IGF1R和EGFR信號(hào)通路的異常對(duì)惡性周圍神經(jīng)鞘瘤細(xì)胞惡性行為的影響
發(fā)布時(shí)間：2018-09-07 08:14
【摘要】：目的:惡性周圍神經(jīng)鞘瘤(Malignant peripheral nerve sheath tumor,MPNST)是指任何起源于外周神經(jīng)或顯示神經(jīng)鞘分化的惡性腫瘤,約占軟組織惡性腫瘤的5-10%。MPNST為高度惡性,復(fù)發(fā)率約45%,轉(zhuǎn)移率約19%,5年生存率為50%左右,而5年無病生存期僅為27%。病變一般進(jìn)展迅速,預(yù)后很差,傳統(tǒng)的手術(shù)、放療、化療綜合治療的有效率不高,未來的獲益很可能來源于在基因及分子水平上明確疾病的發(fā)病機(jī)制并尋找新的預(yù)后指標(biāo)及特異性治療靶點(diǎn)。本研究擬分析MPNST患者的基因組異常、胰島素樣生長(zhǎng)因子1受體(Insulin-like growth factor 1 receptor,IGF1R)及表皮生長(zhǎng)因子受體(Epidermal growth factor receptor,EGFR等重要信號(hào)通路的異常及其生物學(xué)意義,在此基礎(chǔ)上觀察IGF1R及EGFR信號(hào)通路異常對(duì)MPNST細(xì)胞惡性行為的影響。方法:采用微陣列比較基因組雜交(microarray based-comparative genomic hybridization,a CGH)、生物信息統(tǒng)計(jì)(bioinformatics)、熒光原位雜交(Fluorescence in situ hybridization,FISH)、免疫組織化學(xué)(Immnohistochemistry,IHC)、細(xì)胞培養(yǎng)、細(xì)胞生物學(xué)及分子生物學(xué)等方法,進(jìn)行體內(nèi)外的研究。結(jié)果:1.對(duì)來自于天津醫(yī)科大學(xué)腫瘤醫(yī)院(26例)及美國(guó)得克薩斯大學(xué)MD Anderson腫瘤中心(25例)的共51例MPNST患者腫瘤樣本進(jìn)行微陣列-比較基因組雜交(a CGH)分析,結(jié)果發(fā)現(xiàn)發(fā)現(xiàn):(1)對(duì)來自中美兩國(guó)的MPSNT患者的基因組異常對(duì)比后發(fā)現(xiàn)盡管美國(guó)MPNST樣本顯示更多的基因異常,但兩者基因組改變模式無顯著差異,無顯著的異質(zhì)性。(2)MPNST腫瘤中存在大量的染色體異常區(qū)域,共包含了2599個(gè)基因的擴(kuò)增及4901個(gè)基因缺失(至少出現(xiàn)在25%以上的樣品)。常見的改變?nèi)缂s65%的患者中存在9p21.3的缺失(該區(qū)域包含腫瘤抑癌基因CDKN2A和CDKN2B)。其他常見的改變?nèi)缤負(fù)洚悩?gòu)酶(DNA)IIα(topoisomerase(DNA)II alpha,TOP2A)、細(xì)胞周期蛋白依賴性激酶4(cyclin-dependent kinase 4,CDK4)和forkhead box M1(FOXM1)等基因的缺失;表皮生長(zhǎng)因子受體(Epidermal growth factor receptor,EGFR)、胰島素樣生長(zhǎng)因子1受體(Insulin-like growth factor 1 receptor,IGF1R)、細(xì)胞周期蛋白依賴性激酶6(cyclin-dependent kinase 6,CDK6)、鉀通道亞家族成員K 12(potassium channel,subfamily K member 12,KCNK12)、MET原癌基因(met proto-oncogene,MET)和血小板衍生生長(zhǎng)因子受體α(platelet-derived growth factor receptor alpha polypeptide,PDGFRA)等基因的擴(kuò)增;(3)在信號(hào)通路水平,存在11個(gè)信號(hào)通路有顯著的遺傳學(xué)改變?nèi)鏣FF、ERK、ARF、IGF1R及EGFR等信號(hào)通路的基因存在基因拷貝數(shù)顯著擴(kuò)增。2.為了明確IGF1R信號(hào)通路是否為MPNST潛在的治療靶點(diǎn),對(duì)IGF1R信號(hào)通路的基因組學(xué)異常進(jìn)行探討,結(jié)果發(fā)現(xiàn):(1)IGF1R信號(hào)通路的成員存在顯著的基因擴(kuò)增,IGF1R信號(hào)通路基因異常多的患者總生存期顯著降低。(2)通過免疫組織化學(xué)的方法分析發(fā)現(xiàn)IGF1R蛋白的表達(dá)與MPNST的無病生存期和總生存期顯著相關(guān),高表達(dá)IGF1R的MPNST患者無病生存期和總生存期顯著降低。(3)在人MPNST細(xì)胞系ST88-14中,用小干擾RNA抑制IGF1R信號(hào)通路的活性后發(fā)現(xiàn)PI3K/AKT及MAKP信號(hào)途徑受阻而出現(xiàn)腫瘤增殖活性下降、侵襲及遷移能力降低。(4)在人MPNST細(xì)胞系ST88-14中,用特異性IGF1R單克隆抗體MK-0646抑制IGF1R信號(hào)通路的活性后發(fā)現(xiàn)PI3K/AKT及MAKP信號(hào)途徑受阻而出現(xiàn)腫瘤增殖活性下降、侵襲及遷移能力降低。3.對(duì)EGFR信號(hào)通路的分析發(fā)現(xiàn):(1)MPNST中EGFR信號(hào)通路存在顯著的基因異常,包括EGFR基因顯著擴(kuò)增,FISH方法驗(yàn)證了EGFR基因的擴(kuò)增并發(fā)現(xiàn)其擴(kuò)增模式為大片段擴(kuò)增。(2)EGFR蛋白的高表達(dá)與MPNST患者的無病生存和總體生存顯著相關(guān),高表達(dá)EGFR的MPNST患者無病生存期和總生存期顯著降低。(3)在人MPNST細(xì)胞系ST88-14和STS26T中,EGFR si RNA抑制EGFR活性導(dǎo)致細(xì)胞增殖、遷移、侵襲能力的下降,并伴隨有PI3K/Akt和MAPK途徑的活性下降。(4)在人MPNST細(xì)胞系ST88-14和STS26T中,吉非替尼抑制EGFR活性導(dǎo)致細(xì)胞增殖、遷移、侵襲能力的下降,并伴隨有PI3K/Akt和MAPK途徑的活性下降。4.體外實(shí)驗(yàn)在人MPNST細(xì)胞系STS26T和ST88-14中聯(lián)合IGF1R和EGFR si RNA明顯降低細(xì)胞的增殖,但沒有明顯的附加或協(xié)同效應(yīng);聯(lián)合應(yīng)用EGFR和IGF1R抑制劑引起EGFR和IGF1R的活性形式下降并對(duì)細(xì)胞增殖有抑制作用,但沒有明顯的附加或協(xié)同效應(yīng)。結(jié)論:1.MPNST中存在大量的遺傳學(xué)異常,頻發(fā)擴(kuò)增的基因有2599個(gè),頻發(fā)缺失的基因的基因有4901個(gè)。常見的TFF、ERK、ARF、IGF1R及EGFR等信號(hào)通路的基因存在顯著的基因擴(kuò)增。2.MPNST中存在IGF1R信號(hào)通路的遺傳學(xué)異常且對(duì)細(xì)胞的惡性行為產(chǎn)生影響。3.MPNST中存在EGFR信號(hào)通路的遺傳學(xué)異常且對(duì)細(xì)胞的惡性行為產(chǎn)生影響。4.在MPNST中IGF1R和EGFR信號(hào)通路之間可能沒有串?dāng)_(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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.43
本文編號(hào)：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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.43
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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 李鋒;翟勇平;;8p11骨髓增殖綜合征的研究現(xiàn)狀[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2013年04期
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