NU9056 Down-regulates KAT5/c-Myc/miR-202 Pathway To Inhibit The Progression And Inerease The Sensitivity To Chemoradiotherapy Of Anaplastic Thyroid Cancer

Part Ⅰ The Role of KAT5 Inhibitor NU9056 in the Progression and Chemoradiotherapy of Anaplastic Thyroid CancerObjectives:Anaplastic thyroid cancer(ATC)originates from follicular cells,which is rare in clinic.It is highly malignant and grows fast,and often extensively involves thyroid surrounding tissues,and cervical lymph node metastasis and hematogenous metastasis are quite common.Comprehensive treatment is the main treatment method.However,the treatment of ATC is still difficult due to the poor efficacy of various treatments and few lifeprolonging treatments.Therefore,for patients with ATC,it is necessary to explore the molecular mechanism driving the progression of ATC and find better treatments.Previous studies have shown that KAT5 can promote the progression of ATC and may become a potential new target for clinical treatment of ATC.The part of the experiments mainly discussed the effects of KAT5 inhibitor NU9056 on proliferation,migration,invasion in vitro as well as the effects on chemoradiotherapy sensitivity of human ATC cells.The effects of NU9056 on the growth of human ATC cells in nude mice were also investigated.Materials and methods:Firstly,the expression of KAT5 in thyroid cancer and its relationship with the prognosis of patients with thyroid cancer were investigated through literature search and data search in the American Cancer Genome Atlas(TCGA).Western blotting and qRT-PCR were used to detect background KAT5 expression in ATC cells.After NU9056 treatment,the expression of histone H4 and H2A and acetylated histone H4 and H2A were detected in ATC cells to determine whether NU9056 could effectively block KAT5 activity.Then different concentrations of NU9056 were applied to ATC cells 8505C,CAL-62,CAL-62-transfected with KAT5-shRNA,and human normal thyroid follicular epithelial cells Nthy-ori 3-1.Then,the effects of KAT5 inhibitor NU9056 on ATC cell growth,proliferation,invasion,metastasis and sensitivity to chemoradiotherapy were studied by CCK-8 assay,Colony formation assay,Transwell migration and invasion assays.The antitumor activity of NU9056 against ATC in vivo was studied by constructing xenograft tumor model in nude mice.Results:(1)Searching TCGA database showed that thyroid cancer patients with KAT5 abnormal expression had poor prognosis.(2)Western blotting and qRT-PCR assays showed that the expression of KAT5 in human ATC cells was significantly higher than that in welldifferentiated normal human thyroid cells.(3)Western blotting experiments showed that NU9056 played a role by inhibiting the activity of KAT5 acetyltransferase.(4)CCK-8 and Colony formation assays showed that high expression of KAT5 could promote the growth of ATC cells.NU9056 selectively inhibited the activity and proliferation of ATC cells with high expression of KAT5 by inhibiting the acetyltransferase activity of KAT5,and NU9056 weakened or had no inhibitory effect on normal thyroid cells with low expression of KAT5 and ATC cells with low expression of KAT5.(5)Transwell migration and invasion assays showed that NU9056 could specifically inhibit the migration and invasion of ATC cells with high expression of KAT5.(6)CCK-8,Transwell migration and Colony formation experiments showed that NU9056 treatment increased the sensitivity of ATC cells to chemotherapy and radiotherapy,but did not increase the sensitization of normal thyroid cells with low KAT5 expression.(7)The experiments in nude mice showed that NU9056 also inhibited ATC proliferation in vivo.Conclusions:NU9056 could inhibit the growth,proliferation,infiltration and metastasis of ATC cells with high expression of KAT5,and improve the sensitivity of ATC cells to radiotherapy and chemotherapy.However,it had no effect on normal thyroid cells and ATC cells with low expression of KAT5.NU9056 also inhibited the growth of subcutaneous transplanted tumor of ATC cells in nude mice.The high expression of KAT5 promotes the progression of ATC,thus inhibiting KAT5 may be a new decision for future treatment of ATC.Part II The Mechanism of NU9056 Inhibits the Progression and Enhances Sensitivity to Radiochemotherapy of Anaplastic Thyroid CancerPurposes:Based on the research team’s previous results(KAT5 acetylates and promotes the expression of c-Myc protein)and with the help of high-throughput gene sequencing technology,miR-202-5p was significantly up-regulated after KAT5 overexpression in ATC cells.We further hypothesized that KAT5 and its downstream targets such as c-Myc and miR-202-5p play important roles in the progression of ATC.NU9056 may inhibit the progression of ATC and improve the sensitivity of chemoradiotherapy by regulating KAT5,c-Myc and miR-202-5p.Materials and methods:Firstly,through literature search and oncomine database search on ATC and differentiated thyroid cancer data,it was found that c-Myc expression was significantly different in ATC and other differentiated thyroid cancer.The expressions of cMyc in ATC cells and ATC transplanted tumor tissues of nude mice after NU9056 were detected by Western blotting,immunofluorescence and immunohistochemical staining assays.Western blotting was used to detect the effect of Cycloheximide(CHX),a protein synthesis inhibitor,on c-Myc protein expression in ATC cells in order to explore the effect of NU9056 on c-Myc protein and its mechanism.Then,the role of miR-202-5p in the progression of ATC and the effect of NU9056 on its expression were studied by cell transfection,qRT-PCR,CCK-8 and Transwell invasion assays.Finally,J ASPAR prediction program and dual luciferase reporter gene assay were used to predict and verify the transcription factor of miR-202-5p,so as to explore the mechanism of KAT5 regulating the expression of miR-202-5p.Results:(1)Compared with other types of thyroid cancer,c-Myc protein expression was significantly up-regulated in ATC.(2)Western blotting and cell immunofluorescence assays showed that NU9056 reduced c-Myc expression in ATC cells in a dose-dependent manner.Immunohistochemical staining assay further confirmed that NU9056 reduced c-Myc expression in ATC transplanted tumor tissue.(3)Western blotting experiments showed that NU9056 reduced the stability of c-Myc protein by shortening its half-life of ATC cells,thus down-regulating the expression of c-Myc.(4)Cell transfection,miRNAs high-throughput gene sequencing,qRT-PCR,CCK-8 and Transwell invasion assays showed that overexpression of KAT5 significantly increased the expression of miR-202-5p in ATC,while NU9056 inhibition of KAT5 significantly reduced the level of miR-202-5p.It is suggested that miR-202-5p may be the main downstream target of KAT5.(5)Through the JASPAR prediction program,several potential c-Myc binding sites were found in the upstream promoter region of miR-202-5p,suggesting that the transcription factor c-Myc may regulate the expression of miR-202-5p.(6)Double luciferase reporter gene assay showed that transfection of c-Myc significantly increased the luciferase activity of miR-2025p promoter.c-Myc can promote the transcription of miR-202-5p.In conclusion,KAT5 promotes the expression of c-Myc protein through acetylation;KAT5 up-regulated the expression of miR-202-5p at the transcriptional level through c-Myc.KAT5 inhibitor NU9056 down-regulated miR-202-5p expression at the transcriptional level through c-Myc.Conclusions:After the treatment of ATC with KAT5 inhibitor NU9056,the activity of KAT5 acetyltransferase was inhibited,which shortened the half-life and reduced the stability of c-Myc protein,thus down-regulated the expression of miR-202-5p in ATC,inhibiting the progression of ATC and increasing the sensitivity of ATC cells to radiotherapy and chemotherapy.Therefore,the KAT5/c-Myc/miR-202 pathway plays an important role in the progression of ATC.NU9056 can down-regulate the KAT5/c-Myc/miR-202 pathway to inhibit the progression of ATC and increase its sensitivity to radiochemotherapy.