本文關(guān)鍵詞： EGFR 核轉(zhuǎn)位 宮頸癌 放射治療 宮頸癌 EGFR 核轉(zhuǎn)位 輻射 EGFR 宮頸癌 核轉(zhuǎn)位 靶向治療　出處：《廣西醫(yī)科大學》2017年博士論文　論文類型：學位論文

【摘要】：第一部分EGFR核轉(zhuǎn)位對宮頸癌放化療預后的影響背景和目的:表皮生長因子受體(EGFR)具有介導細胞表面信號級聯(lián)和轉(zhuǎn)位到細胞核調(diào)控許多細胞過程包括轉(zhuǎn)錄調(diào)節(jié)和DNA損傷的非同源末端連接修復作用。大量的研究證實,EGFR與肺癌,肝癌,胃癌、頭頸部腫瘤等的發(fā)生、發(fā)展及治療預后有關(guān)。然而,宮頸癌細胞EGFR的表達與預后及對放療反應的相關(guān)性仍然是有爭議或不確定的。最近的研究提示,一些腫瘤細胞核EGFR表達與放化療抵抗和預后差有關(guān)。有研究報道,放射線可誘導蛋白激酶C(Protein Kinase C,PKC)家族分子PKN1活化和EGFR二聚體的形成,以及活化的PKN1使EGFR近末端654位點的蘇氨酸磷酸化(EGFR pThr654),可能是介導EGFR核轉(zhuǎn)位的關(guān)鍵點之一。本文研究了局部中晚期宮頸癌細胞膜EGFR(m EGFR)、磷酸化PKN1(PKN1 pThr774)和EGFR pThr654的表達水平,探討EGFR核轉(zhuǎn)位對中晚期宮頸癌患者放化療的預后作用。方法:收集2007年1月至2012年12月在四川省腫瘤醫(yī)院行根治性同步放化療IIA-IIIB期(FIGO)宮頸癌初治患者129例。用免疫組化法檢測宮頸癌組織標本中m EGFR、EGFR pThr654和PKN1 pThr774蛋白的表達,分析其間表達的相關(guān)性,及其與宮頸癌患者的臨床病理因素和預后的關(guān)系。結(jié)果:顯微觀察顯示,m EGFR在細胞膜上表達,PKN1 pThr774在細胞漿和細胞核內(nèi)均有表達,而EGFR pThr654只在細胞核內(nèi)表達;用X-tile軟件計算m EGFR、EGFR pThr654和PKN1 pThr774表達量的cut-off值,并根據(jù)cut-off值得出,在129例宮頸癌中,EGFR低表達為84例(65.1%,84/129),高表達45例(34.9%,45/129);EGFR pThr654高表達32例(24.8%,32/129),低表達組97例(75.2%,97/129);PKN1 pThr774高表達37例(28.7%,37/129),低表達92例(71.3%,92/129)。斯珀曼(Spearman)相關(guān)系數(shù)的顯著性檢驗顯示,m EGFR與EGFR pThr654顯著正相關(guān)(r=0.256,P=0.015);PKN1 pThr774與EGFR pThr654與顯著正相關(guān)(r=0.217,P=0.04);多因素分析顯示,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)是宮頸癌患者無進展生存期(PFS)獨立預后因素;也是總生存期(OS)的獨立預后因素(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)。結(jié)論:m EGFR和核EGFR pThr654的高表達與宮頸癌初始放化療后獨立的較差的疾病特異性生存期有關(guān),提示這可能是導致放化療抵抗的因素之一。m EGFR、EGFR pThr654和PKN1 pThr774可以作為宮頸癌放化療敏感性及預后的預測指標和治療靶標。第二部分輻射對宮頸癌細胞EGFR核轉(zhuǎn)位及其相關(guān)蛋白的影響背景和目的:表皮生長因子受體(EGFR)可以直接轉(zhuǎn)運至核內(nèi),活化DNA-PK,增強DNA損傷的非同源末端連接(NHEJ)修復,減低放療敏感性,是新近發(fā)現(xiàn)的EGFR傳遞信號的新途徑。我們先前的研究顯示宮頸癌組織中有核EGFR的表達,EGFR和EGFR pThr654、PKN1 pThr774可能是EGFR核轉(zhuǎn)位通路上的相關(guān)蛋白,PDK1是PI3K/AKT通路上的重要蛋白,調(diào)節(jié)DNA-PK的活性。故檢測放療前后EGFR和EGFR pThr654、PKN1和PKN1pThr774、PDK1和PDK1 p Ser241、DNA-PK和DNA-PK pThr2609等蛋白表達量的變化,進一步了解輻射對宮頸癌腫瘤細胞核轉(zhuǎn)運的影響,并探討核轉(zhuǎn)運的機制及在宮頸癌細胞輻射耐受中可能的作用。方法:C33A、Ca Ski、Hela和A431細胞采用6MV X射線進行單次照射,于照射后10、20和40min分離提取細胞質(zhì)、細胞核蛋白,Western blot測定各細胞株EGFR、PDK1、PKN1、非同源末端連接(NHEJ)修復作用相關(guān)的DNA-PK及其相關(guān)磷酸化蛋白放療前后表達水平的變化。結(jié)果:四組細胞株照射之前核內(nèi)均有EGFR表達,人宮頸癌Ca Ski、Hela,C33A細胞株,同人皮膚癌A431細胞株一樣,Ca Ski、Hela,也存在放療后核內(nèi)總EGFR及EGFR pThr654的表達量顯著增加(P0.05),放療后隨核內(nèi)EGFR的增加,DNA-PK pThr2609表達量也明顯增高(P0.05),兩者間有相關(guān)性。Ca Ski細胞內(nèi)PKN1 pThr774放療后也有表達量逐漸增加(P0.05),并與EGFR pThr654放療后變化具有相關(guān)性(P0.05)。Hela核內(nèi)的PDK1及PDK1 p Ser241也有放療后隨時間增加(P0.05),與DNA-PK pThr2609放療后變化有正相關(guān)性(P0.05)。結(jié)論:宮頸癌細胞放療后發(fā)生EGFR核轉(zhuǎn)位,核EGFR激活DNA損傷修復相關(guān)蛋白DNA-PK,可能是其介導Ca Ski、Hela,C33A細胞株放射抵抗的機制之一,并提示EGFR pThr654可能是介導宮頸癌細胞,無論腺癌還是鱗癌,放療后發(fā)生EGFR核轉(zhuǎn)位的關(guān)鍵點之一,PKN1 pThr774在Ca Ski細胞中與EGFR Thr654的磷酸化高度相關(guān)。核PDK1及其磷酸化PDK1Ser241可能與Hela細胞的放療抵抗相關(guān)。本研究還發(fā)現(xiàn)放療后多種蛋白可能發(fā)生核轉(zhuǎn)位,核轉(zhuǎn)位可能是腫瘤的一種防御機制。第三部分抑制EGFR核轉(zhuǎn)位對宮頸癌細胞輻射耐受性的研究背景和目的:前期實驗證實宮頸癌細胞內(nèi)存在放療誘導的EGFR核轉(zhuǎn)位,EGFR pThr654可能是這一過程的關(guān)鍵點,核EGFR激活DNA-PK Thr2609,可能降低放療敏感性。本實驗探討EGFR核轉(zhuǎn)位抑制肽及EGFR靶向藥物:西妥昔單抗、吉非替尼對宮頸癌細胞輻射后核轉(zhuǎn)位的影響,及對輻射耐受性的影響。方法:采用EGFR核轉(zhuǎn)位抑制肽(p T654)及對照肽、西妥昔單抗及吉非替尼預處理人宮頸鱗癌Ca Ski、人宮頸腺癌Hela細胞16h,細胞再暴露于6MVX射線,輻射4Gy后,在0,10,20,40分不同時間點測定(Western blot方法)核EGFR、EGFR pThr654、以及DNA-PK和DNA-PK pThr2609的表達。用γH2AX免疫熒光法分析了西妥昔單抗、吉非替尼對輻射誘導的染色體損傷的影響。采用克隆形成法研究西妥昔單抗/吉非替尼是否增加宮頸癌細胞的放射敏感性,Graph Pad Prism5.0軟件擬合不同藥物組的劑量-生存曲線。采用SPSS17.0軟件進行統(tǒng)計學分析。結(jié)果:EGFR核轉(zhuǎn)位抑制肽p T654、西妥昔單抗及吉非替尼顯著降低了放療后Ca Ski、Hela細胞核EGFR、EGFR pThr654和DNA-PK pThr2609的表達(P0.05),對照肽未有效抑制輻射引起的核轉(zhuǎn)位及相關(guān)蛋白的表達,但西妥昔單抗和吉非替尼也可以誘導EGFR核轉(zhuǎn)位;西妥昔單抗或吉非替尼聯(lián)合放療增加了單純放療后的γ-H2AX foci數(shù)(P0.05);克隆實驗提示西妥昔單抗聯(lián)合放療SER值為1.67,明顯增加了Ca Ski細胞的放療敏感性(P0.05);對于Hela細胞,西妥昔單抗聯(lián)合放療組、吉非替尼聯(lián)合放療組、單純放療組,SER值分別為1.015、1.009、1,均未增加Hela細胞的放療敏感性(P0.05)。結(jié)論:進一步證實EGFR pThr654是EGFR核轉(zhuǎn)位的關(guān)鍵磷酸化位點;抑制EGFR核轉(zhuǎn)位能提高輻射敏感性;西妥昔單抗提高Ca Ski細胞的放療敏感性,但未改善Hela細胞輻射效果,考慮兩種細胞內(nèi)信號通路可能不全相同�；A(chǔ)核EGFR高表達可能是非獲得性治療耐受的誘因,靶向藥或放療誘導的核轉(zhuǎn)位可能是獲得性治療耐受的誘因。
[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閫氳礬涓婄殑閲嶈铔嬬櫧,璋冭妭DNA-PK鐨勬椿鎬，

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