结直肠癌转移差异分泌蛋白质组学研究
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摘要
结直肠癌是严重威胁人类健康的常见恶性肿瘤。2008年,美国结直肠癌的发病率和死亡率在男、女性中均列恶性肿瘤的第三位。数十年来,随着我国人民生活方式和膳食结构的改变,结直肠癌的发病率和死亡率也呈逐年上升的趋势。虽然现代癌症研究和医疗技术不断发展,结直肠癌患者的预后并没有明显改善。转移是影响结直肠癌患者治疗效果和导致死亡的主要原因。因此,研究转移发生过程中的关键分子,早期预测结直肠癌的转移潜能,是提高结直肠癌患者生存率的关键。
目前医疗界公认,检测肿瘤标志物是实现肿瘤转移预测的最佳手段。二十余年以来,在分子生物学和免疫学检测技术不断发展的带动下,已有不少重要的肿瘤标志物如癌胚抗原、甲胎蛋白、前列腺特异性抗原等被开发应用。虽然这些肿瘤标志物已经广泛应用于临床诊断和病情监测。但遗憾的是,它们的敏感性和特异性却往往不尽人意。寻找更加敏感和特异的肿瘤标志物势在必行。
在后基因组时代,逐渐发展成熟的蛋白质组学技术使得大规模的肿瘤标志物筛选成为可能。但是,传统的组织蛋白质组学策略在应用于肿瘤标志物筛选方面存在缺陷。因为,理想的肿瘤标志物理应能够从血液或尿液中被无创性的方法检测出来,而通过组织蛋白质组学策略筛选得到的肿瘤相关分子却不一定能满足这一基本要求。与之相对,近年来兴起的血蛋白质组学策略更加诱人,因为其可以通过对血中蛋白表达谱的比较而直接获取肿瘤相关的血清标志物。不过,该策略却有其难以逾越的技术障碍。这是因为,血液的成份非常复杂,其中大量高丰度蛋白的存在会对其它重要蛋白产生致命的掩盖效应,从而大大降低血蛋白质组学筛选的效能。所以,现在越来越多的研究者开始关注一个全新的领域一肿瘤分泌蛋白质组学。
分泌蛋白质组这一概念最早是由Tjalsma等于2000年在对枯草杆菌分泌蛋白的研究中提出的。从广义上说,分泌蛋白质组是指细胞、组织或器官释放到胞外的全部蛋白质,既包括由经典的蛋白分泌途径所分泌的蛋白质,也包括非经典分泌性蛋白质,还包括通过胞外体(exosome)分泌的蛋白质。肿瘤的形成是一个长期的多因素参与的复杂过程,在该过程中,肿瘤细胞能够分泌多种蛋白质;这些蛋白质不仅能够以自分泌、旁分泌的形式调控肿瘤的发生和发展,还可以进入血液、尿液等体液中,易于被临床无创性的方法检测出来。所以,肿瘤分泌蛋白质组学研究为肿瘤标志物的开发开辟了一条崭新、充满希望的道路。
由于起步较晚,有关肿瘤分泌蛋白质组学的研究目前还为数不多,但取得的成果已经足以证明其是筛选肿瘤标志物的有效方法。迄今为止,与结直肠癌转移相关的分泌蛋白质组学研究尚未有报导。在本研究中,我们利用非标记定量鸟枪法蛋白质组学技术,分析比较了具有相同遗传背景、不同转移潜能的人结直肠癌细胞株SW480和SW620的分泌蛋白质组,并对感兴趣的差异分泌蛋白进行了较为全面的验证,以期发现对结直肠癌转移有预测价值的血清标志物。
首先,我们以来自同一亲本的人结直肠癌原发灶细胞株SW480和淋巴结转移.灶细胞株SW620作为体外研究的模型系统,通过条件的反复优化,从这两株细胞的无血清培养上清中成功的收集了高质量的分泌蛋白样本。随后,用特异性的胰酶消化蛋白质,酶解后的肽段经变性、脱盐等处理后经高效液相色谱分离,最后用Finnigan~(TM) LTQ~(TM)线性离子阱串联质谱进行分析。质谱所采集到的原始数据用SEQUEST程序搜索国际蛋白指数(International Protein Index,IPI)人蛋白非冗余库(Version 3.26)以鉴定蛋白质。利用液相色谱-串联质谱技术,我们从SW480和SW620细胞的分泌蛋白样本中总共鉴定出了910个非冗余蛋白质。由于这些蛋白质的识别均是基于两个及以上独特肽段的,所以更加严格和可靠;据我们所知,这也是迄今为止鉴定蛋白质数目最多的结直肠癌相关研究。
为保证后续非标记定量的质量,我们对液相色谱-串联质谱分析结果的重复性和可靠性进行了评估。结果发现:1、共计6次液相色谱-串联质谱分析的二维图谱具有极高的相似度。2、SW480和SW620各3次重复的液相色谱-串联质谱分析中鉴定到的蛋白质数量稳定。3、SW480和SW620的3次实验所识别蛋白的重复性分别为77%和74%。4、在设定的参数下,肽段错误发现率为3.82%。证明液相色谱-串联质谱分析的稳定性和重复性好,肽段鉴定的可信度较高。
在此基础上,利用DeCyder~(TM) MS差异分析软件(Version 1.0),对液相色谱串联质谱所得的全扫描前体质谱峰图进行检测、谱图比较和定量。所有的检测、匹配及鉴定都在DeCyder MS的全自动模式下进行,无需手动的指定或更正。最后采用统计学组间t检验方法,得到所有P<0.01的极显著差异多肽分子,并搜索数据库鉴定这些多肽分子和他们对应的蛋白质。通过DeCyder MS无标记定量分析,我们一共识别了145个在SW480和SW620分泌蛋白质组中表达差异超过1.5倍的蛋白。其中,75个蛋白在SW620中表达上调,而另外70个蛋白在SW620中表达下降。这也是迄今为止最大的结直肠癌转移相关的差异蛋白表达谱。在这145个差异分泌蛋白中有13个蛋白具有3个或以上的有定量信息的肽段。我们计算了这13个蛋白的肽段定量比值的变异系数,发现变异系数最高的为48.7%,最低的仅3.2%,平均为21%,说明我们无标记定量的准确度高,由此获得的定量信息是可信的。
为了解这些差异分泌蛋白在总体上的分子特征,我们利用多种生物信息学工具对145个差异分泌蛋白的细胞定位、生物学功能、信号通路、组织表达等进行了分析。首先,我们使用HPRD和GO数据库对差异分泌蛋白的细胞定位进行了分析。发现:在145个差异分泌蛋白中,分泌蛋白质组研究的兴趣蛋白-胞外蛋白和膜蛋白共为62个(38个细胞外蛋白,24个膜蛋白),占总数的42%。该比例高于目前有报导的同类研究中的一般水平(约30%),说明我们对分泌蛋白的富集是成功的。除13个未分类蛋白外,在145个差异分泌蛋白中有70个被归入胞内蛋白。为什么分泌蛋白质组中会存在如此高比例(49%)的胞内蛋白呢?我们推测,这些定位于胞内的蛋白中至少有一部分是主动释放至胞外的,而这种主动释放可能通过的是胞外体(exosome)分泌途径和非经典分泌途径。为明确这70个胞内蛋白中是否确实存在非经典分泌蛋白,我们将从IPI人蛋白非冗余库中获取的蛋白质氨基酸序列信息以FASTA格式提交至非经典分泌蛋白的预测软件SecretomeP 2.0和信号肽预测软件SignalP 3.0进行在线分析,结果中SecP score>0.5且排除有信号肽的蛋白即被判定为非经典分泌蛋白。最后,从70个胞内蛋白中成功的预测出了23个(33%)非经典分泌蛋白,证实了我们的推测。
我们利用DAVID和KEGG网站的分析工具获取了差异分泌蛋白的的功能聚类和信号通路分类的重要信息。我们发现,这些蛋白的主要生物学功能涉及细胞分化、细胞骨架组构、凋亡、解剖学结构形态发生、细胞增殖、细胞粘附及外部刺激反应等方面;而肌动蛋白细胞骨架调节通路、细胞外基质-受体相互作用通路、局部粘附通路等都存在着蛋白的富集。这些功能和信号通路在肿瘤发生、发展过程中均起着至关重要的作用,提示我们所识别的差异分泌蛋白是与肿瘤发生、发展密切相关的蛋白,有可能包含重要的肿瘤标志物。利用WebGestalt网站提供的组织表达分析工具,我们还识别了25个在结肠高特异性表达的差异分泌蛋白,它们有成为结直肠癌特异性标志物的潜能。
从145个差异分泌蛋白中,我们选择了7个(TFF3、GDF15、AGR2、LASP1、TGM2、LCN2和IGFBP7)进行荧光定量PCR分析和Western blot验证。荧光定量PCR分析发现,7个蛋白中,除了LASP1,其余6个的mRNA表达的变化趋势均与质谱定量的结果一致。对细胞培养上清和细胞裂解液中差异分泌蛋白的Western blot检测显示:在培养上清中,7个蛋白的相对表达变化均与质谱定量结果完全一致,这就直接证明了我们质谱分析结果的可靠性。在细胞裂解液中,TFF3、AGR2、GDF15、TGM2和LCN2的相对表达也与质谱定量结果一致,说明这些蛋白分泌增加可能是合成增加的结果。但是,LASP1在细胞裂解液中的表达却呈现出与质谱定量以及上清中检测结果相反的变化,而与前面PCR结果一致。所以,我们推测:LASP1存在合成和分泌不一致的情况。即在mRNA和总蛋白水平上,LASP1在SW620细胞中的表达较SW480细胞下调,但其在SW620细胞中分泌量却显著增加,从而使其在SW620细胞培养上清中表达上调。当然,为何会出现蛋白合成与分泌不一致的现象,在SW620细胞中,LASP1又是通过什么机制而分泌增加的,还不得而知,需要后续的实验研究。此外,我们发现,虽然IGFBP7在SW480细胞的培养上清中有较强的表达,却未能在细胞裂解液中检测到其表达,推测该蛋白可能在合成后即迅速、完全的释放到胞外。
这些在结直肠癌原发灶细胞株SW480和转移灶细胞株SW620之间存在差异表达的分泌蛋白可否成为结直肠癌转移相关的血清标志物呢?这是我们最为关注的问题。为此,我们选择了两个在SW620中表达上调的分泌蛋白—TFF3和GDF15进行了更深入的研究。首先,我们用免疫组织化学的方法检测了TFF3与GDF15在38例有淋巴结转移的结直肠癌(每例均有原发灶和配对的转移淋巴结)和31例无转移的结直肠癌中的表达情况。结果发现,TFF3和GDF15的阳性染色主要定位于癌细胞的胞浆中。其中,TFF3在淋巴结转移组的表达水平略强于无转移组,差异无统计学意义(P=0.157);但是,淋巴结转移灶中TFF3的免疫组织化学染色明显强于其配对的原发灶(P<0.0001)。GDF15的表达在无转移的结直肠癌、伴淋巴结转移的结直肠癌原发灶和配对淋巴结转移灶中依次增高,差异均有统计学意义。提示TFF3和GDF15表达与结直肠癌转移密切相关。相关分析还发现GDF15与TFF3在结直肠癌中的表达有低度相关性(r=0.241;P=0.046)。此外,TFF3的表达水平与肿瘤部位有关,结肠癌中TFF3染色强度显著高于直肠癌(P=0.007);其与性别、年龄、肿瘤大小、分级、TNM分期等临床病理参数无统计学相关性(P>0.05)。与TFF3不同,GDF15的表达与TNM分期有关,TNM分期Ⅲ/Ⅳ期的结直肠癌GDF15表达水平显著高于Ⅰ/Ⅱ期(P<0.0001);其与性别、年龄、肿瘤部位、肿瘤大小、组织学类型、分级等临床病理参数之间无统计学相关性。
利用双抗夹心ELISA方法,我们进一步对TFF3和GDF15在144例结直肠癌和156例正常人血清中的表达进行了检测。在正常人群中,TFF3和GDF15均与年龄、性别无统计学相关性。血清TFF3浓度在结直肠癌患者中高于正常对照(结直肠癌VS正常人群:1925±1637 ng/ml VS 824±278 ng/ml),淋巴结转移组中明显高于无转移组(淋巴结转移组VS无转移组:2690±1839 ng/ml VS 1071±727ng/ml),差异均有显著性(P<0.0001)。同TFF3相似,GDF15浓度在结直肠癌患者血清中要显著高于正常人群(结直肠癌VS正常人群:2030±1122 pg/ml VS639±212 pg/ml),而淋巴结转移组GDF15的水平明显高于无转移组(淋巴结转移组VS无转移组:2667±1067 pg/ml VS 1319±663 pg/ml),差异均有显著性(P<0.0001)。
上述结果提示TFF3和GDF15有作为结直肠癌发生和转移血清标志物的可能性。为此,我们采用了ROC曲线分析以评判两者的诊断效能。以结直肠癌为疾病诊断判别模型的ROC曲线分析发现:TFF3的曲线下面积为0.730(95%可信区间:0.670-0.791),在最佳截断浓度取1323 ng/ml时,其诊断结直肠癌的灵敏度和特异度分别53.3%和97.4%;GDF15的曲线下面积为0.897(95%可信区间:0.856-0.938),当最佳截断浓度选择1144 pg/ml时,GDF15诊断结直肠癌的灵敏度和特异度分别77.8%和99.4%。以结直肠癌淋巴结转移为疾病诊断判别模型的ROC曲线分析显示:TFF3的曲线下面积为0.822(95%可信区间:0.750-0.894),取最佳截断浓度1789 ng/ml时,血清TFF3预测结直肠癌转移的灵敏度和特异度分别73.7%和91.2%;GDF15的曲线下面积为0.867(95%可信区间:0.806-0.928),在最佳截断浓度1881 pg/ml处,其预测结直肠癌转移的灵敏度和特异度分别82.9%和82.4%。这些结果说明TFF3与GDF15均可以作为结直肠癌诊断及转移预测的血清标志物。
相关分析发现血清中TFF3与GDF15浓度水平呈正相关(r=0.422;P<0.0001)。两个指标联合诊断的ROC曲线分析发现:对于判别诊断结直肠癌,血清TFF3与GDF15联合(曲线下面积:0.926)较TFF3或GDF15单独诊断时效能增强,差异均有显著性(P<0.0001);而对于预测结直肠癌转移,血清TFF3与GDF15联合(曲线下面积:0.866)较TFF3单独诊断的效能增加,但和GDF15之间无显著性差异。
我们还对结直肠癌血清TFF3、GDF15水平与临床病理特征的关系进行了分析。结果发现两者均随肿瘤TNM分期进展而增高、在高级别的结直肠癌中高于低级别结直肠癌、有远处转移的患者中高于无远处转移的,差异均有统计学意义(P<0.05)。此外,结直肠癌TFF3、GDF15血清浓度与性别、年龄、肿瘤部位等临床病理参数无统计学相关性。以上结果说明TFF3和GDF15和结直肠癌分化、病情进展以及转移相关。
通过上述研究,我们得出以下结论:
运用非标记定量鸟枪法蛋白质组学技术分析肿瘤差异分泌蛋白质组,是寻找肿瘤血清标志物的高效、可行的研究策略。TFF3、GDF15与结直肠癌发生和转移密切相关,可以作为结直肠癌诊断和转移预测的候选血清标志物。
Colorectal cancer(CRC) is one of the most common malignancies worldwide.In American,the incidence and mortality of CRC still ranked the 3rd among all the cancers in both the male and the female in 2008.In China,with the changes of life style and diet structure,the incidence and mortality of CRC continues to increase in recent decades. Despite considerable refinement in therapeutic modalities,almost half of the CRC patients treated with "curative" surgery undergo recurrence within 5 years-mostly with metastatic lesions.Notably,extensive metastasis renders the current treatment ineffective and accounts for most fatalities caused by CRC.Therefore,to predict the metastatic potential of CRC can provide valuable prognostic information as well as opportunities for enhanced intervention,leading to an improved prognosis and increased survival rate.
It is widely accepted that the best ways to predict cancer metastasis is to use cancer-specific biomarkers.Over the past several decades,enormous efforts have been made to screen and characterize useful cancer-specific biomarkers.Some important molecules such as CEA,AFP and PSA are identified as cancer biomarkers and commonly applied in clinical practice.Unfortunately,most currently available cancer biomarkers are not satisfactory because of limited specificity and/or sensitivity, stressing the need to discover clinically valuable biomarkers.
In a post-genome era,proteomic approaches have been introduced to seek novel cancer biomarkers.Nevertheless,the conventional tissue proteomics approaches have been less striking for biomarker discovery.The reason lies in the fact that proteins identified from tissues are not necessarily detectable in serum or plasma.It is conceivable that a biomarker to be useful in cancer screening and monitoring should be measurable in body fluid samples.Accordingly,mining cancer biomarkers from blood proteome is of particular interest.However,the prospects of blood proteomics are challenged by the fact that blood is a very complex body fluid,comprising an enormous diversity of proteins with a large dynamic range.The abundant blood proteins may mask the less abundant proteins,which are usually potential biomarkers.Several procedures have been made to remove these more abundant proteins before proteomic analysis.Nevertheless,these methods may sacrifice other proteins by nonspecific binding and thus lower the screen efficiency.Above-mentioned major limitations of blood proteomics emphasize the need to seek other approaches for cancer biomarker discovery.More recently,there has been an increasing interest in a newly emerging approach-cancer secretome analysis,which is a promising tool for the identification of cancer biomarkers.
The term "secretome" was first proposed by Tjalsma et al.in a genome-based global survey on secreted proteins of Bacillus subtilis.In a broader sense,the secretome harbors proteins released by a cell,tissue or organism through various mechanisms including classical secretion,nonclassical secretion,and secretion through the release of exosomes.It is known that cancer cells interact with their microenvironment by secreting a variety of proteins,including growth factors,extracellular matrix-degrading proteinases,cell motility factors and immunoregulatory cytokines or other bioactive molecules.These cancer-secreted proteins are essential in the processes of differentiation,invasion,and metastasis.More importantly,these cancer secreted proteins or their fragments always enter body fluids such as blood or urine and can be measured via non-invasive assays.Thus,cancer secretome may reflect a broad variety of pathological conditions and represents a more reliable source of biomarkers.
To date,only a minority of studies has analyzed cancer secretomes,however,the results regarding biomarker discovery are exciting.Of note,no differential secretome analysis on CRC metastasis has been reported.In this study we used a liquid chromatography-tandem mass spectrometry(LC-MS/MS) based label-free quantitative shotgun proteomics approach to investigate the secretomes of two human CRC cell lines(SW480 and SW620),in order to seek novel metastasis-associated serum biomarkers of CRC.
First,we used SW480 and SW620 as our model system because this pair of CRC cell lines derived from the same individual but with different metastatic potentials.The secretome samples collected from SW480 and SW620 were digested with trypsin and analyzed by a Finnigan~(TM)LTQ~(TM)MS coupled with a HPLC system.MS/MS spectra were automatically searched against the non-redundant International Protein Index(IPI) human protein database(Version 3.26) using the TurboSEQUEST~(TM)program.The stringent protein identification criteria were based on Delta Cn(≥0.1) and cross-correlation scores(Xcorr,one charge≥1.9,two charges≥2.2,three charges≥3.75).BuildSummary,a in-house tool,was used to combine the peptide sequences into proteins and delete redundant proteins.Finally,a total of 910 nonredundant proteins based on the identification of two or more unique peptides were identified,which,to our knowledge,represents one of the largest protein profiles identified for CRC.
In SW480,588 proteins were identified.Of these,451 proteins were identified in all three replicates,showing a protein identification reproducibility of 77%.The similarly high protein identification reproducibility(74%) was found in SW620,in which 672 proteins were identified and 497 proteins were present in all three replicates. Furthermore,searches against the sequence-reversed decoy IPI human databases using the same search parameters yielded a FDR of 3.82%at the peptide level.The high reproducibility and low FDR highlights the fidelity of LC-MS/MS analysis.
Label-free quantitative comparison between the two cell lines was performed by DeCyder~(TM)MS Differential Analysis Software(Version 1.0).Peptide detection, background subtraction and quantitation were performed on the full scan precursor mass spectra in fully automatic mode.Collectively,145 proteins displayed more than 1.5-fold quantitative alterations(t-test,P<0.01) in the secretomes of SW620 versus SW480.Among the 145 proteins,75 proteins were up-regulated,whereas 70 proteins were down-regulated in SW620 compared with SW480 cells.To date,this is one of the largest qualitative proteome studies for CRC.For the 145 proteins,the number of peptides used for quantitation from each protein varied between 1 and 10.Among these, 13 proteins were comparatively quantified on the basis of change levels of three or more peptides that varied similarly.From these data it is possible to evaluate the confidence of the quantitative approach.The average coefficient of variation(CV) of the fold changes for peptides from these 13 proteins was 21%(range 3.2-48.7%),yielding a reasonable reproducibility of the quantitative data.
Subsequently,overall features for the differential secreted proteins were analyzed by various bioinformatics analytic tools.First,cellular localization of identified proteins was analyzed on the basis of Gene Ontology(GO) and Human Protein Reference Database(HPRD).The localization of 42%differential proteins was classified as extracellular and membrane.The ratio is a little higher than previous secretome studies (30%),which demonstrates the advantage of our enrichment method for secreted proteins.Additionally,70 proteins identified in the CM were assigned to intracellular organelles,cytoskeleton,nucleus and cytoplasm(49%).The identification of a large portion of intracellular proteins is due in part to nonspecific liberation of cytoplasmic proteins as a consequence of cell death.However,we believe that the presence of many putative intracelluar proteins in CM may not merely be caused by cell autolysis,but also due in part to the active release through nonclassical secretion pathway and exosomes. Using SecretomeP 2.0 and SignalP 3.0 software,we analyzed the amino acid sequences of differential secreted proteins.The nonclassical secreted protein identification criteria were based on SecP score > 0.5 and the absence of signal peptides.Finally,we successfully identified 23(33%) nonclassical secreted proteins from the 70 intracellular proteins.
Biological function classifications were performed with the tools on DAVID Bioinformatics Resources 2008,and pathway analysis was done by searching Kyoto Encyclopedia of Genes and Genome(KEGG) database.The top biological functions of differential proteins were cell differentiation followed by cytoskeleton organization, apoptosis,anatomical structure morphogenesis,cell proliferation,cell adhesion, response to external stimulus and cell motility.The top ranked pathways were those involved in regulation of actin cytoskeleton,ECM-receptor interaction and focal adhesion.These findings suggest that the differential secreted proteins identified in SW480 versus SW620 might be implicated in CRC initiation and progression,and include valuable biomarker candidates for CRC.
The relative expression level of proteins determined by DeCyder MS was verified by SYBR Green Q-PCR and Western blot analysis of 7 selected proteins,GDF15,TFF3, AGR2,LASP1,TGM2,LCN2 and IGFBP7.These 7 proteins were selected on the basis of a combination of parameters including level of differential expression in SW620 versus SW480,cellular localization,availability of commercial antibodies,and the potential relevancy with cancer progression.From Q-PCR results,we found that the relative expression level of 6 genes coincided with the results of DeCyder MS;only LASP1 showed reversed fold change between the two cell lines.The expressions of the 7 selected proteins in the CM and in the total cell lysates were assayed by Western blot as well.In the CM harvested from this pair of CRC cells,the changes in protein abundance noted in the Western blot analysis were considerably consistent with the results obtained by proteomic screens,which demonstrates the accuracy of label-free quantitation.For GDF15,TFF3,AGR2,TGM2 and LCN2,similar results were observed in the cell lysates,which suggested the dysregulated secretion of these proteins was likely associated with the altered protein expression between the two cell lines.Notably,the change levels of LASP1 noted in cell lysates were consistent with Q-PCR results,suggesting that the increased expression of this protein in SW620 CM might be the increased secretion.Unfortunately,we could not detect IGFBP7 in cell lysates,although it was clearly present in SW480 CM,indicating rapid and efficient secretion of this protein.
Among the 7 proteins confirmed by Q-PCR and Western blot analysis,we chose two up-regulated proteins in SW620 versus SW480,TFF3 and GDF15,for further estimation in terms of its clinical relevancy in cancer metastasis.Using immunohistochemical staining,we measured TFF3 and GDF15 expression in 38 cases of CRC with lymph node metastasis(LNM),in which each case contains both primary tumor and metastatic lymph node,and 31 cases of non-LNM CRC.Comparison of immunoreactive scores of non-LNM CRC,primary tumors of LNM CRC and matched metastatic lymph nodes demonstrated that TFF3 expression in LNM CRC was slightly up-regulated although not significantly different from that in non-LNM CRC;in contrast,metastatic lymph nodes displayed stronger immunoreactivity of TFF3 than matched primary tumors(P<0.0001).A strong immunoreactivity was detected for GDF15 in LNM CRC,which was significantly higher than that in non-LNM CRC(P < 0.0001).In addition,metastatic lymph nodes exhibited stronger GDF15 expression compared to matched primary tumors(P=0.041).This finding indicated that dysregulation of TFF3 or GDF15 expression in CRC tissues was highly associated with CRC metastasis.Furthermore,No appreciable correlation was demonstrated between TFF3 expression and patient's gender,age,tumor size,histological grade and clinical TNM stage.Notably,however,TFF3 expression was site-related,cancers localized in colon mostly exhibited stronger immunoreactivity than cancers in rectum(P=0.007). There was a significant increase in the imunostaining of GDF15 between TNM tumor stageⅠ/ⅡandⅢ/Ⅳ(P<0.0001).The level of GDF15 expression was not statistically associated with gender,age,tumor size and site.
As immunohistochemical analysis indicated the association between TFF3 and GDF15 dysregulation and CRC metastasis,we speculated they might be serum marker candidates for predicting CRC metastasis.To test this speculation,the levels of TFF3 and GDF15 in serum samples collected from CRC patients(n=144) and healthy controls (n=156) were measured by double-antibody sandwich ELISA system.The serum levels of both proteins were significantly higher in CRC patients than in healthy controls.For details,the mean levels for serum TFF3 and GDF15 in healthy control were 824±278 ng/ml,and 639±212 pg/ml,respectively,whereas in CRC patients serum samples,the corresponding mean were elevated to 1925±1637 ng/ml and 2030±1122 pg/ml, respectively.Then,for analysis purposes,the 144 cases of CRC serum samples were subdivided into non-LNM CRC(n=68) and LNM CRC(n=76) groups.Compared with non-LNM CRC group,LNM CRC group exhibited significantly increased serum levels of both TFF3(2690±1839 ng/ml versus 1071±727 ng/ml) and GDF15(2667±1067 pg/ml versus 1319±663 pg/ml).These findings indicated that the two proteins might be potential serum biomarkers for CRC.
To evaluate the diagnostic performance of serum TFF3 and GDF15,we constructed receiver operating characteristic(ROC) curve by plotting sensitivity versus specificity.The area under the ROC curve(AUC),a commonly used indicator for estimating the diagnostic efficacy of a potential biomarker,was subsequently calculated. For discriminating CRC from healthy controls,the AUC was determined to be 0.730 (95%confidence interval,0.670-0.791) for TFF3 and 0.897(95%confidence interval, 0.856-0.938) for GDF15,respectively.When a cutoff value of 1323 ng/ml was chosen for TFF3,the sensitivity and specificity for differentiating CRC with healthy controls were 53.5 and 97.4%,respectively.As well,using a cutoff value of 1144 pg/ml,GDF15 had a diagnostic sensitivity of 77.8%and specificity of 99.4%in detecting CRC. Notably,the combination of TFF3 and GDF15 showed a higher diagnostic capacity than either marker alone(AUC=0.926;95%confidence interval,0.890-0.961).The ROC curves of serum TFF3 and GDF15 for discerning LNM CRC versus non-LNM CRC were also constructed.The AUC for TFF3 was 0.822(95%confidence interval, 0.750-0.894),at a cutoff value of 1789 ng/ml,its sensitivity and specificity for predicting CRC metastasis reached to 73.7%and 91.2%,respectively.GDF15 had a similar AUC to that of TFF3(0.867,95%confidence interval,0.806-0.928).Setting a cutoff value of 1881 pg/ml,sensitivity and specificity for GDF15 in distinguishing LNM-CRC with non-LNM CRC were 82.9%and 82.4%,respectively.Nevertheless, combining serum TFF3 with GDF15 measurements did not improve the efficacy for predicting CRC metastasis(AUC 0.866,95%confidence interval,0.805-0.927).These results collectively indicated that TFF3 and GDF15 might be valuable serum biomarker for diagnosis and metastasis prediction of CRC.
Analysis of the cohort of 144 CRC patients was carried out to define the relationship between TFF3 and GDF15 serum levels and clinicopathologic features of CRC.The rises in TFF3 or GDF15 serum levels were significantly associated with higher histological grade,increasing clinical TNM stage and the presence of distant metastasis of CRC.Serum level of TFF3 and GDF15 was not statistically correlated with gender,age and site,although cancers localized in colon tended to have higher serum TFF3 level than cancers in rectum(2071±1629 versus 1753±1642 ng/ml, P=0.059).
In conclusion,our data demonstrated that:
Using label-free quantitative shotgun proteomics approach to investigate differential cancer secretomes is a feasible strategy to seek valuable serum biomarkers. TFF3 and GDF15 are associated with the carcinogenesis and metastasis of CRC,and might be clinically useful serum biomarkers for CRC detection and metastasis prediction.
目前医疗界公认,检测肿瘤标志物是实现肿瘤转移预测的最佳手段。二十余年以来,在分子生物学和免疫学检测技术不断发展的带动下,已有不少重要的肿瘤标志物如癌胚抗原、甲胎蛋白、前列腺特异性抗原等被开发应用。虽然这些肿瘤标志物已经广泛应用于临床诊断和病情监测。但遗憾的是,它们的敏感性和特异性却往往不尽人意。寻找更加敏感和特异的肿瘤标志物势在必行。
在后基因组时代,逐渐发展成熟的蛋白质组学技术使得大规模的肿瘤标志物筛选成为可能。但是,传统的组织蛋白质组学策略在应用于肿瘤标志物筛选方面存在缺陷。因为,理想的肿瘤标志物理应能够从血液或尿液中被无创性的方法检测出来,而通过组织蛋白质组学策略筛选得到的肿瘤相关分子却不一定能满足这一基本要求。与之相对,近年来兴起的血蛋白质组学策略更加诱人,因为其可以通过对血中蛋白表达谱的比较而直接获取肿瘤相关的血清标志物。不过,该策略却有其难以逾越的技术障碍。这是因为,血液的成份非常复杂,其中大量高丰度蛋白的存在会对其它重要蛋白产生致命的掩盖效应,从而大大降低血蛋白质组学筛选的效能。所以,现在越来越多的研究者开始关注一个全新的领域一肿瘤分泌蛋白质组学。
分泌蛋白质组这一概念最早是由Tjalsma等于2000年在对枯草杆菌分泌蛋白的研究中提出的。从广义上说,分泌蛋白质组是指细胞、组织或器官释放到胞外的全部蛋白质,既包括由经典的蛋白分泌途径所分泌的蛋白质,也包括非经典分泌性蛋白质,还包括通过胞外体(exosome)分泌的蛋白质。肿瘤的形成是一个长期的多因素参与的复杂过程,在该过程中,肿瘤细胞能够分泌多种蛋白质;这些蛋白质不仅能够以自分泌、旁分泌的形式调控肿瘤的发生和发展,还可以进入血液、尿液等体液中,易于被临床无创性的方法检测出来。所以,肿瘤分泌蛋白质组学研究为肿瘤标志物的开发开辟了一条崭新、充满希望的道路。
由于起步较晚,有关肿瘤分泌蛋白质组学的研究目前还为数不多,但取得的成果已经足以证明其是筛选肿瘤标志物的有效方法。迄今为止,与结直肠癌转移相关的分泌蛋白质组学研究尚未有报导。在本研究中,我们利用非标记定量鸟枪法蛋白质组学技术,分析比较了具有相同遗传背景、不同转移潜能的人结直肠癌细胞株SW480和SW620的分泌蛋白质组,并对感兴趣的差异分泌蛋白进行了较为全面的验证,以期发现对结直肠癌转移有预测价值的血清标志物。
首先,我们以来自同一亲本的人结直肠癌原发灶细胞株SW480和淋巴结转移.灶细胞株SW620作为体外研究的模型系统,通过条件的反复优化,从这两株细胞的无血清培养上清中成功的收集了高质量的分泌蛋白样本。随后,用特异性的胰酶消化蛋白质,酶解后的肽段经变性、脱盐等处理后经高效液相色谱分离,最后用Finnigan~(TM) LTQ~(TM)线性离子阱串联质谱进行分析。质谱所采集到的原始数据用SEQUEST程序搜索国际蛋白指数(International Protein Index,IPI)人蛋白非冗余库(Version 3.26)以鉴定蛋白质。利用液相色谱-串联质谱技术,我们从SW480和SW620细胞的分泌蛋白样本中总共鉴定出了910个非冗余蛋白质。由于这些蛋白质的识别均是基于两个及以上独特肽段的,所以更加严格和可靠;据我们所知,这也是迄今为止鉴定蛋白质数目最多的结直肠癌相关研究。
为保证后续非标记定量的质量,我们对液相色谱-串联质谱分析结果的重复性和可靠性进行了评估。结果发现:1、共计6次液相色谱-串联质谱分析的二维图谱具有极高的相似度。2、SW480和SW620各3次重复的液相色谱-串联质谱分析中鉴定到的蛋白质数量稳定。3、SW480和SW620的3次实验所识别蛋白的重复性分别为77%和74%。4、在设定的参数下,肽段错误发现率为3.82%。证明液相色谱-串联质谱分析的稳定性和重复性好,肽段鉴定的可信度较高。
在此基础上,利用DeCyder~(TM) MS差异分析软件(Version 1.0),对液相色谱串联质谱所得的全扫描前体质谱峰图进行检测、谱图比较和定量。所有的检测、匹配及鉴定都在DeCyder MS的全自动模式下进行,无需手动的指定或更正。最后采用统计学组间t检验方法,得到所有P<0.01的极显著差异多肽分子,并搜索数据库鉴定这些多肽分子和他们对应的蛋白质。通过DeCyder MS无标记定量分析,我们一共识别了145个在SW480和SW620分泌蛋白质组中表达差异超过1.5倍的蛋白。其中,75个蛋白在SW620中表达上调,而另外70个蛋白在SW620中表达下降。这也是迄今为止最大的结直肠癌转移相关的差异蛋白表达谱。在这145个差异分泌蛋白中有13个蛋白具有3个或以上的有定量信息的肽段。我们计算了这13个蛋白的肽段定量比值的变异系数,发现变异系数最高的为48.7%,最低的仅3.2%,平均为21%,说明我们无标记定量的准确度高,由此获得的定量信息是可信的。
为了解这些差异分泌蛋白在总体上的分子特征,我们利用多种生物信息学工具对145个差异分泌蛋白的细胞定位、生物学功能、信号通路、组织表达等进行了分析。首先,我们使用HPRD和GO数据库对差异分泌蛋白的细胞定位进行了分析。发现:在145个差异分泌蛋白中,分泌蛋白质组研究的兴趣蛋白-胞外蛋白和膜蛋白共为62个(38个细胞外蛋白,24个膜蛋白),占总数的42%。该比例高于目前有报导的同类研究中的一般水平(约30%),说明我们对分泌蛋白的富集是成功的。除13个未分类蛋白外,在145个差异分泌蛋白中有70个被归入胞内蛋白。为什么分泌蛋白质组中会存在如此高比例(49%)的胞内蛋白呢?我们推测,这些定位于胞内的蛋白中至少有一部分是主动释放至胞外的,而这种主动释放可能通过的是胞外体(exosome)分泌途径和非经典分泌途径。为明确这70个胞内蛋白中是否确实存在非经典分泌蛋白,我们将从IPI人蛋白非冗余库中获取的蛋白质氨基酸序列信息以FASTA格式提交至非经典分泌蛋白的预测软件SecretomeP 2.0和信号肽预测软件SignalP 3.0进行在线分析,结果中SecP score>0.5且排除有信号肽的蛋白即被判定为非经典分泌蛋白。最后,从70个胞内蛋白中成功的预测出了23个(33%)非经典分泌蛋白,证实了我们的推测。
我们利用DAVID和KEGG网站的分析工具获取了差异分泌蛋白的的功能聚类和信号通路分类的重要信息。我们发现,这些蛋白的主要生物学功能涉及细胞分化、细胞骨架组构、凋亡、解剖学结构形态发生、细胞增殖、细胞粘附及外部刺激反应等方面;而肌动蛋白细胞骨架调节通路、细胞外基质-受体相互作用通路、局部粘附通路等都存在着蛋白的富集。这些功能和信号通路在肿瘤发生、发展过程中均起着至关重要的作用,提示我们所识别的差异分泌蛋白是与肿瘤发生、发展密切相关的蛋白,有可能包含重要的肿瘤标志物。利用WebGestalt网站提供的组织表达分析工具,我们还识别了25个在结肠高特异性表达的差异分泌蛋白,它们有成为结直肠癌特异性标志物的潜能。
从145个差异分泌蛋白中,我们选择了7个(TFF3、GDF15、AGR2、LASP1、TGM2、LCN2和IGFBP7)进行荧光定量PCR分析和Western blot验证。荧光定量PCR分析发现,7个蛋白中,除了LASP1,其余6个的mRNA表达的变化趋势均与质谱定量的结果一致。对细胞培养上清和细胞裂解液中差异分泌蛋白的Western blot检测显示:在培养上清中,7个蛋白的相对表达变化均与质谱定量结果完全一致,这就直接证明了我们质谱分析结果的可靠性。在细胞裂解液中,TFF3、AGR2、GDF15、TGM2和LCN2的相对表达也与质谱定量结果一致,说明这些蛋白分泌增加可能是合成增加的结果。但是,LASP1在细胞裂解液中的表达却呈现出与质谱定量以及上清中检测结果相反的变化,而与前面PCR结果一致。所以,我们推测:LASP1存在合成和分泌不一致的情况。即在mRNA和总蛋白水平上,LASP1在SW620细胞中的表达较SW480细胞下调,但其在SW620细胞中分泌量却显著增加,从而使其在SW620细胞培养上清中表达上调。当然,为何会出现蛋白合成与分泌不一致的现象,在SW620细胞中,LASP1又是通过什么机制而分泌增加的,还不得而知,需要后续的实验研究。此外,我们发现,虽然IGFBP7在SW480细胞的培养上清中有较强的表达,却未能在细胞裂解液中检测到其表达,推测该蛋白可能在合成后即迅速、完全的释放到胞外。
这些在结直肠癌原发灶细胞株SW480和转移灶细胞株SW620之间存在差异表达的分泌蛋白可否成为结直肠癌转移相关的血清标志物呢?这是我们最为关注的问题。为此,我们选择了两个在SW620中表达上调的分泌蛋白—TFF3和GDF15进行了更深入的研究。首先,我们用免疫组织化学的方法检测了TFF3与GDF15在38例有淋巴结转移的结直肠癌(每例均有原发灶和配对的转移淋巴结)和31例无转移的结直肠癌中的表达情况。结果发现,TFF3和GDF15的阳性染色主要定位于癌细胞的胞浆中。其中,TFF3在淋巴结转移组的表达水平略强于无转移组,差异无统计学意义(P=0.157);但是,淋巴结转移灶中TFF3的免疫组织化学染色明显强于其配对的原发灶(P<0.0001)。GDF15的表达在无转移的结直肠癌、伴淋巴结转移的结直肠癌原发灶和配对淋巴结转移灶中依次增高,差异均有统计学意义。提示TFF3和GDF15表达与结直肠癌转移密切相关。相关分析还发现GDF15与TFF3在结直肠癌中的表达有低度相关性(r=0.241;P=0.046)。此外,TFF3的表达水平与肿瘤部位有关,结肠癌中TFF3染色强度显著高于直肠癌(P=0.007);其与性别、年龄、肿瘤大小、分级、TNM分期等临床病理参数无统计学相关性(P>0.05)。与TFF3不同,GDF15的表达与TNM分期有关,TNM分期Ⅲ/Ⅳ期的结直肠癌GDF15表达水平显著高于Ⅰ/Ⅱ期(P<0.0001);其与性别、年龄、肿瘤部位、肿瘤大小、组织学类型、分级等临床病理参数之间无统计学相关性。
利用双抗夹心ELISA方法,我们进一步对TFF3和GDF15在144例结直肠癌和156例正常人血清中的表达进行了检测。在正常人群中,TFF3和GDF15均与年龄、性别无统计学相关性。血清TFF3浓度在结直肠癌患者中高于正常对照(结直肠癌VS正常人群:1925±1637 ng/ml VS 824±278 ng/ml),淋巴结转移组中明显高于无转移组(淋巴结转移组VS无转移组:2690±1839 ng/ml VS 1071±727ng/ml),差异均有显著性(P<0.0001)。同TFF3相似,GDF15浓度在结直肠癌患者血清中要显著高于正常人群(结直肠癌VS正常人群:2030±1122 pg/ml VS639±212 pg/ml),而淋巴结转移组GDF15的水平明显高于无转移组(淋巴结转移组VS无转移组:2667±1067 pg/ml VS 1319±663 pg/ml),差异均有显著性(P<0.0001)。
上述结果提示TFF3和GDF15有作为结直肠癌发生和转移血清标志物的可能性。为此,我们采用了ROC曲线分析以评判两者的诊断效能。以结直肠癌为疾病诊断判别模型的ROC曲线分析发现:TFF3的曲线下面积为0.730(95%可信区间:0.670-0.791),在最佳截断浓度取1323 ng/ml时,其诊断结直肠癌的灵敏度和特异度分别53.3%和97.4%;GDF15的曲线下面积为0.897(95%可信区间:0.856-0.938),当最佳截断浓度选择1144 pg/ml时,GDF15诊断结直肠癌的灵敏度和特异度分别77.8%和99.4%。以结直肠癌淋巴结转移为疾病诊断判别模型的ROC曲线分析显示:TFF3的曲线下面积为0.822(95%可信区间:0.750-0.894),取最佳截断浓度1789 ng/ml时,血清TFF3预测结直肠癌转移的灵敏度和特异度分别73.7%和91.2%;GDF15的曲线下面积为0.867(95%可信区间:0.806-0.928),在最佳截断浓度1881 pg/ml处,其预测结直肠癌转移的灵敏度和特异度分别82.9%和82.4%。这些结果说明TFF3与GDF15均可以作为结直肠癌诊断及转移预测的血清标志物。
相关分析发现血清中TFF3与GDF15浓度水平呈正相关(r=0.422;P<0.0001)。两个指标联合诊断的ROC曲线分析发现:对于判别诊断结直肠癌,血清TFF3与GDF15联合(曲线下面积:0.926)较TFF3或GDF15单独诊断时效能增强,差异均有显著性(P<0.0001);而对于预测结直肠癌转移,血清TFF3与GDF15联合(曲线下面积:0.866)较TFF3单独诊断的效能增加,但和GDF15之间无显著性差异。
我们还对结直肠癌血清TFF3、GDF15水平与临床病理特征的关系进行了分析。结果发现两者均随肿瘤TNM分期进展而增高、在高级别的结直肠癌中高于低级别结直肠癌、有远处转移的患者中高于无远处转移的,差异均有统计学意义(P<0.05)。此外,结直肠癌TFF3、GDF15血清浓度与性别、年龄、肿瘤部位等临床病理参数无统计学相关性。以上结果说明TFF3和GDF15和结直肠癌分化、病情进展以及转移相关。
通过上述研究,我们得出以下结论:
运用非标记定量鸟枪法蛋白质组学技术分析肿瘤差异分泌蛋白质组,是寻找肿瘤血清标志物的高效、可行的研究策略。TFF3、GDF15与结直肠癌发生和转移密切相关,可以作为结直肠癌诊断和转移预测的候选血清标志物。
Colorectal cancer(CRC) is one of the most common malignancies worldwide.In American,the incidence and mortality of CRC still ranked the 3rd among all the cancers in both the male and the female in 2008.In China,with the changes of life style and diet structure,the incidence and mortality of CRC continues to increase in recent decades. Despite considerable refinement in therapeutic modalities,almost half of the CRC patients treated with "curative" surgery undergo recurrence within 5 years-mostly with metastatic lesions.Notably,extensive metastasis renders the current treatment ineffective and accounts for most fatalities caused by CRC.Therefore,to predict the metastatic potential of CRC can provide valuable prognostic information as well as opportunities for enhanced intervention,leading to an improved prognosis and increased survival rate.
It is widely accepted that the best ways to predict cancer metastasis is to use cancer-specific biomarkers.Over the past several decades,enormous efforts have been made to screen and characterize useful cancer-specific biomarkers.Some important molecules such as CEA,AFP and PSA are identified as cancer biomarkers and commonly applied in clinical practice.Unfortunately,most currently available cancer biomarkers are not satisfactory because of limited specificity and/or sensitivity, stressing the need to discover clinically valuable biomarkers.
In a post-genome era,proteomic approaches have been introduced to seek novel cancer biomarkers.Nevertheless,the conventional tissue proteomics approaches have been less striking for biomarker discovery.The reason lies in the fact that proteins identified from tissues are not necessarily detectable in serum or plasma.It is conceivable that a biomarker to be useful in cancer screening and monitoring should be measurable in body fluid samples.Accordingly,mining cancer biomarkers from blood proteome is of particular interest.However,the prospects of blood proteomics are challenged by the fact that blood is a very complex body fluid,comprising an enormous diversity of proteins with a large dynamic range.The abundant blood proteins may mask the less abundant proteins,which are usually potential biomarkers.Several procedures have been made to remove these more abundant proteins before proteomic analysis.Nevertheless,these methods may sacrifice other proteins by nonspecific binding and thus lower the screen efficiency.Above-mentioned major limitations of blood proteomics emphasize the need to seek other approaches for cancer biomarker discovery.More recently,there has been an increasing interest in a newly emerging approach-cancer secretome analysis,which is a promising tool for the identification of cancer biomarkers.
The term "secretome" was first proposed by Tjalsma et al.in a genome-based global survey on secreted proteins of Bacillus subtilis.In a broader sense,the secretome harbors proteins released by a cell,tissue or organism through various mechanisms including classical secretion,nonclassical secretion,and secretion through the release of exosomes.It is known that cancer cells interact with their microenvironment by secreting a variety of proteins,including growth factors,extracellular matrix-degrading proteinases,cell motility factors and immunoregulatory cytokines or other bioactive molecules.These cancer-secreted proteins are essential in the processes of differentiation,invasion,and metastasis.More importantly,these cancer secreted proteins or their fragments always enter body fluids such as blood or urine and can be measured via non-invasive assays.Thus,cancer secretome may reflect a broad variety of pathological conditions and represents a more reliable source of biomarkers.
To date,only a minority of studies has analyzed cancer secretomes,however,the results regarding biomarker discovery are exciting.Of note,no differential secretome analysis on CRC metastasis has been reported.In this study we used a liquid chromatography-tandem mass spectrometry(LC-MS/MS) based label-free quantitative shotgun proteomics approach to investigate the secretomes of two human CRC cell lines(SW480 and SW620),in order to seek novel metastasis-associated serum biomarkers of CRC.
First,we used SW480 and SW620 as our model system because this pair of CRC cell lines derived from the same individual but with different metastatic potentials.The secretome samples collected from SW480 and SW620 were digested with trypsin and analyzed by a Finnigan~(TM)LTQ~(TM)MS coupled with a HPLC system.MS/MS spectra were automatically searched against the non-redundant International Protein Index(IPI) human protein database(Version 3.26) using the TurboSEQUEST~(TM)program.The stringent protein identification criteria were based on Delta Cn(≥0.1) and cross-correlation scores(Xcorr,one charge≥1.9,two charges≥2.2,three charges≥3.75).BuildSummary,a in-house tool,was used to combine the peptide sequences into proteins and delete redundant proteins.Finally,a total of 910 nonredundant proteins based on the identification of two or more unique peptides were identified,which,to our knowledge,represents one of the largest protein profiles identified for CRC.
In SW480,588 proteins were identified.Of these,451 proteins were identified in all three replicates,showing a protein identification reproducibility of 77%.The similarly high protein identification reproducibility(74%) was found in SW620,in which 672 proteins were identified and 497 proteins were present in all three replicates. Furthermore,searches against the sequence-reversed decoy IPI human databases using the same search parameters yielded a FDR of 3.82%at the peptide level.The high reproducibility and low FDR highlights the fidelity of LC-MS/MS analysis.
Label-free quantitative comparison between the two cell lines was performed by DeCyder~(TM)MS Differential Analysis Software(Version 1.0).Peptide detection, background subtraction and quantitation were performed on the full scan precursor mass spectra in fully automatic mode.Collectively,145 proteins displayed more than 1.5-fold quantitative alterations(t-test,P<0.01) in the secretomes of SW620 versus SW480.Among the 145 proteins,75 proteins were up-regulated,whereas 70 proteins were down-regulated in SW620 compared with SW480 cells.To date,this is one of the largest qualitative proteome studies for CRC.For the 145 proteins,the number of peptides used for quantitation from each protein varied between 1 and 10.Among these, 13 proteins were comparatively quantified on the basis of change levels of three or more peptides that varied similarly.From these data it is possible to evaluate the confidence of the quantitative approach.The average coefficient of variation(CV) of the fold changes for peptides from these 13 proteins was 21%(range 3.2-48.7%),yielding a reasonable reproducibility of the quantitative data.
Subsequently,overall features for the differential secreted proteins were analyzed by various bioinformatics analytic tools.First,cellular localization of identified proteins was analyzed on the basis of Gene Ontology(GO) and Human Protein Reference Database(HPRD).The localization of 42%differential proteins was classified as extracellular and membrane.The ratio is a little higher than previous secretome studies (30%),which demonstrates the advantage of our enrichment method for secreted proteins.Additionally,70 proteins identified in the CM were assigned to intracellular organelles,cytoskeleton,nucleus and cytoplasm(49%).The identification of a large portion of intracellular proteins is due in part to nonspecific liberation of cytoplasmic proteins as a consequence of cell death.However,we believe that the presence of many putative intracelluar proteins in CM may not merely be caused by cell autolysis,but also due in part to the active release through nonclassical secretion pathway and exosomes. Using SecretomeP 2.0 and SignalP 3.0 software,we analyzed the amino acid sequences of differential secreted proteins.The nonclassical secreted protein identification criteria were based on SecP score > 0.5 and the absence of signal peptides.Finally,we successfully identified 23(33%) nonclassical secreted proteins from the 70 intracellular proteins.
Biological function classifications were performed with the tools on DAVID Bioinformatics Resources 2008,and pathway analysis was done by searching Kyoto Encyclopedia of Genes and Genome(KEGG) database.The top biological functions of differential proteins were cell differentiation followed by cytoskeleton organization, apoptosis,anatomical structure morphogenesis,cell proliferation,cell adhesion, response to external stimulus and cell motility.The top ranked pathways were those involved in regulation of actin cytoskeleton,ECM-receptor interaction and focal adhesion.These findings suggest that the differential secreted proteins identified in SW480 versus SW620 might be implicated in CRC initiation and progression,and include valuable biomarker candidates for CRC.
The relative expression level of proteins determined by DeCyder MS was verified by SYBR Green Q-PCR and Western blot analysis of 7 selected proteins,GDF15,TFF3, AGR2,LASP1,TGM2,LCN2 and IGFBP7.These 7 proteins were selected on the basis of a combination of parameters including level of differential expression in SW620 versus SW480,cellular localization,availability of commercial antibodies,and the potential relevancy with cancer progression.From Q-PCR results,we found that the relative expression level of 6 genes coincided with the results of DeCyder MS;only LASP1 showed reversed fold change between the two cell lines.The expressions of the 7 selected proteins in the CM and in the total cell lysates were assayed by Western blot as well.In the CM harvested from this pair of CRC cells,the changes in protein abundance noted in the Western blot analysis were considerably consistent with the results obtained by proteomic screens,which demonstrates the accuracy of label-free quantitation.For GDF15,TFF3,AGR2,TGM2 and LCN2,similar results were observed in the cell lysates,which suggested the dysregulated secretion of these proteins was likely associated with the altered protein expression between the two cell lines.Notably,the change levels of LASP1 noted in cell lysates were consistent with Q-PCR results,suggesting that the increased expression of this protein in SW620 CM might be the increased secretion.Unfortunately,we could not detect IGFBP7 in cell lysates,although it was clearly present in SW480 CM,indicating rapid and efficient secretion of this protein.
Among the 7 proteins confirmed by Q-PCR and Western blot analysis,we chose two up-regulated proteins in SW620 versus SW480,TFF3 and GDF15,for further estimation in terms of its clinical relevancy in cancer metastasis.Using immunohistochemical staining,we measured TFF3 and GDF15 expression in 38 cases of CRC with lymph node metastasis(LNM),in which each case contains both primary tumor and metastatic lymph node,and 31 cases of non-LNM CRC.Comparison of immunoreactive scores of non-LNM CRC,primary tumors of LNM CRC and matched metastatic lymph nodes demonstrated that TFF3 expression in LNM CRC was slightly up-regulated although not significantly different from that in non-LNM CRC;in contrast,metastatic lymph nodes displayed stronger immunoreactivity of TFF3 than matched primary tumors(P<0.0001).A strong immunoreactivity was detected for GDF15 in LNM CRC,which was significantly higher than that in non-LNM CRC(P < 0.0001).In addition,metastatic lymph nodes exhibited stronger GDF15 expression compared to matched primary tumors(P=0.041).This finding indicated that dysregulation of TFF3 or GDF15 expression in CRC tissues was highly associated with CRC metastasis.Furthermore,No appreciable correlation was demonstrated between TFF3 expression and patient's gender,age,tumor size,histological grade and clinical TNM stage.Notably,however,TFF3 expression was site-related,cancers localized in colon mostly exhibited stronger immunoreactivity than cancers in rectum(P=0.007). There was a significant increase in the imunostaining of GDF15 between TNM tumor stageⅠ/ⅡandⅢ/Ⅳ(P<0.0001).The level of GDF15 expression was not statistically associated with gender,age,tumor size and site.
As immunohistochemical analysis indicated the association between TFF3 and GDF15 dysregulation and CRC metastasis,we speculated they might be serum marker candidates for predicting CRC metastasis.To test this speculation,the levels of TFF3 and GDF15 in serum samples collected from CRC patients(n=144) and healthy controls (n=156) were measured by double-antibody sandwich ELISA system.The serum levels of both proteins were significantly higher in CRC patients than in healthy controls.For details,the mean levels for serum TFF3 and GDF15 in healthy control were 824±278 ng/ml,and 639±212 pg/ml,respectively,whereas in CRC patients serum samples,the corresponding mean were elevated to 1925±1637 ng/ml and 2030±1122 pg/ml, respectively.Then,for analysis purposes,the 144 cases of CRC serum samples were subdivided into non-LNM CRC(n=68) and LNM CRC(n=76) groups.Compared with non-LNM CRC group,LNM CRC group exhibited significantly increased serum levels of both TFF3(2690±1839 ng/ml versus 1071±727 ng/ml) and GDF15(2667±1067 pg/ml versus 1319±663 pg/ml).These findings indicated that the two proteins might be potential serum biomarkers for CRC.
To evaluate the diagnostic performance of serum TFF3 and GDF15,we constructed receiver operating characteristic(ROC) curve by plotting sensitivity versus specificity.The area under the ROC curve(AUC),a commonly used indicator for estimating the diagnostic efficacy of a potential biomarker,was subsequently calculated. For discriminating CRC from healthy controls,the AUC was determined to be 0.730 (95%confidence interval,0.670-0.791) for TFF3 and 0.897(95%confidence interval, 0.856-0.938) for GDF15,respectively.When a cutoff value of 1323 ng/ml was chosen for TFF3,the sensitivity and specificity for differentiating CRC with healthy controls were 53.5 and 97.4%,respectively.As well,using a cutoff value of 1144 pg/ml,GDF15 had a diagnostic sensitivity of 77.8%and specificity of 99.4%in detecting CRC. Notably,the combination of TFF3 and GDF15 showed a higher diagnostic capacity than either marker alone(AUC=0.926;95%confidence interval,0.890-0.961).The ROC curves of serum TFF3 and GDF15 for discerning LNM CRC versus non-LNM CRC were also constructed.The AUC for TFF3 was 0.822(95%confidence interval, 0.750-0.894),at a cutoff value of 1789 ng/ml,its sensitivity and specificity for predicting CRC metastasis reached to 73.7%and 91.2%,respectively.GDF15 had a similar AUC to that of TFF3(0.867,95%confidence interval,0.806-0.928).Setting a cutoff value of 1881 pg/ml,sensitivity and specificity for GDF15 in distinguishing LNM-CRC with non-LNM CRC were 82.9%and 82.4%,respectively.Nevertheless, combining serum TFF3 with GDF15 measurements did not improve the efficacy for predicting CRC metastasis(AUC 0.866,95%confidence interval,0.805-0.927).These results collectively indicated that TFF3 and GDF15 might be valuable serum biomarker for diagnosis and metastasis prediction of CRC.
Analysis of the cohort of 144 CRC patients was carried out to define the relationship between TFF3 and GDF15 serum levels and clinicopathologic features of CRC.The rises in TFF3 or GDF15 serum levels were significantly associated with higher histological grade,increasing clinical TNM stage and the presence of distant metastasis of CRC.Serum level of TFF3 and GDF15 was not statistically correlated with gender,age and site,although cancers localized in colon tended to have higher serum TFF3 level than cancers in rectum(2071±1629 versus 1753±1642 ng/ml, P=0.059).
In conclusion,our data demonstrated that:
Using label-free quantitative shotgun proteomics approach to investigate differential cancer secretomes is a feasible strategy to seek valuable serum biomarkers. TFF3 and GDF15 are associated with the carcinogenesis and metastasis of CRC,and might be clinically useful serum biomarkers for CRC detection and metastasis prediction.
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