中国NSCLC人群EGFR-TKI治疗预测因子及EGFR下游信号通路机制研究
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摘要
肺癌在中国乃至全世界是癌症死因的首位。肺癌可分为非小细胞肺癌(non-small cell lung cancer, NSCLC)和小细胞肺癌(small cell lung cancer, SCLC)两大类,其中NSCLC占肺癌总发生率的85%,中晚期占70-80%。就晚期肺癌而言,目前一线化疗有效率仅30%左右,对难治和复发的患者,真正有效的治疗措施有限。因此,迫切需要高效低毒的新药物。
表皮生长因子受体(epidermal growth factor receptor, EGFR),是目前备受瞩目的肿瘤治疗靶位。EGFR-酪氨酸激酶(Tyrosine Kinase,TK)区域是调节肿瘤细胞增殖、侵袭、血管生成、粘附、转移和凋亡的重要组成部分。EGFR与配体结合后导致EGFR二聚体化和胞内区磷酸化,进而引发级联放大,激活下游一系列信号通路(主要包括Src/STAT通路、PI3K/Akt通路和Ras-Raf-MEK-Erk/MAPK通路等),从而调控上述一系列肿瘤生物学行为。因为EGFR在NSCLC过表达,且其表达水平和恶性预后相关,因此EGFR酪氨酸激酶抑制剂(Tyrosine Kinase Inhibitor,TKI)被开发为针对NSCLC的新型药物。Gefitinib是首个被FDA批准作为晚期NSCLC的分子靶向药物,它通过竞争性结合于EGFR-TK催化区域中Mg-ATP位点,抑制酪氨酸自身磷酸化,截断来自EGFR配体-受体结合后的信号传导,诱导细胞周期阻滞、增加凋亡和抑制增殖。
2004年,Lynch和Paez等同时报道EGFR-TK区域的突变可以预测NSCLC对gefitinib的敏感性,被认为是通向NSCLC个体化分子靶向治疗的里程碑。随后,大量研究重复和充实着这一观点。在化疗复发的NSCLC,EGFR-TKI的II期临床试验发现其反应率为9%-19% ,中位生存期为7.6-8.4月。III期临床试验BR.21报道erlotinib和安慰剂对比可以显著延长生存;然而ISEL却并未发现gefitinib和安慰剂对比能改善生存,但其中非吸烟和亚裔NSCLC患者,却可以获益于gefitinib。两个设计如此相似的国际多中心临床研究之间的差异,能说明gefitinib的疗效确实不如erlotinib,还是两个研究的设计,入组病人,分子生物学特性和剂量影响到最终结果仍有争论。目前该领域的热点是如何通过临床和分子指标筛选出可能获益于TKI的人群。中国是世界上人口最多的国家,然而仍未见关于中国NSCLC患者EGFR基因突变大样本资料汇总。系统回顾这些数据将对我国NSCLC的个体化分子靶向治疗提供依据,但临床和流行病学数据只能提供一种概率和趋势,同时需要有分子生物学相关理论支持。因此本研究分成两部分:
第一部分通过全面的检索,纳入所有关于中国大陆NSCLC患者EGFR突变状况及和gefitinib疗效关系的文献,联系作者获取个体化病人资料(indivudual patient data, IPD)并重整和提炼,从而筛选中国大陆TKI治疗的获益人群。并通过追踪大样本量连续性的肺癌病例,了解这一组获益人群对规范综合治疗的预后作为基线资料从而筛选出TKI治疗的优势人群和靶向人群。
第二部分采用RNAi技术抑制肺腺癌细胞株的EGFR基因,探讨通过构建质粒载体的方法能否介导肿瘤细胞出现EGFR基因沉默,并应用此技术尝试了解EGFR/HER2基因下游信号通路中各因子的相关性以及与癌细胞增殖、凋亡的相互作用。
第一部分非小细胞肺癌EGFR-TKI分子靶向治疗预测因子
第一章中国大陆NSCLC人群EGFR基因突变特征及与吉非替尼疗效的关系:基于个体化病人资料的荟萃分析
目的
分析中国大陆人群EGFR基因突变和人群特征之间的关系;探讨EGFR突变和TKI治疗的疗效关系以筛选TKI治疗的获益人群。
方法
系统检索2004年4月至2007年5月以下电子数据库:Pubmed、EMBASE, www.cochrane.org、CBMdisc、CNKI、万方、ISI web of science、www.clinicaltrials.gov和近三年国内外肿瘤会议论文中关于中国大陆NSCLC人群通过直接测序法了解EGFR突变状况的研究。对纳入研究行质量评估并联系作者获取IPD,主要观察指标包括: EGFR突变状态;性别、年龄、地域、病理类型、吸烟史、吸烟指数和TNM分期等人群特征;对EGFR-TKI的反应率和预后。
统计分析软件应用RevMan 4.2、SigmaPlot 10.0整合SigmaStat 3.5,以及带有meta插件的STATA 8.0。
结果
最终检索得到来自11个研究机构包括834例患者的30篇符合纳入标准的文献,其中7个研究机构514例患者联系获取了IPD,3个研究机构73例患者提供了gefitinib治疗反应的资料,57例患者有gefitinib治疗后完整的生存数据。11个研究机构的病例来自中国沿海7省和直辖市,从南至北包括云南、广东、东部沿海、北京和吉林5地域,中国NSCLC人群总体EGFR突变率为30.6%,其中19外显子缺失和21外显子L858R占突变总数的94.51%,中国19外显子突变检出21种不同类型,占突变总数54%并存在地域分布差异。IPD荟萃分析显示EGFR突变在女性、腺癌和非吸烟者中比例明显较高,Ⅱ期和Ⅳ期患者突变率有显著差异(16.1% vs. 40.4%, p=0.003)。Logistic回归分析发现腺癌和吸烟史是EGFR突变的独立预测因子。EGFR突变组gefitinib治疗有效率(70.3%, 26/37)显著高于野生组(16.7%, 6/36)(p<0.0001),对反应率的多因素分析发现:“腺癌状态”,“吸烟史”和“EGFR突变状态”三者是独立的预测因子。在gefitinib治疗的生存分析中,突变能预测反应率而并不能预测生存(p=0.110),但19外显子缺失gefitinib治疗预后比21外显子L858R点突变有更好的趋势(619天vs. 182天;HR 2.28;95% CI 0.95-5.49; p=0.057);在Cox多因素分析中,“腺癌状态”是gefitinib治疗的唯一独立预后因子(p<0.00001)。
结论
中国NSCLC人群中的女性、非吸烟腺癌有更高的EGFR突变率,随着病期的进展突变率有增高的趋势,腺癌和吸烟史是EGFR突变的独立预测因子。EGFR突变的病人对TKI的敏感性更高,TKI治疗的临床获益人群是非吸烟腺癌。中国NSCLC人群EGFR突变类型最常见为19外显子缺失并存在地域分布差异,19外显子缺失者TKI治疗预后比21外显子L858R好,因此19外显子缺失是中国NSCLC人群的热点突变。
第二章女性非吸烟周围型肺腺癌的自然病程
目的
评价以女性非吸烟周围型肺腺癌为核心的这一亚群病人对化疗的毒性反应和疗效,以及肺癌切除术后的自然病程,为第一章中所筛选出的TKI临床获益人群提供背景基线资料,以展示EGFR突变和TKI治疗的内在关系。
方法
数据来源于1999年6月至2004年6月一组连续性肺癌患者,该组病例病理诊断明确,TNM分期严格,治疗策略规范,资料齐全。资料初诊时由主管医生录入前瞻性设计的电子病历中,建立随访登记表。2004年9月至2007年4月重新整理、随访及更新于access肺癌数据库中。以住院号和病人姓名为双标识的个体化病人数据从access数据库各子库导出至excel后,通过筛选、排序,导入SPSS匹配整合于同一页面用于数据分析。从性别、吸烟状态、病理类型、肿瘤位置四个方面全面评估不同亚群病人对化疗毒性反应、疗效评价、手术切除情况、胸内区域淋巴结转移、术后并发症发生率、远处转移的部位、无瘤生存期(disease free survival, DFS)和总体生存期(overall survival, OS)的差异。分类资料用Fisher精确检验或者Pearsonχ2检验比较;等级资料用非参数M-W U秩和检验;连续性资料比较用t-检验;生存资料用Kaplan-Meier分析,组间差异用log-rank检验评估,Cox回归用于调查主要预后因子。检验水准p<0.05。
结果
共616例肺癌病人纳入分析,年龄、TNM分期和肿瘤位置在男女间分布无显著性差异;但女性以非吸烟腺癌为主,男性以吸烟鳞癌为主;在总体人群中,腺癌和鳞癌所占的比例分别为42.6%和33.88%,周围型和中央型所占的比例分别为50%和44%。男性化疗非血液学毒性小于女性(p=0.01);男性术后并发症率高于女性(p=0.011);吸烟者术后并发症率高于非吸烟者(p=0.031)。其余关于非吸烟女性周围型腺癌和其对立面之间的比较,均未见在化疗毒性反应、疗效评价、手术切除情况、胸内区域淋巴结转移、术后并发症发生率、出现远处转移的部位等有明显差异。单因素生存分析显示,腺癌的中位DFS长于非腺癌(23.70月vs. 11.37月,p=0.028);女性、非吸烟、腺癌根治术预后比男性、吸烟、非腺癌好,中位OS分别是(52.17月vs. 33.13月,p=0.02;52.17月vs. 33.13月,p=0.006和43.9月vs. 20.37月,p=0.009),非吸烟腺癌的DFS和OS都明显优于吸烟非腺癌(p=0.043和p=0.001); Cox多因素分析中,上述均非独立的预后因子;胸内淋巴结分组的预后多因素分析表明对DFS有影响的转移淋巴结是气管后、主肺动脉窗、叶支气管淋巴结,而对OS有影响的淋巴结转移是叶支气管淋巴结。
结论
女性、非吸烟、周围型腺癌与其对立面相比,未见在化疗毒性反应、疗效评价和手术切除情况等方面有显著优势,在胸内区域淋巴结转移和术后远处转移部位也无明显差异。但腺癌的术后DFS长于非腺癌;女性非吸烟腺癌切除术预后比男性吸烟非腺癌好,这种优势尤其见于非吸烟腺癌。非吸烟腺癌是一个预后良好的亚群。结合第一章的结论,我们认为非吸烟腺癌是TKI治疗的临床优势人群,而EGFR基因突变则是TKI治疗的靶向人群。
第二部分RNA干扰抑制非小细胞肺癌细胞株EGFR/HER2基因表达的研究
第一章RNA干扰技术抑制人肺腺癌A549细胞株EGFR基因表达
目的:
建立质粒表达载体体内转录介导的RNAi技术,为开发阻断EGFR信号通路的新途径和EGFR下游信号传导通路研究提供实验依据。
方法
1.细胞培养:复苏人肺腺癌A549细胞株并用含10%小牛血清的RPMI 1640传代培养。
2.确定siRNA-EGFR序列:从Genebank中检索人EGFR全长cDNA序列,设计siRNA-EGFR序列,具体设计原则根据“Tuschl法则”及参考文献记载,把获取的序列与人类全基因序列进行BLAST比对。设计的siRNAs经化学合成后转染A549腺癌细胞株,通过Western Blot检测确认沉默效果。
3.重组载体Psciencer 2.0 U6-shRNA-EGFR的构建及鉴定:以上述确认的siRNA-EGFR干扰序列为基础,根据Ambion公司Psilencer 2.0-U6载体构建原则设计成特异性shRNA-EGFR单链模板。通过合成shRNA-EGFR单链,PAGE纯化,等比混合退火,Psilencer2.0-U6载体BamHI和HindⅢ双酶切,T4 DNA连接酶连接,重组体转化DH5α,筛选Amp抗性克隆,摇菌扩增,小提质粒,酶切和测序鉴定,中提并纯化重组质粒,分装保存等步骤获取重组载体Psilencer2.0-U6-shRNA-EGFR。
4.瞬时转染:用脂质体lipofectamine 2000介导将重组质粒和对照质粒转染A549人肺腺癌细胞株。
5.基因沉默效果检测:用实时荧光定量RT-PCR检测靶mRNA沉默效率;用Western Blot检测蛋白表达。
6.基因沉默后生物学特性改变:四甲基偶氮唑盐(MTT)比色检测细胞体外增殖率;DAPI染色观察细胞凋亡;流式细胞仪Annexin V/PI双标记法检测细胞的凋亡指数;流式细胞仪测定细胞周期改变。
结果
1. EGFR基因特异性干扰序列:通过文献检索、BLAST比对、化学合成siRNA-EGFR转染A549并用Western Blot检测确定的EGFR有效干扰序列如下:siRNA-EGFR-1‘5–CACAGUGGAGCGAAUUCCUtt-3’;siRNA-EGFR-2‘5–CUCUGGAGGAAAAGAAAGUtt-3’。
2.基因沉默效果检测:用实时荧光定量RT-PCR检测以上述两条干扰序列为模板构建的3种shRNA-EGFR:shRNA-EGFR1, shRNA-EGFR2, shRNA-EGFR3和阴性对照control的EGFR基因表达量对比分别下降92.67%和96.77%和94.79%;用Western Blot检测shRNA-EGFR1和shRNA-EGFR2对EGFR蛋白表达下调分别为54%和72%,选shRNA-EGFR2用于后续研究。
3.生物学特性改变:MTT法检测shRNA-EGFR可抑制细胞增殖;荧光显微镜下见shRNA-EGFR诱发的凋亡细胞核DAPI染色明显增强,胞核缩小,核膜皱缩,染色质凝聚、边缘化、破碎;Annexin V/P I双标记法检测shRNA-EGFR各干扰组48小时第四象限凋亡早期细胞比例增高;流式细胞仪细胞周期分析结果表明shRNA-EGFR各组G0-G1期细胞百分数较对照组增加,进入S期的细胞百分数较对照组减少。
结论
化学合成法和体内转录质粒载体法介导的RNAi可序列特异性下调A549细胞株EGFR基因水平,降低EGFR蛋白表达。shRNA-EGFR重组质粒通过下调EGFR表达可抑制细胞增殖和诱发凋亡,将更多的细胞阻滞在G0-G1期,部分逆转NSCLC细胞的恶性表型,有望成为NSCLC基因治疗和功能研究的工具。
第二章RNA干扰抑制非小细胞肺癌细胞株EGFR/HER2表达及其对相关信号通路的影响
目的:
在第一章建立EGFR RNAi技术的基础上改进转染及干扰条件,进一步探讨EGFR/HER2基因沉默后对非小细胞肺癌EGFR酪氨酸激酶信号传导通路的交互影响及其与细胞周期改变、细胞凋亡和增殖的关系。
方法:
1.细胞培养:人肺腺癌A549、肺腺癌SPC-A-1、肺鳞癌L78、大细胞H460肺癌细胞株用含10%小牛血清的RPMI 1640传代培养。
2.筛选细胞:通过比较四种细胞的转染效率,对转染试剂的耐受性,Western Blot和免疫组化测定细胞EGFR和HER2蛋白表达水平筛选用于进一步RNAi研究的细胞株。
3. EGFR、HER2基因及EGFR/HER2共干扰序列:EGFR干扰序列采用第一章确定的序列;HER2干扰序列用第一章同样的方法确定;EGFR/HER2共干扰序列通过EGFR/HER2基因全长序列比对取其编码区连续同源片段结合“Tuschl法则”寻找。
4.重组载体的构建:以上述确定的干扰序列为模板,由公司协助构建带有GFP和新霉素抗性筛选标记的重组质粒。
5.瞬时转染:方法同第一章,设立空转对照、阴性干扰序列对照、EGFR干扰、HER2干扰、EGFR干扰联合HER2干扰和EGFR-HER2同干扰6组。
6.基因沉默效果检测:用实时荧光定量RT-PCR法检测EGFR/HER2靶mRNA沉默效率;用Western Blot法检测EGFR/HER2蛋白及其磷酸化蛋白表达水平变化。转染后提取细胞蛋白前15min用EGF刺激。
7.基因沉默后生物学特性改变: MTT法检测细胞体外增殖率;流式细胞仪测定凋亡率和细胞周期改变。
8.下游信号通路蛋白检测:用Western Blot检测EGFR下游信号通路蛋白Akt, p-Akt, p-Erk1/2, p-38表达水平变化。
9.统计分析:采用SPSS13.0统计分析软件,组间比较采用随机区组方差分析。两两比较用SNK法,检验水准为p<0.05。
结果
1. SPC-A-1肺腺癌细胞是四种肺癌细胞株中转染效率最高、对转染试剂耐受性好而且EGFR和HER2表达水平相对较高的一种细胞株。
2. EGFR基因干扰序列:见第一章; HER2基因干扰序列:‘5 - GCATACGTGATGGCTGGTG -3’; EGFR/HER2共干扰序列:‘5 - GTCTACATGATCATGGTCAA -3’。
3.基因沉默后生物学特性改变: MTT法检测EGFR干扰组、HER2干扰组、EGFR干扰联合HER2干扰组和EGFR-HER2共干扰组体外增殖率和阴性对照组对比有下降趋势;流式细胞仪检测HER2干扰组(p=0.001)、EGFR干扰+HER2干扰组(p=0.048)和EGFR-HER2共干扰组(p=0.005)的凋亡率和阴性对照比有显著性差异,EGFR干扰组和阴性对照间差异无显著性(p=0.104);流式细胞仪检测EGFR干扰组(p=0.007)、HER2干扰组(p<0.001)、EGFR干扰+HER2干扰组(p=0.001)和EGFR-HER2共干扰组(p<0.001)的细胞周期分布和阴性对照序列对比G1期和S期细胞比例有显著性改变,而阴性对照和空白对照之间细胞增殖、凋亡和周期改变均无显著性差异。
4.基因沉默效果检测:EGFR和HER2基因蛋白水平能被下调。
5.下游信号通路蛋白检测:EGFR下游信号通路蛋白Akt, p-Akt, p-Erk1/2, p-38表达水平和细胞增殖、凋亡以及细胞周期改变之间未发现明显相关规律。结论
单纯EGFR干扰SPC-A-1细胞不能诱发显著凋亡,HER2和EGFR/HER2基因共沉默后诱发的人肺腺癌SPC-A-1细胞凋亡比阴性对照显著增加。
EGFR/HER2基因沉默后诱发的细胞增殖、凋亡和细胞周期改变与EGFR家族下游信号通路蛋白之间未发现显著相关关系。
Lung cancer is the leading cause of cancer deaths in China and worldwide. Non-small-cell lung cancer (NSCLC) accounts for about 85% in which 70-80% are advance cases. Despite treatment advances, chemotherapy is only marginally effective (approximately 30%). For those patients refractory to or intolerant of the current chemotherapy, treatment options are limited. Hence, more effective therapy with fewer side effects is needed.
Epidermal growth factor receptor (EGFR) is one of the focus targets for molecular cancer therapy. The EGFR pathway plays a crucial role in tumor development and progression, including cell proliferation, regulation of apoptotic, angiogenesis, and metastatic spread. Ligand binding to EGFR induces the formation of receptor homo- and heterodimers and the activation of the intrinsic kinase domain, resulting in phosphorylation on specific tyrosine residues within the cytoplasmic tail. These phosphorylated residues serve as docking sites for a range of proteins, the recruitment of which leads to the activation of intracellular signalling pathways including Src/STAT, PI3K/AKT and ras-raf-MEK-Erk/MAPK et al. Since EGFR is commonly overexpressed in NSCLC and the level of EGFR expression correlates with poor prognosis, EGFR inhibitors have been developed as novel therapy for NSCLC. Gefitinib, the first molecular targeted agent approved for the treatment of advanced NSCLC, is a highly effective EGFR tyrosine-kinase inhibitor (TKI) that selectively blocks the signal transduction pathways implicated in cancer growth, cell cycle arrest and apoptosis.
In 2004, Lynch and Paez et al. reported almost simultaneously that mutations of EGFR might predict the sensitivity of NSCLC to gefitinib, which is regarded as a milestone for approaching individualized molecular targeted therapy for NSCLC. Subsequently, many publications reported data consistent with this finding. Phase II trials with EGFR-TKI in chemo-refractory NSCLC patients found response rates of 9%-19% and median survival ranging from 7.6 to 8.4 months. Furthermore, BR21, a phase III study of erlotinib showed a significant survival benefit compared with placebo. However, in a similarly designed study, ISEL, gefitinib did not show any survival gain over placebo. Subsets of patients, those who had never smoked and of Asian origin appeared to benefit from gefitinib. Whether gefitinib is less efficacious than erlotinib, or whether the study design, the patients enrolled, the tumor molecular characteristics, and the dose might have contributed to the different outcome remain unclear. One area of current research focuses on the identification of factors distinguishing those who are more likely to derive benefit from EGFR-TKIs therapy.
China is the largest country in the world. However, data on EGFR mutations in Mainland China are remain scarce. Comprehensive review of existing information regarding EGFR mutations was essential for personalized therapy for advanced NSCLC. While data from clincal and epidemic resourse could only offer a trend and should be combined with the basic research theory, so there would be two part in our article:
In the first part, we included all relevant trials from a comprehensive search to provide evidence on the relationship between EGFR mutations and gefitinib therapy in NSCLC cases from Mainland China, and baseline data from the natural history of large sample consecutive NSCLC patients would be evaluated.
In the second part, RNAi tecnique was applied to explore the relatioship between downstream signal pathway in EGFR family and the cell proliferation, cell cycle alteration, apoptosis after EGFR/HER2 knocked down.
PART 1: Clinical and Molecular Predictors of Response to EGFR-TKI Therapy in NSCLC Patients
CHAPTER 1: EGFR Mutations and Their Correlation with Gefitinib Therapy in Patients with NSCLC: Pooled-analysis Based on Updated Individual Patient Data in Mainland China
Objective:
We aimed to investigate the relevance of demographic characteristics and EGFR mutations, correlations between the efficacy of gefitinib and EGFR mutations in NSCLC, and to identify individuals who would more likely benefit from gefitinib. Methods:
A systematic search was used to identify all relevant trials about EGFR mutations in NSCLC in Mainland China from April 2004 to April 2007.
Computerized bibliographic searches with PubMed, EMBASE, Cochrane Library, Chinese biomedical literature database, CNKI, ISI web of science and www.clinicaltrials.gov were supplemented with hand searches of conferences abstracts. Then quality of inclued studies was evaluated and a pooled-analysis based on updated individual patient data was performed. Outcome measures included the EGFR mutation status, demographic characteristics, response, and survival. The statistical analyses were performed using RevMan 4.2, SigmaPlot 10.0 with SigmaStat 3.5 integration, and STATA 8.0.
Results:
Totally, 834 NSCLC patients from 11 institutions in 30 articles matched the selection criteria. IPD were obtained from 7 institutions including 514 patients which 73 cases received gefitinib therapy and 57 cases have the full survival data. From South to North, cases in the 11 institutions orignated from 5 regions including Yunnan, Guangdong, East coast, Beijing and JiLin. The EGFR mutation rate was 30.6% in Chinese NSCLC. The exon 19 deletions and exon 21 L858R point muation totally accounts for 94.51% of all EGFR mutations cases. 21 types of exon 19 deletions, which account for 54% of total muations cases present difference of geographical distribution in China. Female patients with smoking history and adenocarcinoma had a higher mutation rate than male patients with no smoking history and non-adenocarcinoma. EGFR mutations rate in stageⅡis lower than stageⅣsignificantintly(16.1% vs. 40.4%, p=0.003). Multivariate analysis showed that“adenocarcinoma”and“non-smoker”were independent predictors of EGFR mutations. In a subgroup of gefitinib-treated NSCLC patients, response rate to gefitinib in the EGFR mutant group was 70.3%, significantly higher than that in wild-type EGFR group (16.7%) (p<0.0001).“EGFR mutation”,“adenocarcinoma”and“non-smoker”were independent predictors of response. Overall survival in the EGFR mutant group and the wild-type group did not differ significantly, (hazard ratio=0.60; 95% CI, 0.32-1.12; p=0.110). The prognosis of exon 19 deletions was better than the exon 21 L858R point muation (619天vs. 182 days; pooled HR 2.282; 95% CI 0.95- 5.49; p=0.057) after gefitinib therapy.“Adenocarcinoma status”was an independent prognostic factor for survival (p<0.00001).
Conclusions:
In Mainland China, adenocarcinoma, non-smoker and female patients with NSCLC have a higher EGFR mutations rate. EGFR mutations rate is lower in an earlier TNM stage. Adenocarcinoma and smoking status are independent predictors for the EGFR mutations. Response to gefitinib therapy favors EGFR mutations group. The clinical selected populations for gefitinib are non-smokers with adenocarcinoma. The predominant type of EGFR mutations in China is exon 19 deletions; the prognosis of exon 19 delections after TKI therapy is better than missense mutation in exon 21 L858R. Therefore exon 19 delections is the most important target in Chinsese NSCLC population who receive gefitinib therapy.
CHAPTER 2: Natural History of Peripheral Lung Adenocarcinomas in Female Non-smokers
Objective:
To evaluate the side effect and efficacy of peripheral lung adenocarcinomas in female non-smokers to chemotherapy, and the natural history after thoractomy; to offer a baseline data on the advantage population of TKI therapy and to display the internal relationship between EGFR muations and TKI therapy.
Methods:
A cohort of consecutive patients with NSCLC from June 1999 to June 2004 was included. All the cases were carefully staged, received standardized treatment strategy, closely followed up and recorded. Electronic medical records prospectively designed were finished by doctors in charge at the time of initial dignosis and followed up closely. Data were updated and input in the lung cancer ACCESS database from Sep 2004 to April 2007. IPD bilabelled with patients’name and admission number were exported from ACCESS to excel, filtered, sorted and imported to a single page of SPSS for analysis. Different subgroups of Gender, smoking status, histological type and tumor location were comprehensively assessed from toxicity and response to chemotherapy, complete resection rate, intrathoracic regional lymph node metastasis, postoperative complications, distant metastasis site, disease free survival and overall survival. Characteristics of the patients and treatments were compared by Fisher exact test or Pearson chi suquare test for categoric data, rank data by using non-parametric M-W U test, and t-test for continuous data. Survival estimates were derived by Kaplan-Meier analysis, and log-rank tests were used to assess differences in survival among the groups. Stratified log rank analyses and Cox proportional hazards modeling were used to investigate major prognostic factors. Analysis was performed using SPSS 13.0.
Results:
There were 616 patients inclued. Groups were similar for age, TNM stage and lesion locations between gender. Female had a higher incidence of non-smokers with adenocarcinoma while male had a higher incidence of smokers with squamous cell carcinoma. Adenocarcinoma and squamous cell carcinoma accounts for 42.6% and 33.88% respectively in the total cases. Peripheral and central lung cancer accounts for 50% and 44% respectively in the total cases. The non-hematologic toxicity induced by chemotherapy in male was milder than female (p=0.01); the postoperative complications rate in female is higher than male (p=0.011) and in smokers is higher than non-smokers (p=0.031). In survival analysis, DFS of adenocarcinomas is longer than non-adenocarcinomas (23.70 months vs. 11.37 months,p=0.028); the prognosis of lung adenocarcinomas in female non-smokers is better than non-adenocarcinomas in male smokers after thoracotomy (median survival being 43.9 months vs. 20.37 months, p=0.009; 52.17 months vs. 33.13 months,p=0.02;52.17 months vs. 33.13 months, p=0.006 repectively). Gender, smoking history and adenocarcinomas status were not independend prognositc factor in Cox regression analysis. Multivariate analysis for intrathoracic regional lymph node metastasis showed that retrotracheal (p=0.002), subaortic (p=0.002) and lobar lymph node (p=0.015) affecting DFS, while lobar lymph node(p=0.002) metastasis had influence on OS.
Conclusions:
Compared with their opposite features, peripheral lung adenocarcinomas in female non-smokers have not any advantage on chemotherapy toxicity, response, complete resection rate, complications in thoractomy, and no significant difference on mediastinal lymph node metastasis and distant metastases. While the DFS in adenocarcinomas is longer than non-adenocarcinomas, the prognosis of adenocarcinomas of female non-smokers is better than non-adenocarcinomas male somkers, these advantages present especially in the subgroup of adenocarcinomas in non-smokers. Combined with the conclusions draw from the previous chapter, we deduce adenocarcinomas in non-smokers are the advantage populations in TKI therapy, while EGFR gene mutation is the targeted populations in TKI therapy.
PART 2: Effects of RNA Interference on EGFR/HER2 Expression in Non-Small-Cell Lung Cancer Cell Lines
CHAPTER 1: RNA Interference on EGFR Gene Expression in A549 Cell Line
Objective
To investigate whether RNA interference could induce gene silencing in human lung adenocarcinomas cell line A549, and to evaluate the degree of EGFR gene silencing and its effect on cell biological characteristics. Meterials and methods
1. Cell culture: Recovery A549 lung adenocarcinoma cell line and maintained in RPMI 1640 medium with 10% fetal calf serum.
2. Selection of sequence-specific siRNA-EGFR: Sequence-specific siRNA-EGFR were designed according to previous literatures, ensured by“Tuschl rules”and BLAST, and confirmed by the results of Western Blot after transfection.
3. Construction and identification of Psilencer 2.0 U6-shRNA-EGFR: ShRNA-EGFR oligonucleotide was designed based on the sequence-specific siRNA-EGFR and the online sofeware offered by Ambion company. A series of procudures (including anneal the template oligonucleotides, ligate annealed shRNA template insert into Psilencer, transform E. coli with the ligation products, purify Psilencer plasmid for transfection and identify clones with the shRNA template insert through direct suquencing) were performed to construct and identify the Psilencer 2.0 U6-shRNA-EGFR.
4. Transient transfection: Psilencer 2.0 U6-shRNA-EGFR and the control vector were transfected into A549 cell line with lipofectamine 2000.
5. Effect of EGFR knocked down: Real time Quantitative RT-PCR was used to detect the silencing of the EGFR gene level. Western Blot was used to measure the reduction in the expression of the EGFR protein.
6. Investigation of the alteration of biological features after EGFR RNA interference: MTT analysis, DAPI staining, Annexin V/PI double labeling and flow cytometry were used to evaluate cell proliferation, apoptosis and cell cycle distribution after RNA interference.
Results
1. Sequence-specific siRNA-EGFR: siRNA-EGFR-1‘5 -CACAGUGGAGCGAAUUCCUtt-3’; siRNA-EGFR-2‘5 -CUCUGGAGGAAAAGAAAGUtt-3’。
2. Effect of EGFR knocked down: In A549 cell line, we displayed sequence specific silencing of the EGFR gene with 92.67%, 96.77% and 94.79% in shRNA-EGFR1, shRNA-EGFR2 and shRNA-EGFR3 based on the above sequence compared with the negative control through Real Time Quantitative RT-PCR. The EGFR protein expression was down regulated for 54% and 72% by shRNA-EGFR1and shRNA-EGFR2 respectively through Western Blot.
3. Alteration of biological characteristics after EGFR RNA interference in A549 cells: MTT analysis detected growth inhibition of the cells dealt with shRNA-EGFR. Observation with fluorescent microscope revealed shrunk nuclei staining with DAPI, crimpled nuclear membrane as well as condensation and fragmentation of chromatin induced by shRNA-EGFR. Annexin V/PI double labeling found the sign of cell membrane stained with green fluorescent in the third phase was more significant in shRNA-EGFR group. Cell cycle analysis by flow cytometry showed that shRNA-EGFR induced accumulation of cells in phase G0-G1 with a decrease in the percentage of cells in phase S compared with the negative control.
Conclusion
The sequence specific siRNA-EGFR and shRNA-EGFR showed a remarkable effect in downregulation of EGFR expression, inhibition of the cellular proliferation and motility, and arrestment of the cell cycle in A549 cells. The application of RNA interference to partly reverse the neoplastic phenotype of EGFR overexpressing cells might provide a new approach to gene therapy and research of NSCLC.
CHAPTER 2: Relatioship between EGFR/HER2 Gene Knocked Down and Downstream Signal Pathway in EGFR Family
Objective
RNAi tecnique was applied to explore the relatioship between downstream signal pathway in EGFR family and the cell proliferation, cell cycle alteration, apoptosis after EGFR/HER2 RNA interference.
Meterials and methods
1. Cell culture: Recovery A549 lung adenocarcinoma, SPC-A-1 lung adenocarcinoma, L78 squamous cell carcinoma, H460 large cell lung cancer cell line and maintained in RPMI 1640 medium with 10% fetal calf serum.
2. Cell Screening: The above 4-cell lines were screened by comparision with transfection efficiency, tolerance to transfection reagent and the level of EGFR/HER2 expression for further research on RNAi.
3. Selection of sequence-specific siRNA for EGFR, HER2 and EGFR/HER2 joint interference: Sequence-specific siRNA for EGFR and HER2 were designed on the previous literature;"Tuschl rules" and BLAST on the full length of EGFR/HER2 cDNA were used to ensure the sequence-specific siRNA for EGFR/HER2 joint interference.
4. Construction of recombinant vector: Based on the above sequence-specific siRNA, recombinant plasmids with GFP and neomycin resistance marker were constructed by company.
5. Transient transfection: Six arms including mock, negative control, shRNA-EGFR, shRNA-HER2, shRNA-EGFR/HER2 and shRNA-EGFR+shRNA-HER2 were established.
6. Effect of EGFR kncked down: Real time Quantitative RT-PCR was used to detect the silencing of the EGFR/HER2 gene level. Western Blot was used to measure the levels of EGFR/HER2 protein and protein phosphorylation expression. Transfected cells were stimulated with EGF 15mins before protein extraction.
7. Alteration of biological features: MTT assay and flow cytometry were used to evaluate the cell proliferation, apoptosis and cell cycle distribution after RNAi.
8. Downstream signaling pathway protein detection: the protein expression level of downstream signaling pathway protein including Akt, p-Akt, p-Erk1/2, p-p38, were measured with Real time Quantitative RT-PCR PCR and Western Blot.
9. Statistical Analysis: Randomized block analysis of variance and SNK methods were used to compare the differences between the groups. P<0.05 was considered significant. Analysis was performed using SPSS 13.0. Results
6. SPC-A-1 was seleted for further research on RNAi based on its transfection efficiency, tolerance of transfection reagent and the level of EGFR/HER2 expression.
7. Sequence-specific siRNA: siRNA-HER2‘5 - GCATACGTGATGGCTGGTGTT-3’; siRNA- EGFR/HER2‘5 - GTCTACATGATCATGGTCAATT-3’。
8. Alteration of biological characteristics after EGFR RNA interference: MTT analysis found that cell proliferation was inhibited in the groups of shRNA-EGFR, shRNA-HER2, shRNA-EGFR+shRNA-HER2 and shRNA-EGFR/HER2. Cell cycle analysis by flow cytometry showed that apoptosis ratio in shRNA-HER2 (p=0.001), shRNA-EGFR/HER2 (p=0.005) and shRNA-EGFR+shRNA-HER2 (p=0.048) groups were significantly higher than negative control group, while there was no statistical difference between shRNA-EGFR and negative control (p=0.104), and that the distributions in phase G1 and phase S in shRNA-EGFR (p=0.007), shRNA-HER2 (p<0.001), shRNA-EGFR/HER2 (p< 0.001) and shRNA-EGFR+shRNA-HER2 (p=0.001) were significantly different compared with the negative control.
9. Effect of EGFR/HER2 kncked down: The level of EGFR/HER2 protein and protein phosphorylation expression were down regulated.
10. Downstream signaling pathway protein detection: The cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference have no significant relationship with downstearm signal pathway moleculars in EGFR family.
Conclusion
EGFR gene knockdown may not cause significant apoptosis in SPC-A-1. The variations of cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference were not found to have significant relationship with downstearm signal pathway molecules in EGFR family.
表皮生长因子受体(epidermal growth factor receptor, EGFR),是目前备受瞩目的肿瘤治疗靶位。EGFR-酪氨酸激酶(Tyrosine Kinase,TK)区域是调节肿瘤细胞增殖、侵袭、血管生成、粘附、转移和凋亡的重要组成部分。EGFR与配体结合后导致EGFR二聚体化和胞内区磷酸化,进而引发级联放大,激活下游一系列信号通路(主要包括Src/STAT通路、PI3K/Akt通路和Ras-Raf-MEK-Erk/MAPK通路等),从而调控上述一系列肿瘤生物学行为。因为EGFR在NSCLC过表达,且其表达水平和恶性预后相关,因此EGFR酪氨酸激酶抑制剂(Tyrosine Kinase Inhibitor,TKI)被开发为针对NSCLC的新型药物。Gefitinib是首个被FDA批准作为晚期NSCLC的分子靶向药物,它通过竞争性结合于EGFR-TK催化区域中Mg-ATP位点,抑制酪氨酸自身磷酸化,截断来自EGFR配体-受体结合后的信号传导,诱导细胞周期阻滞、增加凋亡和抑制增殖。
2004年,Lynch和Paez等同时报道EGFR-TK区域的突变可以预测NSCLC对gefitinib的敏感性,被认为是通向NSCLC个体化分子靶向治疗的里程碑。随后,大量研究重复和充实着这一观点。在化疗复发的NSCLC,EGFR-TKI的II期临床试验发现其反应率为9%-19% ,中位生存期为7.6-8.4月。III期临床试验BR.21报道erlotinib和安慰剂对比可以显著延长生存;然而ISEL却并未发现gefitinib和安慰剂对比能改善生存,但其中非吸烟和亚裔NSCLC患者,却可以获益于gefitinib。两个设计如此相似的国际多中心临床研究之间的差异,能说明gefitinib的疗效确实不如erlotinib,还是两个研究的设计,入组病人,分子生物学特性和剂量影响到最终结果仍有争论。目前该领域的热点是如何通过临床和分子指标筛选出可能获益于TKI的人群。中国是世界上人口最多的国家,然而仍未见关于中国NSCLC患者EGFR基因突变大样本资料汇总。系统回顾这些数据将对我国NSCLC的个体化分子靶向治疗提供依据,但临床和流行病学数据只能提供一种概率和趋势,同时需要有分子生物学相关理论支持。因此本研究分成两部分:
第一部分通过全面的检索,纳入所有关于中国大陆NSCLC患者EGFR突变状况及和gefitinib疗效关系的文献,联系作者获取个体化病人资料(indivudual patient data, IPD)并重整和提炼,从而筛选中国大陆TKI治疗的获益人群。并通过追踪大样本量连续性的肺癌病例,了解这一组获益人群对规范综合治疗的预后作为基线资料从而筛选出TKI治疗的优势人群和靶向人群。
第二部分采用RNAi技术抑制肺腺癌细胞株的EGFR基因,探讨通过构建质粒载体的方法能否介导肿瘤细胞出现EGFR基因沉默,并应用此技术尝试了解EGFR/HER2基因下游信号通路中各因子的相关性以及与癌细胞增殖、凋亡的相互作用。
第一部分非小细胞肺癌EGFR-TKI分子靶向治疗预测因子
第一章中国大陆NSCLC人群EGFR基因突变特征及与吉非替尼疗效的关系:基于个体化病人资料的荟萃分析
目的
分析中国大陆人群EGFR基因突变和人群特征之间的关系;探讨EGFR突变和TKI治疗的疗效关系以筛选TKI治疗的获益人群。
方法
系统检索2004年4月至2007年5月以下电子数据库:Pubmed、EMBASE, www.cochrane.org、CBMdisc、CNKI、万方、ISI web of science、www.clinicaltrials.gov和近三年国内外肿瘤会议论文中关于中国大陆NSCLC人群通过直接测序法了解EGFR突变状况的研究。对纳入研究行质量评估并联系作者获取IPD,主要观察指标包括: EGFR突变状态;性别、年龄、地域、病理类型、吸烟史、吸烟指数和TNM分期等人群特征;对EGFR-TKI的反应率和预后。
统计分析软件应用RevMan 4.2、SigmaPlot 10.0整合SigmaStat 3.5,以及带有meta插件的STATA 8.0。
结果
最终检索得到来自11个研究机构包括834例患者的30篇符合纳入标准的文献,其中7个研究机构514例患者联系获取了IPD,3个研究机构73例患者提供了gefitinib治疗反应的资料,57例患者有gefitinib治疗后完整的生存数据。11个研究机构的病例来自中国沿海7省和直辖市,从南至北包括云南、广东、东部沿海、北京和吉林5地域,中国NSCLC人群总体EGFR突变率为30.6%,其中19外显子缺失和21外显子L858R占突变总数的94.51%,中国19外显子突变检出21种不同类型,占突变总数54%并存在地域分布差异。IPD荟萃分析显示EGFR突变在女性、腺癌和非吸烟者中比例明显较高,Ⅱ期和Ⅳ期患者突变率有显著差异(16.1% vs. 40.4%, p=0.003)。Logistic回归分析发现腺癌和吸烟史是EGFR突变的独立预测因子。EGFR突变组gefitinib治疗有效率(70.3%, 26/37)显著高于野生组(16.7%, 6/36)(p<0.0001),对反应率的多因素分析发现:“腺癌状态”,“吸烟史”和“EGFR突变状态”三者是独立的预测因子。在gefitinib治疗的生存分析中,突变能预测反应率而并不能预测生存(p=0.110),但19外显子缺失gefitinib治疗预后比21外显子L858R点突变有更好的趋势(619天vs. 182天;HR 2.28;95% CI 0.95-5.49; p=0.057);在Cox多因素分析中,“腺癌状态”是gefitinib治疗的唯一独立预后因子(p<0.00001)。
结论
中国NSCLC人群中的女性、非吸烟腺癌有更高的EGFR突变率,随着病期的进展突变率有增高的趋势,腺癌和吸烟史是EGFR突变的独立预测因子。EGFR突变的病人对TKI的敏感性更高,TKI治疗的临床获益人群是非吸烟腺癌。中国NSCLC人群EGFR突变类型最常见为19外显子缺失并存在地域分布差异,19外显子缺失者TKI治疗预后比21外显子L858R好,因此19外显子缺失是中国NSCLC人群的热点突变。
第二章女性非吸烟周围型肺腺癌的自然病程
目的
评价以女性非吸烟周围型肺腺癌为核心的这一亚群病人对化疗的毒性反应和疗效,以及肺癌切除术后的自然病程,为第一章中所筛选出的TKI临床获益人群提供背景基线资料,以展示EGFR突变和TKI治疗的内在关系。
方法
数据来源于1999年6月至2004年6月一组连续性肺癌患者,该组病例病理诊断明确,TNM分期严格,治疗策略规范,资料齐全。资料初诊时由主管医生录入前瞻性设计的电子病历中,建立随访登记表。2004年9月至2007年4月重新整理、随访及更新于access肺癌数据库中。以住院号和病人姓名为双标识的个体化病人数据从access数据库各子库导出至excel后,通过筛选、排序,导入SPSS匹配整合于同一页面用于数据分析。从性别、吸烟状态、病理类型、肿瘤位置四个方面全面评估不同亚群病人对化疗毒性反应、疗效评价、手术切除情况、胸内区域淋巴结转移、术后并发症发生率、远处转移的部位、无瘤生存期(disease free survival, DFS)和总体生存期(overall survival, OS)的差异。分类资料用Fisher精确检验或者Pearsonχ2检验比较;等级资料用非参数M-W U秩和检验;连续性资料比较用t-检验;生存资料用Kaplan-Meier分析,组间差异用log-rank检验评估,Cox回归用于调查主要预后因子。检验水准p<0.05。
结果
共616例肺癌病人纳入分析,年龄、TNM分期和肿瘤位置在男女间分布无显著性差异;但女性以非吸烟腺癌为主,男性以吸烟鳞癌为主;在总体人群中,腺癌和鳞癌所占的比例分别为42.6%和33.88%,周围型和中央型所占的比例分别为50%和44%。男性化疗非血液学毒性小于女性(p=0.01);男性术后并发症率高于女性(p=0.011);吸烟者术后并发症率高于非吸烟者(p=0.031)。其余关于非吸烟女性周围型腺癌和其对立面之间的比较,均未见在化疗毒性反应、疗效评价、手术切除情况、胸内区域淋巴结转移、术后并发症发生率、出现远处转移的部位等有明显差异。单因素生存分析显示,腺癌的中位DFS长于非腺癌(23.70月vs. 11.37月,p=0.028);女性、非吸烟、腺癌根治术预后比男性、吸烟、非腺癌好,中位OS分别是(52.17月vs. 33.13月,p=0.02;52.17月vs. 33.13月,p=0.006和43.9月vs. 20.37月,p=0.009),非吸烟腺癌的DFS和OS都明显优于吸烟非腺癌(p=0.043和p=0.001); Cox多因素分析中,上述均非独立的预后因子;胸内淋巴结分组的预后多因素分析表明对DFS有影响的转移淋巴结是气管后、主肺动脉窗、叶支气管淋巴结,而对OS有影响的淋巴结转移是叶支气管淋巴结。
结论
女性、非吸烟、周围型腺癌与其对立面相比,未见在化疗毒性反应、疗效评价和手术切除情况等方面有显著优势,在胸内区域淋巴结转移和术后远处转移部位也无明显差异。但腺癌的术后DFS长于非腺癌;女性非吸烟腺癌切除术预后比男性吸烟非腺癌好,这种优势尤其见于非吸烟腺癌。非吸烟腺癌是一个预后良好的亚群。结合第一章的结论,我们认为非吸烟腺癌是TKI治疗的临床优势人群,而EGFR基因突变则是TKI治疗的靶向人群。
第二部分RNA干扰抑制非小细胞肺癌细胞株EGFR/HER2基因表达的研究
第一章RNA干扰技术抑制人肺腺癌A549细胞株EGFR基因表达
目的:
建立质粒表达载体体内转录介导的RNAi技术,为开发阻断EGFR信号通路的新途径和EGFR下游信号传导通路研究提供实验依据。
方法
1.细胞培养:复苏人肺腺癌A549细胞株并用含10%小牛血清的RPMI 1640传代培养。
2.确定siRNA-EGFR序列:从Genebank中检索人EGFR全长cDNA序列,设计siRNA-EGFR序列,具体设计原则根据“Tuschl法则”及参考文献记载,把获取的序列与人类全基因序列进行BLAST比对。设计的siRNAs经化学合成后转染A549腺癌细胞株,通过Western Blot检测确认沉默效果。
3.重组载体Psciencer 2.0 U6-shRNA-EGFR的构建及鉴定:以上述确认的siRNA-EGFR干扰序列为基础,根据Ambion公司Psilencer 2.0-U6载体构建原则设计成特异性shRNA-EGFR单链模板。通过合成shRNA-EGFR单链,PAGE纯化,等比混合退火,Psilencer2.0-U6载体BamHI和HindⅢ双酶切,T4 DNA连接酶连接,重组体转化DH5α,筛选Amp抗性克隆,摇菌扩增,小提质粒,酶切和测序鉴定,中提并纯化重组质粒,分装保存等步骤获取重组载体Psilencer2.0-U6-shRNA-EGFR。
4.瞬时转染:用脂质体lipofectamine 2000介导将重组质粒和对照质粒转染A549人肺腺癌细胞株。
5.基因沉默效果检测:用实时荧光定量RT-PCR检测靶mRNA沉默效率;用Western Blot检测蛋白表达。
6.基因沉默后生物学特性改变:四甲基偶氮唑盐(MTT)比色检测细胞体外增殖率;DAPI染色观察细胞凋亡;流式细胞仪Annexin V/PI双标记法检测细胞的凋亡指数;流式细胞仪测定细胞周期改变。
结果
1. EGFR基因特异性干扰序列:通过文献检索、BLAST比对、化学合成siRNA-EGFR转染A549并用Western Blot检测确定的EGFR有效干扰序列如下:siRNA-EGFR-1‘5–CACAGUGGAGCGAAUUCCUtt-3’;siRNA-EGFR-2‘5–CUCUGGAGGAAAAGAAAGUtt-3’。
2.基因沉默效果检测:用实时荧光定量RT-PCR检测以上述两条干扰序列为模板构建的3种shRNA-EGFR:shRNA-EGFR1, shRNA-EGFR2, shRNA-EGFR3和阴性对照control的EGFR基因表达量对比分别下降92.67%和96.77%和94.79%;用Western Blot检测shRNA-EGFR1和shRNA-EGFR2对EGFR蛋白表达下调分别为54%和72%,选shRNA-EGFR2用于后续研究。
3.生物学特性改变:MTT法检测shRNA-EGFR可抑制细胞增殖;荧光显微镜下见shRNA-EGFR诱发的凋亡细胞核DAPI染色明显增强,胞核缩小,核膜皱缩,染色质凝聚、边缘化、破碎;Annexin V/P I双标记法检测shRNA-EGFR各干扰组48小时第四象限凋亡早期细胞比例增高;流式细胞仪细胞周期分析结果表明shRNA-EGFR各组G0-G1期细胞百分数较对照组增加,进入S期的细胞百分数较对照组减少。
结论
化学合成法和体内转录质粒载体法介导的RNAi可序列特异性下调A549细胞株EGFR基因水平,降低EGFR蛋白表达。shRNA-EGFR重组质粒通过下调EGFR表达可抑制细胞增殖和诱发凋亡,将更多的细胞阻滞在G0-G1期,部分逆转NSCLC细胞的恶性表型,有望成为NSCLC基因治疗和功能研究的工具。
第二章RNA干扰抑制非小细胞肺癌细胞株EGFR/HER2表达及其对相关信号通路的影响
目的:
在第一章建立EGFR RNAi技术的基础上改进转染及干扰条件,进一步探讨EGFR/HER2基因沉默后对非小细胞肺癌EGFR酪氨酸激酶信号传导通路的交互影响及其与细胞周期改变、细胞凋亡和增殖的关系。
方法:
1.细胞培养:人肺腺癌A549、肺腺癌SPC-A-1、肺鳞癌L78、大细胞H460肺癌细胞株用含10%小牛血清的RPMI 1640传代培养。
2.筛选细胞:通过比较四种细胞的转染效率,对转染试剂的耐受性,Western Blot和免疫组化测定细胞EGFR和HER2蛋白表达水平筛选用于进一步RNAi研究的细胞株。
3. EGFR、HER2基因及EGFR/HER2共干扰序列:EGFR干扰序列采用第一章确定的序列;HER2干扰序列用第一章同样的方法确定;EGFR/HER2共干扰序列通过EGFR/HER2基因全长序列比对取其编码区连续同源片段结合“Tuschl法则”寻找。
4.重组载体的构建:以上述确定的干扰序列为模板,由公司协助构建带有GFP和新霉素抗性筛选标记的重组质粒。
5.瞬时转染:方法同第一章,设立空转对照、阴性干扰序列对照、EGFR干扰、HER2干扰、EGFR干扰联合HER2干扰和EGFR-HER2同干扰6组。
6.基因沉默效果检测:用实时荧光定量RT-PCR法检测EGFR/HER2靶mRNA沉默效率;用Western Blot法检测EGFR/HER2蛋白及其磷酸化蛋白表达水平变化。转染后提取细胞蛋白前15min用EGF刺激。
7.基因沉默后生物学特性改变: MTT法检测细胞体外增殖率;流式细胞仪测定凋亡率和细胞周期改变。
8.下游信号通路蛋白检测:用Western Blot检测EGFR下游信号通路蛋白Akt, p-Akt, p-Erk1/2, p-38表达水平变化。
9.统计分析:采用SPSS13.0统计分析软件,组间比较采用随机区组方差分析。两两比较用SNK法,检验水准为p<0.05。
结果
1. SPC-A-1肺腺癌细胞是四种肺癌细胞株中转染效率最高、对转染试剂耐受性好而且EGFR和HER2表达水平相对较高的一种细胞株。
2. EGFR基因干扰序列:见第一章; HER2基因干扰序列:‘5 - GCATACGTGATGGCTGGTG -3’; EGFR/HER2共干扰序列:‘5 - GTCTACATGATCATGGTCAA -3’。
3.基因沉默后生物学特性改变: MTT法检测EGFR干扰组、HER2干扰组、EGFR干扰联合HER2干扰组和EGFR-HER2共干扰组体外增殖率和阴性对照组对比有下降趋势;流式细胞仪检测HER2干扰组(p=0.001)、EGFR干扰+HER2干扰组(p=0.048)和EGFR-HER2共干扰组(p=0.005)的凋亡率和阴性对照比有显著性差异,EGFR干扰组和阴性对照间差异无显著性(p=0.104);流式细胞仪检测EGFR干扰组(p=0.007)、HER2干扰组(p<0.001)、EGFR干扰+HER2干扰组(p=0.001)和EGFR-HER2共干扰组(p<0.001)的细胞周期分布和阴性对照序列对比G1期和S期细胞比例有显著性改变,而阴性对照和空白对照之间细胞增殖、凋亡和周期改变均无显著性差异。
4.基因沉默效果检测:EGFR和HER2基因蛋白水平能被下调。
5.下游信号通路蛋白检测:EGFR下游信号通路蛋白Akt, p-Akt, p-Erk1/2, p-38表达水平和细胞增殖、凋亡以及细胞周期改变之间未发现明显相关规律。结论
单纯EGFR干扰SPC-A-1细胞不能诱发显著凋亡,HER2和EGFR/HER2基因共沉默后诱发的人肺腺癌SPC-A-1细胞凋亡比阴性对照显著增加。
EGFR/HER2基因沉默后诱发的细胞增殖、凋亡和细胞周期改变与EGFR家族下游信号通路蛋白之间未发现显著相关关系。
Lung cancer is the leading cause of cancer deaths in China and worldwide. Non-small-cell lung cancer (NSCLC) accounts for about 85% in which 70-80% are advance cases. Despite treatment advances, chemotherapy is only marginally effective (approximately 30%). For those patients refractory to or intolerant of the current chemotherapy, treatment options are limited. Hence, more effective therapy with fewer side effects is needed.
Epidermal growth factor receptor (EGFR) is one of the focus targets for molecular cancer therapy. The EGFR pathway plays a crucial role in tumor development and progression, including cell proliferation, regulation of apoptotic, angiogenesis, and metastatic spread. Ligand binding to EGFR induces the formation of receptor homo- and heterodimers and the activation of the intrinsic kinase domain, resulting in phosphorylation on specific tyrosine residues within the cytoplasmic tail. These phosphorylated residues serve as docking sites for a range of proteins, the recruitment of which leads to the activation of intracellular signalling pathways including Src/STAT, PI3K/AKT and ras-raf-MEK-Erk/MAPK et al. Since EGFR is commonly overexpressed in NSCLC and the level of EGFR expression correlates with poor prognosis, EGFR inhibitors have been developed as novel therapy for NSCLC. Gefitinib, the first molecular targeted agent approved for the treatment of advanced NSCLC, is a highly effective EGFR tyrosine-kinase inhibitor (TKI) that selectively blocks the signal transduction pathways implicated in cancer growth, cell cycle arrest and apoptosis.
In 2004, Lynch and Paez et al. reported almost simultaneously that mutations of EGFR might predict the sensitivity of NSCLC to gefitinib, which is regarded as a milestone for approaching individualized molecular targeted therapy for NSCLC. Subsequently, many publications reported data consistent with this finding. Phase II trials with EGFR-TKI in chemo-refractory NSCLC patients found response rates of 9%-19% and median survival ranging from 7.6 to 8.4 months. Furthermore, BR21, a phase III study of erlotinib showed a significant survival benefit compared with placebo. However, in a similarly designed study, ISEL, gefitinib did not show any survival gain over placebo. Subsets of patients, those who had never smoked and of Asian origin appeared to benefit from gefitinib. Whether gefitinib is less efficacious than erlotinib, or whether the study design, the patients enrolled, the tumor molecular characteristics, and the dose might have contributed to the different outcome remain unclear. One area of current research focuses on the identification of factors distinguishing those who are more likely to derive benefit from EGFR-TKIs therapy.
China is the largest country in the world. However, data on EGFR mutations in Mainland China are remain scarce. Comprehensive review of existing information regarding EGFR mutations was essential for personalized therapy for advanced NSCLC. While data from clincal and epidemic resourse could only offer a trend and should be combined with the basic research theory, so there would be two part in our article:
In the first part, we included all relevant trials from a comprehensive search to provide evidence on the relationship between EGFR mutations and gefitinib therapy in NSCLC cases from Mainland China, and baseline data from the natural history of large sample consecutive NSCLC patients would be evaluated.
In the second part, RNAi tecnique was applied to explore the relatioship between downstream signal pathway in EGFR family and the cell proliferation, cell cycle alteration, apoptosis after EGFR/HER2 knocked down.
PART 1: Clinical and Molecular Predictors of Response to EGFR-TKI Therapy in NSCLC Patients
CHAPTER 1: EGFR Mutations and Their Correlation with Gefitinib Therapy in Patients with NSCLC: Pooled-analysis Based on Updated Individual Patient Data in Mainland China
Objective:
We aimed to investigate the relevance of demographic characteristics and EGFR mutations, correlations between the efficacy of gefitinib and EGFR mutations in NSCLC, and to identify individuals who would more likely benefit from gefitinib. Methods:
A systematic search was used to identify all relevant trials about EGFR mutations in NSCLC in Mainland China from April 2004 to April 2007.
Computerized bibliographic searches with PubMed, EMBASE, Cochrane Library, Chinese biomedical literature database, CNKI, ISI web of science and www.clinicaltrials.gov were supplemented with hand searches of conferences abstracts. Then quality of inclued studies was evaluated and a pooled-analysis based on updated individual patient data was performed. Outcome measures included the EGFR mutation status, demographic characteristics, response, and survival. The statistical analyses were performed using RevMan 4.2, SigmaPlot 10.0 with SigmaStat 3.5 integration, and STATA 8.0.
Results:
Totally, 834 NSCLC patients from 11 institutions in 30 articles matched the selection criteria. IPD were obtained from 7 institutions including 514 patients which 73 cases received gefitinib therapy and 57 cases have the full survival data. From South to North, cases in the 11 institutions orignated from 5 regions including Yunnan, Guangdong, East coast, Beijing and JiLin. The EGFR mutation rate was 30.6% in Chinese NSCLC. The exon 19 deletions and exon 21 L858R point muation totally accounts for 94.51% of all EGFR mutations cases. 21 types of exon 19 deletions, which account for 54% of total muations cases present difference of geographical distribution in China. Female patients with smoking history and adenocarcinoma had a higher mutation rate than male patients with no smoking history and non-adenocarcinoma. EGFR mutations rate in stageⅡis lower than stageⅣsignificantintly(16.1% vs. 40.4%, p=0.003). Multivariate analysis showed that“adenocarcinoma”and“non-smoker”were independent predictors of EGFR mutations. In a subgroup of gefitinib-treated NSCLC patients, response rate to gefitinib in the EGFR mutant group was 70.3%, significantly higher than that in wild-type EGFR group (16.7%) (p<0.0001).“EGFR mutation”,“adenocarcinoma”and“non-smoker”were independent predictors of response. Overall survival in the EGFR mutant group and the wild-type group did not differ significantly, (hazard ratio=0.60; 95% CI, 0.32-1.12; p=0.110). The prognosis of exon 19 deletions was better than the exon 21 L858R point muation (619天vs. 182 days; pooled HR 2.282; 95% CI 0.95- 5.49; p=0.057) after gefitinib therapy.“Adenocarcinoma status”was an independent prognostic factor for survival (p<0.00001).
Conclusions:
In Mainland China, adenocarcinoma, non-smoker and female patients with NSCLC have a higher EGFR mutations rate. EGFR mutations rate is lower in an earlier TNM stage. Adenocarcinoma and smoking status are independent predictors for the EGFR mutations. Response to gefitinib therapy favors EGFR mutations group. The clinical selected populations for gefitinib are non-smokers with adenocarcinoma. The predominant type of EGFR mutations in China is exon 19 deletions; the prognosis of exon 19 delections after TKI therapy is better than missense mutation in exon 21 L858R. Therefore exon 19 delections is the most important target in Chinsese NSCLC population who receive gefitinib therapy.
CHAPTER 2: Natural History of Peripheral Lung Adenocarcinomas in Female Non-smokers
Objective:
To evaluate the side effect and efficacy of peripheral lung adenocarcinomas in female non-smokers to chemotherapy, and the natural history after thoractomy; to offer a baseline data on the advantage population of TKI therapy and to display the internal relationship between EGFR muations and TKI therapy.
Methods:
A cohort of consecutive patients with NSCLC from June 1999 to June 2004 was included. All the cases were carefully staged, received standardized treatment strategy, closely followed up and recorded. Electronic medical records prospectively designed were finished by doctors in charge at the time of initial dignosis and followed up closely. Data were updated and input in the lung cancer ACCESS database from Sep 2004 to April 2007. IPD bilabelled with patients’name and admission number were exported from ACCESS to excel, filtered, sorted and imported to a single page of SPSS for analysis. Different subgroups of Gender, smoking status, histological type and tumor location were comprehensively assessed from toxicity and response to chemotherapy, complete resection rate, intrathoracic regional lymph node metastasis, postoperative complications, distant metastasis site, disease free survival and overall survival. Characteristics of the patients and treatments were compared by Fisher exact test or Pearson chi suquare test for categoric data, rank data by using non-parametric M-W U test, and t-test for continuous data. Survival estimates were derived by Kaplan-Meier analysis, and log-rank tests were used to assess differences in survival among the groups. Stratified log rank analyses and Cox proportional hazards modeling were used to investigate major prognostic factors. Analysis was performed using SPSS 13.0.
Results:
There were 616 patients inclued. Groups were similar for age, TNM stage and lesion locations between gender. Female had a higher incidence of non-smokers with adenocarcinoma while male had a higher incidence of smokers with squamous cell carcinoma. Adenocarcinoma and squamous cell carcinoma accounts for 42.6% and 33.88% respectively in the total cases. Peripheral and central lung cancer accounts for 50% and 44% respectively in the total cases. The non-hematologic toxicity induced by chemotherapy in male was milder than female (p=0.01); the postoperative complications rate in female is higher than male (p=0.011) and in smokers is higher than non-smokers (p=0.031). In survival analysis, DFS of adenocarcinomas is longer than non-adenocarcinomas (23.70 months vs. 11.37 months,p=0.028); the prognosis of lung adenocarcinomas in female non-smokers is better than non-adenocarcinomas in male smokers after thoracotomy (median survival being 43.9 months vs. 20.37 months, p=0.009; 52.17 months vs. 33.13 months,p=0.02;52.17 months vs. 33.13 months, p=0.006 repectively). Gender, smoking history and adenocarcinomas status were not independend prognositc factor in Cox regression analysis. Multivariate analysis for intrathoracic regional lymph node metastasis showed that retrotracheal (p=0.002), subaortic (p=0.002) and lobar lymph node (p=0.015) affecting DFS, while lobar lymph node(p=0.002) metastasis had influence on OS.
Conclusions:
Compared with their opposite features, peripheral lung adenocarcinomas in female non-smokers have not any advantage on chemotherapy toxicity, response, complete resection rate, complications in thoractomy, and no significant difference on mediastinal lymph node metastasis and distant metastases. While the DFS in adenocarcinomas is longer than non-adenocarcinomas, the prognosis of adenocarcinomas of female non-smokers is better than non-adenocarcinomas male somkers, these advantages present especially in the subgroup of adenocarcinomas in non-smokers. Combined with the conclusions draw from the previous chapter, we deduce adenocarcinomas in non-smokers are the advantage populations in TKI therapy, while EGFR gene mutation is the targeted populations in TKI therapy.
PART 2: Effects of RNA Interference on EGFR/HER2 Expression in Non-Small-Cell Lung Cancer Cell Lines
CHAPTER 1: RNA Interference on EGFR Gene Expression in A549 Cell Line
Objective
To investigate whether RNA interference could induce gene silencing in human lung adenocarcinomas cell line A549, and to evaluate the degree of EGFR gene silencing and its effect on cell biological characteristics. Meterials and methods
1. Cell culture: Recovery A549 lung adenocarcinoma cell line and maintained in RPMI 1640 medium with 10% fetal calf serum.
2. Selection of sequence-specific siRNA-EGFR: Sequence-specific siRNA-EGFR were designed according to previous literatures, ensured by“Tuschl rules”and BLAST, and confirmed by the results of Western Blot after transfection.
3. Construction and identification of Psilencer 2.0 U6-shRNA-EGFR: ShRNA-EGFR oligonucleotide was designed based on the sequence-specific siRNA-EGFR and the online sofeware offered by Ambion company. A series of procudures (including anneal the template oligonucleotides, ligate annealed shRNA template insert into Psilencer, transform E. coli with the ligation products, purify Psilencer plasmid for transfection and identify clones with the shRNA template insert through direct suquencing) were performed to construct and identify the Psilencer 2.0 U6-shRNA-EGFR.
4. Transient transfection: Psilencer 2.0 U6-shRNA-EGFR and the control vector were transfected into A549 cell line with lipofectamine 2000.
5. Effect of EGFR knocked down: Real time Quantitative RT-PCR was used to detect the silencing of the EGFR gene level. Western Blot was used to measure the reduction in the expression of the EGFR protein.
6. Investigation of the alteration of biological features after EGFR RNA interference: MTT analysis, DAPI staining, Annexin V/PI double labeling and flow cytometry were used to evaluate cell proliferation, apoptosis and cell cycle distribution after RNA interference.
Results
1. Sequence-specific siRNA-EGFR: siRNA-EGFR-1‘5 -CACAGUGGAGCGAAUUCCUtt-3’; siRNA-EGFR-2‘5 -CUCUGGAGGAAAAGAAAGUtt-3’。
2. Effect of EGFR knocked down: In A549 cell line, we displayed sequence specific silencing of the EGFR gene with 92.67%, 96.77% and 94.79% in shRNA-EGFR1, shRNA-EGFR2 and shRNA-EGFR3 based on the above sequence compared with the negative control through Real Time Quantitative RT-PCR. The EGFR protein expression was down regulated for 54% and 72% by shRNA-EGFR1and shRNA-EGFR2 respectively through Western Blot.
3. Alteration of biological characteristics after EGFR RNA interference in A549 cells: MTT analysis detected growth inhibition of the cells dealt with shRNA-EGFR. Observation with fluorescent microscope revealed shrunk nuclei staining with DAPI, crimpled nuclear membrane as well as condensation and fragmentation of chromatin induced by shRNA-EGFR. Annexin V/PI double labeling found the sign of cell membrane stained with green fluorescent in the third phase was more significant in shRNA-EGFR group. Cell cycle analysis by flow cytometry showed that shRNA-EGFR induced accumulation of cells in phase G0-G1 with a decrease in the percentage of cells in phase S compared with the negative control.
Conclusion
The sequence specific siRNA-EGFR and shRNA-EGFR showed a remarkable effect in downregulation of EGFR expression, inhibition of the cellular proliferation and motility, and arrestment of the cell cycle in A549 cells. The application of RNA interference to partly reverse the neoplastic phenotype of EGFR overexpressing cells might provide a new approach to gene therapy and research of NSCLC.
CHAPTER 2: Relatioship between EGFR/HER2 Gene Knocked Down and Downstream Signal Pathway in EGFR Family
Objective
RNAi tecnique was applied to explore the relatioship between downstream signal pathway in EGFR family and the cell proliferation, cell cycle alteration, apoptosis after EGFR/HER2 RNA interference.
Meterials and methods
1. Cell culture: Recovery A549 lung adenocarcinoma, SPC-A-1 lung adenocarcinoma, L78 squamous cell carcinoma, H460 large cell lung cancer cell line and maintained in RPMI 1640 medium with 10% fetal calf serum.
2. Cell Screening: The above 4-cell lines were screened by comparision with transfection efficiency, tolerance to transfection reagent and the level of EGFR/HER2 expression for further research on RNAi.
3. Selection of sequence-specific siRNA for EGFR, HER2 and EGFR/HER2 joint interference: Sequence-specific siRNA for EGFR and HER2 were designed on the previous literature;"Tuschl rules" and BLAST on the full length of EGFR/HER2 cDNA were used to ensure the sequence-specific siRNA for EGFR/HER2 joint interference.
4. Construction of recombinant vector: Based on the above sequence-specific siRNA, recombinant plasmids with GFP and neomycin resistance marker were constructed by company.
5. Transient transfection: Six arms including mock, negative control, shRNA-EGFR, shRNA-HER2, shRNA-EGFR/HER2 and shRNA-EGFR+shRNA-HER2 were established.
6. Effect of EGFR kncked down: Real time Quantitative RT-PCR was used to detect the silencing of the EGFR/HER2 gene level. Western Blot was used to measure the levels of EGFR/HER2 protein and protein phosphorylation expression. Transfected cells were stimulated with EGF 15mins before protein extraction.
7. Alteration of biological features: MTT assay and flow cytometry were used to evaluate the cell proliferation, apoptosis and cell cycle distribution after RNAi.
8. Downstream signaling pathway protein detection: the protein expression level of downstream signaling pathway protein including Akt, p-Akt, p-Erk1/2, p-p38, were measured with Real time Quantitative RT-PCR PCR and Western Blot.
9. Statistical Analysis: Randomized block analysis of variance and SNK methods were used to compare the differences between the groups. P<0.05 was considered significant. Analysis was performed using SPSS 13.0. Results
6. SPC-A-1 was seleted for further research on RNAi based on its transfection efficiency, tolerance of transfection reagent and the level of EGFR/HER2 expression.
7. Sequence-specific siRNA: siRNA-HER2‘5 - GCATACGTGATGGCTGGTGTT-3’; siRNA- EGFR/HER2‘5 - GTCTACATGATCATGGTCAATT-3’。
8. Alteration of biological characteristics after EGFR RNA interference: MTT analysis found that cell proliferation was inhibited in the groups of shRNA-EGFR, shRNA-HER2, shRNA-EGFR+shRNA-HER2 and shRNA-EGFR/HER2. Cell cycle analysis by flow cytometry showed that apoptosis ratio in shRNA-HER2 (p=0.001), shRNA-EGFR/HER2 (p=0.005) and shRNA-EGFR+shRNA-HER2 (p=0.048) groups were significantly higher than negative control group, while there was no statistical difference between shRNA-EGFR and negative control (p=0.104), and that the distributions in phase G1 and phase S in shRNA-EGFR (p=0.007), shRNA-HER2 (p<0.001), shRNA-EGFR/HER2 (p< 0.001) and shRNA-EGFR+shRNA-HER2 (p=0.001) were significantly different compared with the negative control.
9. Effect of EGFR/HER2 kncked down: The level of EGFR/HER2 protein and protein phosphorylation expression were down regulated.
10. Downstream signaling pathway protein detection: The cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference have no significant relationship with downstearm signal pathway moleculars in EGFR family.
Conclusion
EGFR gene knockdown may not cause significant apoptosis in SPC-A-1. The variations of cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference were not found to have significant relationship with downstearm signal pathway molecules in EGFR family.
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