摘要
研究背景和意义
结肠癌是最常见的消化道恶性肿瘤之一,发病率逐年上升且死亡率居高不下,严重危害着人民群众的健康,成为世界肿瘤研究领域的一大难题。结肠癌的侵袭转移是影响其预后的主要因素,表现出明显的组织器官特异性,肝、肺、淋巴结是其主要转移部位,其中肝转移最为常见,是导致结肠癌患者死亡的主要原因。传统观点认为结肠癌细胞经门静脉回流肝脏时运动变慢,容易在此停留和生长。但近年来越来越多的研究表明,这一现象并不能单纯由解剖结构和血液、淋巴引流来解释,应该还有其他因素参与,但这些因素及其具体机制目前还不清楚。
趋化因子是一类具有趋化作用的小分子量细胞因子,其相应受体为G蛋白偶联的7次跨膜受体,二者的相互作用可以介导多种免疫细胞(如淋巴细胞、树突状细胞等)循其浓度梯度做定向迁移,在组织发育、淋巴归巢、机体免疫、炎症反应等诸多过程中发挥重要作用。随着研究的不断深入,越来越多的证据表明趋化因子及其受体与肿瘤细胞的侵袭转移存在密切关系。趋化因子及其受体在肿瘤器官选择性转移中的作用机制类似于其介导的免疫细胞向淋巴组织的归巢过程,即趋化因子与其受体结合后,肿瘤细胞运动能力增强,并循趋化因子浓度梯度向趋化因子富集器官做定向运动,最终完成肿瘤的定向转移。CXCR4是肿瘤细胞中表达最普遍的趋化因子受体之一,在结肠癌、胰腺癌、乳腺癌、肺癌、卵巢癌等多种肿瘤中均有较高的表达率,趋化因子SDF-1是其唯一配体,二者相互作用构成SDF-1/CXCR4生物轴在肿瘤细胞的定向迁移过程中发挥重要作用。而且肝、肺、淋巴结等结肠癌好发转移部位均为SDF-1表达丰富的器官组织,提示SDF-1/CXCR4在结肠癌器官特异性转移中具有重要作用,然而其具体作用机制仍不清楚。
结肠癌的组织器官特异性转移是一个多步骤的复杂过程。一方面,结肠癌细胞的定向转移需要有一定的“方向性”。而SDF-1的浓度梯度可以为其提供“方向导航”,即CXCR4阳性的结肠癌细胞可以循着SDF-1的浓度梯度,向富含SDF-1的组织器官定向迁移；另一方面,结肠癌细胞的定向转移需要一定的“动力”。既往研究表明SDF-1/CXCR4本身并不直接参与细胞的粘附及迁移运动,而可能通过向细胞内转导信号,激活其他分子,促进细胞的迁移粘附。因此我们认为,与细胞迁移、粘附直接相关的粘附分子可能在其中发挥关键作用。
整合素属于细胞表面粘附分子家族,是一类由α亚基和p亚基以非共价键结合组成的跨膜糖蛋白受体。整合素可以介导细胞间及细胞与细胞外基质的粘附反应,通过向细胞内外传导生物学信号,调控细胞的生物学行为。整合素αvβ6是一类仅存在于上皮源性恶性肿瘤的特殊整合素亚型,与多种上皮性肿瘤的发生发展和侵袭转移密切相关,而其作为一种抗恶性上皮性肿瘤的靶目标,已成为该领域的研究热点之一。课题组通过十余年的系统研究表明：整合素αvβ6与结肠癌和胃癌的病理类型、分化程度和病理分期密切相关,可作为结肠癌和胃癌的独立不良预后指标；αvβ6与ERK之间存在直接连接,结肠癌细胞通过αvβ6-ERK信号通路调控uPA、MMP-9和MMP-2的分泌及活性,激活纤溶酶原系统降解细胞外基质,使肿瘤细胞获得高侵袭性表型；在结肠癌细胞中,整合素αvβ6持续的进行内吞胞吐循环,而αvβ6正是通过自身的内吞胞吐循环,直接参与肿瘤细胞的迁移运动。
由此可见,SDF-1/CXCR4介导的结肠癌定向转移是多因素相互作用的结果,而目前关于其作用机制的研究多局限于SDF-1/CXCR4生物轴本身及其下游信号通路,SDF-1/CXCR4与其他分子特别是粘附分子相互作用的研究则很少,尤其与结肠癌转移密切相关的αvβ6在其中的作用目前更是未见相关研究。
本项目拟通过临床标本和细胞实验,明确CXCR4和avβ6在结肠癌组织和不同转移潜能的结肠癌细胞中的表达情况及其与结肠癌临床病理特征特别是器官特异性转移的关系,分析二者之间的表达相关性,并在此基础上进一步探讨αvβ6在SDF-1/CXCR4介导的结肠癌定向转移中的可能作用及具体分子机制,从而为解释结肠癌细胞的肝脏特异性转移提供一种新型的理论机制。如能在这一新型调节机制中找到一个或多个关键作用靶点,进行针对性干预,将会有效减少结肠癌的远处转移,改善患者预后,对于结肠癌的综合治疗具有十分重要的实际意义和临床应用前景。
第一部分αvβ6和CXCR4在结肠癌组织及结肠癌细胞中的表达情况
目的
明确整合素αvβ6和CXCR4对结肠癌肝转移的影响及二者表达情况的相关性。
方法收集2005年～2008年间在山东大学齐鲁医院由同一手术团队行结肠癌根治术的结肠癌患者结肠癌组织及肝转移组织标本,并进行相关资料的采集和随访；资料采集内容包括：性别和年龄等一般资料,肿瘤大小、病理类型、分化程度,TNM分期,手术方式,以及脏器和淋巴结转移等预后情况；然后利用免疫组织化学方法检测病理组织中αvβ6、CXCR4的表达部位、阳性表达率及表达强度,并进行免疫组化评分；在此基础上结合相关临床随访资料,统计分析avβ6和CXCR4在结肠癌组织和肝转移组织中的表达情况及其对肝转移等结肠癌预后情况的影响；并进一步利用Mann-Whitney test和Spearman等级相关分析等统计方法初步探讨αvβ6和CXCR4的表达相关性。选取高转移潜能的结肠癌细胞系HT-29和WiDr以及非转移性结肠癌细胞系Caco-2作为研究对象；逆转录PCR (RT-PCR)检测β6亚基和CXCR4在各细胞系中的mRNA表达水平；Western blot检测β6亚基和CXCR4在各细胞系中的蛋白表达水平；流式细胞术检测整合素αvβ6和CXCR4在各细胞表面的表达情况。
结果
159例符合要求的结肠癌患者被纳入本研究,共收集了159例原发结肠癌组织和21例肝转移组织。通过对159例原发结肠癌组织进行免疫组化检测发现,CXCR4和avβ6分别在在107例(67.3%)和73例(45.9%)结肠癌组织中表达阳性,而二者在正常结肠组织中几乎无表达；通过对21例肝转移组织进行免疫组化检测发现,CXCR4和βvβ6分别在19例(90.5%)和16例(76.2%)肝转移组织中表达阳性,而二者在正常肝脏组织中几乎无表达；CXCR4表达与结肠癌的淋巴结转移(p<0.05)、TNM分期p<0.05)和肝转移p<0.01)密切相关；avβ6表达与结肠癌的淋巴结转移(p<0.05)、TNM分期(p<0.05)、淋巴血管侵犯((p<0.05)和肝转移密切(p<0.01)相关；αvβ6和CXCR4均高表达的患者,与二者表达情况的其他组合相比,更容易出现淋巴结转移(p<0.05)和肝脏转移(p<0.01)；Mann-Whitney test和Spearman等级相关分析表明,αvβ6和CXCR4的表达呈正相关性。细胞实验表明,p6亚基和CXCR4的mRNA和蛋白表达水平在HT-29和WiDr细胞系中均相对较高,而在Caco-2细胞系中的表达水平很低；整合素αvβ6和CXCIR4在HT-29和WiDr细胞表面的表达水平相对较高,而在Caco-2细胞表面几乎无表达或表达较低。
结论
整合素αvβ6和CXCR4与结肠癌的肝转移密切相关,且二者的表达具有一定的相关性。二者均在高转移潜能的结肠癌细胞中高表达,而在非转移性的细胞中低表达,而且二者均高表达的患者更容易发生肝转移。
意义该部分证实了整合素avβ6和CXCR4与结肠癌肝转移的密切关系,为进一步探讨αvβ6和CXCR4参与结肠癌肝转移的具体机制奠定了实验基础并提供了一定的理论前提。
第二部分SDF-1/CXCR4对αvβ6及其他整合素表达的影响
目的
探讨SDF-1/CXCR4对αvβ6及其他常见整合素表达的影响及其具体机制。
方法利用实时定量PCR、western blot检测SDF-1/CXCR4对HT-29和WiDr细胞中av、β1、β3、(35和β6亚基表达的影响；流式细胞术检测SDF-1/CXCR4对细胞表面αvβ6表达的影响；western blot检测SDF-1/CXCR4对ERK1/2磷酸化的影响,并观察ERK抑制剂U0126对αvβ6表达和HT-29细胞定向迁移的影响；western blot、共聚焦显微镜检测SDF-1/CXCR4对Ets-1活化的影响,并观察Ets-1特异性siRNA对αvβ6表达和HT-29细胞定向迁移的影响。
结果
实时定量PCR、western blot结果表明SDF-1/CXCR4在HT-29和WiDr细胞中可明显增加β6亚基的表达、轻度增加β1亚基的表达,而对αv、β3、β5亚基的表达无影响；流式细胞术显示SDF-1/CXCR4可明显增加HT-29和WiDr细胞表面αvβ6的表达；SDF-1/CXCR4可促进ERK1/2的磷酸化,而ERK抑制剂U0126则可显著抑制SDF-1/CXCR4介导的αvβ6表达上调和HT-29细胞定向迁移；SDF-1/CXCR4可促进Ets-1的活化,而Ets-1特异性siRNA则可显著抑制SDF-1/CXCR4介导的αvβ6表达上调和HT-29细胞定向迁移。
结论
SDF-1/CXCR4可通过ERK-Ets-1通路促进整合素αvβ6的表达增加。
意义
明确了SDF-1/CXCR4对αvβ6表达的影响及具体机制,从而为下一步明确αvβ6在SDF-1/CXCR4介导的定向转移和侵袭中的作用奠定了基础。
第三部分αvβ6在SDF-1/CXCR4介导的结肠癌细胞定向迁移中的作用
目的
明确整合素αvβ6在SDF-1/CXCR4介导的结肠癌细胞定向迁移中的作用。方法
在Transwell小室的下室加入一定浓度的SDF-1作为趋化物,分别检测HT-29和WiDr细胞在纤连蛋白表面的定向迁移能力；对HT-29和WiDr细胞分别给予三种干预手段,即αvβ6功能阻断抗体10D5、αvβ6特异性siRNA以及CXCR4特异性阻断剂AMD3100,观察各干预手段对结肠癌细胞定向迁移能力的影响。CXCR4过表达质粒pcDNA-CXCR4转染非转移性细胞系Caco-2;利用含绿色荧光蛋白的表达质粒pcDNA-GFP验证转染效率,利用western blot和流式细胞术验证CXCR4的过表达效果；200ng/ml SDF-1刺激转染了pcDNA-CXCR4的Caco-2细胞,利用实时定量PCR、western blot和流式细胞术检测SDF-1对αvβ6表达的影响；迁移实验检测过表达CXCR4对Caco-2细胞定向迁移能力的影响,并通过干扰αvβ6观察αvβ6在此过程中的作用。
结果
与对照组(Transwell小室的下室不加趋化物SDF-1)相比,SDF-1能明显增加HT-29和WiDr细胞在纤连蛋白表面的定向迁移能力；10D5、αvβ6特异性siRNA、 AMD3100可显著抑制SDF-1诱导的定向迁移。pcDNA的转染效率大于80%,pcDNA-CXCR4可有效增加Caco-2细胞中CXCR4的表达水平；SDF-1可明显上调转染pcDNA-CXCR4的Caco-2细胞中αvβ6的表达,而CXCR4特异性抑制剂AMD3100可有效抑制SDF-1诱导的avβ6表达上调；过表达CXCR4可显著增加Caco-2细胞的定向迁移能力,而同时应用αvβ6功能阻断抗体10D5则可有效抑制Caco-2细胞定向迁移能力的增加。
结论
SDF-1可显著增加HT-29和WiDr细胞的定向迁移能力,而αvβ6是这一过程所必需的。而非转移性结肠癌细胞系Caco-2在过表达CXCR4后,可通过上调αvβ6表达而获得较高的定向迁移能力。
意义
进一步明确了SDF-1/CXCR4可通过上调αvβ6表达促进结肠癌细胞的定向转移。
Background:
Colon cancer is one of the most common gastrointestinal cancer with increasing incidence and mortality rates, which seriously endangers people's health has become a major problem in the world. Invasion and metastasis is a major factor affecting the prognosis of colon cancer. Colon cancer displays a predilection for metastasis to liver, lung and lymph node, especially liver, which is the most common leading cause of colon cancer. common liver metastases, patients with colon cancer is the leading cause of death. The traditional view shows that, when passing liver in portal venous, colon cancer cells migrate slowly and are easy to stay and grow in this site. However, recent studies show that this phenomenon can not be explained simply by the anatomical structures and vascular and lymphatic drainage, and there should be other factors involved. But the factors and the specific mechanism is unclear.
Chemokines are a class of small molecular weight of cytokines, and their receptors are seven transmembrane G protein-coupled receptors. Interaction between such receptors and their respective chemokines can induce directional migration of cells towards a gradient of chemokines (namely chemotaxis), which plays an important role in tissue development, lymphatic homing, immune and inflammation. With the deepening of research, more and more evidence shows that chemokines and their receptors are closely associated with invasion and metastasis of tumor cells. Considering many similarities shared by cancer cell metastasis and leukocyte trafficking, the binding of chemokines to their receptors can induce directional migration of tumor cells towards the organs with abundant chemokines. CXCR4is a commonly expressed chemokine receptor in different tumors. Previous studies reported that CXCR4together with its unique ligand stromal cell-derived factor-1(SDF-1or CXCL12) played a significant role in the progression and metastasis of colorectal cancer, breast cancer, prostate cancer, lung cancer, ovarian cancer and laryngeal carcinoma. SDF-1is the only ligand of CXCR4, and SDF-1/CXCR4axis plays an important role directional migration of tumor cells. Intriguingly, SDF-1is highly expressed in liver, lung, bone marrow and lymph nodes, which just represent the most common sites for CRC metastasis, which suggests the important role of SDF-1/CXCR4axis in organ-specific metastasis of colon cancer. However, the underlying mechanism is still unclear.
The process of cancer metastasis consists of a series of sequential and interrelated steps, including invasion, detachment from the primary sites, intravasation, survival in the circulation and translocation to microvessels of target organs, extravasation and clonization. The definition of directional migration consists of two principles. First, organ-specific metastasis requires a complex signalling mechanism to guide CRC cells to the liver, which can be liken to a "navigation system". As a major chemokine receptor on CRC cells, CXCR4has been proved to induce chemotaxis of cells towards a gradient of SDF-1(5,17). Furthermore, high levels of SDF-1was detected in liver, lung and bone marrow, representing the most common target organs for CRC metastasis (17). This suggests that SDF-1/CXCR4axis could function as a "navigation system" for CRC cells, guiding cancer cell migration from their original site to final destination. Indeed, SDF-1/CXCR4axis has been reported to play a significant role in liver metastasis of CRC (9-11). Second, the ability of migration or motility is also required. A keystone of cell migration is the formation and regulation of adhesion contacts, especially integrin contacts, which are dynamically disassembled at the rear and reassembled at the leading edge (29,42). However, previous studies demonstrated that CXCR4itself didn't directly contribute to adhesion of prostrate and renal carcinoma cells to extracellular matrix, but served as signal transmitters to modulate integrin α5and β3-mediated adhesion (18,19). Therefore, it is possible that SDF-1/CXCR4axis may promote CRC cell migration through cross talk with other receptors, especially the adhesion receptors.
Integrins belong to superfamily of cell surface adhesion molecule, and are heterodimeric trans-membrane receptors comprising alpha and beta subunits. Integrins serve as anchoring molecules by mediating the adhesion of the cellular cytoskeleton to the extracellular matrix (ECM). They also serve as bidirectional signaling molecules by mediating outside-in and inside-out signaling. The integrin αvβ6is unique in that it is expressed exclusively in epithelial cells, and β6partners only with av forming a single heterodimer. We previously demonstrated that:integrin αvβ6was closely related with pathologic type, differentiation and stage of colon cancer and gastric cancer, and could function as an independent indicator for prognosis; there was a direct binding between integrin αvβ6and ERK, and colon cancer cells could induce secretion of uPA, MMP-9and MMP-2and promote degradation of ECM via the direct binding of αvβ6-ERK, which rendered colon cancer cells more invasive. Recently we have verified that avP6can promote colon cancer cell migration in a mechanism of integrin trafficking.
Thus, SDF-1/CXCR4axis-induced invasion and metastasis of colon cancer is the result of interaction of multiple factors, and current research on the mechanism of this process was restricted to SDF-1/CXCR4axis itself and its downstream signaling pathways. However, the research on the interaction of SDF-1/CXCR4axis with other molecules, especially adhesion molecules. Indeed, there are none studies about the crosstalk between SDF-1/CXCR4axis and integrin αvβ6.
This study aims to investigate the expression profiles of integrin avP6and CXCR4in colon cancer specimens and colon cancer cell lines and the relationship between their expression and clinicopathological features of colon cancer, and also to analyse the correlation between expression of integrin αvβ6and CXCR4. In addition, we further investigate the potential role and underlying mechanism of αvβ6in SDF-1/CXCR4-mediated CRC cell migration. This study will present a novel mechanism for SDF-1/CXCR4-induced organ-specific metastasis and shed light on effective therapeutic approaches for liver metastasis of CRC.
Part Ⅰ
Expression of αvβ6and CXCR4in colon cancer tissue and colon cancer cell lines with different metastatic potential
Objective
To explore the effect of the expression of integrin αvβ6and CXCR4on livr metastasis of colon cancer, and to investigate the correlation between the the expression of integrin αvβ6and CXCR4
Methods
Colon cancer patients who underwent surgery by the same surgical team at the Department of General Surgery of Qilu hospital (Shandong University, china) were selected consecutively from2005to2008; Patients were regularly followed up and clinical data was collected, including gender, age, tumor size, pathological type, degree of differentiation, TNM stage, surgical methods as well as organic and lymphonodus metastasis; Then immunohistochemistry was carried out to detect the expression of αvβ6and CXCR4and the final immunohistochemistry score was also calculated; Expression of CXCR4and αvβ6in colon cancer tissues and associated liver metastatic tissues and their impact on the prognosis were statistically analysed; Furthermore, Mann-Whitney test and Spearman rank correlation analysis were both used to investigate the correlation between the expression of CXCR4and αvβ6. Highly metastatic colon cancer cell lines HT-29and WiDr and non-metastatic cell line Caco-2were used in this study; Reverse transcription PCR (RT-PCR) was used to detect the mRNA expression of β6subunit and CXCR4in these cell lines; Western blot was used to detect the protein expression of β6subunit and CXCR4; Flow cytometry was used to detect the surface expression of integrin avP6and CXCR4.
Results
159colon cancer patients meeting the requirements were finally included in the study. And159cases of primary colon cancer tissue and21cases of liver metastatic tissue were collected. Immunohistochemistry analysis of159primary CRC samples demonstrated that CXCR4was positive in107tumors (67.3%) and αvβ6was positive in73tumors (45.9%), but there were no or equivocal staining of CXCR4and αvβ6in normal colon tissues. In the21liver metastatic samples,19cases (90.5%) showed positive CXCR4staining and16(76.2%) cases showed positive αvβ6staining, but none to equivocal staining of CXCR4and αvβ6were observed in normal liver specimens; Expression of CXCR4was significantly associated with lymph node metastasis (p<0.05), TNM stage (p<0.05) and liver metastasis (p<0.01); Expression of αvβ6was significantly associated with lymph node metastasis (p<0.05), TNM stage (p<0.05), lymphovascular invasion (p<0.05) and liver metastasis (p<0.01); Notably, high αvβ6and high CXCR4expressions, when compared with other combinations, were significantly associated with lymph node metastasis (p<0.05) and liver metastasis (p<0.01); Mann-Whitney test and Spearman rank correlation analysis demonstrated that expression of αvβ6was positively correlated with expression of CXCR4. The relative mRNA and protein levels of06subunit (in affiliation only with the αv subunit) and CXCR4were high in metastatic cell lines HT-29and WiDr, while non-metastatic cell line Caco-2exhibited very low levels of both CXCR4and β6. With respect to cell surface presentation, flow cytometry showed that high membrane levels of both integrin αvβ6and CXCR4were detected in HT-29and WiDr cells, while lower levels of CXCR4and hardly any αvβ6in Caco-2
Conclusion
Expression of integrin αvβ6and CXCR4was significantly associated with liver metastasis of colon cancer. Moreover, expression of αvβ6is significantly correlated with that of CXCR4. They were highly expressed in highly metastatic colon cancer cells and lowly expressed in non-metastatic colon cancer cells, and patients with both high expression of αvβ6and CXCR4are more prone to develop liver metastasis.
Significance
This section confirms the close relationship between the expression of integrin αvβ6and CXCR4and the liver metastasis of colon cancer, which lays a foundation for further study of the mechanisms of αvβ6and CXCR4in liver metastasis of colon cancer.
Part II Effect of SDF-1/CXCR4on the expression of integrin αvβ6and other integrins
Objective
To investigate the effect of SDF-1/CXCR4on the expression of αvβ6and other integrins and to explore the potential mechanism.
Methods
Real-time quatitative PCR and western blot were used to detect the effect of SDF-1/CXCR4on the expression of av, β1, β3, β5and β6subunits in HT-29and WiDr cells; Flow cytometry was used to detect the effect of SDF-1/CXCR4on the surface expression of αvβ6; Western blot was used to detect the effect of SDF-1/CXCR4on ERK1/2phosphorylation, and the effect of ERK specific inhibitor U0126on the expression of αvβ6and the directional migration of HT-29cells was also observed; Western blot and confocal microscopy were used to detect the effect of SDF-1/CXCR4on the activation of Ets-1, and the effect of Ets-1specific siRNA on the expression of αvβ6and the directional migration of HT-29cells was also observed.
Results
Real-time quatitative PCR and western blot demonstrate that SDF-1/CXCR4could induce significant upregulation of β6subunit and slight upregulation of β1subunit, but has no effect on the expression of αv,β3and (35; Flow cytometry demonstrates that SDF-1/CXCR4could significantly increase the surface expression of integrin αvβ6; SDF-1/CXCR4could promote phosphorylation of ERK1/2, and ERK specific inhibitor U0126could significantly inhibit SDF-1/CXCR4-induced upregulation of αvβ6and directional migration of HT-29cells.
Conclusion
SDF-1/CXCR4could upregulate expresion of αvβ6via ERK-Ets-1pathway.
Significance This section demonstrates the effect of SDF-1/CXCR4on the expression of αvβ6and the underlying mechanism, which further confirms the important role of αvβ6in SDF-1/CXCR4-induced directional migration.
Part Ⅲ Role of αvβ6in SDF-1/CXCR4axis-induced directional migration of colon cancer cells
Objective
To verify the role of integrin αvβ6in SDF-1/CXCR4axis-induced directional migration of colon cancer cells.
Methods
Serum-free medium containing200ng/ml SDF-la was used as a chemoattractant in the lower chamber, and the ability of directional migration on fibronectin were detected in HT-29and WiDr cells; Cells were pretreated with αvβ6-functional blocking antibody10D5,αvβ6specific siRNA and CXCR4specific inhibitor AMD3100, and effect of these interventions on directional migration of colon cancer cells was detected. Non-metastatic Caco-2cells were transfected with human CXCR4-expressed plasmid (pcDNA-CXCR4); Green fluorescence protein-expressed plasmid (pcDNA-GFP) was used as a control to demonstrate transfection efficiency, and western blot and flow cytometry were used to detect overexpression effect of CXCR4; pcDNA-CXCR4transfected Caco-2cells were incubated with SDF-la (200ng/ml), and real-time PCR, western blot and flow cytometry were used to detect effect of SDF-1on avP6expression; Migration assay were used to detect effect of CXCR4overexpression on directional migration ability of Caco-2cells, and to observe role of avP6in this process by interfering αvβ6.
Results
Compared with control group (without chemoattractant SDF-1in the lower chamber), SDF-1could significantly increase the directional migration on fibronectin of HT-29and WiDr cells;10D5, αvβ6specific siRNA and AMD3100could significantly inhibit SDF-1-induced increase in directional migration. Transfection efficiency was more than80%in our experiment, and pcDNA-CXCR4could significantly overexpress CXCR4in Caco-2cells; Furthermore pcDNA-CXCR4transfected Caco-2cells were incubated with SDF-la and, as a result, SDF-la induced a significant increase in mRNA and protein levels of β6integrin which was evidently attenuated by CXCR4inhibitor AMD3100; SDF-la dramatically increased the migration of pcDNA-CXCR4but not mock vector transfected Caco-2cells, Moreover inhibition of αvβ6by neutralizing antibody10D5significantly alleviated SDF-1a-induced migration of pcDNA-CXCR4transfected Caco-2cells.
Conclusion
SDF-1could significantly increase the ability of directional migration of HT-29and WiDr cells, and avP6is essential for this process. Caco-2cells were rendered migrating after overexpression of CXCR4, treatment with SDF-la and subsequent upregulation of integrin αvβ6.
Significance
This sectin verifies the important role of integrin αvβ6in SDF-1/CXCR4-induced directional migration and further confirms that SDF-1/CXCR4promotes directional metastasis of colon cancer cells through upregulation of αvβ6.
引文
1.吴孟超,吴在德,黄家驷外科学(第七版).北京,人民卫生出版社,2008：p.12.
2. Steinert, G., et al., Biology and significance of circulating and disseminated tumour cells in colorectal cancer. Langenbecks Arch Surg,2012.397(4):p. 535-42.
3. Taylor, I., Liver metastases from colorectal cancer:lessons from past and present clinical studies. Br J Surg,1996.83(4):p.456-60.
4. Nguyen, D.X., P.D. Bos, and J. Massague, Metastasis:from dissemination to organ-specific colonization. Nat Rev Cancer,2009.9(4):p.274-84.
5. Wang, Y., et al., Gene expression profiles and molecular markers to predict recurrence of Dukes'B colon cancer. J Clin Oncol,2004.22(9):p.1564-71.
6. Fidler, I.J., The pathogenesis of cancer metastasis:the 'seed and soil' hypothesis revisited. Nat Rev Cancer,2003.3(6):p.453-8.
7. Koizumi, K., et al., Chemokine receptors in cancer metastasis and cancer cell-derived chemokines in host immune response. Cancer Sci,2007.98(11):p. 1652-8.
8. Lira, S.A. and G.C. Furtado, The biology of chemokines and their receptors. Immunol Res,2012.54(1-3):p.111-20.
9. Yoshie, O., T. Imai, and H. Nomiyama, Novel lymphocyte-specific CC chemokines and their receptors. J Leukoc Biol,1997.62(5):p.634-44.
10. Zlotnik, A. and O. Yoshie, Chemokines:a new classification system and their role in immunity. Immunity,2000.12(2):p.121-7.
11. Kim, J., et al., Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival. J Clin Oncol, 2005.23(12):p.2744-53.
12. Ottaiano, A., et al., Overexpression of both CXC chemokine receptor 4 and vascular endothelial growth factor proteins predicts early distant relapse in stage Ⅱ-Ⅲ colorectal cancer patients. Cin Cancer Res,2006.12(9):p. 2795-803.
13. Yoshitake, N., et al., Expression of SDF-1 alpha and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer. Br J Cancer,2008.98(10):p. 1682-9.
14. Muller, A., et al., Involvement of chemokine receptors in breast cancer metastasis. Nature,2001.410(6824):p.50-6.
15. Taichman, R.S., et al., Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res,2002.62(6):p. 1832-7.
16. Su, L., et al., Differential expression of CXCR4 is associated with the metastatic potential of human non-small cell lung cancer cells. Clin Cancer Res,2005.11(23):p.8273-80.
17. Kajiyama, H., et al., Involvement of SDF-1 alpha/CXCR4 axis in the enhanced peritoneal metastasis of epithelial ovarian carcinoma. Int J Cancer,2008. 122(1):p.91-9.
18. Tan, C.T., et al., CXCL12/CXCR4 promotes laryngeal and hypopharyngeal squamous cell carcinoma metastasis through MMP-13-dependent invasion via the ERK1/2/AP-1 pathway. Carcinogenesis,2008.29(8):p.1519-27.
19. Domanska, U.M., et al., A review on CXCR4/CXCL12 axis in oncology:no place to hide. Eur J Cancer,2013.49(1):p.219-30.
20. Engl, T., et al., CXCR4 chemokine receptor mediates prostate tumor cell adhesion through alpha5 and beta3 integrins. Neoplasia,2006.8(4):p. 290-301.
21. Jones, J., et al., CXCR4 chemokine receptor engagement modifies integrin dependent adhesion of renal carcinoma cells. Exp Cell Res,2007.313(19):p. 4051-65.
22. Hynes, R.O., Integrins:bidirectional, allosteric signaling machines. Cell, 2002.110(6):p.673-87.
23. Bandyopadhyay, A. and S. Raghavan, Defining the role of integrin alphavbeta6 in cancer. Curr Drug Targets,2009.10(7):p.645-52.
24. Hecht, J.L., et al., Overexpression of the alphavbeta6 integrin in endometrial cancer. Appl Immunohistochem Mol Morphol,2008.16(6):p.543-7.
25. Marsh, D., et al., alpha vbeta 6 Integrin promotes the invasion of morphoeic basal cell carcinoma through stromal modulation. Cancer Res,2008.68(9):p. 3295-303.
26. Yang, G Y., et al., Integrin alpha v beta 6 mediates the potential for colon cancer cells to colonize in and metastasize to the liver. Cancer Sci,2008.99(5): p.879-87.
27. Bates, R.C., et al., Transcriptional activation of integrin beta6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma. J Clin Invest,2005.115(2):p.339-47.
28. Zhang, Z.Y., et al., Integrin alphanvbeta6 acts as a prognostic indicator in gastric carcinoma. Clin Oncol (R Coll Radiol),2008.20(1):p.61-6.
29. Hazelbag, S., et al., Overexpression of the alpha v beta 6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival. J Pathol,2007.212(3):p.316-24.
30. Hamidi, S., et al., Expression of alpha(v)beta6 integrin in oral leukoplakia. Br J Cancer,2000.82(8):p.1433-40.
31. Sipos, B., et al., Immunohistochemical screening for beta6-integrin subunit expression in adenocarcinomas using a novel monoclonal antibody reveals strong up-regulation in pancreatic ductal adenocarcinomas in vivo and in vitro. Histopathology,2004.45(3):p.226-36.
32. Ahmed, N., et al., Overexpression of alpha(v)beta6 integrin in serous epithelial ovarian cancer regulates extracellular matrix degradation via the plasminogen activation cascade. Carcinogenesis,2002.23(2):p.237-44.
33. Gu, X., et al., Integrin alpha(v)beta6-associated ERK2 mediates MMP-9 secretion in colon cancer cells. Br J Cancer,2002.87(3):p.348-51.
34. Wang, J., et al., Suppression of integrin alphaupsilonbeta6 by RNA interference in colon cancer cells inhibits extracellular matrix degradation through the MAPK pathway. Int J Cancer,2008.123(6):p.1311-7.
35. Ahmed, N., et al., Direct integrin alphavbeta6-ERK binding:implications for tumour growth. Oncogene,2002.21(9):p.1370-80.
36. Liu, S., et al., The beta6-integrin-ERK/MAP kinase pathway contributes to chemo resistance in colon cancer. Cancer Lett,2013.328(2):p.325-34.
37. Wang, J., et al., PKC promotes the migration of colon cancer cells by regulating the internalization and recycling of integrin alphavbeta6. Cancer Lett,2011.311(1):p.38-47.
38. Sgambato, A. and A. Cittadini, Inflammation and cancer:a multifaceted link. Eur Rev Med Pharmacol Sci,2010.14(4):p.263-8.
39. Cojoc, M., et al., Emerging targets in cancer management:role of the CXCL12/CXCR4 axis. Onco Targets Ther,2013.6:p.1347-1361.
40. Aiuti, A., et al., The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med,1997. 185(1):p.111-20.
41. Ma, Q., et al., Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4-and SDF-1-deficient mice. Proc Natl Acad Sci U S A,1998.95(16):p.9448-53.
42. Zou, Y.R., et al., Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature,1998.393(6685):p. 595-9.
43. Kucia, M., et al., CXCR4-SDF-1 signalling, locomotion, chemotaxis and adhesion. J Mol Histol,2004.35(3):p.233-45.
44. Balkwill, F., The significance of cancer cell expression of the chemokine receptor CXCR4. Semin Cancer Biol,2004.14(3):p.171-9.
45. Vandercappellen, J., J. Van Damme, and S. Struyf, The role of CXC chemokines and their receptors in cancer. Cancer Lett,2008.267(2):p. 226-44.
46. Zlotnik, A., New insights on the role of CXCR4 in cancer metastasis. J Pathol, 2008.215(3):p.211-3.
47. Darash-Yahana, M., et al., Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis. FASEB J,2004.18(11):p.1240-2.
48. Marechal, R., et al., High expression of CXCR4 may predict poor survival in resected pancreatic adenocarcinoma. Br J Cancer,2009.100(9):p.1444-51.
49. Zirvi, K.A., T.A. Najjar, and B.L. Slomiany, Sensitivity of human colon tumor metastases to anticancer drugs in athymic (nude) mice. Cancer Lett,1993. 72(1-2):p.39-44.
50. Sato, M., et al., The association of sialyl Lewis(a) antigen with the metastatic potential of human colon cancer cells. Anticancer Res,1997.17(5A):p. 3505-11.
51. Hamada, K., et al., Liver metastasis models of colon cancer for evaluation of drug efficacy using NOD/Shi-scid IL2Rgammanull (NOG) mice. Int J Oncol, 2008.32(1):p.153-9.
52. Tammali, R., et al., Inhibition of aldose reductase prevents colon cancer metastasis. Carcinogenesis,2011.32(8):p.1259-67.
53. Mollevi, D.G, et al., Mutations in TP53 are a prognostic factor in colorectal hepatic metastases undergoing surgical resection. Carcinogenesis,2007.28(6): p.1241-6.
54. Sarvaiya, P.J., et al., Chemokines in tumor progression and metastasis. Oncotarget,2013.4(12):p.2171-85.
55. Helbig, G, et al., NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. J Biol Chem,2003.278(24):p.21631-8.
56. Libura, J., et al., CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion. Blood,2002. 100(7):p.2597-606.
57. Zhao-Yang, Z., et al., Signaling and regulatory mechanisms of integrin alphavbeta6 on the apoptosis of colon cancer cells. Cancer Lett,2008.266(2): p.209-15.
58. Niu, J., et al., The alphaVbeta6 integrin regulates its own expression with cell crowding:implications for tumour progression. Int J Cancer,2001.92(1):p. 40-8.
59. Scala, S., et al., Expression of CXCR4 predicts poor prognosis in patients with malignant melanoma. Clin Cancer Res,2005.11(5):p.1835-41.
60. Burger, M., et al., Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation, and adhesion to stromal cells. Oncogene,2003.22(50):p.8093-101.
61. Engl, T., et al., Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix. Life Sci,2006.78(16):p. 1784-93.
62. Spano, J.P., et al., Chemokine receptor CXCR4 and early-stage non-small cell lung cancer:pattern of expression and correlation with outcome. Ann Oncol, 2004.15(4):p.613-7.
63. Sun, Y.X., et al., Expression and activation of alpha v beta 3 integrins by SDF-1/CXC12 increases the aggressiveness of prostate cancer cells. Prostate, 2007.67(1):p.61-73.
64. Lai, T.H., et al., Stromal cell-derived factor-1 increase alphavbeta3 integrin expression and invasion in human chondrosarcoma cells. J Cell Physiol,2009. 218(2):p.334-42.
65. Huang, Y.C., et al., Stromal cell-derived fact or-1 enhances motility and integrin up-regulation through CXCR4, ERK and NF-kappaB-dependent pathway in human lung cancer cells. Biochem Pharmacol,2007.74(12):p. 1702-12.
66. Niu, J., et al., Integrin expression in colon cancer cells is regulated by the cytoplasmic domain of the beta6 integrin subunit. Int J Cancer,2002.99(4):p. 529-37.
67. Huang, W.S., et al., Stromal cell-derived factor-1/CXC receptor 4 and beta1 integrin interaction regulates urokinase-type plasminogen activator expression in human colorectal cancer cells. J Cell Physiol,2012.227(3):p.1114-22.
68. Teicher, B.A. and S.P. Fricker, CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res,2010.16(11):p.2927-31.
69. Oikawa, T. and T. Yamada, Molecular biology of the Ets family of transcription factors. Gene,2003.303:p.11-34.
70. Callaway, K.A., et al., Properties and regulation of a transiently assembled ERK2.Ets-1 signaling complex. Biochemistry,2006.45(46):p.13719-33.
71. Milkiewicz, M., et al., Shear stress-induced Ets-1 modulates protease inhibitor expression in microvascular endothelial cells. J Cell Physiol,2008.217(2):p. 502-10.
