【作者】 欧娟娟；

【导师】 梁后杰；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2014， 博士

【摘要】 背景：肿瘤的发生发展受到基因的不稳定性及肿瘤微环境的双重调控。许多证据已经表明肿瘤微环境在恶性肿瘤的发生发展中发挥重要作用，但其作用机制仍不清楚。纤连蛋白是肿瘤微环境中重要的细胞外基质糖蛋白，存在不同的可变剪接变异体，其中EDA+FN特异性的高表达于多种恶性肿瘤，且与多种肿瘤的恶性表型相关，但EDA对肿瘤生物学特性的影响及其具体分子机制尚不清楚。代谢是机体生命活动的基本特征，正常细胞向恶性细胞的转化过程中发生多种生物学特性的改变，其中代谢重编程是其最显著的特征之一。近年来研究发现，某些癌基因的激活或抑癌基因的失活可调控代谢稳态发挥其促癌或抑癌作用，且某些代谢酶本身即可作为癌基因或抑癌基因参与肿瘤的发生发展。肿瘤有氧糖酵解是最早被发现和研究的肿瘤代谢重编程，即肿瘤细胞即使在有氧环境下，也主要以糖酵解而非产能效率更高的线粒体氧化磷酸化的方式供能（Warburg效应）。葡萄糖和脂肪酸代谢是能量的主要来源，既往关于肿瘤代谢的研究主要集中在糖代谢，近年来的研究发现，脂代谢异常也参与调控了多种肿瘤的恶性表型，但脂代谢在恶性肿瘤中的具体作用及机制仍知之甚少。结肠癌已被证实为糖酵解异常增强的肿瘤，而肿瘤组织中脂质的异常沉积也是其重要表型，但结肠癌中这种糖脂代谢紊乱的原因及机制尚不明确。研究证实，肿瘤微环境参与调控了肿瘤代谢的重编程，而肿瘤微环境成分EDA是否参与了对结肠癌代谢的调控仍有待于进一步探讨。本研究通过慢病毒载体建立EDA过表达及干扰表达的细胞模型，研究了EDA对结肠癌及鼻咽癌生物学特性的影响及分子机制。同时，我们发现结肠癌中EDA的过表达可显著抑制脂代谢相关基因CGI-58的表达活性，结肠癌中CGI-58呈现出特异性的表达缺失。既往研究表明，CGI-58是脂质分解的一个强有力的活化因子，其缺乏导致脂滴大量沉积，且CGI-58缺乏的小鼠对葡萄糖利用显著增加。因此我们猜测，结肠癌中CGI-58缺乏是导致肿瘤组织中脂质异常沉积和糖酵解增强的重要原因。为进一步明确CGI-58在结肠癌生物学特性中的作用，我们通过建立肠道CGI-58特异性敲除的ApcMin/+小鼠，CGI-58敲低和CGI-58过表达细胞，联合人结肠癌组织标本，鉴定了CGI-58缺失在诱导代谢重编程促进结肠癌发生发展中的作用。Wnt通路的激活与结肠癌发生发展密切相关，CGI-58缺失导致Wnt通路的靶基因c-MET表达显著上调，提示Wnt通路被激活。我们的研究表明，CGI-58敲低细胞及CGI-58敲除小鼠肠道肿瘤中激活Wnt通路的经典分子β-catenin的表达及核转位并无增加。因此，我们进一步探讨了CGI-58缺失经非β-catenin依赖途径诱导YAP/TAZ复合物核转位，激活Wnt信号通路的分子机制。为进一步探讨CGI-58在其他恶性肿瘤中是否同样具有抑癌基因功能，我们根据肿瘤突变基因数据库COSMIC的统计发现，CGI-58在神经来源肿瘤中也呈现显著缺失。黑色素瘤是神经来源的具有高度侵袭性的恶性肿瘤，已有研究报道脂代谢相关基因MAGL可显著促进黑色素瘤细胞的侵袭转移能力，为进一步明确CGI-58在其他恶性肿瘤中是否同样具有抑癌基因作用，通过慢病毒载体建立的CGI-58敲低及过表达黑色素瘤细胞模型，结合黑色素瘤临床肿瘤组织标本，我们继而研究了CGI-58在黑色素瘤分化及侵袭转移中的作用及机制。目的：1、阐明结肠癌微环境成分EDA对结肠癌生物学特性的影响及作用机制；2、验证EDA相关脂代谢基因CGI-58在结肠癌发生发展中作用及分子机制；3、验证CGI-58在恶性黑色素瘤侵袭转移中的作用及分子机制；4、揭示CGI-58作为抑癌基因的功能，进一步阐释肿瘤微环境与肿瘤代谢之间相互调控促进肿瘤恶性表型的作用及具体机制。材料与方法：1、肿瘤组织芯片免疫组化染色用于临床病理特征相关性分析；2、慢病毒载体建立EDA及CGI-58敲低及过表达的细胞模型用于研究EDA和CGI-58功能；3、 CGI-58肠道特异性敲除APCmin/+小鼠用于研究肠道肿瘤发生发展。结果：1、EDA在结肠癌及鼻咽癌中发挥促癌作用。EDA通过维持CD133+/CD44+结肠癌亚群细胞的特性促进结肠癌恶性表型，Integrin/FAK/ERK信号途径介导的Wnt/β-catenin通路激活是EDA促进结肠癌“干性”特征的重要分子机制。结肠癌细胞分泌型EDA通过与淋巴管内皮细胞整合素受体Integrinα9相互作用促进出芽诱导因子Prox1的表达和细胞微丝蛋白的极性排列，显著促进淋巴管内皮细胞的迁移、出芽和成管。血管内皮细胞分泌型EDA通过诱导结肠癌细胞发生上皮-间质转化促进结肠癌的侵袭转移，FAK/Src/Snail信号途径介导了血管内皮细胞分泌型EDA与结肠癌细胞表面integrinα9β1受体相互作用促进细胞EMT和侵袭转移的效应。此外，鼻咽癌组织中EDA促进细胞放射抵抗，FAK/Akt/JNK信号途径介导了EDA对鼻咽癌放疗敏感性的影响。此外，我们的研究还表明，EDA可显著调控脂代谢相关基因CGI-58的表达。利用基因芯片对EDA过表达和对照细胞进行比较筛查发现，EDA过表达细胞中葡萄糖转运蛋白GLUT1及多种糖酵解关键酶显著上调，而脂代谢相关基因比较基因组学鉴定蛋白58（CGI-58）表达明显收到抑制。Western blots检测进一步验证发现，EDA过表达细胞中CGI-58蛋白表达水平显著下降。2、CGI-58缺失启动代谢重编程诱导细胞恶变及发生上皮-间质转化，进而促进结肠癌发生发展。在结肠癌组织中CGI-58的表达显著低于癌旁组织和正常结肠组织，在结肠癌发生谱中，相比正常结肠粘膜组织，CGI-58的表达从炎症到不典型增生无显著变化，而在腺瘤中开始出现部分缺失，且缺失率与腺瘤癌变密切相关。通过将CGI-58肠道特异性敲除小鼠模型与结肠癌发生发展研究的经典模型APCmin/+小鼠（肠道多发腺瘤小鼠模型）进行杂交发现，肠道特异性CGI-58敲除的APCmin/+小鼠肠道肿瘤数量及体积较对照小鼠显著增加，且肠道腺瘤发生明显的恶性转化。体外实验研究进一步证实，正常结肠粘膜上皮细胞CGI-58表达显著高于结肠癌细胞，利用慢病毒载体敲低正常结肠粘膜上皮细胞中CGI-58表达后，细胞发生明显的上皮-间质转化（EMT），侵袭能力显著增强，且可在裸鼠体内成瘤，而在结肠癌细胞中回复CGI-58的表达可显著逆转其成瘤及侵袭能力。进一步研究表明，CGI-58缺失的肠道上皮细胞中酯酶水解活性下降，胞内出现大量中性脂质沉积，脂肪酸线粒体氧化明显受到抑制，同时细胞对葡萄糖的摄取明显增加，糖酵解关键酶表达上调，且糖酵解中间产物明显堆积。我们的研究还发现，CGI-58敲低的细胞内AMPK/p53活性受到抑制，而PI3K/Akt/mTOR信号通路被显著激活，细胞自噬水平降低。结肠癌临床标本研究进一步证实，CGI-58在结肠癌中的缺失与结肠癌的分期分级、转移及复发率呈显著正相关，与结肠癌患者的预后呈负相关。3. GI-58缺失诱导YAP/TAZ核转位激活Wnt信号通路。Wnt通路的激活与结肠癌发生发展密切相关，CGI-58缺失的结肠癌细胞Wnt通路靶基因c-MET表达显著上调，提示Wnt通路被显著激活。CGI-58缺失细胞中β-catenin表达略有下降，且核转位并无明显增强，提示CGI-58通过非β-catenin途径激活了Wnt通路。进一步研究发现，CGI-58敲低细胞中TAZ表达增强，YAP/TAZ复合物磷酸化显著降低，核转位明显，且其共激活转录调节因子TEAD活性显著上调，提示YAP/TAZ促转录活性增强。同时CGI-58肠道敲除小鼠模型研究结果也证实，CGI-58敲除小鼠肠道肿瘤中TAZ表达增强，且YAP/TAZ核转位明显强于对照组。免疫荧光及免疫共沉淀证实CGI-58与YAP/TAZ并无直接结合，而脂滴相关蛋白ADRP与YAP/TAZ之间有共定位及结合，提示CGI-58通过蛋白-蛋白相互作用促进YAP/TAZ复合物去磷酸化而发生核转位，进而通过增强共激活转录因子TEAD转录活性，上调Wnt靶基因c-MET表达，促进细胞上皮-间质转化和侵袭转移。4. CGI-58缺失诱导黑色素瘤细胞去分化而促进其侵袭转移能力CGI-58在恶性黑色素瘤肿瘤组织中的表达与黑色素瘤生物学特性显著相关。高侵袭黑色素瘤细胞中CGI-58表达显著低于低侵袭黑色素瘤细胞。在低侵袭力黑色素瘤细胞中敲低CGI-58表达显著促进其侵袭转移能力，同时细胞色素出现显著丢失。代谢产物分析发现CGI-58敲低的黑色素瘤细胞中溶血磷脂酸（LPA）含量升高，且LPA受体表达显著上调，利用LPA受体阻断剂可有效逆转CGI-58敲低引起的细胞侵袭转移及去分化。进一步研究发现，CGI-58敲低的黑色素瘤细胞中与细胞分化密切相关的Wnt5A表达显著上调，证实CGI-58引起的磷脂代谢重编程可诱导细胞去分化而促进黑色素细胞侵袭转移能力。结论：1、结肠癌微环境中，结肠癌细胞分泌型EDA和内皮细胞分泌型EDA均显著促进结肠癌的恶性表型。EDA/整合素信号途径在结肠癌发生发展中扮演重要角色，是结肠癌靶向治疗的特异性潜在靶标；EDA在鼻咽癌中的高表达显著促进了鼻咽癌放疗抵抗，因此阻断EDA相关信号途径是提高鼻咽癌放疗敏感性的有效手段；EDA可能通过显著调控代谢基因CGI-58的表达参与肿瘤代谢调控；2、CGI-58缺失导致的细胞供能方式从线粒体氧化转化为有氧糖酵解，可能是结肠癌糖酵解增强重要原因；结肠癌发生发展过程中CGI-58特异性的表达活性缺失启动的结肠癌代谢重编程激活下游PI3K/Akt/mTOR促癌信号途径，诱导细胞恶性转化及促进细胞侵袭转移，是结肠癌发生发展的重要分子机理。4、CGI-58缺失导致Wnt信号通路显著激活，但CGI-58缺失通过非经典β-catenin途径，而通过诱导YAP/TAZ复合物入核激活Wnt信号，进一步揭示了CGI-58缺失促进结肠癌发生发展的重要分子机制；5、CGI-58在黑色素瘤细胞中的缺失通过调控磷脂代谢诱导细胞去分化而促进其侵袭转移能力，揭示了脂代谢重编程促进黑色素瘤侵袭转移的作用和机制，首次揭示了脂代谢重编程在黑色素瘤细胞分化中的重要作用。6、通过研究结肠癌微环境成分纤维粘连蛋白EDA片段对结肠癌及鼻咽癌生物学特性的影响，以及EDA相关脂代谢相关基因CGI-58在结肠癌发生发展和黑色素瘤侵袭转移中的作用，进一步揭示了肿瘤微环境与代谢重编程在肿瘤发生发展中的重要作用。首次提出CGI-58是一具有酶活性的抑癌基因，其相关代谢途径及代谢产物为多种恶性肿瘤的诊断和治疗提供了新的潜在生物标记物和治疗靶标。更多还原

【Abstract】 Background：The etiology of cancer attributes to both genetic and environmental factors. Numerousevidence has been indicating the critical role of tumor microenvironment in regulatingcancer pathogenesis, but the underlying mechanism remains elusive. Cancer cells interactwith their extracellular matrix (ECM) during proliferation and migration. Fibronectin is animportant ECM glycoprotein in tumor microenvironment with several alternatively splicedvariants, including extra domain A (EDA). Previous studies have reported that theexpression levels of EDA are associated with the malignancy in several cancers, includingcolon cancer (CRC) as well as head and neck cancer, but the precise effect and molecularmechanism is still unknown.Metabolic reprogramming is a hallmark of malignant tumors. Many oncogenes andtumor suppressor genes have positioned at critical nodes of important metabolic networks,and their activities are hyper-responsive to metabolic perturbations. Additionally, inborn oracquired mutations of several metabolic enzymes are associated with development andprogression of several types of cancer, highlighting important roles of altered cellmetabolism in cancer etiology. A distinct metabolic phenotype possess during thedevelopment and progression of cancer, which is weakly associated with cancer subtype,but more strongly associated with tumor microenvironment-derived signature. Glycolyticbreakdown of glucose for ATP production in the presence of ample oxygen to fuelmitochondrial oxidative phosphorylation is a characteristic metabolic reprogramming inmultiple malignant tumors, and is known as “Warburg effect”. Although the Warburg effecthas been demonstrated to critically contribute to cancer pathogenesis, it is largely unknownhow cancer cells shift their energy metabolism to aerobic glycolysis.It has been revealed that tumor microenvironment is involved in regulating tumor metabolic reprogramming. Therefore, we presumed that EDA may play an important role infacilitating the development an progression of cancer via manipulating metabolicreprogramming. In this study, we established both full length form and shRNA formplasmid of EDA, transfected them into CRC cells, endothelial cells and nasopharyngealcarcinoma (NPC) cells, and the effect of EDA on the cancrt pathogenesis was investigated.In addition, we identified the lipid metabolism associated gene, CGI-58as a critical targetof EDA, and further used CGI-58intestine-specific knockout ApcMin/+mice,CGI-58-silenced and CGI-58-forced cells, and human CRC and melanoma tissues to revealthe novel role of CGI-58as a tumor suppressor in CRC and melanoma, and also as a switchof “Warburg effect”.Wnt signaling critically contributes to the development and progression of CRC, and isthe driven cause of the tumorigenesis of ApcMin/+mice. We further revealed the underlyingmechanism of the activation of Wnt signaling in the absence of CGI-58.To further investigate the casual role of CGI-58as a tumor suppressor, we checked thedatabase of Catalogue of Somatic Mutations in Cancer (COSMIC), and found that anotherstriking frequent loss is reported in tumors derived from nervous system. Melanoma is acommon type of nervous system-derived cancer displaying highly aggressive malignancy.Little is known about the mechanism underlying the highly aggressive behavior ofmelanoma. It was shown that another lipolytic enzyme, MAGL, promots invasion andmetastasis of multiple cancers, including melanoma. Due to the abundant expression ofCGI-58in normal skin and the striking phenotype of skin lesions in CGI-58deficientpatients, we then throw an appreciation in investigating the potential role of CGI-58inmelanoma pathogenesis.Objective：To investigate the molecular mechanism underlying the oncogenic role of EDA incancer pathogenesis; To reveal the novel tumor suppressor role of EDA associatedmetabolic gene CGI-58in the development and progression of CRC, and further identifythe casual role of CGI-58as a tumor suppressor in regulating the differentiation ofmelanoma.Materials and methods：1. Tissue chips consisting of human CRC and melanoma specimens and human normal colon tissues were used for statistical analysis of clinicopathological features;2. EDA or CGI-58manipulations in cells were established by lentiviral particles;3. Intestine-specific CGI-58knockout mice were generated by mating CGI-58-floxedmice with B6.SJL-Tg(Vil-cre)977Gum/J mice, followed by crossing CGI-58f/+/Vil-cremicewith CGI-58f/+mice to get homozygous CGI-58floxed mice with Vil-cre transgene,Results：1. EDA promotes the pathogenesis of CRC as well as NPCWe found that tumor tissue and serum EDA levels are substantially higher in advancedversus early stage human CRC, and EDA levels are significantly correlated with the clinicalfeatures of CRC patients. We further demonstrated that EDA sustains the properties ofCD133+/CD44+CRC cells via activating integrin/FAK/ERK pathway to driveWnt/β-catenin signaling. Furthermore, we assert that CRC cell-derived EDA significantlyfacilitates migration, sprouting and tubulogenesis of co-cultured lymphatic endothelial cells,indicating an important role of EDA in lymphangiogenesis of CRC. Intriguingly, we alsofound that endothelial cell-secreted-EDA promotes the metastatic capacity CRC cells viainducing an epithelial-mesenchymal-transition (EMT). In addition, our findingsdemonstrated that EDA levels are significantly correlated with the clinical features of NPCpatients. EDA substantially aggravates the radioresistance of NPC cells, and FAK/Akt/JNKsignaling was found to be a critical signaling mediating EDA function in regulating NPCradiosensitivity. Very impressively, we revealed that EDA suppresses a well establishedintracellular lipolytic activator, Comparative Gene Identification-58(CGI-58).2. CGI-58deficiency promotes CRC development and metastasis by inducingaerobic glycolysis.Extensive loss of CGI-58is a hallmark of CRCs and is significantly correlated with theclinical features of CRCs. Silencing of CGI-58in normal fibroblasts induces malignanttransformation. Intestine-specific knockout of CGI-58in ApcMin/+mice robustly increasestumorigenesis and malignant transformation of adenomatous polyps. In colon cancer cells,CGI-58deficiency induces epithelial-mesenchymal transition by suppressing theAMPKα-p53pathway, which is attributable to increased aerobic glycolysis. In humanCRCs, CGI-58expression falls substantially and correlates negatively with malignantfeatures. Our study is the first to link CGI-58to CRC pathogenesis. It suggests that cancer cells may develop aerobic glycolysis by suppressing CGI-58-mediated intracellularlipolysis.3. CGI-58deficiency activates Wnt signaling in CRC via inducing the nucleartranslocation of YAP/TAZ independent of β-catenin.The phenotype of CGI-58knockout in ApcMin/+mice indicates an activation of Wntsignaling, we then sought to explore the underlying molecular mechanism. Wnt/β-catenin isa classic signaling involved in CRC development and progression. We found that theexpression and the translocation of β-catenin showed no changes in CGI-58deficient cells,while the expression and the translocation of YAP/TAZ were substantially increased inCGI-58deficient cells. Furthermore, the protein-protein interaction between CGI-58/ADRPand YAP/TAZ was demonstrated to mediate the activation of Wnt signaling in CGI-58deficient CRC cells.4. Melanoma silences CGI-58to promote invasion capacity via inducing celldedifferentiationThe expression level of CGI-58is significantly correlated with the clinical features ofmelanoma. Aggressive melanoma cells showed substantially lower expression of CGI-58relative to non-aggressive melanoma cells. CGI-58deficiency induced reprogramming oflysophophatidic acid metabolism and the activation of Wnt5A signaling mediate thededifferentiation of melanoma cells, and therefore increased their metastatic capacity.Conclusions：1. Both CRC cell derived-EDA and endothelial cell derived-EDA significantlypromote the pathogenesis of CRC. EDA/integrin signaling are substantially attributable tothe malignancy of CRC, and may be potential specific targets in CRC therapy. EDA alsostrongly aggravates the radiosensitivity of NPC cells, and FAK/Akt/JNK signaling wasfound to critically mediates EDA effect on NPC radiosensitivity. EDA substantiallysuppresses the expression of a well-established cellular lipolytic activator, CGI-582. CGI-58deficiency robustly induces tumorigenesis and malignant transformation bypromoting aerobic glycolysis, highlighting a key role of blockade of cellular fat catabolismin driving cancer development and progression, and revealing the switch role of CGI-58inWarburg effect. The strong negative correlation between CGI-58expression levels andmalignant features of human colorectal carcinomas suggest that CGI-58and the associated metabolites may serve as biomarkers for colorectal cancer metastasis and prognosis.3.CGI-58deficiency induced Wnt activation is YAP/TAZ translocation dependentinstead of β-catenin dependent. Our findings showed a novel correlation between lipolyticgene and Wnt signaling.4. CGI-58plays a casual tumor suppressor role in melanoma. CGI-58deficiencyinduces dedifferentiation of melanoma cells via triggering LPA metabolic reprogramming toactivate Wnt5A signaling, and critically contributes to the invasion and metastasis capacityof melanoma.5.Collectively our results establish a regulatory network between tumormicrocnviroment and tumor metabolism to further explain the oncogenic mechanismunderlying the development and progression of cancer.更多还原