宫颈癌细胞EGFR核转位介导的放射抵抗的潜在机制及临床研究

发布时间：2018-02-13 22:41

  本文关键词： EGFR 核转位 宫颈癌 放射治疗 宫颈癌 EGFR 核转位 辐射 EGFR 宫颈癌 核转位 靶向治疗　出处：《广西医科大学》2017年博士论文　论文类型：学位论文

【摘要】：第一部分EGFR核转位对宫颈癌放化疗预后的影响背景和目的:表皮生长因子受体(EGFR)具有介导细胞表面信号级联和转位到细胞核调控许多细胞过程包括转录调节和DNA损伤的非同源末端连接修复作用。大量的研究证实,EGFR与肺癌,肝癌,胃癌、头颈部肿瘤等的发生、发展及治疗预后有关。然而,宫颈癌细胞EGFR的表达与预后及对放疗反应的相关性仍然是有争议或不确定的。最近的研究提示,一些肿瘤细胞核EGFR表达与放化疗抵抗和预后差有关。有研究报道,放射线可诱导蛋白激酶C(Protein Kinase C,PKC)家族分子PKN1活化和EGFR二聚体的形成,以及活化的PKN1使EGFR近末端654位点的苏氨酸磷酸化(EGFR pThr654),可能是介导EGFR核转位的关键点之一。本文研究了局部中晚期宫颈癌细胞膜EGFR(m EGFR)、磷酸化PKN1(PKN1 pThr774)和EGFR pThr654的表达水平,探讨EGFR核转位对中晚期宫颈癌患者放化疗的预后作用。方法:收集2007年1月至2012年12月在四川省肿瘤医院行根治性同步放化疗IIA-IIIB期(FIGO)宫颈癌初治患者129例。用免疫组化法检测宫颈癌组织标本中m EGFR、EGFR pThr654和PKN1 pThr774蛋白的表达,分析其间表达的相关性,及其与宫颈癌患者的临床病理因素和预后的关系。结果:显微观察显示,m EGFR在细胞膜上表达,PKN1 pThr774在细胞浆和细胞核内均有表达,而EGFR pThr654只在细胞核内表达;用X-tile软件计算m EGFR、EGFR pThr654和PKN1 pThr774表达量的cut-off值,并根据cut-off值得出,在129例宫颈癌中,EGFR低表达为84例(65.1%,84/129),高表达45例(34.9%,45/129);EGFR pThr654高表达32例(24.8%,32/129),低表达组97例(75.2%,97/129);PKN1 pThr774高表达37例(28.7%,37/129),低表达92例(71.3%,92/129)。斯珀曼(Spearman)相关系数的显著性检验显示,m EGFR与EGFR pThr654显著正相关(r=0.256,P=0.015);PKN1 pThr774与EGFR pThr654与显著正相关(r=0.217,P=0.04);多因素分析显示,m EGFR(HR=28.469;95%CI=4.626-86.348;P=0.019)和EGFR pThr654(HR=45.137,95%CI=6.312-110.850;P=0.000)是宫颈癌患者无进展生存期(PFS)独立预后因素;也是总生存期(OS)的独立预后因素(m EGFR:HR=32.278,95%CI=6.615-89.134;P=0.001和EGFR pThr654:HR=40.009;95%CI=4.425-100.962;P=0.000)。结论:m EGFR和核EGFR pThr654的高表达与宫颈癌初始放化疗后独立的较差的疾病特异性生存期有关,提示这可能是导致放化疗抵抗的因素之一。m EGFR、EGFR pThr654和PKN1 pThr774可以作为宫颈癌放化疗敏感性及预后的预测指标和治疗靶标。第二部分辐射对宫颈癌细胞EGFR核转位及其相关蛋白的影响背景和目的:表皮生长因子受体(EGFR)可以直接转运至核内,活化DNA-PK,增强DNA损伤的非同源末端连接(NHEJ)修复,减低放疗敏感性,是新近发现的EGFR传递信号的新途径。我们先前的研究显示宫颈癌组织中有核EGFR的表达,EGFR和EGFR pThr654、PKN1 pThr774可能是EGFR核转位通路上的相关蛋白,PDK1是PI3K/AKT通路上的重要蛋白,调节DNA-PK的活性。故检测放疗前后EGFR和EGFR pThr654、PKN1和PKN1pThr774、PDK1和PDK1 p Ser241、DNA-PK和DNA-PK pThr2609等蛋白表达量的变化,进一步了解辐射对宫颈癌肿瘤细胞核转运的影响,并探讨核转运的机制及在宫颈癌细胞辐射耐受中可能的作用。方法:C33A、Ca Ski、Hela和A431细胞采用6MV X射线进行单次照射,于照射后10、20和40min分离提取细胞质、细胞核蛋白,Western blot测定各细胞株EGFR、PDK1、PKN1、非同源末端连接(NHEJ)修复作用相关的DNA-PK及其相关磷酸化蛋白放疗前后表达水平的变化。结果:四组细胞株照射之前核内均有EGFR表达,人宫颈癌Ca Ski、Hela,C33A细胞株,同人皮肤癌A431细胞株一样,Ca Ski、Hela,也存在放疗后核内总EGFR及EGFR pThr654的表达量显著增加(P0.05),放疗后随核内EGFR的增加,DNA-PK pThr2609表达量也明显增高(P0.05),两者间有相关性。Ca Ski细胞内PKN1 pThr774放疗后也有表达量逐渐增加(P0.05),并与EGFR pThr654放疗后变化具有相关性(P0.05)。Hela核内的PDK1及PDK1 p Ser241也有放疗后随时间增加(P0.05),与DNA-PK pThr2609放疗后变化有正相关性(P0.05)。结论:宫颈癌细胞放疗后发生EGFR核转位,核EGFR激活DNA损伤修复相关蛋白DNA-PK,可能是其介导Ca Ski、Hela,C33A细胞株放射抵抗的机制之一,并提示EGFR pThr654可能是介导宫颈癌细胞,无论腺癌还是鳞癌,放疗后发生EGFR核转位的关键点之一,PKN1 pThr774在Ca Ski细胞中与EGFR Thr654的磷酸化高度相关。核PDK1及其磷酸化PDK1Ser241可能与Hela细胞的放疗抵抗相关。本研究还发现放疗后多种蛋白可能发生核转位,核转位可能是肿瘤的一种防御机制。第三部分抑制EGFR核转位对宫颈癌细胞辐射耐受性的研究背景和目的:前期实验证实宫颈癌细胞内存在放疗诱导的EGFR核转位,EGFR pThr654可能是这一过程的关键点,核EGFR激活DNA-PK Thr2609,可能降低放疗敏感性。本实验探讨EGFR核转位抑制肽及EGFR靶向药物:西妥昔单抗、吉非替尼对宫颈癌细胞辐射后核转位的影响,及对辐射耐受性的影响。方法:采用EGFR核转位抑制肽(p T654)及对照肽、西妥昔单抗及吉非替尼预处理人宫颈鳞癌Ca Ski、人宫颈腺癌Hela细胞16h,细胞再暴露于6MVX射线,辐射4Gy后,在0,10,20,40分不同时间点测定(Western blot方法)核EGFR、EGFR pThr654、以及DNA-PK和DNA-PK pThr2609的表达。用γH2AX免疫荧光法分析了西妥昔单抗、吉非替尼对辐射诱导的染色体损伤的影响。采用克隆形成法研究西妥昔单抗/吉非替尼是否增加宫颈癌细胞的放射敏感性,Graph Pad Prism5.0软件拟合不同药物组的剂量-生存曲线。采用SPSS17.0软件进行统计学分析。结果:EGFR核转位抑制肽p T654、西妥昔单抗及吉非替尼显著降低了放疗后Ca Ski、Hela细胞核EGFR、EGFR pThr654和DNA-PK pThr2609的表达(P0.05),对照肽未有效抑制辐射引起的核转位及相关蛋白的表达,但西妥昔单抗和吉非替尼也可以诱导EGFR核转位;西妥昔单抗或吉非替尼联合放疗增加了单纯放疗后的γ-H2AX foci数(P0.05);克隆实验提示西妥昔单抗联合放疗SER值为1.67,明显增加了Ca Ski细胞的放疗敏感性(P0.05);对于Hela细胞,西妥昔单抗联合放疗组、吉非替尼联合放疗组、单纯放疗组,SER值分别为1.015、1.009、1,均未增加Hela细胞的放疗敏感性(P0.05)。结论:进一步证实EGFR pThr654是EGFR核转位的关键磷酸化位点;抑制EGFR核转位能提高辐射敏感性;西妥昔单抗提高Ca Ski细胞的放疗敏感性,但未改善Hela细胞辐射效果,考虑两种细胞内信号通路可能不全相同。基础核EGFR高表达可能是非获得性治疗耐受的诱因,靶向药或放疗诱导的核转位可能是获得性治疗耐受的诱因。
[Abstract]:The first part of the nuclear translocation of EGFR in cervical cancer chemotherapy and prognostic impact of background and purpose: the epidermal growth factor receptor (EGFR) is mediated by cell surface signaling cascade and translocation to nucleus and regulates many cellular processes including non homologous end joining repair damage and transcriptional regulation of DNA. A large number of studies confirmed that EGFR and lung cancer, liver cancer. Gastric cancer, head and neck cancer development, prognosis and treatment. However, the expression of EGFR in cervical carcinoma cells and the correlation between prognosis and response to radiotherapy is still controversial and uncertain. Recent studies suggest that some tumor cells EGFR expression is associated with resistance to chemotherapy and poor prognosis. Studies have reported that radiation the line can be induced by protein kinase C (Protein Kinase C, PKC) the formation and activation of PKN1 family molecules EGFR two dimers, and the activation of PKN1 EGFR threonine phosphorylation sites near the end of 654 (EGFR pT Hr654), may be one of the key points is mediated by the nuclear translocation of EGFR. This paper studies the local advanced cervical cancer cell membrane EGFR (m EGFR), phosphorylated PKN1 (PKN1 pThr774) expression of EGFR and pThr654, to investigate the prognostic role of chemotherapy and nuclear translocation of EGFR in patients with advanced cervical cancer. Methods: to collect from January 2007 to December 2012, radical chemoradiation IIA-IIIB in the tumor hospital of Sichuan province (FIGO) for cervical cancer patients. 129 cases were detected in cervical cancer m EGFR immunohistochemistry, the expression of EGFR pThr654 and PKN1 pThr774 protein, correlation analysis between the expression of the relationship between clinicopathological factors and prognosis of with cervical cancer patients. Results: the microscopic observation showed that the expression of M EGFR in PKN1 pThr774 in the cell membrane, cytoplasm and nucleus were expressed, while EGFR pThr654 expression only in the nucleus; calculation of M EGFR with X-tile software, EGF R pThr654 and PKN1 pThr774 expression in cut-off, and according to the cut-off value, in 129 cases of cervical carcinoma, low expression of EGFR in 84 cases (65.1%, 84/129), high expression in 45 cases (34.9%, 45/129); EGFR pThr654 high expression in 32 cases (24.8%, 32/129), 97 cases (low expression group 75.2%, 97/129); PKN1 pThr774 high expression in 37 cases (28.7%, 37/129), low expression in 92 cases (71.3%, 92/129). Shipman (Spearman) significant correlation coefficient showed a significant positive correlation between M EGFR and EGFR pThr654 (r=0.256, P=0.015); PKN1 pThr774 and EGFR pThr654 with significant positive correlation (r=0.217, P=0.04); multivariate analysis showed that m EGFR (HR=28.469; 95%CI=4.626-86.348; P=0.019) and EGFR pThr654 (HR=45.137,95%CI=6.312-110.850; P=0.000) is cervical cancer progression free survival (PFS) is an independent prognostic factor; overall survival (OS) independent prognostic factor (m EGFR:HR=32.278,95%CI=6.615-89.134; P=0.001 and EGFR PThr654:HR=40.009; 95%CI=4.425-100.962; P=0.000). Conclusion: high expression of EGFR and m in cervical cancer with initial nuclear EGFR pThr654 after radiotherapy and chemotherapy independent poor disease-specific survival, suggesting that this may be the cause of.M EGFR is one of the factors of resistance to chemotherapy, predictive biomarkers and therapeutic targets in EGFR pThr654 and PKN1 pThr774 can be used as cervical cancer chemotherapy sensitivity and prognosis. The second part radiation on human cervical cancer cells EGFR nuclear translocation and its related protein background and purpose: the epidermal growth factor receptor (EGFR) can be directly transported to the nucleus, live DNA-PK, enhance the non homologous end joining (NHEJ) DNA damage repair, reduce the radiation sensitivity, is a new way the newly discovered EGFR signal. Our previous studies showed that the expression of nuclear EGFR in cervical carcinoma, EGFR and EGFR pThr654, PKN1 pThr774 may be EGFR nuclear translocation pathway 涓婄殑鐩稿叧铔嬬櫧,PDK1鏄疨I3K/AKT閫氳矾涓婄殑閲嶈�佽泲鐧�,璋冭妭DNA-PK鐨勬椿鎬，

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