【作者】 孙颖；

【导师】 高明；

【作者基本信息】 天津医科大学 ， 临床医学（专业学位）， 2023， 博士

【摘要】 背景:甲状腺未分化癌(Anaplastic thyroid carcinoma,ATC)具有侵袭性高、转移早、预后差的特点,是恶性程度最高的肿瘤之一,也是甲状腺癌中预后最差的一种类型,即使采用广泛的多模式治疗(手术、外照射放疗和化疗),生存期也仅为数周或数月,到目前为止,还没有统一的治疗方案能提高ATC的总生存期(OS)。近年来,研究肿瘤的分子生物学越来越丰富,以及信号转导通路的深入挖掘,靶向治疗将基因突变作为切入点,为晚期肿瘤提供了新的治疗方案。ATC常由分化型甲状腺癌(DTC)引起,但也可从头出现。ATC的典型基因突变包括TP53、TERT、KRAS/NRAS、BRAF等以及WNT信号通路中的基因突变。BRAF/MEK抑制剂组合(达拉非尼/曲美替尼)已被批准用于BRAF V600E突变的ATC,并显着提高生存率,但10年生存率也低于10%。在寻找新的治疗策略时,一些研究集中在表观遗传修饰在甲状腺肿瘤发生中的作用,而表观遗传调节剂本身已引起人们的广泛兴趣,作为癌症中可治疗的新型治疗靶标。那么,开发新的表观遗传调节剂有望成为治疗ATC的新思路。表观遗传失调是诱发肿瘤发生发展的关键因素之一,但其作用机制仍不十分清楚。DNA甲基化、染色质重塑、组蛋白修饰的改变以及非编码RNA丰度的变化等表观遗传的异常是多种癌变的重要标志,整个表观基因组的组蛋白赖氨酸甲基化动态在时间和空间上的精确协调,几乎调节着所有的DNA模板化过程。已在人类的多种恶性肿瘤中检测到组蛋白赖氨酸去甲基酶(KDM)突变或失调,包括尿路上皮癌、乳腺癌和T细胞急性淋巴细胞白血病等。已知组蛋白赖氨酸去甲基酶抑制剂可抑制肺癌、结直肠癌和前列腺癌细胞系的生长及诱导细胞凋亡,但目前尚无实验证据表明该类抑制剂对ATC细胞具有类似作用。甲状腺未分化癌干细胞具有侵袭性,对包括放疗和化疗在内的常规疗法具有高度抵抗力,即使原发病灶已被根除,也会导致疾病复发。因此,靶向甲状腺未分化细胞可能代表一种有效的治疗侵袭性肿瘤的策略。目的:通过本实验的开展,我们希望寻找到一种新型靶向小分子药物,能抑制肿瘤干性,并有效抑制甲状腺未分化癌的发生发展,为临床药物治疗甲状腺未分化癌提供新的实验依据。方法:1.以高通量测序检测ATC细胞系在接受177种小分子抑制剂处理后筛选出杀伤作用都可以达到的50%以上的药物,本课题选择其中之一JIB-04进行实验。2.使用流式细胞术检测ATC细胞系的细胞周期与凋亡,CCK-8实验检测细胞增殖能力,单克隆形成实验检测克隆形成能力以及免疫印迹法检测组蛋白赖氨酸三甲基化表达水平进行检测。3.利用ATC异种移植小鼠模型进行体内实验确认JIB-04疗效,评估药物安全性,并利用免疫组化染色小鼠皮下肿瘤标本Ki67确定体内肿瘤增殖能力。4.基因富集分析ATC细胞系中的JIB-04作用通路,并以实时定量PCR、以及免疫印迹法分别对ATC细胞系的信号通路中特色指标(p-Smad2、p-Smad3等)进行分析,通过细胞侵袭和迁移试验、免疫印迹法进行回复实验,反向验证JIB-04通过TGF-β通路在甲状腺未分化癌中发挥作用。5.免疫印迹法检测ATC细胞系中干性指标,检验JIB-04能否抑制ATC干性。结果:1.高通量药物筛选现发现Jumonji组蛋白去甲基化酶抑制剂JIB-04可以有效杀伤多种ATC。2.ATC细胞系在接受Jumonji组蛋白去甲基化酶抑制剂JIB-04处理后,细胞克隆形成及增殖能力明显被抑制,且存在时间和剂量依赖性。3.与对照组相比,JIB-04处理的ATC细胞的迁移和侵袭能力均明显下降,细胞周期更为显著地阻滞在S期,JIB-04处理让ATC细胞的组蛋白赖氨酸三甲基转移酶水平上调,体内条件下JIB-04有效抑制小鼠肿瘤生长,且小鼠体重无明显变化,未见明显毒性反应。4.基因富集分析ATC细胞系中的JIB-04作用通路可能与TGF-β相关,在TGF-β通路中的p-Smad2/3、TGF-β蛋白表达水平明显降低,实时定量PCR检测ATC细胞系中TGF-β的转录水平经JIB-04处理后也受到抑制。5.JIB-04明显下调ATC细胞系干性指标。6.回复实验证实外源性补充TGF-β后,被JIB-04处理的ATC细胞的迁移、侵袭能力均有恢复,p-Smad2/3蛋白水平也有所增加。结论:在本研究中,我们旨在通过筛选各种表观遗传途径的抑制剂来鉴定选择性靶向ATC细胞的小分子化合物,JIB-04在体外及体内实验中显著减弱了ATC的发生和发展,并抑制了细胞的干性,作用机制可能与TGF-β通路相关。总体而言,我们的结果表明,JIB-04可能是ATC的新型治疗剂。更多还原

【Abstract】 BackgroundAnaplastic thyroid carcinoma(ATC)is one of the most malignant tumors and one of the worst prognosis types of thyroid cancer,to date,there is no uniform treatment regimen that can improve the overall survival(OS)of ATC.Epigenetic dysregulation is one of the key factors that induce tumor development,but its mechanism is still not very clear.The precise coordination of the histone lysine methylation dynamics of the entire epigenome in time and space regulates almost all DNA tematization processes.JIB-04 is a small molecule that inhibits the demethylase activity of the Jumonji family of histone lysine demethylases by a novel mechanism.JIB-04 is a fully selective inhibitor that is known to inhibit the growth and induce apoptosis of lung cancer,colorectal cancer and prostate cancer cell lines,but no experimental evidence has shown that JIB-04 has a similar effect on ATC cells.ObjectiveBy conducting this experiment,we hope to find a novel targeted small molecule drug that can inhibit tumor stemness and effectively inhibit the development of undifferentiated thyroid cancer,providing a new experimental basis for clinical drug treatment of undifferentiated thyroid cancer.Methods1.ATC cell lines were detected by high-throughput sequencing.After 177 kinds of small molecule inhibitors were treated,more than 50%of the drugs that could achieve killing effect were screened out.2.Clonal formation ability,cell proliferation ability,cell cycle and histone lysine trimethylation expression level of ATC cell lines were detected by monoclonal formation assay,flow cytometry and so on.3.A xenograft mouse model was established for in vivo experiments to confirm the efficacy of JIB-04,and the immunohistochemical staining index Ki67 was used to determine the proliferation ability of tumor in vivo.4.Gene enrichment analysis of JIB-04 action pathway in ATC cell lines,and real-time quantitative PCR and western blotting were used to analyze the characteristic indicators(p-Smad2,p-Smad3,etc.)in the signaling pathway of ATC cell lines,and cell invasion and migration tests and western blotting were used to perform recovery experiments.The role of JIB-04 in undifferentiated thyroid carcinoma through TGF-βpathway was reversely verified.5.Immunoblotting assay was performed to detect stemness indicators in ATC cell lines and to test whether JIB-04 could inhibit ATC stemness.Results1.High-throughput drug screening now revealed that Jumonji histone demethylase inhibitor JIB-04 could effectively kill a variety of ATCs.2.ATC cell lines treated with JIB-04 showed significant inhibition of cell clone formation and proliferation ability in a time-and dose-dependent manner.3.The migration and invasion ability of JIB-04-treated ATC cells were decreased significantly,and the cell cycle was blocked in the S phase more significantly.JIB-04treatment upregulated the histone lysine trimethyltransferase level of ATC cells,and JIB-04 effectively inhibited tumor growth in mice under in vivo conditions,and no significant toxic reactions were observed.4.Gene enrichment analysis of JIB-04 pathway in ATC cell lines may be related to TGF-β.The expression levels of p-Smad2/3 and TGF-β1 proteins in the TGF-βpathway were significantly reduced,and the transcript levels of TGF-β1 in ATC cell lines were also inhibited after JIB-04 treatment by real-time quantitative PCR.5.JIB-04 significantly down-regulated the stemness of ATC cell lines.6.Response experiments confirmed that exogenous supplementation of TGF-βrestored the migratory and invasive abilities of ATC cells treated with JIB-04,and p-Smad2/3 protein levels were increased.ConclusionIn this study,we found that JIB-04 significantly attenuated the occurrence and development of ATC and inhibited cell stemness in in vitro and in vivo experiments.Overall,our results suggest that JIB-04 may be a novel therapeutic agent for ATC.更多还原