A Preliminary Study On The Regulation Of Aldehyde Dehydrogenase 18 Member A1 On Self-renewal Of Ewing’s Sarcoma Cells And Its Therapeutic Significance

Background:Ewing sarcoma is a highly aggressive malignant tumor with the second highest incidence of bone tumors in children.The treatment of Ewing sarcoma still relies on a multidisciplinary combination of intense chemotherapy and surgery/radiotherapy to control the primary and metastatic lesions.Although the 5-year survival rate for patients with localized Ewing sarcoma family tumors has reached 70-80%,the 5-year survival rate for patients with metastases and recurrences is still less than 30%.As a highly aggressive malignancy with a high recurrence rate,Ewing sarcoma urgently needs to find new therapeutic targets.As a pediatric tumor with clear oncogenic factors,the oncogenic EWS-FLI1 fusion gene of Ewing sarcoma activates downstream target genes in a phase-separated form,leading to extensive epigenetic remodeling and driving extensive metabolic reprogramming processes.As a tumor with a low mutation rate,Ewing sarcoma lacks viable alternative therapeutic targets.Therefore,its oncogenic fusion protein-driven genetic transcriptional regulation is a new direction for targeted therapy of Ewing sarcoma.Epigenetics and tumor metabolism as a pair of mutually crosstalking and inextricably important features in tumors have received much attention in recent years and may be a new direction for targeted therapy in Ewing sarcoma.It has been suggested that parabolic effects in proline metabolism mediated by massive prolyl hydroxylation play an important role in epigenetic modifications,while the role of aldehyde dehydrogenase 18 family member A1（ALDH18A1）,a key enzyme in glutamate,arginine and proline metabolism,in the progression of Ewing sarcoma is unclear and remains to be investigated.In this study,we found that high expression of ALDH18A1 was an independent predictor of poor prognosis in patients with Ewing sarcoma;ALDH18A1 regulates self-renewal,proliferation and tumorigenic capacity of Ewing sarcoma cells in vivo;ALDH18A1 may promote self-renewal and proliferation of Ewing sarcoma cells by promoting Poly（ADP-ribose）polymerase 1（PARP1）expression and thereby regulating EWS-FLI1 downstream target gene expression;ALDH18A1 inhibitor YG1702 may be a potential candidate small molecule for Ewing sarcoma treatment.Methods and Results:1.By using the Gene Expression Omnibus（GEO）gene expression profile dataset（GSE17674）,the expression of ALDH18A1 in human Ewing sarcoma tissue and human normal muscle tissue was compared.The results showed that ALDH18A1 was highly expressed in Ewing sarcoma tissues((Tumor_mean（n=44）vs.Normal_mean（n=18）:6.94vs.4.77,P=2.46×10^-34)).2.Using the GEO dataset,the prognostic significance of ALDH18A1 in Ewing sarcoma patients was analyzed by Kaplan-Meier and Cox regression,and the results showed that high ALDH18A1 expression could be used as a poor prognostic marker in Ewing sarcoma patients.In the GSE17674 dataset:survival time was significantly longer in patients with low ALDH18A1 expression（Mean survival time:67.11±48.05 months,n=34）compared to those with high ALDH18A1 expression（Mean survival time:22.67±13.21 months,n=10）(P=1.00×10^-3);in the GSE17679 dataset:survival time was significantly longer in patients with low ALDH18A1 expression（Mean survival time:65.87±47.07 months,n=71）compared to those with high ALDH18A1 expression（Mean survival time:20.01±11.36months,n=17）(P=1.00×10^-4);Univariate and multifactorial regression analysis in GSE17679 showed that ALDH18A1 expression levels were associated with prognosis in patients with Ewing sarcoma(P=1.00×10^-4)and were independent prognostic predictors(P=1.46×10^-7,risk factor:5.57,95%confidence interval:2.94-10.55).3.The WGCNA（weighted gene co-expression network analysis）algorithm was used to find gene sets closely associated with ALDH18A1 high expression in the GSE17618 dataset,and GO enrichment analysis was performed to screen the enrichment of the pathways（screening criteria:FDR<0.25,P<0.05）.The results showed that high ALDH18A1expression was closely associated with processes such as cell division and DNA replication,with cell division(100 genes enriched,P=2.33×10^-37)and DNA replication(67 genes enriched,P=1.13×10^-33).4.After knockdown of ALDH18A1 expression by ShRNA,Cell Counting Kit-8（CCK-8）,plate clone formation assay,sphere-forming assay and subcutaneous transplantation tumor assay in nude mice were used to detect changes in proliferation,self-renewal and tumorigenic ability of Ewing sarcoma cells in vivo.The results show that:Proliferation capacity is significantly inhibited(CCK8 proliferation assay:A673:shALD H18A1-NC vs.shALDH18A1-1,n=5,P=7.00×10^-3;shALDH18A1-NC vs.shALDH18A1-2;n=5;P=7.50×10^-3;SK-N-MC:shALDH18A1-NC vs.shALDH18A1-1,n=5,P=4.00×10^-6;shALDH18A1-NC vs.shALDH18A1-2,n=5,P=1.40×10^-5).Clonogenic capacity is significantly inhibited(plate clone formation assay:A673:shAL DH18A1-NC vs.shALDH18A1-1,n=3,P=2.90×10^-5;shALDH18A1-NC vs.shALDH18A1-2,n=3,P=4.00×10^-6;SK-N-MC:shALDH18A1-NC vs.shALDH18A1-1,n=3,P=5.14×10^-4;shALDH18A1-NC vs.shALDH18A1-2,n=3,P=4.77×10^-4).Sphere-forming ability is significantly inhibited(sphere-forming assay:A673:shALDH18A1-NC vs.shALDH18A1-1,n=6,P=1.35×10^-3;shALDH18A1-NC vs.shALDH18A1-2,n=6,P=1.35×10^-3;SK-N-MC:shALDH18A1-NC vs.shALDH18A1-1,n=6,P=0.02;shALD H18A1-NC vs.shALDH18A1-2,n=6,P=0.02).Tumorigenic capacity in vivo is significantly inhibited(subcutaneous transplantation tum or experiment in nude mice:A673:Mean tumor volume:shALDH18A1-NC vs.shALDH18A1-1=957.60±161.40mm³ vs.355.20±101.60mm³,n=6,P=7.90×10^-5,Average tumor weigh t:shALDH18A1-NC vs.shALDH18A1-1=0.99±0.23 g vs.0.42±0.16 g,n=6,P=5.37×10^-4).5.After knockdown of ALDH18A1 expression by ShRNA,changes in the expression lev els of EWS-FLI1 downstream target genes were detected by RT-q PCR.The results showed th at the expression of EWS-FLI1 downstream target genes was significantly downregulated in Ewing sarcoma cells after knockdown of ALDH18A1:A673:PARP1 mRNA levels:shALDH18A1-NC vs.shALDH18A1-1,n=3,P=2.18×10^-5;shALDH18A1-NC vs.shALDH18A1-2,n=3,P=5.56×10^-5,EWS-FLI1 downstream target genes as EZH2 mRNA levels:shALDH18A1-NC vs.shALDH18A1-1,n=3,P=7.89×10^-3;shALDH18A1-NC vs.shALDH18A1-2,n=3,P=7.92×10^-3.SK-N-MC:PARP1 mRNA levels:shALDH18A1-NC vs.shALDH18A1-1,n=3,P=9.03×10^-4;shALDH18A1-NC vs.shALDH18A1-2,n=3,P=1.58×10^-3,EWS-FLI1 downstream ta rget genes as NR0B1 mRNA levels:shALDH18A1-NC vs.shALDH18A1-1,n=3,P=5.29×10^-4;shALDH18A1-NC vs.shALDH18A1-2,n=3,P=3.70×10^-3.6.After knockdown of ALDH18A1 expression by ShRNA,Mito-Tracker Red immunofl uorescence staining was performed to detect the number of mitochondria.The results showed that knockdown of ALDH18A1 reduced the number of mitochondria in Ewing sarcoma cells(Mito-Tracker relative fluorescence intensity:A673:shALDH18A1-NC（n=25）vs.shALDH18A1-1（n=25）,P=3.00×10^-4;shALDH18A1-NC（n=25）vs.shALDH18A1-2（n=25）,P=3.00×10^-4;SK-N-MC:shALDH18A1-NC（n=39）vs.shALDH18A1-1（n=36）,P=8.00×10^-3;shALDH18A1-NC（n=39）vs.shALDH18A1-2（n=60）,P=5.59×10^-11).7.Using the Rcis Target R package in the GSE17679 dataset,GSE63155 dataset and GSE63156 dataset,transcription factor enrichment analysis for differential genes in patients with high and low expression of ALDH18A1 showed significant enrichment of PARP1(NES=4.47,AUC=9.29×10^-2).A positive correlation between ALDH18A1 and PARP1 expression was found in the GSE17679 dataset,GSE63155 dataset,and GSE63156 dataset(R=0.44,P=8.00×10^-9).8.The inhibitory effect of YG1702,a small molecule inhibitor of ALDH18A1,on the proliferation of Ewing sarcoma cells was determined by the CCK8 assay for cell viability;further,the inhibitory effect of YG1702 on the self-renewal ability of Ewing sarcoma cells was verified by plate cloning and sphere-forming assays.The results show that:YG1702significantly inhibits the proliferation of Ewing sarcoma cells in vitro(A673:WT vs.YG1702,n=5,P=9.94×10^-3;SK-N-MC:WT vs.YG1702,n=5,P=2.52×10^-7),cloning formation capability(A673:WT vs.YG1702,n=3,P=7.80×10^-5;SK-N-MC:WT vs.YG1702,n=3,P=1.86×10^-4),and sphere-forming ability(A673:WT vs.YG1702,n=6,P=0.03;SK-N-MC:WT vs.YG1702,n=6,P=6.96×10^-4).9.The changes in the expression levels of PARP1 and EWS-FLI1 downstream target genes were detected by RT-q PCR after YG1702 treatment of Ewing sarcoma cells.The results showed that the expression of PARP1 and EWS-FLI1 downstream target genes in Ewing sarcoma cells were significantly downregulated after YG1702 treatment:A673:PARP1 mRNA level:Control vs.IC50,n=3,P=2.04×10^-2;Control vs.IC90,n=3,P=3.22×10^-3,EWS-FLI1 downstream target genes such as EZH2 mRNA level:Control vs.IC50,n=3,P=4.47×10^-6;Control vs.IC90,n=3,P=3.69×10^-8.SK-N-MC:PARP1 mRNA level:Control vs.IC50,n=3,P=5.00×10^-6;Control vs.IC90,n=3,P=2.00×10^-6,EWS-FLI1 downstream target genes such as EZH2 mRNA level:Control vs.IC50,n=3,P=3.00×10^-6;Control vs.IC90,n=3,P=1.08×10^-7.10.A subcutaneous transplantation tumor model of Ewing sarcoma cells（A673）was constructed in nude mice.After YG1702 was administered intraperitoneally,the body weight and tumor growth of the nude mice were observed.The results showed that the in vivo tumorigenic capacity was significantly inhibited(mean tumor volume:Control:883.90±209.80 mm³,YG1702:387.10±72.90 mm³,n=6,P=2.70×10^-4;mean tumor weight:Control:0.99±0.27 g,YG1702:0.28±0.04 g,n=6,P=2.03×10^-3).Conclusion:1.ALDH18A1 is highly expressed in Ewing sarcoma and is closely associated with poor patient prognosis,suggesting that ALDH18A1 may play an important role in the progression of Ewing sarcoma.2.Knockdown of ALDH18A1 significantly inhibited the abilities of proliferation,clone formation,spheroid formation and tumorigenic ability in xenografts in Ewing sarcoma cell.It is suggested that high ALDH18A1 expression is associated with the abilities of self-renewal,proliferation and tumor initiation in Ewing sarcoma cell.3.The expression of PARP1 and EWS-FLI1 downstream target genes appeared to be downregulated after knockdown of ALDH18A1.It suggests that ALDH18A1 may inhibit Ewing sarcoma cell self-renewal through the EWS-FLI1-PARP1 positive feedback loop.4.YG1702,a small molecule inhibitor of ALDH18A1,significantly reduces the ability of Ewing sarcoma to self-renew and proliferate in vivo and in vitro,and may be a candidate small molecule for Ewing sarcoma treatment.