IGF1R和EGFR信号通路的异常对恶性周围神经鞘瘤细胞恶性行为的影响
发布时间：2018-09-07 08:14
【摘要】：目的:恶性周围神经鞘瘤(Malignant peripheral nerve sheath tumor,MPNST)是指任何起源于外周神经或显示神经鞘分化的恶性肿瘤,约占软组织恶性肿瘤的5-10%。MPNST为高度恶性,复发率约45%,转移率约19%,5年生存率为50%左右,而5年无病生存期仅为27%。病变一般进展迅速,预后很差,传统的手术、放疗、化疗综合治疗的有效率不高,未来的获益很可能来源于在基因及分子水平上明确疾病的发病机制并寻找新的预后指标及特异性治疗靶点。本研究拟分析MPNST患者的基因组异常、胰岛素样生长因子1受体(Insulin-like growth factor 1 receptor,IGF1R)及表皮生长因子受体(Epidermal growth factor receptor,EGFR等重要信号通路的异常及其生物学意义,在此基础上观察IGF1R及EGFR信号通路异常对MPNST细胞恶性行为的影响。方法:采用微阵列比较基因组杂交(microarray based-comparative genomic hybridization,a CGH)、生物信息统计(bioinformatics)、荧光原位杂交(Fluorescence in situ hybridization,FISH)、免疫组织化学(Immnohistochemistry,IHC)、细胞培养、细胞生物学及分子生物学等方法,进行体内外的研究。结果:1.对来自于天津医科大学肿瘤医院(26例)及美国得克萨斯大学MD Anderson肿瘤中心(25例)的共51例MPNST患者肿瘤样本进行微阵列-比较基因组杂交(a CGH)分析,结果发现发现:(1)对来自中美两国的MPSNT患者的基因组异常对比后发现尽管美国MPNST样本显示更多的基因异常,但两者基因组改变模式无显著差异,无显著的异质性。(2)MPNST肿瘤中存在大量的染色体异常区域,共包含了2599个基因的扩增及4901个基因缺失(至少出现在25%以上的样品)。常见的改变如约65%的患者中存在9p21.3的缺失(该区域包含肿瘤抑癌基因CDKN2A和CDKN2B)。其他常见的改变如拓扑异构酶(DNA)IIα(topoisomerase(DNA)II alpha,TOP2A)、细胞周期蛋白依赖性激酶4(cyclin-dependent kinase 4,CDK4)和forkhead box M1(FOXM1)等基因的缺失;表皮生长因子受体(Epidermal growth factor receptor,EGFR)、胰岛素样生长因子1受体(Insulin-like growth factor 1 receptor,IGF1R)、细胞周期蛋白依赖性激酶6(cyclin-dependent kinase 6,CDK6)、钾通道亚家族成员K 12(potassium channel,subfamily K member 12,KCNK12)、MET原癌基因(met proto-oncogene,MET)和血小板衍生生长因子受体α(platelet-derived growth factor receptor alpha polypeptide,PDGFRA)等基因的扩增;(3)在信号通路水平,存在11个信号通路有显著的遗传学改变如TFF、ERK、ARF、IGF1R及EGFR等信号通路的基因存在基因拷贝数显著扩增。2.为了明确IGF1R信号通路是否为MPNST潜在的治疗靶点,对IGF1R信号通路的基因组学异常进行探讨,结果发现:(1)IGF1R信号通路的成员存在显著的基因扩增,IGF1R信号通路基因异常多的患者总生存期显著降低。(2)通过免疫组织化学的方法分析发现IGF1R蛋白的表达与MPNST的无病生存期和总生存期显著相关,高表达IGF1R的MPNST患者无病生存期和总生存期显著降低。(3)在人MPNST细胞系ST88-14中,用小干扰RNA抑制IGF1R信号通路的活性后发现PI3K/AKT及MAKP信号途径受阻而出现肿瘤增殖活性下降、侵袭及迁移能力降低。(4)在人MPNST细胞系ST88-14中,用特异性IGF1R单克隆抗体MK-0646抑制IGF1R信号通路的活性后发现PI3K/AKT及MAKP信号途径受阻而出现肿瘤增殖活性下降、侵袭及迁移能力降低。3.对EGFR信号通路的分析发现:(1)MPNST中EGFR信号通路存在显著的基因异常,包括EGFR基因显著扩增,FISH方法验证了EGFR基因的扩增并发现其扩增模式为大片段扩增。(2)EGFR蛋白的高表达与MPNST患者的无病生存和总体生存显著相关,高表达EGFR的MPNST患者无病生存期和总生存期显著降低。(3)在人MPNST细胞系ST88-14和STS26T中,EGFR si RNA抑制EGFR活性导致细胞增殖、迁移、侵袭能力的下降,并伴随有PI3K/Akt和MAPK途径的活性下降。(4)在人MPNST细胞系ST88-14和STS26T中,吉非替尼抑制EGFR活性导致细胞增殖、迁移、侵袭能力的下降,并伴随有PI3K/Akt和MAPK途径的活性下降。4.体外实验在人MPNST细胞系STS26T和ST88-14中联合IGF1R和EGFR si RNA明显降低细胞的增殖,但没有明显的附加或协同效应;联合应用EGFR和IGF1R抑制剂引起EGFR和IGF1R的活性形式下降并对细胞增殖有抑制作用,但没有明显的附加或协同效应。结论:1.MPNST中存在大量的遗传学异常,频发扩增的基因有2599个,频发缺失的基因的基因有4901个。常见的TFF、ERK、ARF、IGF1R及EGFR等信号通路的基因存在显著的基因扩增。2.MPNST中存在IGF1R信号通路的遗传学异常且对细胞的恶性行为产生影响。3.MPNST中存在EGFR信号通路的遗传学异常且对细胞的恶性行为产生影响。4.在MPNST中IGF1R和EGFR信号通路之间可能没有串扰(crosstalk)。
[Abstract]:Objective: Malignant peripheral nerve sheath tumor (MPNST) is any malignant tumor originating from peripheral nerve or showing nerve sheath differentiation, accounting for about 5-10% of soft tissue malignancies. MPNST is highly malignant, with a recurrence rate of about 45%, a metastasis rate of about 19%, a 5-year survival rate of about 50%, and a 5-year disease-free survival rate of only 27%. In general, the disease progresses rapidly and the prognosis is poor. The efficiency of traditional surgery, radiotherapy and chemotherapy is not high. Future benefits may come from identifying the pathogenesis of the disease at the genetic and molecular level and looking for new prognostic indicators and specific therapeutic targets. The abnormalities of important signaling pathways such as Insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR) and their biological significance were observed. The effects of abnormalities of IGF1R and EGFR signaling pathways on malignant behavior of MPNST cells were observed. Microarray based-comparative genomic hybridization (a CGH), bioinformatics, fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), cell culture, cell biology and molecular biology were used for in vivo and in vitro studies. Microarray-comparative genomic hybridization (a CGH) analysis of 51 MPNST patients from Tianjin Medical University Cancer Hospital (26 cases) and MD Anderson Cancer Center (25 cases) of the University of Texas revealed that: (1) Abnormal genomic comparisons were made between MPNST patients from China and the United States, although MPNST patients from the United States were found. Samples showed more genetic abnormalities, but there was no significant difference in the pattern of genomic alterations between the two groups, and no significant heterogeneity. Deletions (this region contains tumor suppressor genes CDKN2A and CDKN2B). Other common changes include deletions of topoisomerase (DNA) II alpha (TOP2A), cyclin-dependent kinase 4 (CDK4) and forkhead box M1 (FOXM1), and deletions of epidermal growth factor receptor (Epidermal growth factor) Receptor, EGFR, Insulin-like growth factor 1 receptor (IGF1R), cyclin-dependent kinase 6 (CDK6), potassium channel subfamily K member 12 (KCNK12), MET proto-oncogene (MET) and platelet-derived growth Amplification of genes such as platelet-derived growth factor receptor alpha polypeptide (PDGFRA); (3) Gene copies of 11 signaling pathways with significant genetic changes such as TFF, ERK, ARF, IGF1R and EGFR were significantly amplified at the signaling level. Genomic abnormalities in the IGF1R signaling pathway were investigated. The results showed: (1) There was significant gene amplification in the members of the IGF1R signaling pathway, and the overall survival time of patients with abnormal IGF1R signaling pathway was significantly reduced. (2) The expression of IGF1R protein and the absence of MPNST were detected by immunohistochemical analysis. There was a significant correlation between disease survival and overall survival, and a significant decrease in disease-free survival and overall survival in MPNST patients with high expression of IGF1R. (3) Inhibition of IGF1R signaling pathway by small interfering RNA in human MPNST cell line ST88-14 revealed a decrease in tumor proliferation, invasion and migration when PI3K/AKT and MAKP signaling pathways were blocked. Inhibition of IGF1R signaling pathway by specific IGF1R monoclonal antibody MK-0646 in human MPNST cell line ST88-14 revealed a decrease in tumor proliferation, invasion and migration due to blocked PI3K/AKT and MAKP signaling pathways. Including significant amplification of EGFR gene, FISH method confirmed the amplification of EGFR gene and found that the amplification pattern was large fragment amplification. (2) The high expression of EGFR protein was significantly associated with disease-free survival and overall survival of MPNST patients, and the disease-free survival and overall survival of MPNST patients with high expression of EGFR were significantly reduced. (3) Human MPNST cell lines ST88-14 and STS2 were significantly reduced. In 6T, EGFR Si RNA inhibited EGFR activity, resulting in a decrease in cell proliferation, migration and invasiveness, accompanied by a decrease in PI3K/Akt and MAPK pathways. (4) In human MPNST cell lines ST88-14 and STS26T, gefitinib inhibited EGFR activity leading to a decrease in cell proliferation, migration and invasiveness, accompanied by a decrease in PI3K/Akt and MAPK pathways. In vitro, the combination of IGF1R and EGFR Si RNA in human MPNST cell lines STS26T and ST88-14 significantly decreased cell proliferation, but there was no obvious additional or synergistic effect. The combination of EGFR and IGF1R inhibitors decreased the activity of EGFR and IGF1R and inhibited cell proliferation, but no obvious additional or synergistic effect. There are a lot of genetic abnormalities in MPNST. There are 2599 frequently amplified genes and 4901 frequently deleted genes. Genes of common TFF, ERK, ARF, IGF1R and EGFR signaling pathways have significant gene amplification. 2. There are genetic abnormalities in IGF1R signaling pathway in MPNST, which affect the malignant behavior of cells. There is genetic abnormality of EGFR signaling pathway and it affects the malignant behavior of cells. 4. There may be no cross talk between IGF1R and EGFR signaling pathways in MPNST.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.43
本文编号：2227714
[Abstract]:Objective: Malignant peripheral nerve sheath tumor (MPNST) is any malignant tumor originating from peripheral nerve or showing nerve sheath differentiation, accounting for about 5-10% of soft tissue malignancies. MPNST is highly malignant, with a recurrence rate of about 45%, a metastasis rate of about 19%, a 5-year survival rate of about 50%, and a 5-year disease-free survival rate of only 27%. In general, the disease progresses rapidly and the prognosis is poor. The efficiency of traditional surgery, radiotherapy and chemotherapy is not high. Future benefits may come from identifying the pathogenesis of the disease at the genetic and molecular level and looking for new prognostic indicators and specific therapeutic targets. The abnormalities of important signaling pathways such as Insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR) and their biological significance were observed. The effects of abnormalities of IGF1R and EGFR signaling pathways on malignant behavior of MPNST cells were observed. Microarray based-comparative genomic hybridization (a CGH), bioinformatics, fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), cell culture, cell biology and molecular biology were used for in vivo and in vitro studies. Microarray-comparative genomic hybridization (a CGH) analysis of 51 MPNST patients from Tianjin Medical University Cancer Hospital (26 cases) and MD Anderson Cancer Center (25 cases) of the University of Texas revealed that: (1) Abnormal genomic comparisons were made between MPNST patients from China and the United States, although MPNST patients from the United States were found. Samples showed more genetic abnormalities, but there was no significant difference in the pattern of genomic alterations between the two groups, and no significant heterogeneity. Deletions (this region contains tumor suppressor genes CDKN2A and CDKN2B). Other common changes include deletions of topoisomerase (DNA) II alpha (TOP2A), cyclin-dependent kinase 4 (CDK4) and forkhead box M1 (FOXM1), and deletions of epidermal growth factor receptor (Epidermal growth factor) Receptor, EGFR, Insulin-like growth factor 1 receptor (IGF1R), cyclin-dependent kinase 6 (CDK6), potassium channel subfamily K member 12 (KCNK12), MET proto-oncogene (MET) and platelet-derived growth Amplification of genes such as platelet-derived growth factor receptor alpha polypeptide (PDGFRA); (3) Gene copies of 11 signaling pathways with significant genetic changes such as TFF, ERK, ARF, IGF1R and EGFR were significantly amplified at the signaling level. Genomic abnormalities in the IGF1R signaling pathway were investigated. The results showed: (1) There was significant gene amplification in the members of the IGF1R signaling pathway, and the overall survival time of patients with abnormal IGF1R signaling pathway was significantly reduced. (2) The expression of IGF1R protein and the absence of MPNST were detected by immunohistochemical analysis. There was a significant correlation between disease survival and overall survival, and a significant decrease in disease-free survival and overall survival in MPNST patients with high expression of IGF1R. (3) Inhibition of IGF1R signaling pathway by small interfering RNA in human MPNST cell line ST88-14 revealed a decrease in tumor proliferation, invasion and migration when PI3K/AKT and MAKP signaling pathways were blocked. Inhibition of IGF1R signaling pathway by specific IGF1R monoclonal antibody MK-0646 in human MPNST cell line ST88-14 revealed a decrease in tumor proliferation, invasion and migration due to blocked PI3K/AKT and MAKP signaling pathways. Including significant amplification of EGFR gene, FISH method confirmed the amplification of EGFR gene and found that the amplification pattern was large fragment amplification. (2) The high expression of EGFR protein was significantly associated with disease-free survival and overall survival of MPNST patients, and the disease-free survival and overall survival of MPNST patients with high expression of EGFR were significantly reduced. (3) Human MPNST cell lines ST88-14 and STS2 were significantly reduced. In 6T, EGFR Si RNA inhibited EGFR activity, resulting in a decrease in cell proliferation, migration and invasiveness, accompanied by a decrease in PI3K/Akt and MAPK pathways. (4) In human MPNST cell lines ST88-14 and STS26T, gefitinib inhibited EGFR activity leading to a decrease in cell proliferation, migration and invasiveness, accompanied by a decrease in PI3K/Akt and MAPK pathways. In vitro, the combination of IGF1R and EGFR Si RNA in human MPNST cell lines STS26T and ST88-14 significantly decreased cell proliferation, but there was no obvious additional or synergistic effect. The combination of EGFR and IGF1R inhibitors decreased the activity of EGFR and IGF1R and inhibited cell proliferation, but no obvious additional or synergistic effect. There are a lot of genetic abnormalities in MPNST. There are 2599 frequently amplified genes and 4901 frequently deleted genes. Genes of common TFF, ERK, ARF, IGF1R and EGFR signaling pathways have significant gene amplification. 2. There are genetic abnormalities in IGF1R signaling pathway in MPNST, which affect the malignant behavior of cells. There is genetic abnormality of EGFR signaling pathway and it affects the malignant behavior of cells. 4. There may be no cross talk between IGF1R and EGFR signaling pathways in MPNST.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.43
【参考文献】
相关期刊论文 前2条
1 Masakazu Yashiro;Tasuku Matsuoka;;Fibroblast growth factor receptor signaling as therapeutic targets in gastric cancer[J];World Journal of Gastroenterology;2016年08期
2 李锋;翟勇平;;8p11骨髓增殖综合征的研究现状[J];中国实验血液学杂志;2013年04期
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