mTOR经HIF-1α调控脑胶质瘤血管生成拟态的机制研究
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摘要
脑胶质瘤是最常见的颅内原发性恶性肿瘤,占成人所有恶性肿瘤的2%,具有发病率高、预后差和复发率高等特点。尽管目前临床上联合手术切除病灶、术后放化疗,甚至免疫治疗和血管靶向治疗等手段,在恶性脑胶质瘤的治疗方面已取得较大的进展,但现有治疗措施尚不能很好地控制恶性肿瘤的发展,亟需对其进行深入研究,寻找新的治疗靶点,从而延长患者生命直至完全治愈。因此,寻找脑胶质瘤诊断和治疗的新靶点,可为其临床诊治提供理论基础,具有非常重要的意义。
恶性肿瘤的存活和生长离不开充足有效血液供应。长期以来,人们一直认为肿瘤血管重要来源于两种途径：血管发生(angiogenesis)和血管生成(vasculogenesis)。直到血管生成拟态(vasculogenic mimicry, VM)概念的提出,人们才逐渐意识到肿瘤细胞尚有其他血供形式,这种血供不需要内皮细胞的参与,但其内可含有红细胞和血浆,并且还可以与常规的内皮血管相沟通,构成网络状的血液输送循环。已经有越来越多的的研究表明,不同来源的恶性肿瘤组织中均有血管生成拟态的存在,包括脑胶质瘤中的髓母细胞瘤和胶质母细胞瘤。Maniotis等在首次定义血管生成拟态的同时,探讨了血管生成拟态与恶性黑色素瘤患者预后之间的关系,结果表明血管生成拟态与肿瘤转移及患者死亡存在很强的关联,Kaplan-Meier生存分析显示肿瘤中存在和缺乏血管生成拟态结构的患者之间的曲线明显分离。之后越来越多的研究证实,血管生成拟态的存在标志着患者预后不良。所以,对脑胶质瘤血管生成拟态的深入研究和有针对性的治疗具有重要的现实意义。
传统的血管靶向治疗恶性肿瘤旨在抑制内皮细胞,进而阻止肿瘤细胞的血液供应,达到遏制肿瘤生长的目的。近年来,血管靶向治疗已逐渐被人们所接受。抗血管生成药物阿瓦斯汀(贝伐单抗)曾经成为一些肿瘤最流行的血管靶向治疗药物,其中就包括脑胶质瘤。然而进一步的研究表明该治疗方案未能很有效的治愈肿瘤,甚至会加速肿瘤转移并伴有明显的缺氧形成和血管生成拟态的生成。和常规内皮血管相比,血管生成拟态作为肿瘤的另一供血途径,为肿瘤提供有效的营养和代谢,甚至形成了抗化疗作用并促进了肿瘤的转移。为了防止内皮血管靶向治疗后导致缺血缺氧,更容易形成血管生成拟态并加速转移,有学者建议抗血管生成药物(如贝伐单抗)小剂量、多次给药效果更好。血管生成拟态的发现加深了人们对肿瘤微循环和转移机制的认识,所以有学者提出,正是因为内皮血管靶向治疗诱发了肿瘤内部的逃逸机制,即无需内皮细胞参与进而形成的血管生成拟态,这种情况下单纯抗内皮血管治疗可能无意义,应考虑联合抗血管生成拟态治疗。因此,血管靶向治疗肿瘤不仅仅针对内皮细胞,同时针对血管生成拟态可能效果更好,抑制血管生成拟态形成可能成为肿瘤血管靶向治疗的新方向。
很多实验室均对血管生成拟态形成的信号转导机制进行了探讨。到目前为止,已经在不同的肿瘤中证实了一些分子参与了血管生成拟态的形成,包括HIF-1α, VE-cadherin, EphA2, MMP-14, MMP-2和层黏连蛋白等。随着上述涉及血管生成拟态的相关分子被发现,经典的血管生成拟态信号传导通路模型逐渐形成。在这个模型中,缺氧被认为是血管生成拟态形成的“启动开关(switch)",能够直接调控EphA2的基因表达(通过HIF-1α)或间接调节VE-cadherin的表达(通过激活中介蛋白),然后推动了其余的信号级联反应。在血管生成拟态信号通路的最后阶段,MMP-14的表达和活化能够激活MMP-2, MMP-2再与活化的MMP-14结合,促进层黏连蛋白分解,层黏连蛋白分解的片段释放进入细胞外微环境中收缩基质形成所谓的管道,即导致血管生成拟态的形成,同时也增加肿瘤细胞的迁移和侵袭能力。该经典通路虽然能够较好的阐释血管生成拟态的形成过程,但是仍有部分机制还不清楚,尤其是肿瘤细胞是如何感受细胞外微环境的缺血缺氧进而向细胞内传递,以及通路中尚有一些分子如何向下传导信号等一直未能明确。
肿瘤细胞的恶性增殖是肿瘤最主要的特征,不断增多的肿瘤细胞导致局部耗氧量增加,易造成肿瘤内部缺氧微环境的形成。这在恶性程度高的实体肿瘤中表现的尤为突出。已证实缺氧能够诱导肝癌、Ewing肉瘤、胶质母细胞瘤以及恶性黑色素瘤中血管生成拟态的形成。缺氧诱导因子-1(hypoxia inducible factor-1, HIF-1)是存在于哺乳动物和人体内一种转录因子,其表达和稳定受氧分压调节。它以异二聚体形式存在,由一个a和β亚单位组成。其中,HIF-lα蛋白常氧状态下极不稳定,在细胞内表达量维持在较低水平,易被蛋白酶水解,但能在缺氧条件下保持稳定,是组织缺氧的内在标志。具体到血管生成拟态,当氧分压较低时,HIF-1α进入细胞核内,在效应基因的启动子和增强子部位与缺氧反应元件(hypoxia response elements, HRE)结合,进而直接调节VEGF-A、 VEGFR、EphA2等基因的表达或者间接调控VE-cadherin和TF表达(通过激活中介蛋白)。由此可见,缺氧能够诱导血管生成拟态形成和/或血管生成拟态相关基因的表达,那么就不难想象为什么使用抗血管生成药物治疗可能促进肿瘤的可塑性和转移进展。
除了上述机制以外,近年来还发现了多种与血管生成拟态形成有关的调控因子。研究发现,组织因子途径抑制因子-2(tissue factor pathway inhibitor-2, TFPI-2)的相关基因表达水平在高侵袭性黑色素瘤细胞中可明显增高,同时,将低度侵袭性黑色素瘤细胞培养在含有重组TFPI-2的三维基质中,低侵袭性肿瘤细胞能够转换为高度侵袭性细胞并呈现出内皮细胞的表型,产生一些血管样通道；另外,环氧合酶-2(cyclooxygenase-2, COX-2)参与调节了乳腺癌血管生成拟态的形成。这些研究证实,肿瘤血管生成拟态形成的分子机制十分复杂,是一个多因子参与调控、多路径发展的过程。
对血管生成拟态形成机制进行探讨的同时,有研究者也探索了一些有针对性的治疗措施。目前针对血管生成拟态的抗肿瘤治疗方案主要是通过抑制相关分子,然而,仅仅阻断下游因子(如VE-cadherin, EphA2等)会造成肿瘤细胞内部进一步的缺氧,从而更有利于血管生成拟态的形成。所以对于血管生成拟态形成的起始因素HIF-1α的阻断就显得尤为重要。
TOR基因于1991年在酵母菌中作为雷帕霉素(rapamycin)的靶蛋白基因被发现,之后人们在哺乳动物细胞内同样发现了类似结构的哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)。mTOR是一种分子量为289kDa的丝氨酸-苏氨酸激酶,是调控蛋白翻译起始的一个中心分子,在包括细胞的生长、增殖、分化和凋亡等许多生理过程中起着重要的调控作用,并可能成为脑胶质瘤治疗的一个靶点。
研究表明,mTOR；号的过度激活在脑胶质瘤的发生发展中起了非常重要的作用。mTOR信号途径是最近新出现的细胞内重要信号途径,这一途径在进化上高度保守,主要通过控制蛋白合成来调节细胞生长。在mTOR信号通路中,各种细胞信号包括促有丝分裂原、生长因子、细胞能量水平甚至缺氧均能对mTOR信号通路产生影响。已经有研究证实,mTOR与肿瘤血管生成密切相关。同时,mTOR与血管生成拟态形成信号通路中多种分子之间的关系也很密切,尤其是参与缺氧诱导因子(hypoxia-inducible factor, HIF)信号通路,在缺氧的作用下可促进HIF-1α基因表达VEGF-A、VEGFR-1、EphA2等；最为重要的是,有学者使用mTOR抑制剂rapamycin抑制HIF信号时发现血管生成拟态形成同时受到抑制。由此可见,mTOR与血管生成拟态形成的信号通路中多种分子之间存在交叉(crosstalk),然而其与血管生成拟态之间的确切关系尚不明确。如果能进一步明确mTOR在脑胶质瘤血管生成拟态形成中所起作用、确定mTOR与血管生成拟态形成信号通路之间的相关性,既有利于确定血管生成拟态形成的新机制,又有望获得新的治疗靶点和方法,对于改善脑胶质瘤患者预后具有非常重要的意义。
第一章脑胶质瘤血管生成拟态的临床意义及其与mTOR表达的相关性分析
目的验证脑胶质瘤中血管生成拟态的存在,并初步探讨mTOR与脑胶质瘤中血管生成拟态的相关性。
方法收集南方医科大学珠江医院病理科127例脑胶质瘤临床病理组织,均来自2009至2012年在神经外科行手术治疗,术后病理诊断为胶质瘤的病例标本。手术后所有标本都按标准程序包埋于石蜡中保存。所有病理标本需进行再次切片行免疫组织化学染色,然后由两位对患者的临床资料不知情的病理学专家根据2007年世界卫生组织关于中枢神经系统肿瘤分类方法,再次验证脑胶质细胞瘤的诊断。病例具体的纳入标准为：1、患者行手术治疗,在手术前未经放疗和化疗；2、术后病理确诊为脑胶质细胞瘤；3、可以获得完整的病人信息。整理病人的临床资料包括性别、年龄、KPS评分标准、肿瘤大小和肿瘤分级等。将所有的病理切片进行CD34-PAS染色,根据结果将患者分为两组,分别是血管生成拟态阳性组和血管生成拟态阴性组,利用SPSS13.0统计软件对实验数据进行处理,采用χ2检验比较血管生成拟态阳性和阴性两组患者的临床病理特点包括性别、年龄、KPS评分、肿瘤大小、病理分级和mTOR表达水平等的差异,进一步分析血管生成拟态形成与mTOR是否有关联。统计学分析过程中,P<0.05被认为差异具有统计学意义；
结果127例脑胶质瘤中,共有34例发现有血管生成拟态结构,占全部的26.8%。和低级别脑胶质瘤相比,血管生成拟态结构在高级别脑胶质瘤中较为常见(Ⅲ级和Ⅳ级分别为41.2%和38.2%,而Ⅰ级和Ⅱ级分别为0.0%和20.6%),经Mann-Whitney非参数检验结果证实,血管生成拟态阳性和阴性两组中肿瘤分级有显著差异(Z=2.902, P=0.004),且血管生成拟态阳性组的病理级别较高。血管生成拟态结构较多的脑胶质瘤组织切片中mTOR表达明显高于血管生成拟态阴性组(-/+为3.0%,++为52.9%,而+++为44.1%)。经Mann-Whitney非参数检验结果表明血管生成拟态阳性和阴性两组中mTOI表达水平有显著差异(Z=7.748,P=0.006),且血管生成拟态阳性组的mTOR表达水平较高。统计结果表明血管生成拟态与脑胶质瘤中mTOR表达水平有关(P<0.05)。
结论本研究验证了血管生成拟态存在于脑胶质瘤中；提示血管生成拟态不仅与肿瘤分级呈正相关,也与脑胶质瘤中mTOI表达水平有关,而与患者的一般临床病理特征,如性别、年龄、KPS评分、肿瘤大小等无关。
第二章缺氧及mTOR特异性阻断剂rapamycin对恶性胶质瘤血管生成拟态形成的影响
目的建立脑胶质瘤细胞株U87-MG的三维培养模型,观察缺氧及mTOR特异性阻断剂rapamycin对其血管生成拟态的体外形成的影响。
方法用三维培养及管道计数实验研究经常氧及缺氧条件下U87-MG细胞体外血管生成拟态形成能力的差别；用不同浓度的mTOR特异性阻断剂rapamycin处理U87-MG细胞,观察其对血管生成拟态的体外形成的影响,利用Western Blotting检测常氧及缺氧条件下mTOR被抑制后HIF-1α的表达差异。
结果恶性胶质瘤细胞U87-MG能够在Matrigel基质胶上形成类似于内皮细胞的管状结构,但该结构在常氧条件下欠完整,部分管腔没有闭合。而在缺氧条件下能够形成更为完整的管腔结构。常氧条件下,mTOR特异性阻断剂rapamycin均能够抑制U87-MG在体外形成的血管生成拟态,并且随着rapamycin浓度的逐渐增加,恶性胶质瘤细胞U87-MG的血管生成拟态形成能力也逐渐减弱,且相互之间具有显著性差异(F=222.341,P=0.000)；同时,缺氧条件下也能够得到类似的结果(F=393.436,P=0.000)。Western Blotting结果显示,rapamycin既能够抑制常氧条件下也能够抑制缺氧条件下mTOR及HIF-1α的表达,通过对目的蛋白条带的灰度值进行统计分析,可以发现常氧条件下mTOR(F=198.179,P=0.000)和HIF-1α(F=60.991, P=0.000)以及缺氧条件下mTOR(F=365.728,P=0.000)和HIF-1α(F=60.478, P=0.000)相互之间均具有显著性差异。
结论通过体外三维培养,脑胶质瘤细胞株U87-MG能够在Matrigel基质胶上形成类似于HUVEC的圆孔状管腔结构,从而从体外实验的角度证实恶性胶质瘤细胞具有形成血管生成拟态的能力；缺氧能够促进脑胶质瘤细胞株U87-MG形成血管生成拟态,mTOR能够通过HIF-1α信号通路影响血管生成拟态的形成。
第三章mTOR参与恶性胶质瘤血管生成拟态的分子生物学机制研究
目的利用mTOR基因干扰技术研究mTOR siRNA对U87-MG血管生成拟态相关蛋白表达的影响。
方法设计并合成4条mTOR siRNA,从中筛选出一条干扰效率最高的mTOR siRNA;利用Western Blotting技术和划痕实验检测各组血管生成拟态相关基因的表达差异。
结果成功设计、合成并筛选出一条干扰效率最高的mTOR siRNA；利用mTOR siRNA同样能够抑制常氧和缺氧条件下的mTOR及HIF-1α的表达(P<0.01),通过对目的蛋白条带的灰度值进行统计分析,空白组及阴性对照组相关蛋白的表达之间无明显差异(P>0.05)。血管生成拟态相关信号通路中的分子(如MMP-14、MMP-2)的表达也同样能被干扰。划痕实验结果显示在缺氧条件相对于常氧条件下,脑胶质瘤细胞株U87-MG细胞迁移能力增强,而经mTOR siRNA干扰后在常氧和缺氧条件下脑胶质瘤细胞株U87-MG细胞迁移能力均明显减弱。
结论利用基因干扰技术沉默mTOR表达之后,无论是常氧还是缺氧条件下,HIF-1α及血管生成拟态相关基因(如MMP-14和MMP-2)的表达均被抑制；mTOR是血管生成拟态信号通路中的一个很重要的分子,其通过调控HIF-1α参与了胶质瘤血管生成拟态的形成过程。
Glioma is one of the most common intracranial primary malignant tumors, accounting for2%of all adult malignant tumors, with high incidence rate, high recurrence rate and poor prognosis. Despite the current clinical joint treatments including operation, postoperative radiotherapy and chemotherapy, and even immunotherapy and vascular targeting therapy, have been made great progress in the treatment of malignant glioma, the malignant tumor can not be controlled to stop developing. We need to research it on and seek new targets for treatment, so as to prolong life of patient until gliomas could be completely cured. Therefore, a new target for diagnosis and treatment of gliomas can provide a theoretical basis for the clinic with very important significance.
Malignant tumors cannot survive and grow without sufficient blood supply. For a long time, new blood vessels in tumor were believed to exclusively occur through the mechanism of angiogenesis and vasculogenesis. Until vasculogenic mimicry (VM) proposed, it is gradually realized that there is another form of blood supply without endothelial cells involved, but containing red blood cells and plasma, which could be an alternative therapeutic target for cancer. There have been an increasing number of studies have shown that vasculogenic mimicry exists in tumor tissues from different tumors. While Maniotis and his colleagues first defined vasculogenic mimicry, they explored the relationship between vasculogenic mimicry and tumor prognosis, and there was a strong statistical separation in survival between patients whose tumors lacked loops and networks and those whose tumors contained these structures by the Kaplan-Meier survival analysis. Then, growing researches confirm that the existence of vasculogenic mimicry marks a poor prognosis for the patients. Therefore, a depth study on the vasculogenic mimicry and the targeted therapy has important practical significance.
Traditional vascular targeting therapies for the treatment of malignant tumor are aimed to inhibit endothelial cells, which will prevent tumor blood supply and suppress the tumor growth. In recent years, vascular targeting therapies have been gradually accepted. The anti-angiogenesis drug, Avastin (bevacizumab) has become one of the most popular vascular targeting therapy drugs for some tumors, including glioma. However, studies show that the treatment is not effective in the treatment of tumors, and will accelerate to metastasis and the formation of vasculogenic mimicry with hypoxia. Compared with the normal endothelium, vasculogenic mimicry, as an alternative tumor blood supply, can supply nutrition and metabolism for tumors. It forms a resistance to chemotherapy and promotes tumor metastasis. In order to prevent the ischemia and hypoxia after endothelial vascular targeting therapy, which is prone to form vasculogenic mimicry and to accelerate the metastasis, some scholars suggest that therapy with small and multiple doses of anti-angiogenic drugs (such as bevacizumab) would get good effect. Vasculogenic mimicry has deepened the understanding of tumor microcirculation and transfer mechanism, therefore, some scholars considered that it was because of endothelial vascular targeting therapy, which induced a tumor escape mechanism without endothelial cells participation in the formation of vasculogenic mimicry, the simple anti-endothelial vascular targeting therapies may be meaningless, and the anti-vasculogenic mimicry should be considered. Therefore, inhibition of vasculogenic mimicry formation may be one of novel directions for the tumor vascular targeting therapies.
Many laboratories have been discussed the mechanism of signal transduction of mimicry formation. So far, a number of molecules involved in the formation of vasculogenic mimicry have been confirmed in different tumors, including HIF-1α, VE-cadherin, EphA2, MMP-14, MMP-2and Laminin. Along with the related molecules involved in vasculogenic mimicry above found, a classical model of vasculogenic mimicry signal pathway formed gradually. In this model, hypoxia is considered to be a switch of vasculogenic mimicry formation, which can directly regulate the gene expression of EphA2(through HIF-1α) or regulating the expression of VE-cadherin indirectly (through the activation of intermediary protein), then activated the rest of the signaling cascade. In the final stage of vasculogenic mimicry signaling pathway, expression and activation of MMP-14can activate MMP-2. MMP-2binding with the activated MMP-14promote the decomposition of laminin, resulting that the extracellular matrix contract and form the so-called lumens, which are the vasculogenic mimicry. Simultaneously it also increases the ability of migration and invasion of tumor cells. Although the classical pathway interprets the formation process of vasculogenic mimicry well, the specific mechanism is still unclear.
Hypoxia, either persistent or transient, is a hallmark of most tumors and has been shown to regulate pathways in the maintenance of the stem cell-like phenotype, cellular differentiation, invasion, metastasis, apoptotic resistance, genomic instability, angiogenesis, and VM. Molecularly, protein stabilization and nuclear localization of hypoxiainducible factor-la (HIF-1α)/HIF-2a transcription factors and binding to hypoxia response elements (HRE) in promoter and enhancers of effector genes occurs in response to low oxygen, oncogenes, or inactivated tumor suppressor genes. Hypoxia has been shown to induce VM in hepatocellular carcinoma, Ewing sarcoma, and melanoma. Moreover, hypoxia can induce a dedifferentiated phenotype in breast carcinoma. Pertinent to VM, hypoxia has been shown to either directly modulate VEGF-A, VEGFR, EphA2, Twist, Nodal, and COX-2gene expression (via HIF-1/HRE binding) or indirectly modulate VE-cadherin and TF expression (via activation of an intermediary protein). Hypoxia can also modulate the expression of Notch-responsive genes; specifically, hypoxia stabilizes the NICD protein, which interacts with HIF-1α and activates genes with Notch-responsive promoters, including Nodal. This noncanonical cross-talk between HIF-la and Notch-signaling pathways is thought to promote an undifferentiated cell state, further illuminating the possible etiology of tumor cell plasticity underlying VM. Based on the numerous studies showing hypoxia-induced VM and/or VM-associated genes, it is conceivable that therapeutic use of antiangiogenic agents may promote tumor plasticity and metastatic progression.
In addition to the above mechanism, in recent years, a variety of regulatory factors of vasculogenic mimicry formation are found. For example, the expression of tissue factor pathway inhibitor-2(TFPI-2) gene is significantly higher in invasive melanoma cells than the non invasive ones, while the low invasive melanoma cell cultured in a three-dimensional matrix containing the recombinant TFPI-2can be converted into highly invasive cells and exhibit phenotypes like endothelial cells, which form some vessel-like channel. In addition, cyclooxygenase-2(COX-2) is involved in the regulation of vasculogenic mimicry formation in breast cancer. These studies confirmed that the molecular mechanisms of tumor vasculogenic mimicry formation are very complex, which is a process with multi-factor involved and multi-path regulated.
In addition to discuss on the mechanism of vasculogenic mimicry formation, at the same time, researchers also explored some targeting therapies for the vasculogenic mimicry. Inhibition of moleculars involved in vasculogenic mimicry formation can reduce the blood supply of tumor, which provides more theoretical basis for the treatment of vasculogenic mimicry with a good clinical significance.
However, the molecular mechanism of tumor vasculogenic mimicry formation is very complex, multi path is a multi factor involved in the regulation of, development. The mechanism of formation of vasculogenic mimicry is not completely understood; for the treatment of vasculogenic mimicry has certain effect, still need to be further studied.
TOR is found as a target gene of rapamycin in yeast in1991. Then people also discovered this gene in mammalian cells and called it the mammalian target of rapamycin (mTOR). mTOR is a molecular weight of289kDa serine-threonine kinase, which is a central molecule regulating the initiation of protein translation and plays an important role in many physiological processes such as cell growth, proliferation, differentiation and apoptosis, and could become a target for glioma therapy.
Studies have shown that excessive activation of mTOR signaling plays a very important role in the development of gliomas. mTOR signaling pathways is an important signal pathway within cells found recently, which is highly conserved in evolution, and regulates cell growth mainly by controlling protein synthesis. In the mTOR signaling pathway, various cellar signals including the mitogen, growth factor, hypoxia or cellular energy level can affect the mTOR signaling pathway. Studies have confirmed that, mTOR is closely related to tumor angiogenesis. However, the relationship between mTOR and vasculogenic mimicry is unclear. In addition, mTOR is close related to multiple moleculars in the formation of vasculogenic mimicry, especially the hypoxia-inducible factor (HIF). Hypoxia can promote HIF-la to express VEGF-A, VEGFR-1, EphA2, etc. Most importantly, some scholars using mTOR inhibitor rapamycin to inhibite HIF1α discovered that vasculogenic mimicry was also inhibited. In summary, mTOR are closely related to the formation of vasculogenic mimicry, and there is a crosstalk between signal pathways in a variety of molecules involved in vasculogenic mimicry formation. If we can further clarify that the role of mTOR in in the formation of vasculogenic mimicry in glioma, and identify the correlation between mTOR and vasculogenic mimicry formation, it will be beneficial for identifying new mechanisms of vasculogenic mimicry formation and new therapeutic targets and methods, which has a very important significance in improving the prognosis of patients with glioma.
Chapter one:Clinical significance of vasculogenic mimicry and the correlation analysis between mTOR expression and vasculogenic mimicry in glioma
Objective To verify the presence of vasculogenic mimicry in glioma, and investigate the relationship between mTOR and vasculogenic mimicry in glioma.
Methods127cases of glioma were collected from Department of Pathology, Zhujiang Hospital, Southern Medical University between2009and2012. All specimens are preserved in paraffin embedded by standard procedures after surgery. And all pathological specimens are need to be re-sliced and stained by immunohistochemistry, then diagnosis by two pathologists according to the2007World Health Organization classification of tumors of the central nervous system without knowledge of the patient's clinical data. Cases inclusion criteria:1) Patients underwent surgery without radiation and chemotherapy before.2) Postoperative pathological diagnosis is glioma.3) Complete information of patient can be collected. Clinical data of patients including gender, age, KPS score, tumor size and tumor grading were sorted out. All slices would be stained by CD34-PAS, and according to the results, patients were divided into two groups, which are vasculogenic mimicry positive group and vasculogenic mimicry negative group. For experimental data processing, χ2test was carried out to compare the clinical and pathological features of patients in two groups, including gender, age, KPS score, tumor size, histological grade and level of expression of mTOR using SPSS13.0statistical software. In the statistical analysis process, P<0.05was considered as statistically significant difference.
Results In the total127cases of glioma, vasculogenic mimicry structures were found in34cases, accounting for26.8%. Compared to low-grade gliomas, vasculogenic mimicry structures are more iditified in high-grade gliomas (gradeⅢ and grade IV were33.3%and39.4%, while grade I and II were0.0%and15.6%)(χ2=9.051, P=0.029). In the glioma slices with more vasculogenic mimicry structures, mTOR expression was significantly higher than that of vasculogenic mimicry negative (-/+10.0%++21.7%, and+++was44.1%)(χ2=7.748, P=0.021). These results suggest that vasculogenic mimicry in gliomas is related to the level of mTOR expression (P<0.05).
Conclusion This study verified the presence of vasculogenic mimicry in gliomas and suggested that vasculogenic mimicry is not only positively correlated with tumor grade, but also with the level of mTOR expression in gliomas, and there is no correlation with patient's general clinicopathological features such as gender, age, KPS score, tumor size, etc.
Chapter two:The effect of hypoxia and mTOR-specific inhibitor rapamycin on the formation of vasculogenic mimicry of malignant glioma
Objective To establish dimensional culture model of U87malignant glioma (U87-MG), and observe the effect of hypoxia and the specific mTOR inhibitor rapamycin on the formation of vasculogenic mimicry in vitro.
Methods the ability of vasculogenic mimicry formation by U87-MG cells was studied under normoxia and hypoxia condition in vitro using three-dimensional culture and tube formation assay. Vasculogenic mimicry formation by U87-MG cells was observed with different concentrations of mTOR specific inhibitor rapamycin treatment in vitro. The expression of HIF-1α was studied after mTOR inhibition under normoxic and hypoxic conditions by technique of Western Blotting.
Results U87-MG cells are capable of forming a kind of tubular structures analogous to the endothelial cells on Matrigel. However, this structure is incomplete under normoxic condition, some lumens is not closed, whereas a more complete lumen structure formed under hypoxic condition. The mTOR-specific inhibitor rapamycin were able to decrease the structures of vasculogenic mimicry formed by U87-MG in vitro whether under normoxic or hypoxic condition. Furthermore, with rapamycin concentrations increasing, the ability of vasculogenic mimicry formed U87-MG cells was gradually weakened. Western Blotting showed that rapamycin can inhibit both mTOR and HIF-la expression under normoxic condition and hypoxic condition.
Conclusion Through three-dimensional culture in vitro, U87-MG cells can form tubular structure-like similar to the HUVEC on Matrigel, which confirmed that malignant glioma cells have the ability to form vasculogenic mimicry in vitro. Hypoxia promotes U87-MG to form vasculogenic mimicry. mTOR was involved in vasculogenic mimicry signaling pathways via HIF-1α.
Chapter three:Study on the molecular mechanisms of mTOR involved in vasculogenic mimicry of malignant glioma
Objective To study the effect of the vasculogenic mimicry related molecules expression in U87-MG by mTOR gene interference technology.
Methods Four mTOR siRNAs were designed and synthesized, and one of which knockdown maximally mTOR expression was chosen to screen the downstream signaling of VM. The differential expressions of vasculogenic mimicry-related molecules were detected by Western Blotting technique and Cell migration assay.
Results We successfully designed and synthesized four mTOR siRNA, and chose one which knockdown maximally mTOR expression to screen the downstream signaling of VM. When mTOR expression significantly was decreased by the siRNA, as expected, HIF-la expression was also significantly downregulated, either under normoxic or hypoxic condition. By western blotting, both MMP-14and MMP-2expressions were extremely lower in U87-MG cells transfected with mTOR siRNA than the control cells, even under hypoxia. MMP-2was associated with cell migration. Migration abilities of U87-MG cells increased after hypoxia for24h. However, this increase did not recur after siRNA interference.
Conclusion HIF-la and vasculogenic mimicry-related genes (such as MMP-14and MMP-2) expression were inhibited after mTOR gene interference, either under normoxic or hypoxic conditions. Through mediating HIF-la, mTOR is one of important molecules in the vasculogenic mimicry signaling pathway of glioma.
恶性肿瘤的存活和生长离不开充足有效血液供应。长期以来,人们一直认为肿瘤血管重要来源于两种途径：血管发生(angiogenesis)和血管生成(vasculogenesis)。直到血管生成拟态(vasculogenic mimicry, VM)概念的提出,人们才逐渐意识到肿瘤细胞尚有其他血供形式,这种血供不需要内皮细胞的参与,但其内可含有红细胞和血浆,并且还可以与常规的内皮血管相沟通,构成网络状的血液输送循环。已经有越来越多的的研究表明,不同来源的恶性肿瘤组织中均有血管生成拟态的存在,包括脑胶质瘤中的髓母细胞瘤和胶质母细胞瘤。Maniotis等在首次定义血管生成拟态的同时,探讨了血管生成拟态与恶性黑色素瘤患者预后之间的关系,结果表明血管生成拟态与肿瘤转移及患者死亡存在很强的关联,Kaplan-Meier生存分析显示肿瘤中存在和缺乏血管生成拟态结构的患者之间的曲线明显分离。之后越来越多的研究证实,血管生成拟态的存在标志着患者预后不良。所以,对脑胶质瘤血管生成拟态的深入研究和有针对性的治疗具有重要的现实意义。
传统的血管靶向治疗恶性肿瘤旨在抑制内皮细胞,进而阻止肿瘤细胞的血液供应,达到遏制肿瘤生长的目的。近年来,血管靶向治疗已逐渐被人们所接受。抗血管生成药物阿瓦斯汀(贝伐单抗)曾经成为一些肿瘤最流行的血管靶向治疗药物,其中就包括脑胶质瘤。然而进一步的研究表明该治疗方案未能很有效的治愈肿瘤,甚至会加速肿瘤转移并伴有明显的缺氧形成和血管生成拟态的生成。和常规内皮血管相比,血管生成拟态作为肿瘤的另一供血途径,为肿瘤提供有效的营养和代谢,甚至形成了抗化疗作用并促进了肿瘤的转移。为了防止内皮血管靶向治疗后导致缺血缺氧,更容易形成血管生成拟态并加速转移,有学者建议抗血管生成药物(如贝伐单抗)小剂量、多次给药效果更好。血管生成拟态的发现加深了人们对肿瘤微循环和转移机制的认识,所以有学者提出,正是因为内皮血管靶向治疗诱发了肿瘤内部的逃逸机制,即无需内皮细胞参与进而形成的血管生成拟态,这种情况下单纯抗内皮血管治疗可能无意义,应考虑联合抗血管生成拟态治疗。因此,血管靶向治疗肿瘤不仅仅针对内皮细胞,同时针对血管生成拟态可能效果更好,抑制血管生成拟态形成可能成为肿瘤血管靶向治疗的新方向。
很多实验室均对血管生成拟态形成的信号转导机制进行了探讨。到目前为止,已经在不同的肿瘤中证实了一些分子参与了血管生成拟态的形成,包括HIF-1α, VE-cadherin, EphA2, MMP-14, MMP-2和层黏连蛋白等。随着上述涉及血管生成拟态的相关分子被发现,经典的血管生成拟态信号传导通路模型逐渐形成。在这个模型中,缺氧被认为是血管生成拟态形成的“启动开关(switch)",能够直接调控EphA2的基因表达(通过HIF-1α)或间接调节VE-cadherin的表达(通过激活中介蛋白),然后推动了其余的信号级联反应。在血管生成拟态信号通路的最后阶段,MMP-14的表达和活化能够激活MMP-2, MMP-2再与活化的MMP-14结合,促进层黏连蛋白分解,层黏连蛋白分解的片段释放进入细胞外微环境中收缩基质形成所谓的管道,即导致血管生成拟态的形成,同时也增加肿瘤细胞的迁移和侵袭能力。该经典通路虽然能够较好的阐释血管生成拟态的形成过程,但是仍有部分机制还不清楚,尤其是肿瘤细胞是如何感受细胞外微环境的缺血缺氧进而向细胞内传递,以及通路中尚有一些分子如何向下传导信号等一直未能明确。
肿瘤细胞的恶性增殖是肿瘤最主要的特征,不断增多的肿瘤细胞导致局部耗氧量增加,易造成肿瘤内部缺氧微环境的形成。这在恶性程度高的实体肿瘤中表现的尤为突出。已证实缺氧能够诱导肝癌、Ewing肉瘤、胶质母细胞瘤以及恶性黑色素瘤中血管生成拟态的形成。缺氧诱导因子-1(hypoxia inducible factor-1, HIF-1)是存在于哺乳动物和人体内一种转录因子,其表达和稳定受氧分压调节。它以异二聚体形式存在,由一个a和β亚单位组成。其中,HIF-lα蛋白常氧状态下极不稳定,在细胞内表达量维持在较低水平,易被蛋白酶水解,但能在缺氧条件下保持稳定,是组织缺氧的内在标志。具体到血管生成拟态,当氧分压较低时,HIF-1α进入细胞核内,在效应基因的启动子和增强子部位与缺氧反应元件(hypoxia response elements, HRE)结合,进而直接调节VEGF-A、 VEGFR、EphA2等基因的表达或者间接调控VE-cadherin和TF表达(通过激活中介蛋白)。由此可见,缺氧能够诱导血管生成拟态形成和/或血管生成拟态相关基因的表达,那么就不难想象为什么使用抗血管生成药物治疗可能促进肿瘤的可塑性和转移进展。
除了上述机制以外,近年来还发现了多种与血管生成拟态形成有关的调控因子。研究发现,组织因子途径抑制因子-2(tissue factor pathway inhibitor-2, TFPI-2)的相关基因表达水平在高侵袭性黑色素瘤细胞中可明显增高,同时,将低度侵袭性黑色素瘤细胞培养在含有重组TFPI-2的三维基质中,低侵袭性肿瘤细胞能够转换为高度侵袭性细胞并呈现出内皮细胞的表型,产生一些血管样通道；另外,环氧合酶-2(cyclooxygenase-2, COX-2)参与调节了乳腺癌血管生成拟态的形成。这些研究证实,肿瘤血管生成拟态形成的分子机制十分复杂,是一个多因子参与调控、多路径发展的过程。
对血管生成拟态形成机制进行探讨的同时,有研究者也探索了一些有针对性的治疗措施。目前针对血管生成拟态的抗肿瘤治疗方案主要是通过抑制相关分子,然而,仅仅阻断下游因子(如VE-cadherin, EphA2等)会造成肿瘤细胞内部进一步的缺氧,从而更有利于血管生成拟态的形成。所以对于血管生成拟态形成的起始因素HIF-1α的阻断就显得尤为重要。
TOR基因于1991年在酵母菌中作为雷帕霉素(rapamycin)的靶蛋白基因被发现,之后人们在哺乳动物细胞内同样发现了类似结构的哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)。mTOR是一种分子量为289kDa的丝氨酸-苏氨酸激酶,是调控蛋白翻译起始的一个中心分子,在包括细胞的生长、增殖、分化和凋亡等许多生理过程中起着重要的调控作用,并可能成为脑胶质瘤治疗的一个靶点。
研究表明,mTOR；号的过度激活在脑胶质瘤的发生发展中起了非常重要的作用。mTOR信号途径是最近新出现的细胞内重要信号途径,这一途径在进化上高度保守,主要通过控制蛋白合成来调节细胞生长。在mTOR信号通路中,各种细胞信号包括促有丝分裂原、生长因子、细胞能量水平甚至缺氧均能对mTOR信号通路产生影响。已经有研究证实,mTOR与肿瘤血管生成密切相关。同时,mTOR与血管生成拟态形成信号通路中多种分子之间的关系也很密切,尤其是参与缺氧诱导因子(hypoxia-inducible factor, HIF)信号通路,在缺氧的作用下可促进HIF-1α基因表达VEGF-A、VEGFR-1、EphA2等；最为重要的是,有学者使用mTOR抑制剂rapamycin抑制HIF信号时发现血管生成拟态形成同时受到抑制。由此可见,mTOR与血管生成拟态形成的信号通路中多种分子之间存在交叉(crosstalk),然而其与血管生成拟态之间的确切关系尚不明确。如果能进一步明确mTOR在脑胶质瘤血管生成拟态形成中所起作用、确定mTOR与血管生成拟态形成信号通路之间的相关性,既有利于确定血管生成拟态形成的新机制,又有望获得新的治疗靶点和方法,对于改善脑胶质瘤患者预后具有非常重要的意义。
第一章脑胶质瘤血管生成拟态的临床意义及其与mTOR表达的相关性分析
目的验证脑胶质瘤中血管生成拟态的存在,并初步探讨mTOR与脑胶质瘤中血管生成拟态的相关性。
方法收集南方医科大学珠江医院病理科127例脑胶质瘤临床病理组织,均来自2009至2012年在神经外科行手术治疗,术后病理诊断为胶质瘤的病例标本。手术后所有标本都按标准程序包埋于石蜡中保存。所有病理标本需进行再次切片行免疫组织化学染色,然后由两位对患者的临床资料不知情的病理学专家根据2007年世界卫生组织关于中枢神经系统肿瘤分类方法,再次验证脑胶质细胞瘤的诊断。病例具体的纳入标准为：1、患者行手术治疗,在手术前未经放疗和化疗；2、术后病理确诊为脑胶质细胞瘤；3、可以获得完整的病人信息。整理病人的临床资料包括性别、年龄、KPS评分标准、肿瘤大小和肿瘤分级等。将所有的病理切片进行CD34-PAS染色,根据结果将患者分为两组,分别是血管生成拟态阳性组和血管生成拟态阴性组,利用SPSS13.0统计软件对实验数据进行处理,采用χ2检验比较血管生成拟态阳性和阴性两组患者的临床病理特点包括性别、年龄、KPS评分、肿瘤大小、病理分级和mTOR表达水平等的差异,进一步分析血管生成拟态形成与mTOR是否有关联。统计学分析过程中,P<0.05被认为差异具有统计学意义；
结果127例脑胶质瘤中,共有34例发现有血管生成拟态结构,占全部的26.8%。和低级别脑胶质瘤相比,血管生成拟态结构在高级别脑胶质瘤中较为常见(Ⅲ级和Ⅳ级分别为41.2%和38.2%,而Ⅰ级和Ⅱ级分别为0.0%和20.6%),经Mann-Whitney非参数检验结果证实,血管生成拟态阳性和阴性两组中肿瘤分级有显著差异(Z=2.902, P=0.004),且血管生成拟态阳性组的病理级别较高。血管生成拟态结构较多的脑胶质瘤组织切片中mTOR表达明显高于血管生成拟态阴性组(-/+为3.0%,++为52.9%,而+++为44.1%)。经Mann-Whitney非参数检验结果表明血管生成拟态阳性和阴性两组中mTOI表达水平有显著差异(Z=7.748,P=0.006),且血管生成拟态阳性组的mTOR表达水平较高。统计结果表明血管生成拟态与脑胶质瘤中mTOR表达水平有关(P<0.05)。
结论本研究验证了血管生成拟态存在于脑胶质瘤中；提示血管生成拟态不仅与肿瘤分级呈正相关,也与脑胶质瘤中mTOI表达水平有关,而与患者的一般临床病理特征,如性别、年龄、KPS评分、肿瘤大小等无关。
第二章缺氧及mTOR特异性阻断剂rapamycin对恶性胶质瘤血管生成拟态形成的影响
目的建立脑胶质瘤细胞株U87-MG的三维培养模型,观察缺氧及mTOR特异性阻断剂rapamycin对其血管生成拟态的体外形成的影响。
方法用三维培养及管道计数实验研究经常氧及缺氧条件下U87-MG细胞体外血管生成拟态形成能力的差别；用不同浓度的mTOR特异性阻断剂rapamycin处理U87-MG细胞,观察其对血管生成拟态的体外形成的影响,利用Western Blotting检测常氧及缺氧条件下mTOR被抑制后HIF-1α的表达差异。
结果恶性胶质瘤细胞U87-MG能够在Matrigel基质胶上形成类似于内皮细胞的管状结构,但该结构在常氧条件下欠完整,部分管腔没有闭合。而在缺氧条件下能够形成更为完整的管腔结构。常氧条件下,mTOR特异性阻断剂rapamycin均能够抑制U87-MG在体外形成的血管生成拟态,并且随着rapamycin浓度的逐渐增加,恶性胶质瘤细胞U87-MG的血管生成拟态形成能力也逐渐减弱,且相互之间具有显著性差异(F=222.341,P=0.000)；同时,缺氧条件下也能够得到类似的结果(F=393.436,P=0.000)。Western Blotting结果显示,rapamycin既能够抑制常氧条件下也能够抑制缺氧条件下mTOR及HIF-1α的表达,通过对目的蛋白条带的灰度值进行统计分析,可以发现常氧条件下mTOR(F=198.179,P=0.000)和HIF-1α(F=60.991, P=0.000)以及缺氧条件下mTOR(F=365.728,P=0.000)和HIF-1α(F=60.478, P=0.000)相互之间均具有显著性差异。
结论通过体外三维培养,脑胶质瘤细胞株U87-MG能够在Matrigel基质胶上形成类似于HUVEC的圆孔状管腔结构,从而从体外实验的角度证实恶性胶质瘤细胞具有形成血管生成拟态的能力；缺氧能够促进脑胶质瘤细胞株U87-MG形成血管生成拟态,mTOR能够通过HIF-1α信号通路影响血管生成拟态的形成。
第三章mTOR参与恶性胶质瘤血管生成拟态的分子生物学机制研究
目的利用mTOR基因干扰技术研究mTOR siRNA对U87-MG血管生成拟态相关蛋白表达的影响。
方法设计并合成4条mTOR siRNA,从中筛选出一条干扰效率最高的mTOR siRNA;利用Western Blotting技术和划痕实验检测各组血管生成拟态相关基因的表达差异。
结果成功设计、合成并筛选出一条干扰效率最高的mTOR siRNA；利用mTOR siRNA同样能够抑制常氧和缺氧条件下的mTOR及HIF-1α的表达(P<0.01),通过对目的蛋白条带的灰度值进行统计分析,空白组及阴性对照组相关蛋白的表达之间无明显差异(P>0.05)。血管生成拟态相关信号通路中的分子(如MMP-14、MMP-2)的表达也同样能被干扰。划痕实验结果显示在缺氧条件相对于常氧条件下,脑胶质瘤细胞株U87-MG细胞迁移能力增强,而经mTOR siRNA干扰后在常氧和缺氧条件下脑胶质瘤细胞株U87-MG细胞迁移能力均明显减弱。
结论利用基因干扰技术沉默mTOR表达之后,无论是常氧还是缺氧条件下,HIF-1α及血管生成拟态相关基因(如MMP-14和MMP-2)的表达均被抑制；mTOR是血管生成拟态信号通路中的一个很重要的分子,其通过调控HIF-1α参与了胶质瘤血管生成拟态的形成过程。
Glioma is one of the most common intracranial primary malignant tumors, accounting for2%of all adult malignant tumors, with high incidence rate, high recurrence rate and poor prognosis. Despite the current clinical joint treatments including operation, postoperative radiotherapy and chemotherapy, and even immunotherapy and vascular targeting therapy, have been made great progress in the treatment of malignant glioma, the malignant tumor can not be controlled to stop developing. We need to research it on and seek new targets for treatment, so as to prolong life of patient until gliomas could be completely cured. Therefore, a new target for diagnosis and treatment of gliomas can provide a theoretical basis for the clinic with very important significance.
Malignant tumors cannot survive and grow without sufficient blood supply. For a long time, new blood vessels in tumor were believed to exclusively occur through the mechanism of angiogenesis and vasculogenesis. Until vasculogenic mimicry (VM) proposed, it is gradually realized that there is another form of blood supply without endothelial cells involved, but containing red blood cells and plasma, which could be an alternative therapeutic target for cancer. There have been an increasing number of studies have shown that vasculogenic mimicry exists in tumor tissues from different tumors. While Maniotis and his colleagues first defined vasculogenic mimicry, they explored the relationship between vasculogenic mimicry and tumor prognosis, and there was a strong statistical separation in survival between patients whose tumors lacked loops and networks and those whose tumors contained these structures by the Kaplan-Meier survival analysis. Then, growing researches confirm that the existence of vasculogenic mimicry marks a poor prognosis for the patients. Therefore, a depth study on the vasculogenic mimicry and the targeted therapy has important practical significance.
Traditional vascular targeting therapies for the treatment of malignant tumor are aimed to inhibit endothelial cells, which will prevent tumor blood supply and suppress the tumor growth. In recent years, vascular targeting therapies have been gradually accepted. The anti-angiogenesis drug, Avastin (bevacizumab) has become one of the most popular vascular targeting therapy drugs for some tumors, including glioma. However, studies show that the treatment is not effective in the treatment of tumors, and will accelerate to metastasis and the formation of vasculogenic mimicry with hypoxia. Compared with the normal endothelium, vasculogenic mimicry, as an alternative tumor blood supply, can supply nutrition and metabolism for tumors. It forms a resistance to chemotherapy and promotes tumor metastasis. In order to prevent the ischemia and hypoxia after endothelial vascular targeting therapy, which is prone to form vasculogenic mimicry and to accelerate the metastasis, some scholars suggest that therapy with small and multiple doses of anti-angiogenic drugs (such as bevacizumab) would get good effect. Vasculogenic mimicry has deepened the understanding of tumor microcirculation and transfer mechanism, therefore, some scholars considered that it was because of endothelial vascular targeting therapy, which induced a tumor escape mechanism without endothelial cells participation in the formation of vasculogenic mimicry, the simple anti-endothelial vascular targeting therapies may be meaningless, and the anti-vasculogenic mimicry should be considered. Therefore, inhibition of vasculogenic mimicry formation may be one of novel directions for the tumor vascular targeting therapies.
Many laboratories have been discussed the mechanism of signal transduction of mimicry formation. So far, a number of molecules involved in the formation of vasculogenic mimicry have been confirmed in different tumors, including HIF-1α, VE-cadherin, EphA2, MMP-14, MMP-2and Laminin. Along with the related molecules involved in vasculogenic mimicry above found, a classical model of vasculogenic mimicry signal pathway formed gradually. In this model, hypoxia is considered to be a switch of vasculogenic mimicry formation, which can directly regulate the gene expression of EphA2(through HIF-1α) or regulating the expression of VE-cadherin indirectly (through the activation of intermediary protein), then activated the rest of the signaling cascade. In the final stage of vasculogenic mimicry signaling pathway, expression and activation of MMP-14can activate MMP-2. MMP-2binding with the activated MMP-14promote the decomposition of laminin, resulting that the extracellular matrix contract and form the so-called lumens, which are the vasculogenic mimicry. Simultaneously it also increases the ability of migration and invasion of tumor cells. Although the classical pathway interprets the formation process of vasculogenic mimicry well, the specific mechanism is still unclear.
Hypoxia, either persistent or transient, is a hallmark of most tumors and has been shown to regulate pathways in the maintenance of the stem cell-like phenotype, cellular differentiation, invasion, metastasis, apoptotic resistance, genomic instability, angiogenesis, and VM. Molecularly, protein stabilization and nuclear localization of hypoxiainducible factor-la (HIF-1α)/HIF-2a transcription factors and binding to hypoxia response elements (HRE) in promoter and enhancers of effector genes occurs in response to low oxygen, oncogenes, or inactivated tumor suppressor genes. Hypoxia has been shown to induce VM in hepatocellular carcinoma, Ewing sarcoma, and melanoma. Moreover, hypoxia can induce a dedifferentiated phenotype in breast carcinoma. Pertinent to VM, hypoxia has been shown to either directly modulate VEGF-A, VEGFR, EphA2, Twist, Nodal, and COX-2gene expression (via HIF-1/HRE binding) or indirectly modulate VE-cadherin and TF expression (via activation of an intermediary protein). Hypoxia can also modulate the expression of Notch-responsive genes; specifically, hypoxia stabilizes the NICD protein, which interacts with HIF-1α and activates genes with Notch-responsive promoters, including Nodal. This noncanonical cross-talk between HIF-la and Notch-signaling pathways is thought to promote an undifferentiated cell state, further illuminating the possible etiology of tumor cell plasticity underlying VM. Based on the numerous studies showing hypoxia-induced VM and/or VM-associated genes, it is conceivable that therapeutic use of antiangiogenic agents may promote tumor plasticity and metastatic progression.
In addition to the above mechanism, in recent years, a variety of regulatory factors of vasculogenic mimicry formation are found. For example, the expression of tissue factor pathway inhibitor-2(TFPI-2) gene is significantly higher in invasive melanoma cells than the non invasive ones, while the low invasive melanoma cell cultured in a three-dimensional matrix containing the recombinant TFPI-2can be converted into highly invasive cells and exhibit phenotypes like endothelial cells, which form some vessel-like channel. In addition, cyclooxygenase-2(COX-2) is involved in the regulation of vasculogenic mimicry formation in breast cancer. These studies confirmed that the molecular mechanisms of tumor vasculogenic mimicry formation are very complex, which is a process with multi-factor involved and multi-path regulated.
In addition to discuss on the mechanism of vasculogenic mimicry formation, at the same time, researchers also explored some targeting therapies for the vasculogenic mimicry. Inhibition of moleculars involved in vasculogenic mimicry formation can reduce the blood supply of tumor, which provides more theoretical basis for the treatment of vasculogenic mimicry with a good clinical significance.
However, the molecular mechanism of tumor vasculogenic mimicry formation is very complex, multi path is a multi factor involved in the regulation of, development. The mechanism of formation of vasculogenic mimicry is not completely understood; for the treatment of vasculogenic mimicry has certain effect, still need to be further studied.
TOR is found as a target gene of rapamycin in yeast in1991. Then people also discovered this gene in mammalian cells and called it the mammalian target of rapamycin (mTOR). mTOR is a molecular weight of289kDa serine-threonine kinase, which is a central molecule regulating the initiation of protein translation and plays an important role in many physiological processes such as cell growth, proliferation, differentiation and apoptosis, and could become a target for glioma therapy.
Studies have shown that excessive activation of mTOR signaling plays a very important role in the development of gliomas. mTOR signaling pathways is an important signal pathway within cells found recently, which is highly conserved in evolution, and regulates cell growth mainly by controlling protein synthesis. In the mTOR signaling pathway, various cellar signals including the mitogen, growth factor, hypoxia or cellular energy level can affect the mTOR signaling pathway. Studies have confirmed that, mTOR is closely related to tumor angiogenesis. However, the relationship between mTOR and vasculogenic mimicry is unclear. In addition, mTOR is close related to multiple moleculars in the formation of vasculogenic mimicry, especially the hypoxia-inducible factor (HIF). Hypoxia can promote HIF-la to express VEGF-A, VEGFR-1, EphA2, etc. Most importantly, some scholars using mTOR inhibitor rapamycin to inhibite HIF1α discovered that vasculogenic mimicry was also inhibited. In summary, mTOR are closely related to the formation of vasculogenic mimicry, and there is a crosstalk between signal pathways in a variety of molecules involved in vasculogenic mimicry formation. If we can further clarify that the role of mTOR in in the formation of vasculogenic mimicry in glioma, and identify the correlation between mTOR and vasculogenic mimicry formation, it will be beneficial for identifying new mechanisms of vasculogenic mimicry formation and new therapeutic targets and methods, which has a very important significance in improving the prognosis of patients with glioma.
Chapter one:Clinical significance of vasculogenic mimicry and the correlation analysis between mTOR expression and vasculogenic mimicry in glioma
Objective To verify the presence of vasculogenic mimicry in glioma, and investigate the relationship between mTOR and vasculogenic mimicry in glioma.
Methods127cases of glioma were collected from Department of Pathology, Zhujiang Hospital, Southern Medical University between2009and2012. All specimens are preserved in paraffin embedded by standard procedures after surgery. And all pathological specimens are need to be re-sliced and stained by immunohistochemistry, then diagnosis by two pathologists according to the2007World Health Organization classification of tumors of the central nervous system without knowledge of the patient's clinical data. Cases inclusion criteria:1) Patients underwent surgery without radiation and chemotherapy before.2) Postoperative pathological diagnosis is glioma.3) Complete information of patient can be collected. Clinical data of patients including gender, age, KPS score, tumor size and tumor grading were sorted out. All slices would be stained by CD34-PAS, and according to the results, patients were divided into two groups, which are vasculogenic mimicry positive group and vasculogenic mimicry negative group. For experimental data processing, χ2test was carried out to compare the clinical and pathological features of patients in two groups, including gender, age, KPS score, tumor size, histological grade and level of expression of mTOR using SPSS13.0statistical software. In the statistical analysis process, P<0.05was considered as statistically significant difference.
Results In the total127cases of glioma, vasculogenic mimicry structures were found in34cases, accounting for26.8%. Compared to low-grade gliomas, vasculogenic mimicry structures are more iditified in high-grade gliomas (gradeⅢ and grade IV were33.3%and39.4%, while grade I and II were0.0%and15.6%)(χ2=9.051, P=0.029). In the glioma slices with more vasculogenic mimicry structures, mTOR expression was significantly higher than that of vasculogenic mimicry negative (-/+10.0%++21.7%, and+++was44.1%)(χ2=7.748, P=0.021). These results suggest that vasculogenic mimicry in gliomas is related to the level of mTOR expression (P<0.05).
Conclusion This study verified the presence of vasculogenic mimicry in gliomas and suggested that vasculogenic mimicry is not only positively correlated with tumor grade, but also with the level of mTOR expression in gliomas, and there is no correlation with patient's general clinicopathological features such as gender, age, KPS score, tumor size, etc.
Chapter two:The effect of hypoxia and mTOR-specific inhibitor rapamycin on the formation of vasculogenic mimicry of malignant glioma
Objective To establish dimensional culture model of U87malignant glioma (U87-MG), and observe the effect of hypoxia and the specific mTOR inhibitor rapamycin on the formation of vasculogenic mimicry in vitro.
Methods the ability of vasculogenic mimicry formation by U87-MG cells was studied under normoxia and hypoxia condition in vitro using three-dimensional culture and tube formation assay. Vasculogenic mimicry formation by U87-MG cells was observed with different concentrations of mTOR specific inhibitor rapamycin treatment in vitro. The expression of HIF-1α was studied after mTOR inhibition under normoxic and hypoxic conditions by technique of Western Blotting.
Results U87-MG cells are capable of forming a kind of tubular structures analogous to the endothelial cells on Matrigel. However, this structure is incomplete under normoxic condition, some lumens is not closed, whereas a more complete lumen structure formed under hypoxic condition. The mTOR-specific inhibitor rapamycin were able to decrease the structures of vasculogenic mimicry formed by U87-MG in vitro whether under normoxic or hypoxic condition. Furthermore, with rapamycin concentrations increasing, the ability of vasculogenic mimicry formed U87-MG cells was gradually weakened. Western Blotting showed that rapamycin can inhibit both mTOR and HIF-la expression under normoxic condition and hypoxic condition.
Conclusion Through three-dimensional culture in vitro, U87-MG cells can form tubular structure-like similar to the HUVEC on Matrigel, which confirmed that malignant glioma cells have the ability to form vasculogenic mimicry in vitro. Hypoxia promotes U87-MG to form vasculogenic mimicry. mTOR was involved in vasculogenic mimicry signaling pathways via HIF-1α.
Chapter three:Study on the molecular mechanisms of mTOR involved in vasculogenic mimicry of malignant glioma
Objective To study the effect of the vasculogenic mimicry related molecules expression in U87-MG by mTOR gene interference technology.
Methods Four mTOR siRNAs were designed and synthesized, and one of which knockdown maximally mTOR expression was chosen to screen the downstream signaling of VM. The differential expressions of vasculogenic mimicry-related molecules were detected by Western Blotting technique and Cell migration assay.
Results We successfully designed and synthesized four mTOR siRNA, and chose one which knockdown maximally mTOR expression to screen the downstream signaling of VM. When mTOR expression significantly was decreased by the siRNA, as expected, HIF-la expression was also significantly downregulated, either under normoxic or hypoxic condition. By western blotting, both MMP-14and MMP-2expressions were extremely lower in U87-MG cells transfected with mTOR siRNA than the control cells, even under hypoxia. MMP-2was associated with cell migration. Migration abilities of U87-MG cells increased after hypoxia for24h. However, this increase did not recur after siRNA interference.
Conclusion HIF-la and vasculogenic mimicry-related genes (such as MMP-14and MMP-2) expression were inhibited after mTOR gene interference, either under normoxic or hypoxic conditions. Through mediating HIF-la, mTOR is one of important molecules in the vasculogenic mimicry signaling pathway of glioma.
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