.MDA-231:0 vs 30μg mL,P=4.280×10-2;0 vs 60μg/mL,P=8.000×10-4;30μg/m L vs60μg/m L,P=2.600×10-3.MDA-468:0 vs 30μg/mL,P=3.144×10-9;0 vs 60μg/m L,P=6.793×10-10;30μg/mL vs 60μg/m L,P=4.390×10-2).Transwell invasion experiment results show that YD1701 can significantly inhibit the invasion ability of TNBC cell lines and has concentration dependence(MDA-231:0 vs 30μg/m L,P=7.678×10-5;0vs 60μg/m L,P=1.196×10-5;30μg/m L vs 60μg/m L,P=4.452×10-2.HCC38:0 vs30μg/m L,P=3.133×10-6;0 vs 60μg/m L,P=1.867×10-6;30μg/m L vs 60μg/mL,P=1.112×10-2).SiRNA interference experiments showed that siRNA knockdown of MDA-468 cells ALDH1A3 expression can significantly inhibit the migration ability of MDA-468 cells(WT vs NC,P=5.087×10-1;NC vs siRNA-1,P=2.454×10-7;NC vs siRNA-2,P=5.924×10-7;NC vs siRNA-3,P=1.139×10-6).Stem cell enrichment experiments on Aldefluor+MDA-468 cells which was treated with different concentrations of YD1701 showed that YD1701 can inhibit the tumorsphering ability of Aldefluor+MDA-468 cells(0 vs 30μg/mL,P=9.438×10-3;0 vs 60μg/mL,P=3.835×10-10;30μg/m L vs 60μg/mL,P=3.077×10-2).Conclusions:1.High expression of ALDH1A3 is closely related to metastasis in TNBC patients,and is an independent prognostic factor.2.ALDH1A3 targeted small molecule inhibitor YD1701 can bind to ALDH1A3 in TNBC cells and inhibit tumor cell migration,invasion and tumorsphering ability. |
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