【作者】 虞舒静；

【导师】 郑树；

【作者基本信息】 浙江大学 ， 肿瘤学， 2009， 博士

【摘要】 大肠癌是一种常见的恶性肿瘤,在我国及西方发达国家其发病率及死亡率均居恶性肿瘤谱前列,且发病率有逐年增高趋势。近年来,传统治疗手段不断发展,生物靶向治疗新药不断研发,但大肠癌的生存率仍改善不明显,五年生存率仅63.4%左右。影响大肠癌预后的因素,主要是局部复发和远处转移。在大肠癌远处转移事件中,肝脏是最主要的转移部位,肝转移也是大肠癌病人治疗失败的主要原因之一。如何临床预测及早期干预肝转移的发生发展,是一个亟待解决的问题。近年来Vogelstein等对大肠癌组织进行了DNA突变分析,发现其中突变较高的几个基因都属于PI3K这条信号传导通路,首次启示我们,肿瘤的基因改变是以通路为单位的,只有从通路出发,才能更科学、更系统、更准确的寻找到合适的标志物。而后在《SCIENCE》上,Vogelstein等又公布了其检测的大肠癌组织中38个组或通路共约140个基因的突变频率,这为我们后续研究大肠癌肿瘤标志物提供了选择的依据。研究目的:1.寻找大肠癌预后相关的标志物,为后续研究大肠癌转移提供线索,并作为潜在的干预靶点2.建立大肠癌预后相关标志物组合优化模型,预测病人预后,指导临床诊治3.明确大肠癌预后及转移相关标志物SPARCL1（Secreted protein acidic and rich incysteine like 1）和其相关分子OPN（Osteopontin）、SPARC（Secreted protein acidicand rich in cysteine）在大肠癌肝转移过程中的作用4.明确SPARCL1重组蛋白抑制大肠癌细胞的作用及机制,能否作为大肠癌抗转移治疗的候选靶点基于信号通路的大肠癌预后相关标志物的寻找基于通路水平,选择上述Vogelstein文中所列大肠癌中基因突变频率较高的3个基因（P53、APC、KRAS）及11条通路（cadherins,axonal guidance,skeletal muscledevelopment,cell-matrix interactions,attractive and repulsive receptors,cytoskeletalremodeling,FSH-beta signaling pathway,Synaptogenesis,GnRH signaling pathway,connective tissue degradation,response to DNA damage stimulus）作为基础,确定了与这些通路及肿瘤均有一定相关的14个标志物(P53、APC、P21^ras、E-cadherin、endothelin B receptor、Shp2、ADCY-2、SPARCL1、neuroligin1、hsp27、mmp-9、MAPK（ERK）、MSH2、Rho)作为研究对象;对156例大肠癌病人进行了三年以上的随访,并将其手术切除的大肠癌组织制备成组织芯片,应用免疫组化的方法研究上述标志物的表达水平,结合分析其与患者的预后生存情况及多个影响预后的临床病理特征之间的相关性。分析结果显示,与大肠癌不同生物学特征显著相关的标志物有所重复又略有不同:与生存情况显著相关的标志物是SPARCL1、Shp2、MSH2、E-cadherin、P53、ADCY-2、MAPK。这7个标志物中,SPARCL1、Shp2和MSH2与绝大多数临床病理特征及预后情况均呈显著相关,它们的高表达提示着病人较好的预后;SPARCL1是唯一与远处转移显著负相关的标志物;P53主要与TNM分期显著相关;E-cadherin和MAPK主要与术后复发转移显著相关。而其它标志物,如endothelin B receptor、APC和Rho,仅与分化程度或分期相关。大肠癌预后相关标志物的组合及优化将前一部分中SPARCL1、MSH2、P53、MAPK（ERK）、E-cadherin、ADCY-2、Shp2这7个与预后显著相关的标志物,用生物信息学（支持向量机）分析方法对预后标志物进行组合及优化。结果发现,SPARCL1和P53的组合是相对最佳的预后模型,其判别结果是独立于TNM分期的提示病人预后的因素（P＜0.001）,尤其在Ⅱ、Ⅲ期病人中具有比传统分期更强的判断预后作用。SPARCL1及相关蛋白在大肠癌肝转移组织中的表达及作用本课题组之前对大肠癌原发灶与肝转移灶组织的基因表达谱芯片数据,应用整合基因染色体区带的生物信息学分析方法,得到差异最显著的染色体区带4q22,其中高表达的主要贡献基因为骨桥蛋白（Osteopontin,OPN）,低表达的主要贡献基因为SPARCL1（Secreted protein acidic and rich in cysteine like 1）。关于骨桥蛋白基因,目前已有较多报道与大肠癌的肝转移关系密切。而SPARCL1的功能目前尚未完全明确。前一部分研究也发现,SPARCL1与大肠癌远处转移呈显著负相关,提示其可能在肝转移过程中发挥重要的作用。首先用免疫组化和实时荧光定量PCR方法检测了SPARCL1、OPN和SPARC在非转移性大肠癌、肝转移性大肠癌原发灶及配对的肝转移灶组织中的表达情况。免疫组化的检测发现,SPARCL1的表达在非转移性大肠癌明显高于肝转移性大肠癌原发灶组织（P＜0.05）,而在大肠癌原发灶及其配对的肝转移灶组织间无显著性差异;OPN的表达在肝转移灶明显高于其配对的大肠癌原发灶组织（P＜0.05）,而在非转移性大肠癌及肝转移性大肠癌原发灶组织间无显著性差异;SPARC的表达在非转移性大肠癌明显低于肝转移性大肠癌原发灶组织（P＜0.05）,而在肝转移灶明显低于其配对的大肠癌原发灶组织（P＜0.05）。经实时荧光定量PCR验证,上述SPARCL1的差异在mRNA水平亦具有显著性意义,但SPARC、OPN的mRNA表达在各组间的差异尚不具显著性意义。另外,应用ELISA的方法检测了大肠癌病人血清中OPN、SPARC的浓度,发现血清中OPN和SPARC的含量在伴肝转移的大肠癌病人中均略高于不伴转移的病人,但其差异均无显著性意义。SPARCL1重组蛋白体外干预实验首先,使用western blot和RT-PCR方法证实三种大肠癌细胞系RKO、SW480、SW620中无SPARCL1蛋白及mRNA的表达。然后,将特定浓度的SPARCL1重组蛋白加入细胞培养液中,用MTT法及细胞划痕实验分别检测SPARCL1重组蛋白对这三种大肠癌细胞的增殖和迁移能力的影响。结果显示,SPARCL1重组蛋白对三种大肠癌细胞增殖能力无显著性改变,但RKO细胞的迁移能力明显减弱（P＜0.05）,而这种抑制作用在SW480、SW620中未能观察到。研究结论:1.基于肿瘤信号通路寻找标志物的思路具有科学性、系统性,且切实可行。2.从通路出发,发现SPARCL1、MSH2、P53、MAPK（ERK）、E-cadherin、ADCY-2、Shp2是与大肠癌预后及多种临床病理特征显著相关的肿瘤标志物。3.生物信息学方法分析证实,SPARCL1和P53的组合模型判别结果是独立于分期的判断病人预后的因素,而且在Ⅱ、Ⅲ期病人中具有比传统分期更强的判断预后作用。4.在组织中,SPARCL1的表达差异主要在伴/不伴肝转移的大肠癌原发灶组织中,提示SPARCL1表达的降低可能是发生在大肠癌肝转移过程中的早期事件,可以作为早期预测大肠癌肝转移的标志物。5.SPARCL1、OPN和SPARC在大肠癌原发灶及肝转移灶组织中的表达水平各异,提示这三者在大肠癌肝转移过程中发挥着不同的作用。6.在体外实验中,观察到SPARCL1重组蛋白具有抑制大肠癌细胞迁移的能力,但对大肠癌细胞的增殖能力抑制不明显,提示SPARCL1是大肠癌的候选抗转移靶点。更多还原

【Abstract】 Colorectal carcinoma is one of the most common cancers in the world.The incidence and mortality of colorectal cancer are in the forefront of all cancers in both China and western developed countries.In recent years,tradional therapies have developed quickly,and many new biological targeted drugs in the treatment of carcinoma have been exploited,however,the survival of colorectal carcinoma patients has been improved only a little,with a 5-year survival of 63.4%.Local recurrence and distant metastasis are major factors related with the poor prognosis of colorectal cancer patients.In distant metastasis of colorectal carcinoma,liver metastasis is the most common events and was also the most common deaths of these patients.How to predict and intervene liver metastasis at an early stage is an urgent question.In recent years,Vogelstein et al analysed DNA mutations of colorectal carcinoma, and found that several highly mutated genes were all belonged to the PI3K signal transductional pathway.It reminded us that the genetic changes of tumors are based on pathways.Only by pathways can we find some meaningful tumor markers scientifically, systematically and exactly.Then in the journal of SCIENCE,Vogelstein et al listed the number of mutations of all 140 genes included in 38 groups or pathways,which provided the evidence for the ongoing research on tumor markers in colorectal carcinoma.Objective:1.Identifying prognosis related markers of colorectal carcinoma,to provide evidences for the ongoing research on metastasis of colorectal cancer and potential targets for therapeutic interventions.2.Establishing a prognostic model by combining and optimizing markers,to evaluate the prognosis for individual patients and to guide the diagnosis and treatment of colorectal cancers.3.Exploring the function of SPARCL1（Secreted protein acidic and rich in cysteine like 1） and related molecules OPN（Osteopontin）,SPARC（Secreted protein acidic and rich in cysteine） in liver metastasis of colorectal carcinoma.4.Exploring the function and mechanism of recombinant SPARCL1 protein on the inhibition of colorectal cancer cell lines and its use as a candidate target for anti-metastasis therapy.Search for prognostic markers based on signal pathways in colorectal carcinomaThree top mutated genes（APC,TP53,KRAS） and 11 groups based on signal pathways（cadherins,axonal guidance,skeletal muscle development,cell-matrix interactions,attractive and repulsive receptors,cytoskeletal remodeling,FSH-beta signaling pathway,synaptogenesis,GnRH signaling pathway,connective tissue degradation,response to DNA damage stimulus） listed in the article of Vogelstein were selected first 14 pathway related markers,also associated with cancer were chosen for this part of the present study.They were P53,APC,P21^ras,E-cadherin,endothelin B receptor,Shp2,ADCY-2,SPARCL1,neuroliginl,hsp27,mmp-9,MAPK（ERK1,2）, MSH2 and Rho.156 colorectal cancer patients were followed up for at least 3 years after surgery,their tissue samples were made into tissue arrays,and immunohistochemical analysis were performed to detect the expression of these 14 markers in the specimens of colorectal carcinoma,and their relationship with the survival and many clinical pathological parameters of the patients was analyzed.Finally 7 tumor markers were found significantly related with prognosis and many clinical pathological parameters of these patients.The results suggested that different biological features shared several significantly associated markers:Markers significantly related with survival were SPARCL1,Shp2,MSH2, E-cadherin,P53,ADCY-2 and MAPK.Among these seven markers,SPARCL1,Shp2 and MSH2 were noticeable ones associated with good prognosis and nearly all clinical pathological features of colorectal cancer patients;SPARCL1 was the only marker negatively related with distant metastasis;P53 was mainly related with TNM staging; E-cadherin and MAPK were mainly related with post-surgery recurrence and metastasis. However,other markers,such as endothelin B receptor,APC and Rho,were just related with differentiation or stages.Optimization of prognostic markers in colorectal carcinomaBioinformatical analysis,support vector machine,was then used to combine and optimize seven prognostic markers（SPARCL1,MSH2,P53,MAPK（ERK）,E-cadherin, ADCY-2,Shp2）.And the combination of P53 and SPARCL1 was proved to be relatively the best prognostic model.The prediction result of this model was a significantly prognostic factor independent from TNM staging（P＜0.001）,and even better than the traditional staging especially in patients of stageⅡandⅢ. Expression and function of SPARCL1 and its related molecules OPN and SPARC in the liver metastasis of colorectal carcinomaOur research group had analyzed the gene expression profiles data of the primary colorectal cancer and liver metastasis tissue by integrating with cytobands information, and confirmed that cytoband 4q22 was the greatest differential one.In this cytoband, osteopontin（OPN） was found to be the highest regulated dominant gene,and SPARCL1 was found to be the lowest regulated dominant gene.OPN has been indicated to be involved in liver metastasis of colorectal carcinoma in several studies.However,the function of SPARCL1 has not been clearly known.The results of the former part indicated that SPARCL1 was a significant tumor marker negatively related with distant metastasis of colorectal carcinoma,suggesting that SPARCL1 may probably play a very important role in the process of liver metastasis of colorectal carcinoma.First,the protein and mRNA expression of SPARCL1 and its related proteins OPN and SPARC was detected by immunohistochemistry and quantitative real- time polymerase chain reaction（QRT-PCR） in colorectal cancer specimens,including primary tumors with and without metastasis,and their corresponding liver metastases.In immunohistochemistry analysis,SPARCL1 expression level in colorectal tumor specimens without metastasis was significantly higher than in those with liver metastasis（P＜0.05）,but there was no significant difference in SPARCL1 expression between colorectal tumor specimens with liver metastasis and their corresponding liver metastases.SPARC expression level in colorectal tumor specimens with liver metastasis was significantly higher than in those without metastasis（P＜0.05）,and was also significantly higher than in their liver metastases counterparts（P＜0.05）.OPN expression level was significantly higher in liver metastases than in their corresponding primary colorectal tumor specimens（P＜0.05）.However,there was no significant difference in OPN expression between colorectal tumor specimens with and without liver metastasis.In quantitative real- time polymerase chain reaction（QRT-PCR） analysis,the differential expression of SPARCL1 described above was confirmed in mRNA level,too.However,no significant difference in mRNA expression of OPN and SPARC was detected in colorectal carcinoma tissue and liver metastases.Additionally,serum concentrations of OPN and SPARC were also detected by Enzyme-Linked Immunosorbnent Assay（ELISA） in colorectal cancer patients with and without liver metastasis.Serum concentrations of OPN and SPARC were both a bit higher in patients with liver metastasis and those without metastasis,however,these differences had no statistical significance.Recombinant SPARCL1 intervening experiments in colorectal carcinoma cells in vitroFirst,by western blot and RT-PCR,we found the protein and mRNA expression of SPARCL1 was under the level of detection in three colorectal cancer cell lines,RKO, SW480,SW620.Then recombinant SPARCL1 protein at a specific concentration was added to the culture liquid of these cells,and found that cell proliferation was not changed in all three cell lines by MTT analysis.However,reduced cell migration was found in RKO cells by scar healing assay,without such inhibition effects observed in two other cell lines,SW480 and SW620.Conclusion:1.Tumor signal pathway based search for tumor marker is scientific,systematic and feasible. 2.Seven tumor markers（SPARCL1、MSH2、P53、ERK、E-cadherin、ADCY-2、Shp2） were found significantly related with prognosis and many clinical pathological parameters of these patients.3.The combination of P53 and SPARCL1 was relatively the best prognostic model by bioinformatical analysis.The prediction result of the model was a significantly prognostic factor independent from TNM staging,and even better than the traditional staging especially in patients of stageⅡandⅢ.4.The differential expression of SPARCL1 in tissues was mainly between primary tumors with and without liver metastasis,and down regulation of SPARCL1 is probably an early event in liver metastasis of colorectal carcinoma.Therefore, SPARCL1 can be used as a marker for the prediction of liver metastasis at an early time.5.The expression level of SPARCL1,OPN and SPARC were different from each other in primary and liver metastases of colorectal cancers,suggested that these three molecules may play different parts in the process of liver metastasis.6.In vitro,recombinant SPARCL1 protein has been observed to inhibit the migration of colorectal cancer cells,rather than the inhibition of cell proliferation,suggested that SPARCL1 may be a candidate target for anti-metastasis study of colorectal carcinoma.更多还原