【作者】 刘志勇；

【导师】 葛红；

【作者基本信息】 郑州大学 ， 肿瘤学（专业学位）， 2022， 博士

【摘要】 研究背景:骨肉瘤是一类最常见的原发骨恶性肿瘤,晚期患者预后差。安罗替尼是我国开发的一种酪氨酸激酶抑制剂,鉴于我国尚未批准任何治疗晚期骨肉瘤的靶向药物,部分治疗中心初步探索了安罗替尼在晚期骨肉瘤中的应用。因此,亟需总结安罗替尼治疗骨肉瘤的临床数据,并对骨肉瘤的发生发展及耐药深入研究。有研究表明,PSMD14（26S proteasome non-ATPase regulatory subunit 14,26S蛋白酶体非ATP酶调节亚基14）基因表达与骨肉瘤患者不良预后有关,可能是不良预后的一个预测指标,是治疗骨肉瘤的潜在靶点,但其作用机制尚不清楚。有研究显示,PSMD14可通过AKT、Snail、m TOR、E2F1和TGF-β等多种关键因子促进肿瘤增殖,侵袭,迁移及耐药等。因此,有必要深入探讨PSMD14对骨肉瘤发生发展的影响和作用机制,以及PSMD14作为骨肉瘤安罗替尼耐药后治疗靶点的可行性。本文拟从骨肉瘤的临床和基础开展研究:1)分析安罗替尼在不可切除或转移性骨肉瘤中的临床疗效和安全性,为靶向治疗提供临床证据;2)PSMD14促进骨肉瘤进展和机制及促进骨肉瘤安罗替尼耐药的研究,为骨肉瘤的发生发展及耐药提供理论基础。第一部分安罗替尼治疗晚期骨肉瘤的安全性和有效性研究目的:评估安罗替尼超适应症治疗不可切除或转移性骨肉瘤的临床疗效和安全性。方法:回顾性分析2018年6月至2020年12月期间经安罗替尼治疗不可切除或转移性进展期骨肉瘤患者的临床资料、疾病转归和预后,统计分析客观缓解率（objective response rate,ORR）、无疾病进展期（progression-free survival,PFS）和不良事件发生等指标。结果:纳入患者27例,其中经典型骨肉瘤25例,其他2例。男性17例,女性10例,中位年龄20（16～68）岁。中位随访5个月,部分缓解2例,疾病稳定16例,ORR为7.4%,中位PFS为4.7个月（95%CI;1.3～16.6个月）,3个月的无进展生存率和6个月的无进展生存率分别为75%和37%。气胸是最常见的3/4级不良事件,其他依次是手足综合征和胆固醇升高等,无药物相关死亡事件。第二部分PSMD14促进骨肉瘤进展及机制研究目的:探讨PSMD14对骨肉瘤进展的影响,并探讨可能的相关机制。方法:1.RT-PCR和Western blot检测13例骨肉瘤组织和非瘤瘤旁骨组织中PSMD14表达;免疫组织化学法检测76例骨肉瘤组织中PSMD14表达,探讨PSMD14表达与临床病理特征和预后的关系。2.筛选Saos-2和U2OS作为研究对象,分别采用sh RNA和过表达质粒慢病毒感染骨肉瘤细胞,RT-PCR和Western blot检测干扰效果。TUNEL实验检测骨肉瘤细胞凋亡;CCK-8实验和克隆形成实验分别检测细胞活力和增殖;划痕实验和Transwell实验分别检测细胞迁移和侵袭;Western blot方法检测PSMD14对上皮间质转化（Epithelial-mesenchymal transition,EMT）的影响。筛选PSMD14敲降稳定克隆细胞,裸鼠成瘤实验观察骨肉瘤细胞体内生长。3.Western blot方法检测PSMD14对PI3K/AKT/m TOR信号通路的影响。结果:1.PSMD14与骨肉瘤预后的相关性RT-PCR和Western blot检测显示骨肉瘤组织中PSMD14的表达显著高于非瘤瘤旁正常骨组织（P<0.05）,PSMD14高表达与肿瘤病理分级和肿瘤分期密切相关,多变量分析显示PSMD14高表达（HR=3.632,P<0.05）和临床分期（HR=3.991,P<0.05）是总生存期的独立危险因素。2.PSMD14对骨肉瘤进展的影响RT-PCR和Western blot检测显示经转染sh RNA和过表达质粒慢病毒感染的Saos-2和U2OS中PSMD14表达分别显著降低和升高（均P<0.05）。TUNEL实验显示敲降PSMD14后Saos-2和U2OS凋亡比例均显著升高（均P<0.05）;CCK-8和克隆形成实验显示敲降PSMD14后Saos-2和U2OS的增殖均显著降低（均P<0.05）;Transwell实验显示敲降PSMD14后Saos-2和U2OS的细胞迁移和侵袭均显著降低（均P<0.05）;敲降PSMD14后Saos-2和U2OS中E-cad表达均显著增加,而Vimentin和Snail表达均显著降低（均P<0.05）。过表达PSMD14的Saos-2和U2OS;Transwell实验显示细胞迁移和侵袭均显著增强（均P<0.05）;E-cad表达均显著下降,而Vimentin和Snail表达均显著增加（均P<0.05）。裸鼠成瘤试验显示敲降PSMD14后骨肉瘤细胞移植瘤体积和重量均显著减少（均P<0.05）。3.PSMD14对PI3K/AKT/m TOR信号通路的影响Western blot方法显示敲降PSMD14后细胞中PI3K、AKT和m TOR蛋白磷酸化水平均显著降低（均P<0.05）,而过表达PSMD14后PI3K、AKT和m TOR蛋白磷酸化水平均显著增加（均P<0.05）,提示PSMD14可促进PI3K/AKT/m TOR信号通路激活。第三部分PSMD14促进骨肉瘤安罗替尼耐药的体外研究目的:探讨PSMD14对骨肉瘤安罗替尼耐药的体外影响。方法:1.采用药物连续诱导法筛选骨肉瘤安罗替尼耐药株（Saos-2-R和U2OS-R）,并通过IC₅₀和TUNEL实验验证耐药株,Western blot方法检测PSMD14在耐药株中表达,筛选敲降PSMD14的稳转耐药株。2.将耐药株分为对照组、安罗替尼组、敲降PSMD14组和敲降PSMD14联合安罗替尼组（联合组）。TUNEL、CCK-8实验和Transwell实验分别检测各组耐药株的凋亡,增殖及迁移和侵袭能力。结果:1.PSMD14在耐药株中高表达Saos-2-R和U2OS-R的IC₅₀分别为亲本细胞的3.05倍（81.77μM对比26.84μM）和3.03倍（142.7μM对比47.05μM）,TUNEL实验显示亲本细胞凋亡率显著高于耐药株（均P<0.05）,提示耐药株构建成功。Western blot方法检测耐药株中PSMD14较亲本细胞高表达。2.敲降PSMD14可逆转耐药株对安罗替尼耐药相对于对照组,TUNEL实验显示安罗替尼组细胞凋亡均无显著差异（均P>0.05）,敲降PSMD14组和联合组凋亡均显著增高（均P<0.05）,联合组凋亡率更高;CCK-8实验显示安罗替尼组细胞活力均无显著差异（均P>0.05）,敲降PSMD14组和联合组细胞活力均显著下降（均P<0.05）,联合组下降更显著;Transwell实验显示安罗替尼组细胞侵袭和迁移均无显著差异（均P>0.05）,敲降PSMD14组和联合组细胞侵袭和迁移能力均显著下降（均P<0.05）,其中联合组细胞侵袭和迁移下降更显著。结论:PSMD14在骨肉瘤中高表达,是患者不良预后的一个独立危险因素,PSMD14可促进骨肉瘤进展和对安罗替尼耐药,并可能通过调控PI3K/AKT/m TOR信号通路促进骨肉瘤进展。更多还原

【Abstract】 Background:Osteosarcoma is one of the most common primary bone malignancies with poor prognosis in advanced patients.In recent years,tyrosine kinase inhibitors have achieved promising results in advanced osteosarcoma.Anlotinib is a multi-target tyrosine kinase inhibitor independently developed in China.In view of the fact that no targeted drugs for advanced osteosarcoma have been approved in China,some treatment centers began to explore the application value of anlotinib in advanced osteosarcoma.It is urgent to summarize the clinical data of the off-label treatment of anlotinib on patients with osteosarcoma who failed standard treatment,and to further study the pathogenesis and drug resistance in osteosarcoma.Studies showed that PSMD14（26S Proteasome non-ATPase regulatory subunit14,26S proteasome non-ATPase regulatory subunit 14）gene expression was associated with poor prognosis in patients with osteosarcoma,and PSMD14 may be a predictor of poor prognosis and a potential target for the treatment of osteosarcoma.However,the effect of PSMD14 in osteosarcoma and its mechanism remains unclear.Studies showed that PSMD14 could promote tumor proliferation,invasion,migration and resistance through various key factors such as AKT,Snail,m TOR,E2F1 and TGF-β.Therefore,it is necessary to further explore the effect and mechanism of PSMD14on the oncogenesis and development of osteosarcoma,and the feasibility of PSMD14as a therapeutic target in anlotinib-resistant osteosarcoma.This study is intended to carry out the research from the preclinical and clinical levels:1)The clinical efficacy and safety of anlotinib in unresectable or metastatic osteosarcoma were analyzed to provide more clinical evidence for targeted treatment of osteosarcoma patients;2)Study on PSMD14 promoting progression and its mechanism and promoting resistance to anlotinib in osteosarcoma to provide theoretical basis for the oncogenesis and resistance in osteosarcoma.Part I:Efficacy and safety of anlotinib in the treatment of advanced osteosarcomaPurpose:To evaluate the clinical efficacy and safety of anlotinib in the off-label treatment of unresectable or metastatic advanced osteosarcoma.Methods:Clinical data and survival of patients with unresectable or metastatic advanced osteosarcoma treated with anlotinib from June 2018 to December 2020 were retrospectively analyzed,and the objective response rate,progression-free survival,and adverse events were statistically analyzed.Result:Twenty-seven patients were included in the study with 25 patients with classical osteosarcoma and 2 others.There were 17 males and 10 females with a median age of20（16-68）years.The median follow-up was 5 months.After treatment,partial response in 2 patients,stable disease in 16 patients,the ORR of 7.4%and median PFS was 4.7months（95%CI;1.3-16.6 months）,the 3-month and 6-month progression-free survival rates were 75%and 37%,respectively.Pneumothorax was the most common grade 3/4adverse events,followed by hand-foot syndrome and elevated cholesterol.No treatment-related deaths were observed.Part II:The study of PSMD14 promoting osteosarcoma progression and its mechanismObjective:To investigate the effect of PSMD14 on osteosarcoma progression and its possible molecular mechanism.Methods:1.RT-PCR and Western blot were used to detect the expression of PSMD14 in 13osteosarcoma tissues and non-tumor adjacent bone tissues.Immunohistochemistry was used to detect the expression of PSMD14 in 76 osteosarcoma patients,and to explore the relationship between the expression of PSMD14 and clinicopathological features and prognosis in osteosarcoma patients.2.The osteosarcoma cell lines Saos-2 and U2OS were screened as the research objects.The osteosarcoma cell lines were infected with lentivirus transfected with sh RNA and overexpressed plasmid,respectively,and the interference effect was detected by RT-PCR and Western blot.TUNEL assay was used to detect the effect of PSMD14 on osteosarcoma cell apoptosis.CCK-8 assay and clone formation assay were used to detect the effects on cell viability and proliferation,respectively.The effects of wound healing assay and transwell assay on cell migration and invasion were detected,respectively.Western blot was used to detect the effect of PSMD14 on epithelial-mesenchymal transition（EMT）.PSMD14 knockdown stable clone cells were screened and tumorigenesis experiments were conducted in nude mice to observe the effect of PSMD14 on the growth of osteosarcoma cells in vivo.3.Western blot was used to detect the effect of PSMD14 on PI3K/AKT/m TOR signaling pathway.Results:1.Expression of PSMD14 and its correlation with poor prognosis in patients with osteosarcomaRT-PCR and Western blot results showed that PSMD14 expression in osteosarcoma tissues was significantly higher than that in non-tumor adjacent normal bone tissues（P<0.05）.It was found that the high expression of PSMD14 was closely related to the pathological grade and tumor stage.Multivariate analysis showed that high PSMD14 expression（HR=3.632,P<0.05）and clinical stage（HR=3.632,P<0.05）were independent risk factors for overall survival.2.Effects of PSMD14 on osteosarcoma progressionRT-PCR and Western blot showed that,compared with the control group,PSMD14 expression was significantly decreased and increased in SAOS-2 and U2OS transfected with sh RNA and overexpressed plasmid lentivirus（all P<0.05）,respectively.TUNEL assay showed that the apoptotic rates of Saos-2 and U2OS were significantly increased after PSMD14 knockdown（all P<0.05）.CCK-8 and colony formation assay showed that the cell viability and proliferation ability of Saos-2 and U2OS were significantly reduced after PSMD14 knockdown（all P<0.05）,respectively.Transwell assay showed that the migration and invasion ability of Saos-2and U2OS were significantly reduced after PSMD14 knockdown（all P<0.05）,respectively.Meanwhile,the expression of E-cadherin（E-Cad）protein in Saos-2 and U2OS was significantly increased after PSMD14 knockdown（all P<0.05）,respectively,while the expression of Vimentin and Snail protein was significantly decreased（all P<0.05）,respectively.After overexpression of PSMD14 in osteosarcoma cellls,the transwell assay showed that the migration and invasion abilities of Saos-2 and U2OS were significantly enhanced（all P<0.05）.E-cad protein expression in Saos-2 and U2OS decreased significantly after PSMD14 overexpression（all P<0.05）,respectively,while the expression of Vimentin and Snail protein increased significantly（all P<0.05）,respectively.Compared with the control group,the volume and weight of transplanted osteosarcoma cells after PSMD14 knockdown were significantly reduced in vivo（all P<0.05）.3.Effect of PSMD14 on PI3K/AKT/m TOR signaling pathway in osteosarcoma cellsWestern blot analysis showed that,compared with the control group,the phosphorylation levels of PI3K,AKT and m TOR proteins in osteosarcoma cells in the knockdown PSMD14 group were significantly decreased（all P<0.05）,respectively,while the phosphorylation levels of PI3K,AKT and m TOR proteins in the overexpression PSMD14 group were significantly increased（all P<0.05）,respectively.These results suggested that PSMD14 may promote the activation of PI3K/AKT/m TOR signaling pathway in osteosarcoma cells.PartⅢ:Study on PSMD14 promoting the resistance to anlotinib in anlotinib-resistant osteosarcoma in vitroObjective:To investigate the effect of PSMD14 on anlotinib-resistant osteosarcoma in vitro.Methods:1.We adopted the continuous drug induction method to gradually induce and screen the osteosarcoma cell lines resistant to anlotinib（Saos-2-R and U2OS-R）,and then the induced anlotinib-resistant osteosarcoma cell lines were verified by IC₅₀ and TUNEL assay,the expression of PSMD14 in anlotinib-resistant cells was used to detect by Western blot.2.The cell lines of anlotinib-resistant osteosarcoma were divided into the control group,the anlotinib group,the knockdown PSMD14 group and the knockdown PSMD14 combined with anlotinib group（combined group）.TUNEL,CCK-8 and Transwell assay was used to detect the apoptosis,proliferation,migration and invasion ability of cell lines of anlotinib-resistant osteosarcoma in each group.Results:1.High expression of PSMD14 in anlotinib-resistant osteosarcoma cell linesThe IC₅₀ values in Saos-2-R and U2OS-R were 3.05（81.77μM vs 26.84μM）and3.03 times（142.7μM vs 47.05μM）higher than those in sensitive cell lines,respectively.TUNEL assay showed that the apoptosis rate of sensitive cell lines was higher than that of resistant cell lines（all P<0.05）,respectively.which suggested that anlotinib-resistant cell lines were induced.Western blot showed that the higher expression of PSMD14 in resistant cell lines was detected than in sensitive cell lines.2.Knockdown PSMD14 could reverse the resistance to anlotinib in anlotinib-resistant osteosarcoma cell linesTUNEL assay showed that there were no significant differences in apoptosis rate between the anlotinib group and the control group in resistant cell lines（all P>0.05）,respectively,while the apoptosis rate of the knockdown PSMD14 group and combined group significantly increased compared with the control group（all P<0.05）,respectively,and the combined group had a higher apoptosis rate.CCK-8 assay showed that there were no significant differences in cell viability in the anlotinib group compared with the control group（all P>0.05）,respectively,while the cell viability was significantly decreased in the knockdown PSMD14 group and the combination group compared with the control group（all P<0.05）,respectively,and the decrease of cell viability was more significant in the combined group.Transwell assay showed that there were no significant differences in the invasion and migration rates in the anlotinib group compared with the control group（all P>0.05）,respectively,while the invasion and migration rates in the knockdown PSMD14 group and the combined group were significantly increased compared with the control group（all P<0.05）,respectively,and the decrease of the invasion and migration rates in the combined group was more significant.Conclusion:PSMD14 is highly expressed in osteosarcoma and is an independent risk factor for poor prognosis.It can promote progression and resistance to anlotinib in osteosarcoma,and may promote osteosarcoma progression by regulating PI3K/AKT/m TOR signaling pathway.更多还原