CCR7对非小细胞肺癌侵袭转移的作用研究
摘要
前言
肺癌是一种常见的恶性肿瘤。近二十年来,肺癌在我国的发病率总体上呈上升趋势,并且随着人口老龄化和人类生活环境的污染与破坏,特别是吸烟人口的不断增加,肺癌的发病和死亡率还将继续攀升。癌细胞的器官播散是肺癌患者死亡的主要原因,临床和病理观察表明淋巴结转移是肺癌转移的早期特征,因此对肺癌侵袭和转移过程中发挥重要作用的因子的研究对于肺癌的防治具有很重要的现实意义。
CCR7(CC chemokine receptor 7)是CC类趋化因子受体之一,CCL21(Chemokine ligand 21)为其配体。生理条件下,CCR7在幼稚T细胞,一些记忆T细胞,B细胞和成熟的树突状细胞表面表达;CCL21特异的高表达于淋巴结、扁桃体、及脾的T细胞富集区及淋巴内皮中;CCR7/CCL21在淋巴细胞的趋化和淋巴结归巢中起重要作用。然而近来研究发现CCR7在多种肿瘤细胞中表达,如乳腺癌、卵巢癌、胃癌、食管癌、非小细胞肺癌、头颈部鳞癌、黑色素瘤等,并且CCR7过表达与这些肿瘤的淋巴结转移密切相关。
CCR7在肿瘤侵袭转移中的作用机制日益受到关注,在乳腺癌的研究中发现,CCR7/CCL21可以通过促进肌动蛋白的聚集及伪足形成诱导肿瘤细胞趋化和侵袭;头颈部鳞癌中,CCR7/CCL21可通过PI3K/Akt信号通路促进肿瘤细胞的迁移和侵袭;通过结肠癌SW460细胞的裸鼠移植实验,发现CCR7可以诱导肿瘤内淋巴管形成。但迄今为止CCR7表达上调及其促进肿瘤内淋巴管形成机制的研究则鲜有报道。仅有文献报道脱氧氮杂胞苷可以上调黑素瘤细胞CCR7表达;内皮素能够通过内皮素受体和HIF-1上调乳腺癌MCF-7细胞CCR7表达。
本研究旨在探讨缺氧对非小细胞肺癌CCR7表达影响,分析CCR7上调机制及CCR7/CCL21调控肺癌细胞迁移侵袭机制;同时本实验通过研究CCR7对淋巴管形成因子VEGF-D的表达调控作用,探讨CCR7对肺癌淋巴管形成的影响,为CCR7促进肿瘤淋巴结转移提供理论依据。
材料与方法
NSCLC组织标本:(1)94例组织标本均来自中国医科大学病理教研室。患者术前均未接受过任何化学或放射治疗。福尔马林固定石蜡包埋的肿瘤组织切片常规进行HE染色,分别由两名病理诊断医师,根据WHO肺及胸膜肿瘤分类标准(2004)和TNM分期系统(1997),确定肿瘤的组织学分型以及临床病理分期。94例NSCLC包括:男性60例,女性34例;年龄18～72岁(平均54岁±19岁)。鳞癌41例,腺癌53例;临床病理分期显示Ⅰ期、Ⅱ期共37例,Ⅲ期、Ⅳ期共57例;淋巴结转移56例,无淋巴结转移38例。常规病理检查手术切除的肺门、纵隔以及肺内淋巴结,用于确定肺癌的淋巴结转移情况。(2)90例组织标本均来标本均来自1980年到2005年在中国医科大学第一附属医院实行手术的病人,患者术前均未接受过任何化学或放射治疗。福尔马林固定石蜡包埋的肿瘤组织切片常规进行HE染色,分别由两名病理诊断医师,根据WHO肺及胸膜肿瘤分类标准(2004)和TNM分期系统(1997),确定肿瘤的组织学分型以及临床病理分期。90例NSCLC包括:年龄≤50岁者50例,≥50岁者40例;鳞癌55例,腺癌35例;临床病理分期显示Ⅰ期、Ⅱ期共39例,Ⅲ期、Ⅳ期共51例;淋巴结转移54例,无淋巴结转移36例。常规病理检查手术切除的肺门、纵隔以及肺内淋巴结,用于确定肺癌的淋巴结转移情况。随访时间截止至2006年12月。
NSCLC细胞系培养及处理:分别用含10%新鲜胎牛血清的RPMI1640或DMEM培养基,在37℃、5%CO_2的条件下培养A549,BE1,SK,QG,H661和H460肺癌细胞。细胞培养至亚融合状态后,培养基中加入抗CCR7抗体、ERK_(1/2)抑制剂PD98059、Akt抑制剂LY294002,使它们的终浓度为50μg/ml。缺氧处理实验中应用Cocl2模拟化学缺氧,24小时后收集细胞,提取蛋白和DNA;物理缺氧实验将细胞置于37℃、1%O_2、5%CO_2、94%N_2的条件下分别培养4h、12h、24h。细胞实验均重复3次。
Western blot:收集细胞提取总蛋白。采用考马斯亮蓝法进行蛋白定量。取等量蛋白,进行SDS-聚丙烯酰胺凝胶电泳和转印,一抗(anti-CCR7 1:200稀释,anti-HIF-1α1:500稀释,anti-HIF-2α1:1000稀释,anti-VEGF-D 1:300稀释,anti-ERK_(1/2) 1:200稀释,anti-p-ERK_(1/2) 1:200稀释,anti-Akt 1:300稀释,anti-p-Akt1:1000稀释,anti-β-actin 1:200稀释)4℃孵育过夜,各自对应二抗(1:3000稀释)37℃孵育2h,最后ECL发光。EC3 Imaging System凝胶成像系统采集图像,Image J软件用于半定量分析特异性条带的光密度值,将目的蛋白与β-actin光密度比值作为相对表达量。实验至少重复3次,取平均值。
免疫组化染色SP法:标本用中性福尔马林溶液固定,石蜡包埋,制成4μm切片,采用链霉素抗生物素蛋白-过氧化物酶免疫组化法(S-P法)检测组织中CCR7、HIF-1α、HIF-2α、VEGF-D、D2-40的蛋白表达,最后DAB显色,苏木素复染细胞核。同时以PBS代替一抗作为阴性对照。两名病理诊断医师分别对组织切片染色情况进行评分。结果判定:CCR7以胞浆和/或胞膜出现棕黄色颗粒视为阳性细胞;HIF-1α和HIF-2α以胞核和/或胞浆出现棕黄色颗粒视为阳性细胞;VEGF-D以胞浆出现棕黄色颗粒视为阳性细胞。至少5个高倍视野下(400×)评价CCR7染色强度(1=弱,2=强)和阳性肿瘤细胞百分比(0=阴性,1.50%=1,51-75%=2,≥76%=3)。上述两项评分的乘积作为每个标本染色的最终评分,最后确定肺癌标本的染色情况分别为阴性:0分;低表达:评分≤3;或高表达:评分＞3。HIF-1α和HIF-2α染色评分同参考文献。淋巴管判定标准为:低倍光镜下确定3个淋巴管高密度区域(热点),然后在高倍镜下分别计数每个热点中3个区域的D2-40染色阳性管腔数均值。当计数相差10%以上时则重新计数。
RNA干扰:依据siRNA设计原则,选取人HIF-1αmRNA、HIF-2αmRNA和CCR7中的特异性核苷酸片断为靶标,应用Ambion公司在线siRNA设计软件设计HIF-1α、HIF-2α和CCR7的RNAi序列,经Blast确定所选靶向的基因是特异的,序列由上海吉凯基因化学技术有限公司合成。实验分三组:空白对照组、非特异性siRNA转染组、特异性siRNA(20nm/L)转染组,每个实验均重复三次,转染具体步骤按Lip2000(Invitrogen,USA)试剂说明书进行。HIF-1αRNAi序列:sense5'-CUG AUG ACC AGC AAC UUG AdTdT-3',antisense 5'-UCA AGU UGC UGGUCA UCA GdTdT-3'。HIF-2αRNAi序列:sense 5'-CAG CAU CUU UGA UAG CAGUdTdT-3',antisense 5'-ACU GCU AUC AAA GAU GCU GdTdT-3'。HIF-1α和HIF-2α非特异性siRNA序列:sense 5'-AGU UCA ACG ACC AGU AGUCdTdT-3',antisense 5'-GAC UAC UGG UCG UUG AdTdT-3'。CCR7 RNAi序列:sense 5'-GGACGT GCG GAA CTT TAA AdTdT-3',antisense 5'-TTT AAA GTT CCG CAC GTCCdTdT-3'。CCR7非特异性siRNA序列:5'-GAC TTC ATA AGG CGC ATGCdTdT-3',antisense 5'-GCA TGC GCC TTA TGAAGT CdTdT-3'。
细胞转染:pLNCX2-CCR7质粒由Sam T.Hwang(National Institutes of Health,USA)惠赠。pLNCX2-CCR7质粒稳定转染CCR7低表达BE1细胞(称为CCR7-BE1),具体转染步骤按脂质体LipofectamineTM2000试剂说明书进行,含500μg/ml G418的RPMI1640筛选培养基培养转染细胞。以未转染的BE1细胞和转染空载体细胞(称为Empty vector)作为对照。
Transwell细胞迁移和侵袭实验:用Matrigel基质胶包被Transwell小室后检测细胞侵袭能力。BE1和CCR7-BE1细胞分别用PD98059处理4h或抗CCR7抗体处理24h后,将2.5×10~4个细胞接种在孔径为8μm的Transwell小室的上层,下层小室加入不同浓度的趋化因子CCL21(50,100,200,300ng/ml),然后将细胞分别置于常氧或缺氧条件下培养24h。用棉签小心擦去膜上表面未发生迁移的细胞,迁移至膜下表面的细胞经4%多聚甲醛固定后用苏木素染色。随机选取5个视野(200×)计数迁移细胞数。实验至少3次,取平均值。细胞迁移实验在无Matrigel基质胶包被Transwell小室进行,方法同细胞侵袭试验,将细胞分别置于常氧或缺氧条件下培养8h。
RT-PCR:取对数生长期的细胞用TRIZOL(Invitrogen,USA)提取总RNA。逆转录条件为30℃10 min,42℃40min,99℃5min,5℃5min。PCR反应条件为:94℃5min,94℃40sec,53℃(HIF-1α、CCR7)/60℃(HIF-2α,VEGF-*)40sec,72℃40sec,72℃5min。取扩增产物5μl,1.5%琼脂糖凝胶电泳,用Image J软件进行表达强度分析。引物均由辽宁博春天生物技术有限公司合成。PCR引物为:CCR7上游5'-GAG GCTATT GTC CCC TAAACC-3',下游5'-TGG AGG ACA GTG AAGAAAACG-3',304bp。HIF-1α上游5'-TAA GAAACC ACC TAT GAC CTG C-3',下游5'-GTC GTG CTG AAT AAT ACC ACT C-3',411bp。HIF-2α上游5'-GAAAACGAG TCC GAA GCC-3',下游5'-CCC AAA ACC AGA GCC ATT-3',242bp。VEGF-D上游5'-TCA GCA TCC CAT CGG TCC ACT AG-3',下游5'-CAA CAGCCA CCA CAT CGG AAC AC-3',223bp。β-actin上游5'-AAA TCG TGC GTGACA TTA A-3',下游5'-CTC GTC ATA CTC CTG CTT G-3',513bp。
统计学分析:SPSS 13.0统计学软件用于数据分析及处理。Pearson相关分析用于评价CCR7与HIF-1α、HIF-2α表达的关系。免疫组化结果应用χ~2检验对蛋白表达与临床病理特征之间的关系进行分析。其它实验采用t检验;用mean±SD表示,P＜0.05为有统计学意义。
实验结果
1、缺氧通过HIF-1α和HIF-2α诱导CCR7表达,促进非小细胞肺癌的侵袭和转移。
1.1非小细胞肺癌组织中CCR7和HIF-1α、HIF-2α表达相关:通过免疫组化方法检测94例非小细胞肺癌组织标本中CCR7和HIF-1α、HIF-2α表达。CCR7主要表达于肿瘤细胞的胞膜和(或)胞质,HIF-1α、HIF-2α蛋白则定位在肿瘤细胞的胞核和胞质。94例非小细胞肺癌组织标本中CCR7阳性率为75.53%(71/94),HIF-1α阳性率为54.25%(51/94),HIF-2α阳性率为70.21%(66/94)。统计学分析显示:CCR7与HIF-1α(r=0-315,P=0.002)、HIF-2α(r=0.247,P=0.017)表达呈正相关;CCR7、HIF-1α和HIF-2α表达均与非小细胞肺癌的临床病理分期(P_(CCR7)=0.027;P_(1α)=0.011;P_(2α)=0.015)和淋巴结转移(P_(CCR7)=0.001;P_(1α)=0.001;P_(2α)=0.015)呈正相关。
1.2六种肺癌细胞中CCR7、HIF-1α和HIF-2αmRNA表达相关:RT-PCR方法检测六种肺癌细胞(A549、BE1、SK、QG、H661、H460)中CCR7、HIF-1α和HIF-2α表达,结果显示三者在六种肺癌细胞中均有表达,且CCR7和HIF-1α、HIF-2αmRNA表达相关,如BE1细胞中CCR7 mRNA低表达,HIF-1α和HIF-2αmRNA表达水平也明显低于其它细胞系,这表明CCR7和HIF-1α、HIF-2α表达相关是不同肺癌细胞中普遍存在的。
1.3缺氧可通过诱导HIF-1α和HIF-2α上调CCR7表达:CCR7低表达的BE1细胞分别经物理缺氧(94%N2,5%CO2,1%O2)及化学缺氧(Cocl_2)处理后,通过RT-PCR和Western blotting方法检测不同时间点CCR7mRNA和蛋白表达情况,结果显示,与常氧培养相比,缺氧后CCR7表达显著上调,同时HIF-1α和HIF-2α表达也明显增加。通过siRNA方法分别抑制BE1细胞中HIF-1α(siHIF-1α)和HIF-2α(siHIF-2α)表达后,CCR7mRNA和蛋白表达水平与对照组相比明显降低(P＜0.05)。
1.4缺氧通过HIF-1α/HIF-2α促进CCL21诱导的BE1细胞迁移和侵袭:缺氧培养BE1细胞后,Transwell侵袭实验检测不同浓度CCL21诱导的细胞侵袭能力。结果表明,CCL21以剂量依赖的方式诱导BE1细胞侵袭,在CCL21浓度为200ng/ml时侵袭的细胞数达到峰值;与常氧相比,不同浓度CCL21诱导细胞侵袭数均比常氧高,然而阻断HIF-1α或HIF-2α表达后,缺氧条件下CCL21诱导细胞的迁移和侵袭数目降低,尤其是干扰HIF-1α后。
1.5 CCR7通过ERK_(1/2)促进BE1细胞迁移和侵袭:CCR7低表达的肺癌细胞BE1细胞稳定转染正义CCR7基因后,CCR7 mRNA和蛋白含量显著增加,获得CCR7高表达的细胞株CCR7-BE1细胞。CCR7-BE1经CCL21刺激后p-ERK_(1/2)蛋白表达水平增加,总ERK_(1/2)未变化;同时细胞迁移和侵袭实验中CCR7-BE1细胞通过Matrigel基质胶的细胞数目(51.29±4.89;74.57±4.69),与空白对照组(20.57±4.2;25.43±3.99)和空质粒转染组(21.7±3.2;26±3.96)比较,迁移和侵袭细胞数目显著增多(P＜0.001)。
CCR7-BE1细胞经抗CCR7抗体或ERK_(1/2)特异性抑制剂PD98059处理后,p-ERK_(1/2)表达水平降低,总ERK_(1/2)未变化。CCL21诱导的细胞侵袭数目也明显减少,结果表明CCR7调控BE1细胞侵袭需要ERK_(1/2)的活化。
2、CCL21/CCR7通过ERK_(1/2)和Akt途径上调肺癌细胞VEGF-D表达,诱导淋巴管形成,促进肿瘤淋巴结转移。
2.1 CCL21/CCR7上调肺癌细胞中VEGF-D表达:A549细胞加入趋化因子CCL21后,通过RT-PCR和Western blotting方法检测VEGF-D mRNA和蛋白表达情况,结果显示随着加药时间的增加,VEGF-D mRNA和蛋白表达显著上调。用CCR7抗体处理A549细胞后VEGF-D的蛋白表达水平降低。在三种检测的肺癌细胞系中,我们都发现了这种现象。以上研究表明CCR7可以调控非小细胞肺癌VEGF-D表达。
2.2 ERK_(1/2)和Akt参与CCL21/CCR7介导的VEGF-D上调:肺癌A549细胞加入趋化因子CCL21后,p-ERK_(1/2)和p-Akt表达上调,总ERK_(1/2)和Akt蛋白表达却没有未改变。加入CCR7抗体阻断A549细胞中CCR7表达后,我们发现p-ERK_(1/2)和p-Akt表达降低。通过PD98059和LY294002分别抑制A549细胞中p-ERK_(1/2)和p-Akt表达后,VEGF-D mRNA和蛋白表达水平明显降低。
2.3非小细胞肺癌组织中CCR7表达与VEGF-D表达水平、淋巴管密度、临床分期、淋巴结转移和患者预后相关:免疫组化方法分析90例NSCLC组织标本显示VEGF-D和CCR7表达呈正相关(r=0.455,P＜0.001)。我们通过D2-40抗体检测了CCR7和VEGF-D高或低表达的NSCLC组织中淋巴管密度。通过定量肿瘤内D2-40阳性的脉管表明,CCR7高表达组LVD为28.09±10.57,CCR7低表达组LVD为20.58±7.6,两者相比差异显著,VEGF-D高表达的肿瘤LVD(30.14±9.87)高于VEGF-D低表达的肿瘤LVD(9.38±7.22,P＜0.001)。
CCR7高表达组患者与低表达组相比进展快(P=0.003),更容易发生淋巴结转移(P=0.004)。VEGF-D高表达的患者与低表达的相比进展快(P=0.005)、易发生淋巴结转移(P=0.001)。CCR7低表达患者的生存周期要比CCR7高表达的患者长(P=0.036)。VEGF-D高表达组患者的平均生存时间为16±3.8月;95%可信区间为8.6-23.4月,明显短于VEGF-D低表达患者(P=0.009)。这些结果表明CCR7可能上调非小细胞肺癌VEGF-D表达,促进肿瘤淋巴管形成,影响肿瘤淋巴结转移,因此CCR7高表达患者预后较差。
结论
1、非小细胞肺癌组织标本中,CCR7与HIF-1α、HIF-2α表达呈正相关,并且它们均与非小细胞肺癌的淋巴结转移和临床病理分期密切相关。
2、缺氧可通过HIF-1α和HIF-2α上调CCR7表达,CCR7表达上调后通过ERK_(1/2)信号通路促进肺癌细胞的迁移和侵袭,提示缺氧-HIF-1α,2α-CCR7-ERK_(1/2)信号通路可以调控肺癌细胞的迁移和侵袭。
3、本研究证实肺癌细胞中VEGF-D是CCR7重要下游基因,CCL21/CCR7通过ERK_(1/2)和Akt途径上调肺癌细胞VEGF-D表达,我们还发现CCR7介导的VEGF-D上调与肺癌患者的临床分期、淋巴管密度、淋巴结转移和生存时间相关。
Introduction
Lung cancer is one of the most common malignant tumor.In China,Lung cancer has become the first cause of death in malignant tumors.Tumor invasion and metastasis were the critical steps in determining the aggressive phenotype of human cancers. Mortality of tumor patients principally resulted from cancer cells spread to distant organs.Clinical and pathological observations had shown that lymph node involvement was one of the earliest features of metastatic cancers.Therefore,study on the factors acting important role in the invasion and metastasis course will be contributed to providing new ideas to make out clinical therapy strategy.
CC Chemokine Receptor 7(CCR7),is expressed on all naive T cells,some memory T cells,B cells and mature dendrite cells and plays a central role in lymphocyte trafficking and homing to lymph nodes.The current evidence shows that CCR7 is highly expressed in human breast cancer cells and metastases,and ligand binding to CCR7 induces chemotactic and invasive responses,including actin polymerization and pseudopodia formation.Moreover,several other tumor types express CCR7,and CCR7 plays a critical role in cancer metastasis of many organs as well as regional lymph nodes,such as breast cancer,cervical cancer,gastric cancer,esophageal squamous cell carcinoma,squamous cell carcinoma of the head and neck,melanoma cells and so on.
The role of CCR7 in tumor cells invasion and metastasis becomes important day by day.However,the factors those regulate CCR7 expression and the mechanisms that how CCR7 promote tumor metastasis remains unclear.In the present study,we investigated whether hypoxia could regulate the expression of CCR7 in non-small cell lung cancer,and analysis the mechanism of CCR7 up-regulation and increased invasion ability of lung cancer cell.In addition,the effects of CCR7 on the lymphangiogenic properties of lung cancer cells were studied by investigating its effects on VEGF-D expression.This provide basis for CCR7 promote tumor cells lymph node metastasis.
Materials and Methods
Sample preparation:A total of 94 cases of NSCLC tissues were obtained from the January 2001 to the April 2006 at the First Affiliated Hospital of China Medical University.The tumor tissues in this study were from NSCLC patients proved by pathological diagnosis.None of the 94 patients had received radiation therapy or chemotherapy before surgery.Tissue samples to be used for immunohistochemistry were fixed in formalin and paraffin embedded.The TNM staging system of the UICC (1997) was used to classify the specimens.The study has been approved by hospital ethical committee.
Immunohistochemical staining:4μm thick sections were prepared from the paraffin-embedded tissues.Immunostaining was performed by the streptavidin-peroxidase(S-P) method(MaiXin,China).The primary antibodies were anti-CCR7 at 1:200 dilution(Santa Cruz,USA),anti-HIF-1αat 1:100 dilution(BD,San Diego,CA) and anti-HIF-2αat 1:150 dilution(Novus Biological Science),respectively. The peroxidase reaction was developed with DAB staining(Maixin,China).For negative control,the primary antibodies were replaced by non-immune serum.All the immunoreactions were separately evaluated by two senior pathologists.The intensity of CCR7 immunostaining(1=weak,2=intense) and the percentage of positive tumor cells(0%=negative,1-50%=1,51-75%=2,≥76%=3) were assessed in at least 5 high power fields(×400 magnification).The tumors were finally determined as low expression:score＜3;or high expression:score≥3.The staining of HIF-1αand HIF-2αwere scored as previous study.~(17) If the difference between the numbers counted by two pathologists was more than 10%,the lumens were recounted and a consensus between each observer was reached.
Cell culture and hypoxic treatment:The lung cell lines A549,BE1,SK,QG,H661 and H460 were used in this study.Cells were maintained in DMEM or RPMI1640 (both from Gibco,Invitrogen Corporation) containing 10%FCS together with 100 U/ml penicillin,100 U/ml streptomycin.All cells were cultured at 37℃in an air/5% CO2 incubator.Hypoxic exposure was carried out under 1%oxygen,5%carbon dioxide,and 94%nitrogen(Galaxy R CO2 incubator,RS Biotech) at 37℃.Cells were incubated in normoxic or hypoxic condition for 4h,12h and 24h respectively.
RT-PCR:Total RNA was isolated from cells in the logarithmic growth phase using TRIZOL(Invitrogen).The PCR primers were as follows:CCR7 F:5'-GAG GCT ATT GTC CCC TAA ACC -3',R:5'-TGG AGG ACA GTG AAG AAA ACG -3'.The length of PCR product was 304bp.HIF-1αF:5'-TAA GAA ACC ACC TAT GAC CTG C-3',R:5'-GTC GTG CTG AAT AAT ACC ACT C -3'.The length of PCR product was 411bp.HIF-2αF:5'-GAA AAC GAG TCC GAA GCC -3',R:5'-CCC AAA ACC AGA GCC ATT -3'.The length of PCR product was 242bp.β-actin F:5'-AAA TCG TGC GTG ACA TTA A-3',R:5'-CTC GTC ATA CTC CTG CTT G-3'.The length was: 513bp.Thirty cycles of 40 seconds denaturation at 95℃,40 seconds annealing at 53℃(HIF-1α,CCR7,β-actin) or 60℃(HIF-2α) and 40 seconds extension at 72℃were then performed.
siRNA Treatment of Cells:RNA interference and siRNA preparation was performed as described previously for HIF-1α~(18) and HIF-2α.~(19) Effects of siRNA for HIF-1α(siHIF-1α) or HIF-2α(siHIF-2α) were compared with those of a random siRNA sequence.~(18) Cells were plated onto 10-cm~2 cell culture dishes and grown to 30-50%confluence before transfection.The duplexes were diluted to give a final concentration of 20nM.Transfections were performed with Lipofectamine 2000 (Invitrogen) and by following the manufacturer's instructions.Twenty-four hours later, the transfection procedure was repeated,and cells were subjected to hypoxia treatment the day after the second transfection.
Stable Transfection:The plasmid pLNCX2-CCR7 was kindly provided by Sam T.Hwang(National Institutes of Health,USA).BE1 cells were transfected pLNCX2-CCR7 in the presence of Lipofectamine 2000(Invitrogen) following the manufacturer's instructions and placed under G418(500μg/ml) selection for 4 weeks. Surviving colonies were isolated and named CCR7-BE1 cells.BE1 cells were transfected empty vector as control and named pLNCX2-BE1 cells.
Western blotting:Cells were serum starved for 24h and then treated as described in "Results".After treatment cells were extracted with lysis buffer(150mMNaCl,1% NP-40,0.1%SDS,2μg/ml aprotinin,1mM PMSF) for 30 min at 4℃.The supernatants were centrifuged at 12000×g for 30 min at 4℃.The supernatant containing total protein was harvested.Aliquots containing 100μg of proteins were separated on a 7.5%SDS-PAGE and transferred to PVDF membranes at 100 V for 2 h at low temperature.The membranes were blocked with 5%skimmed milk.Subsequently, mouse anti-human HIF-1α(BD,San Diego,CA) monclonal antibody was used at 1:500 dilution;mouse anti-human HIF-2α(NB100-132ss) monclonal antibody(Novus Biological Science) was used at 1:1000 dilution;goat anti-human CCR7 polyclonal antibody(Santa Cruz,USA) was used at 1:200 dilution;and anti-β-actin antibody (Santa Cruz,USA) was used at 1:400 dilution.After washing with TBST for three times,immunodetection was performed.The EC3 Imaging System(UVP Inc.) was used to catch up the specific bands,and the optical density to interest proteins andβ-actin of the same sample was calculated as relative content and expressed graphically.
Cell invasion and migration assays:Cell invasion was performed using Matrigel -coated 24-well Transwell chamber(Costar).BE1 cells and CCR7-BE1 cells were preincubated in the presence/absent of PD9059 for 4h or anti-CCR7 antibody for 24h. Cells(2.5×10~4) were seeded in the upper chamber of 8μm pore size insert in the 24-well plate and cultured in hypoxic or normoxic environment for another 24h.Cells were allowed to migrate forward to RPMI1640 containing different concentration (50,100,200,300ng/ml) of recombinant human CCL21(PEPROTECH ASIA) in the bottom chamber.The non-invasive cells on the upper membrane surface were removed with a cotton tip,and the invasive cells attached to the lower membrane surface were fixed with 4%paraformaldehyde and stained with hematoxylin.The number of invasive cells was counted under inverted microscope(200×).Data presented are representative of three individual wells.Cell migration was assayed using a similar approach without Matrigel coating,and cells in migration chamber were incubated under normoxic(N) or hypoxic(H) conditions for 8h.
Statistical analysis:The SPSS 13.0 software was applied to complete data processing.X~2-test was applied to analyze the correlation between the expressions of HIF-1α,HIF-2α,CCR7 and clinicopathological characteristics for the results of immunohistochemistry;Correlation between HIF-1α,HIF-2αand CCR7 expressions was analysed by Spearman's test;Independent-samples t-test was used to evaluate the differences of optical density(OD) values or numbers of migrated cells between groups with various treatments.All data were represented as mean±SD of three independent experiments.Results were considered statistically significant when the p-value was less than 0.05.
Results
1.1 Correlation of CCR7 with HIF-1αand HIF-2αexpression:The predominant staining pattern of CCR7 was cytoplasmic and /or plasma membranes in 94 cases NSCLC tissues.The CCR7 protein was detected in part of the endothelial cells.The high expression rates of CCR7 in the 94 cases were 75.53%(71/94).Both HIF-1αand HIF-2αprotein showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells,HIF-1αand HIF-2αprotein were detected in part of the tumoural vessels and turnout-infiltrating inflammatory cells,while normal lung components showed negative staining.The high expression rates of HIF-1αand HIF-2αin the 94 cases were 54.25% (51/94) and 70.21%(66/94),respectively.Statistical analysis showed that CCR7 expression was correlated positively with the expression of HIF-1α(r=0.315,P=0.002) and HIF-2α(r=0.247,P=0.017).CCR7 expression was significantly associated with lymph node metastasis(P=0.001) and clinical pathological stage(P=0.027).Both HIF-1αand HIF-2αexpression were significantly associated with lymph node metastasis(P=0.001),(P=0.015) and clinical pathological stage(P=0.011),(P=0.015).
All the six cell lines we tested were positive for CCR7,HIF-1αand HIF-2αmRNA,and cell lines expressing higher levels of CCR7 also displayed greater abundance of HIF-1αand HIF-2α.The above data clearly demonstrate that the correlation of CCR7 with HIF-1αand HIF-2αexpression was commonly observed in the different human lung cancer cells.
1.2 Hypoxia promotes CCR7 expression by HIF-1αand HIF-2αin BE1 cells:We exposed BE1 cells under hypoxia conditions(physical hypoxia,chemical hypoxia),the expression of CCR7 mRNA and protein were strongly elevated,and meanwhile the expression of HIF-1αand HIF-2αwas up-regulated.To investigate the role of HIF-1αand HIF-2αin the hypoxic induction of CCR7,we established siRNA for the inhibition of HIF-1αand HIF-2αin BE1 cells.Then,cells were incubated under hypoxia conditions.The results showed that CCR7 mRNA and protein levels were reduced more significantly in siHIF-1αthan siHIF-2αtansfected cells(P＜0.05).
1.3 HIF-1αand HIF-2αregulate CCL21-induced chemotaxis,migration and invasion of BE1 cells:Migration and invasion activity of BE1 cells were analysized by Transwell assay.Chemotaxis toward different concentrations of CCL21 was greatly changes in a dose-dependent manner.Invaded cells achieved the most at the concentration of 200ng/ml CCL21 in BE1 cells.The number of migration and invasive cells was increased under hypoxia compared with normoxia,decreased after down-regulation of HIF-1αor HIF-2α,which was more significant in siHIF-1αtransfected cells.
1.4 ERK_(1/2) activation is required for CCR7-mediated migration and invasion of BE1 cells:we transfected BE1 cells with vectors carrying CCR7(CCR7-BElcells) or empty vector,and observed the transfection efficiency by RT-PCR and western blotting methods.The results showed that CCR7-BE1 cells expressed more CCR7 both at mRNA and protein levels;simultaneously the expression of p-ERK_(1/2) protein was increased.Transwell migration assays showed that the number of cells in control group and empty vector group migrate through the microporous membrane were(20.57±4.2) and(21.7±3.2).The number of the cells(51.29±4.89,P=0.000) that migrate through microporous membrane increased significantly in CCR7-BE1 group.In the invasion assay,the number of cells in control group and empty vector group that invade through Matrigel were(25.43±3.99) and(26±3.96).In CCR7-BE1 cells group the number of the cells that invade through Matrigel increased significantly(74.57±4.69,P=0.000). P-ERK_(1/2) protein expression and the number of invasion cells decreased significantly in the presence of either PD98059 or anti-CCR7 antibody.The results showed that CCR7-mediated migration and invasion of BE1 cells dependent on ERK_(1/2) activation.
2.1 CCR7 Up-Regulated VEGF-D in NSCLC Cell Lines:treatment of A549 cells with human CCL21 increase the expression' of VEGF-D mRNA and protein.Treatment of A549 cells with CCR7 antibody significantly attenuated the endogenous VEGF-D protein level.
Of the three cell lines tested,we found that CCL21 increased the expression of VEGF-D,and blocking CCR7 decreased the expression of VEGF-D,indicating that CCL21 could regulate VEGF-D via CCR7.
2.2 Involvement of ERK_(1/2) and Akt in CCR7-Mediated VEGF-D Up-Regulation: we detected p-ERK_(1/2),ERK_(1/2),p-Akt,Akt protein expressions in A549 cells.Western blotting showed that incubation of A549 cells with CCL21 increased the expressions of p-ERK_(1/2) and p-Akt,while exhibiting no effect on the total protein levels of ERK_(1/2) or Akt.Furthermore,Specific blocking CCR7 expression by anti-CCR7 Ab inhibited the expression of p-ERK_(1/2) and p- Akt.Furthermore,the VEGF-D mRNA and protein expression in A549 cells were decreased significantly after incubation with PD98059 or LY294002,respectively.However,in A549 cells,LY294002 did not inhibit ERK_(1/2) phosphorylation,and PD98059 did not affect phosphorylation of Akt under basal condition.
2.3 CCR7 Expression Correlate with VEGF-D Level,Lymphatic Vessel Density, Clinical Stages,Lymph Node Metastasis,and Patient Survival in NSCLC: Immunohistochemical analysis of 90 NSCLC specimens revealed that the VEGF-D expression was significantly associated with the expression of CCR7(r =0.455,P＜0.001).Quantification of the D2-40-positive vessels in the tumors indicated that tumor with high expression of CCR7 had a higher LVD than in low CCR7 expressing tumors (28.09±10.57 for CCR7 high expression tumors,n=63,and 20.58±7.6 for CCR7 low expression tumors,n=27,P=0.001).Tumors with VEGF-D high expression had a higher LVD than in low VEGF-D expressing tumors 30.14±9.87 for VEGF-D high expression tumors,n=54,and 19.38±7.22 for VEGF-D low expression tumors,n=36, P＜0.001).Patients with CCR7 high expression were more likely than low expression patients to have advanced disease(P=0.003) and lymph node metastasis(P=0.004). VEGF-D high expression patients were more likely than low expression patients to have advanced disease(P=0.005) and lymph node metastasis(P=0.001).For CCR7 low expression patients had a statistically significantly longer survival than CCR7 high expression patients(P=0.036).The median survival time for patients with tumors with high VEGF-D was 16±3.8 months;95%confidence interval 8.6-23.4 months, significantly shorter than that for patients with tumors with low VEGF-D expression (P=0.009).
Conclusions
1、CCR7 expression was correlated positively with the expressions of HIF-1αand HIF-2αin 94 cases NSCLC tissues.Each of them was significantly associated with lymph node metastasis and clinical pathological stage.
2、Hypoxia- HIF-1α,2α-CCR7-ERK1/2 pathway is likely to regulate the invasion and migration of lung cancer cells under hypoxic tissue microenvironments.HIF-1α, HIF-2αand CCR7 activation might play a role in promoting a more malignant phenotype of NSCLC and represents novel therapeutic strategies.
3、Acause-effect link between CCR7 over-expression and tumor lymphangiogenesis through VEGF-D activity was demonstrated in lung cancer.CCR7 could regulate VEGF-D expression through ERK1/2 and Akt signaling pathway.Our findings open newer therapeutic modalities against lymphangiogenesis and lymphatic borne。
肺癌是一种常见的恶性肿瘤。近二十年来,肺癌在我国的发病率总体上呈上升趋势,并且随着人口老龄化和人类生活环境的污染与破坏,特别是吸烟人口的不断增加,肺癌的发病和死亡率还将继续攀升。癌细胞的器官播散是肺癌患者死亡的主要原因,临床和病理观察表明淋巴结转移是肺癌转移的早期特征,因此对肺癌侵袭和转移过程中发挥重要作用的因子的研究对于肺癌的防治具有很重要的现实意义。
CCR7(CC chemokine receptor 7)是CC类趋化因子受体之一,CCL21(Chemokine ligand 21)为其配体。生理条件下,CCR7在幼稚T细胞,一些记忆T细胞,B细胞和成熟的树突状细胞表面表达;CCL21特异的高表达于淋巴结、扁桃体、及脾的T细胞富集区及淋巴内皮中;CCR7/CCL21在淋巴细胞的趋化和淋巴结归巢中起重要作用。然而近来研究发现CCR7在多种肿瘤细胞中表达,如乳腺癌、卵巢癌、胃癌、食管癌、非小细胞肺癌、头颈部鳞癌、黑色素瘤等,并且CCR7过表达与这些肿瘤的淋巴结转移密切相关。
CCR7在肿瘤侵袭转移中的作用机制日益受到关注,在乳腺癌的研究中发现,CCR7/CCL21可以通过促进肌动蛋白的聚集及伪足形成诱导肿瘤细胞趋化和侵袭;头颈部鳞癌中,CCR7/CCL21可通过PI3K/Akt信号通路促进肿瘤细胞的迁移和侵袭;通过结肠癌SW460细胞的裸鼠移植实验,发现CCR7可以诱导肿瘤内淋巴管形成。但迄今为止CCR7表达上调及其促进肿瘤内淋巴管形成机制的研究则鲜有报道。仅有文献报道脱氧氮杂胞苷可以上调黑素瘤细胞CCR7表达;内皮素能够通过内皮素受体和HIF-1上调乳腺癌MCF-7细胞CCR7表达。
本研究旨在探讨缺氧对非小细胞肺癌CCR7表达影响,分析CCR7上调机制及CCR7/CCL21调控肺癌细胞迁移侵袭机制;同时本实验通过研究CCR7对淋巴管形成因子VEGF-D的表达调控作用,探讨CCR7对肺癌淋巴管形成的影响,为CCR7促进肿瘤淋巴结转移提供理论依据。
材料与方法
NSCLC组织标本:(1)94例组织标本均来自中国医科大学病理教研室。患者术前均未接受过任何化学或放射治疗。福尔马林固定石蜡包埋的肿瘤组织切片常规进行HE染色,分别由两名病理诊断医师,根据WHO肺及胸膜肿瘤分类标准(2004)和TNM分期系统(1997),确定肿瘤的组织学分型以及临床病理分期。94例NSCLC包括:男性60例,女性34例;年龄18～72岁(平均54岁±19岁)。鳞癌41例,腺癌53例;临床病理分期显示Ⅰ期、Ⅱ期共37例,Ⅲ期、Ⅳ期共57例;淋巴结转移56例,无淋巴结转移38例。常规病理检查手术切除的肺门、纵隔以及肺内淋巴结,用于确定肺癌的淋巴结转移情况。(2)90例组织标本均来标本均来自1980年到2005年在中国医科大学第一附属医院实行手术的病人,患者术前均未接受过任何化学或放射治疗。福尔马林固定石蜡包埋的肿瘤组织切片常规进行HE染色,分别由两名病理诊断医师,根据WHO肺及胸膜肿瘤分类标准(2004)和TNM分期系统(1997),确定肿瘤的组织学分型以及临床病理分期。90例NSCLC包括:年龄≤50岁者50例,≥50岁者40例;鳞癌55例,腺癌35例;临床病理分期显示Ⅰ期、Ⅱ期共39例,Ⅲ期、Ⅳ期共51例;淋巴结转移54例,无淋巴结转移36例。常规病理检查手术切除的肺门、纵隔以及肺内淋巴结,用于确定肺癌的淋巴结转移情况。随访时间截止至2006年12月。
NSCLC细胞系培养及处理:分别用含10%新鲜胎牛血清的RPMI1640或DMEM培养基,在37℃、5%CO_2的条件下培养A549,BE1,SK,QG,H661和H460肺癌细胞。细胞培养至亚融合状态后,培养基中加入抗CCR7抗体、ERK_(1/2)抑制剂PD98059、Akt抑制剂LY294002,使它们的终浓度为50μg/ml。缺氧处理实验中应用Cocl2模拟化学缺氧,24小时后收集细胞,提取蛋白和DNA;物理缺氧实验将细胞置于37℃、1%O_2、5%CO_2、94%N_2的条件下分别培养4h、12h、24h。细胞实验均重复3次。
Western blot:收集细胞提取总蛋白。采用考马斯亮蓝法进行蛋白定量。取等量蛋白,进行SDS-聚丙烯酰胺凝胶电泳和转印,一抗(anti-CCR7 1:200稀释,anti-HIF-1α1:500稀释,anti-HIF-2α1:1000稀释,anti-VEGF-D 1:300稀释,anti-ERK_(1/2) 1:200稀释,anti-p-ERK_(1/2) 1:200稀释,anti-Akt 1:300稀释,anti-p-Akt1:1000稀释,anti-β-actin 1:200稀释)4℃孵育过夜,各自对应二抗(1:3000稀释)37℃孵育2h,最后ECL发光。EC3 Imaging System凝胶成像系统采集图像,Image J软件用于半定量分析特异性条带的光密度值,将目的蛋白与β-actin光密度比值作为相对表达量。实验至少重复3次,取平均值。
免疫组化染色SP法:标本用中性福尔马林溶液固定,石蜡包埋,制成4μm切片,采用链霉素抗生物素蛋白-过氧化物酶免疫组化法(S-P法)检测组织中CCR7、HIF-1α、HIF-2α、VEGF-D、D2-40的蛋白表达,最后DAB显色,苏木素复染细胞核。同时以PBS代替一抗作为阴性对照。两名病理诊断医师分别对组织切片染色情况进行评分。结果判定:CCR7以胞浆和/或胞膜出现棕黄色颗粒视为阳性细胞;HIF-1α和HIF-2α以胞核和/或胞浆出现棕黄色颗粒视为阳性细胞;VEGF-D以胞浆出现棕黄色颗粒视为阳性细胞。至少5个高倍视野下(400×)评价CCR7染色强度(1=弱,2=强)和阳性肿瘤细胞百分比(0=阴性,1.50%=1,51-75%=2,≥76%=3)。上述两项评分的乘积作为每个标本染色的最终评分,最后确定肺癌标本的染色情况分别为阴性:0分;低表达:评分≤3;或高表达:评分＞3。HIF-1α和HIF-2α染色评分同参考文献。淋巴管判定标准为:低倍光镜下确定3个淋巴管高密度区域(热点),然后在高倍镜下分别计数每个热点中3个区域的D2-40染色阳性管腔数均值。当计数相差10%以上时则重新计数。
RNA干扰:依据siRNA设计原则,选取人HIF-1αmRNA、HIF-2αmRNA和CCR7中的特异性核苷酸片断为靶标,应用Ambion公司在线siRNA设计软件设计HIF-1α、HIF-2α和CCR7的RNAi序列,经Blast确定所选靶向的基因是特异的,序列由上海吉凯基因化学技术有限公司合成。实验分三组:空白对照组、非特异性siRNA转染组、特异性siRNA(20nm/L)转染组,每个实验均重复三次,转染具体步骤按Lip2000(Invitrogen,USA)试剂说明书进行。HIF-1αRNAi序列:sense5'-CUG AUG ACC AGC AAC UUG AdTdT-3',antisense 5'-UCA AGU UGC UGGUCA UCA GdTdT-3'。HIF-2αRNAi序列:sense 5'-CAG CAU CUU UGA UAG CAGUdTdT-3',antisense 5'-ACU GCU AUC AAA GAU GCU GdTdT-3'。HIF-1α和HIF-2α非特异性siRNA序列:sense 5'-AGU UCA ACG ACC AGU AGUCdTdT-3',antisense 5'-GAC UAC UGG UCG UUG AdTdT-3'。CCR7 RNAi序列:sense 5'-GGACGT GCG GAA CTT TAA AdTdT-3',antisense 5'-TTT AAA GTT CCG CAC GTCCdTdT-3'。CCR7非特异性siRNA序列:5'-GAC TTC ATA AGG CGC ATGCdTdT-3',antisense 5'-GCA TGC GCC TTA TGAAGT CdTdT-3'。
细胞转染:pLNCX2-CCR7质粒由Sam T.Hwang(National Institutes of Health,USA)惠赠。pLNCX2-CCR7质粒稳定转染CCR7低表达BE1细胞(称为CCR7-BE1),具体转染步骤按脂质体LipofectamineTM2000试剂说明书进行,含500μg/ml G418的RPMI1640筛选培养基培养转染细胞。以未转染的BE1细胞和转染空载体细胞(称为Empty vector)作为对照。
Transwell细胞迁移和侵袭实验:用Matrigel基质胶包被Transwell小室后检测细胞侵袭能力。BE1和CCR7-BE1细胞分别用PD98059处理4h或抗CCR7抗体处理24h后,将2.5×10~4个细胞接种在孔径为8μm的Transwell小室的上层,下层小室加入不同浓度的趋化因子CCL21(50,100,200,300ng/ml),然后将细胞分别置于常氧或缺氧条件下培养24h。用棉签小心擦去膜上表面未发生迁移的细胞,迁移至膜下表面的细胞经4%多聚甲醛固定后用苏木素染色。随机选取5个视野(200×)计数迁移细胞数。实验至少3次,取平均值。细胞迁移实验在无Matrigel基质胶包被Transwell小室进行,方法同细胞侵袭试验,将细胞分别置于常氧或缺氧条件下培养8h。
RT-PCR:取对数生长期的细胞用TRIZOL(Invitrogen,USA)提取总RNA。逆转录条件为30℃10 min,42℃40min,99℃5min,5℃5min。PCR反应条件为:94℃5min,94℃40sec,53℃(HIF-1α、CCR7)/60℃(HIF-2α,VEGF-*)40sec,72℃40sec,72℃5min。取扩增产物5μl,1.5%琼脂糖凝胶电泳,用Image J软件进行表达强度分析。引物均由辽宁博春天生物技术有限公司合成。PCR引物为:CCR7上游5'-GAG GCTATT GTC CCC TAAACC-3',下游5'-TGG AGG ACA GTG AAGAAAACG-3',304bp。HIF-1α上游5'-TAA GAAACC ACC TAT GAC CTG C-3',下游5'-GTC GTG CTG AAT AAT ACC ACT C-3',411bp。HIF-2α上游5'-GAAAACGAG TCC GAA GCC-3',下游5'-CCC AAA ACC AGA GCC ATT-3',242bp。VEGF-D上游5'-TCA GCA TCC CAT CGG TCC ACT AG-3',下游5'-CAA CAGCCA CCA CAT CGG AAC AC-3',223bp。β-actin上游5'-AAA TCG TGC GTGACA TTA A-3',下游5'-CTC GTC ATA CTC CTG CTT G-3',513bp。
统计学分析:SPSS 13.0统计学软件用于数据分析及处理。Pearson相关分析用于评价CCR7与HIF-1α、HIF-2α表达的关系。免疫组化结果应用χ~2检验对蛋白表达与临床病理特征之间的关系进行分析。其它实验采用t检验;用mean±SD表示,P＜0.05为有统计学意义。
实验结果
1、缺氧通过HIF-1α和HIF-2α诱导CCR7表达,促进非小细胞肺癌的侵袭和转移。
1.1非小细胞肺癌组织中CCR7和HIF-1α、HIF-2α表达相关:通过免疫组化方法检测94例非小细胞肺癌组织标本中CCR7和HIF-1α、HIF-2α表达。CCR7主要表达于肿瘤细胞的胞膜和(或)胞质,HIF-1α、HIF-2α蛋白则定位在肿瘤细胞的胞核和胞质。94例非小细胞肺癌组织标本中CCR7阳性率为75.53%(71/94),HIF-1α阳性率为54.25%(51/94),HIF-2α阳性率为70.21%(66/94)。统计学分析显示:CCR7与HIF-1α(r=0-315,P=0.002)、HIF-2α(r=0.247,P=0.017)表达呈正相关;CCR7、HIF-1α和HIF-2α表达均与非小细胞肺癌的临床病理分期(P_(CCR7)=0.027;P_(1α)=0.011;P_(2α)=0.015)和淋巴结转移(P_(CCR7)=0.001;P_(1α)=0.001;P_(2α)=0.015)呈正相关。
1.2六种肺癌细胞中CCR7、HIF-1α和HIF-2αmRNA表达相关:RT-PCR方法检测六种肺癌细胞(A549、BE1、SK、QG、H661、H460)中CCR7、HIF-1α和HIF-2α表达,结果显示三者在六种肺癌细胞中均有表达,且CCR7和HIF-1α、HIF-2αmRNA表达相关,如BE1细胞中CCR7 mRNA低表达,HIF-1α和HIF-2αmRNA表达水平也明显低于其它细胞系,这表明CCR7和HIF-1α、HIF-2α表达相关是不同肺癌细胞中普遍存在的。
1.3缺氧可通过诱导HIF-1α和HIF-2α上调CCR7表达:CCR7低表达的BE1细胞分别经物理缺氧(94%N2,5%CO2,1%O2)及化学缺氧(Cocl_2)处理后,通过RT-PCR和Western blotting方法检测不同时间点CCR7mRNA和蛋白表达情况,结果显示,与常氧培养相比,缺氧后CCR7表达显著上调,同时HIF-1α和HIF-2α表达也明显增加。通过siRNA方法分别抑制BE1细胞中HIF-1α(siHIF-1α)和HIF-2α(siHIF-2α)表达后,CCR7mRNA和蛋白表达水平与对照组相比明显降低(P＜0.05)。
1.4缺氧通过HIF-1α/HIF-2α促进CCL21诱导的BE1细胞迁移和侵袭:缺氧培养BE1细胞后,Transwell侵袭实验检测不同浓度CCL21诱导的细胞侵袭能力。结果表明,CCL21以剂量依赖的方式诱导BE1细胞侵袭,在CCL21浓度为200ng/ml时侵袭的细胞数达到峰值;与常氧相比,不同浓度CCL21诱导细胞侵袭数均比常氧高,然而阻断HIF-1α或HIF-2α表达后,缺氧条件下CCL21诱导细胞的迁移和侵袭数目降低,尤其是干扰HIF-1α后。
1.5 CCR7通过ERK_(1/2)促进BE1细胞迁移和侵袭:CCR7低表达的肺癌细胞BE1细胞稳定转染正义CCR7基因后,CCR7 mRNA和蛋白含量显著增加,获得CCR7高表达的细胞株CCR7-BE1细胞。CCR7-BE1经CCL21刺激后p-ERK_(1/2)蛋白表达水平增加,总ERK_(1/2)未变化;同时细胞迁移和侵袭实验中CCR7-BE1细胞通过Matrigel基质胶的细胞数目(51.29±4.89;74.57±4.69),与空白对照组(20.57±4.2;25.43±3.99)和空质粒转染组(21.7±3.2;26±3.96)比较,迁移和侵袭细胞数目显著增多(P＜0.001)。
CCR7-BE1细胞经抗CCR7抗体或ERK_(1/2)特异性抑制剂PD98059处理后,p-ERK_(1/2)表达水平降低,总ERK_(1/2)未变化。CCL21诱导的细胞侵袭数目也明显减少,结果表明CCR7调控BE1细胞侵袭需要ERK_(1/2)的活化。
2、CCL21/CCR7通过ERK_(1/2)和Akt途径上调肺癌细胞VEGF-D表达,诱导淋巴管形成,促进肿瘤淋巴结转移。
2.1 CCL21/CCR7上调肺癌细胞中VEGF-D表达:A549细胞加入趋化因子CCL21后,通过RT-PCR和Western blotting方法检测VEGF-D mRNA和蛋白表达情况,结果显示随着加药时间的增加,VEGF-D mRNA和蛋白表达显著上调。用CCR7抗体处理A549细胞后VEGF-D的蛋白表达水平降低。在三种检测的肺癌细胞系中,我们都发现了这种现象。以上研究表明CCR7可以调控非小细胞肺癌VEGF-D表达。
2.2 ERK_(1/2)和Akt参与CCL21/CCR7介导的VEGF-D上调:肺癌A549细胞加入趋化因子CCL21后,p-ERK_(1/2)和p-Akt表达上调,总ERK_(1/2)和Akt蛋白表达却没有未改变。加入CCR7抗体阻断A549细胞中CCR7表达后,我们发现p-ERK_(1/2)和p-Akt表达降低。通过PD98059和LY294002分别抑制A549细胞中p-ERK_(1/2)和p-Akt表达后,VEGF-D mRNA和蛋白表达水平明显降低。
2.3非小细胞肺癌组织中CCR7表达与VEGF-D表达水平、淋巴管密度、临床分期、淋巴结转移和患者预后相关:免疫组化方法分析90例NSCLC组织标本显示VEGF-D和CCR7表达呈正相关(r=0.455,P＜0.001)。我们通过D2-40抗体检测了CCR7和VEGF-D高或低表达的NSCLC组织中淋巴管密度。通过定量肿瘤内D2-40阳性的脉管表明,CCR7高表达组LVD为28.09±10.57,CCR7低表达组LVD为20.58±7.6,两者相比差异显著,VEGF-D高表达的肿瘤LVD(30.14±9.87)高于VEGF-D低表达的肿瘤LVD(9.38±7.22,P＜0.001)。
CCR7高表达组患者与低表达组相比进展快(P=0.003),更容易发生淋巴结转移(P=0.004)。VEGF-D高表达的患者与低表达的相比进展快(P=0.005)、易发生淋巴结转移(P=0.001)。CCR7低表达患者的生存周期要比CCR7高表达的患者长(P=0.036)。VEGF-D高表达组患者的平均生存时间为16±3.8月;95%可信区间为8.6-23.4月,明显短于VEGF-D低表达患者(P=0.009)。这些结果表明CCR7可能上调非小细胞肺癌VEGF-D表达,促进肿瘤淋巴管形成,影响肿瘤淋巴结转移,因此CCR7高表达患者预后较差。
结论
1、非小细胞肺癌组织标本中,CCR7与HIF-1α、HIF-2α表达呈正相关,并且它们均与非小细胞肺癌的淋巴结转移和临床病理分期密切相关。
2、缺氧可通过HIF-1α和HIF-2α上调CCR7表达,CCR7表达上调后通过ERK_(1/2)信号通路促进肺癌细胞的迁移和侵袭,提示缺氧-HIF-1α,2α-CCR7-ERK_(1/2)信号通路可以调控肺癌细胞的迁移和侵袭。
3、本研究证实肺癌细胞中VEGF-D是CCR7重要下游基因,CCL21/CCR7通过ERK_(1/2)和Akt途径上调肺癌细胞VEGF-D表达,我们还发现CCR7介导的VEGF-D上调与肺癌患者的临床分期、淋巴管密度、淋巴结转移和生存时间相关。
Introduction
Lung cancer is one of the most common malignant tumor.In China,Lung cancer has become the first cause of death in malignant tumors.Tumor invasion and metastasis were the critical steps in determining the aggressive phenotype of human cancers. Mortality of tumor patients principally resulted from cancer cells spread to distant organs.Clinical and pathological observations had shown that lymph node involvement was one of the earliest features of metastatic cancers.Therefore,study on the factors acting important role in the invasion and metastasis course will be contributed to providing new ideas to make out clinical therapy strategy.
CC Chemokine Receptor 7(CCR7),is expressed on all naive T cells,some memory T cells,B cells and mature dendrite cells and plays a central role in lymphocyte trafficking and homing to lymph nodes.The current evidence shows that CCR7 is highly expressed in human breast cancer cells and metastases,and ligand binding to CCR7 induces chemotactic and invasive responses,including actin polymerization and pseudopodia formation.Moreover,several other tumor types express CCR7,and CCR7 plays a critical role in cancer metastasis of many organs as well as regional lymph nodes,such as breast cancer,cervical cancer,gastric cancer,esophageal squamous cell carcinoma,squamous cell carcinoma of the head and neck,melanoma cells and so on.
The role of CCR7 in tumor cells invasion and metastasis becomes important day by day.However,the factors those regulate CCR7 expression and the mechanisms that how CCR7 promote tumor metastasis remains unclear.In the present study,we investigated whether hypoxia could regulate the expression of CCR7 in non-small cell lung cancer,and analysis the mechanism of CCR7 up-regulation and increased invasion ability of lung cancer cell.In addition,the effects of CCR7 on the lymphangiogenic properties of lung cancer cells were studied by investigating its effects on VEGF-D expression.This provide basis for CCR7 promote tumor cells lymph node metastasis.
Materials and Methods
Sample preparation:A total of 94 cases of NSCLC tissues were obtained from the January 2001 to the April 2006 at the First Affiliated Hospital of China Medical University.The tumor tissues in this study were from NSCLC patients proved by pathological diagnosis.None of the 94 patients had received radiation therapy or chemotherapy before surgery.Tissue samples to be used for immunohistochemistry were fixed in formalin and paraffin embedded.The TNM staging system of the UICC (1997) was used to classify the specimens.The study has been approved by hospital ethical committee.
Immunohistochemical staining:4μm thick sections were prepared from the paraffin-embedded tissues.Immunostaining was performed by the streptavidin-peroxidase(S-P) method(MaiXin,China).The primary antibodies were anti-CCR7 at 1:200 dilution(Santa Cruz,USA),anti-HIF-1αat 1:100 dilution(BD,San Diego,CA) and anti-HIF-2αat 1:150 dilution(Novus Biological Science),respectively. The peroxidase reaction was developed with DAB staining(Maixin,China).For negative control,the primary antibodies were replaced by non-immune serum.All the immunoreactions were separately evaluated by two senior pathologists.The intensity of CCR7 immunostaining(1=weak,2=intense) and the percentage of positive tumor cells(0%=negative,1-50%=1,51-75%=2,≥76%=3) were assessed in at least 5 high power fields(×400 magnification).The tumors were finally determined as low expression:score＜3;or high expression:score≥3.The staining of HIF-1αand HIF-2αwere scored as previous study.~(17) If the difference between the numbers counted by two pathologists was more than 10%,the lumens were recounted and a consensus between each observer was reached.
Cell culture and hypoxic treatment:The lung cell lines A549,BE1,SK,QG,H661 and H460 were used in this study.Cells were maintained in DMEM or RPMI1640 (both from Gibco,Invitrogen Corporation) containing 10%FCS together with 100 U/ml penicillin,100 U/ml streptomycin.All cells were cultured at 37℃in an air/5% CO2 incubator.Hypoxic exposure was carried out under 1%oxygen,5%carbon dioxide,and 94%nitrogen(Galaxy R CO2 incubator,RS Biotech) at 37℃.Cells were incubated in normoxic or hypoxic condition for 4h,12h and 24h respectively.
RT-PCR:Total RNA was isolated from cells in the logarithmic growth phase using TRIZOL(Invitrogen).The PCR primers were as follows:CCR7 F:5'-GAG GCT ATT GTC CCC TAA ACC -3',R:5'-TGG AGG ACA GTG AAG AAA ACG -3'.The length of PCR product was 304bp.HIF-1αF:5'-TAA GAA ACC ACC TAT GAC CTG C-3',R:5'-GTC GTG CTG AAT AAT ACC ACT C -3'.The length of PCR product was 411bp.HIF-2αF:5'-GAA AAC GAG TCC GAA GCC -3',R:5'-CCC AAA ACC AGA GCC ATT -3'.The length of PCR product was 242bp.β-actin F:5'-AAA TCG TGC GTG ACA TTA A-3',R:5'-CTC GTC ATA CTC CTG CTT G-3'.The length was: 513bp.Thirty cycles of 40 seconds denaturation at 95℃,40 seconds annealing at 53℃(HIF-1α,CCR7,β-actin) or 60℃(HIF-2α) and 40 seconds extension at 72℃were then performed.
siRNA Treatment of Cells:RNA interference and siRNA preparation was performed as described previously for HIF-1α~(18) and HIF-2α.~(19) Effects of siRNA for HIF-1α(siHIF-1α) or HIF-2α(siHIF-2α) were compared with those of a random siRNA sequence.~(18) Cells were plated onto 10-cm~2 cell culture dishes and grown to 30-50%confluence before transfection.The duplexes were diluted to give a final concentration of 20nM.Transfections were performed with Lipofectamine 2000 (Invitrogen) and by following the manufacturer's instructions.Twenty-four hours later, the transfection procedure was repeated,and cells were subjected to hypoxia treatment the day after the second transfection.
Stable Transfection:The plasmid pLNCX2-CCR7 was kindly provided by Sam T.Hwang(National Institutes of Health,USA).BE1 cells were transfected pLNCX2-CCR7 in the presence of Lipofectamine 2000(Invitrogen) following the manufacturer's instructions and placed under G418(500μg/ml) selection for 4 weeks. Surviving colonies were isolated and named CCR7-BE1 cells.BE1 cells were transfected empty vector as control and named pLNCX2-BE1 cells.
Western blotting:Cells were serum starved for 24h and then treated as described in "Results".After treatment cells were extracted with lysis buffer(150mMNaCl,1% NP-40,0.1%SDS,2μg/ml aprotinin,1mM PMSF) for 30 min at 4℃.The supernatants were centrifuged at 12000×g for 30 min at 4℃.The supernatant containing total protein was harvested.Aliquots containing 100μg of proteins were separated on a 7.5%SDS-PAGE and transferred to PVDF membranes at 100 V for 2 h at low temperature.The membranes were blocked with 5%skimmed milk.Subsequently, mouse anti-human HIF-1α(BD,San Diego,CA) monclonal antibody was used at 1:500 dilution;mouse anti-human HIF-2α(NB100-132ss) monclonal antibody(Novus Biological Science) was used at 1:1000 dilution;goat anti-human CCR7 polyclonal antibody(Santa Cruz,USA) was used at 1:200 dilution;and anti-β-actin antibody (Santa Cruz,USA) was used at 1:400 dilution.After washing with TBST for three times,immunodetection was performed.The EC3 Imaging System(UVP Inc.) was used to catch up the specific bands,and the optical density to interest proteins andβ-actin of the same sample was calculated as relative content and expressed graphically.
Cell invasion and migration assays:Cell invasion was performed using Matrigel -coated 24-well Transwell chamber(Costar).BE1 cells and CCR7-BE1 cells were preincubated in the presence/absent of PD9059 for 4h or anti-CCR7 antibody for 24h. Cells(2.5×10~4) were seeded in the upper chamber of 8μm pore size insert in the 24-well plate and cultured in hypoxic or normoxic environment for another 24h.Cells were allowed to migrate forward to RPMI1640 containing different concentration (50,100,200,300ng/ml) of recombinant human CCL21(PEPROTECH ASIA) in the bottom chamber.The non-invasive cells on the upper membrane surface were removed with a cotton tip,and the invasive cells attached to the lower membrane surface were fixed with 4%paraformaldehyde and stained with hematoxylin.The number of invasive cells was counted under inverted microscope(200×).Data presented are representative of three individual wells.Cell migration was assayed using a similar approach without Matrigel coating,and cells in migration chamber were incubated under normoxic(N) or hypoxic(H) conditions for 8h.
Statistical analysis:The SPSS 13.0 software was applied to complete data processing.X~2-test was applied to analyze the correlation between the expressions of HIF-1α,HIF-2α,CCR7 and clinicopathological characteristics for the results of immunohistochemistry;Correlation between HIF-1α,HIF-2αand CCR7 expressions was analysed by Spearman's test;Independent-samples t-test was used to evaluate the differences of optical density(OD) values or numbers of migrated cells between groups with various treatments.All data were represented as mean±SD of three independent experiments.Results were considered statistically significant when the p-value was less than 0.05.
Results
1.1 Correlation of CCR7 with HIF-1αand HIF-2αexpression:The predominant staining pattern of CCR7 was cytoplasmic and /or plasma membranes in 94 cases NSCLC tissues.The CCR7 protein was detected in part of the endothelial cells.The high expression rates of CCR7 in the 94 cases were 75.53%(71/94).Both HIF-1αand HIF-2αprotein showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells,HIF-1αand HIF-2αprotein were detected in part of the tumoural vessels and turnout-infiltrating inflammatory cells,while normal lung components showed negative staining.The high expression rates of HIF-1αand HIF-2αin the 94 cases were 54.25% (51/94) and 70.21%(66/94),respectively.Statistical analysis showed that CCR7 expression was correlated positively with the expression of HIF-1α(r=0.315,P=0.002) and HIF-2α(r=0.247,P=0.017).CCR7 expression was significantly associated with lymph node metastasis(P=0.001) and clinical pathological stage(P=0.027).Both HIF-1αand HIF-2αexpression were significantly associated with lymph node metastasis(P=0.001),(P=0.015) and clinical pathological stage(P=0.011),(P=0.015).
All the six cell lines we tested were positive for CCR7,HIF-1αand HIF-2αmRNA,and cell lines expressing higher levels of CCR7 also displayed greater abundance of HIF-1αand HIF-2α.The above data clearly demonstrate that the correlation of CCR7 with HIF-1αand HIF-2αexpression was commonly observed in the different human lung cancer cells.
1.2 Hypoxia promotes CCR7 expression by HIF-1αand HIF-2αin BE1 cells:We exposed BE1 cells under hypoxia conditions(physical hypoxia,chemical hypoxia),the expression of CCR7 mRNA and protein were strongly elevated,and meanwhile the expression of HIF-1αand HIF-2αwas up-regulated.To investigate the role of HIF-1αand HIF-2αin the hypoxic induction of CCR7,we established siRNA for the inhibition of HIF-1αand HIF-2αin BE1 cells.Then,cells were incubated under hypoxia conditions.The results showed that CCR7 mRNA and protein levels were reduced more significantly in siHIF-1αthan siHIF-2αtansfected cells(P＜0.05).
1.3 HIF-1αand HIF-2αregulate CCL21-induced chemotaxis,migration and invasion of BE1 cells:Migration and invasion activity of BE1 cells were analysized by Transwell assay.Chemotaxis toward different concentrations of CCL21 was greatly changes in a dose-dependent manner.Invaded cells achieved the most at the concentration of 200ng/ml CCL21 in BE1 cells.The number of migration and invasive cells was increased under hypoxia compared with normoxia,decreased after down-regulation of HIF-1αor HIF-2α,which was more significant in siHIF-1αtransfected cells.
1.4 ERK_(1/2) activation is required for CCR7-mediated migration and invasion of BE1 cells:we transfected BE1 cells with vectors carrying CCR7(CCR7-BElcells) or empty vector,and observed the transfection efficiency by RT-PCR and western blotting methods.The results showed that CCR7-BE1 cells expressed more CCR7 both at mRNA and protein levels;simultaneously the expression of p-ERK_(1/2) protein was increased.Transwell migration assays showed that the number of cells in control group and empty vector group migrate through the microporous membrane were(20.57±4.2) and(21.7±3.2).The number of the cells(51.29±4.89,P=0.000) that migrate through microporous membrane increased significantly in CCR7-BE1 group.In the invasion assay,the number of cells in control group and empty vector group that invade through Matrigel were(25.43±3.99) and(26±3.96).In CCR7-BE1 cells group the number of the cells that invade through Matrigel increased significantly(74.57±4.69,P=0.000). P-ERK_(1/2) protein expression and the number of invasion cells decreased significantly in the presence of either PD98059 or anti-CCR7 antibody.The results showed that CCR7-mediated migration and invasion of BE1 cells dependent on ERK_(1/2) activation.
2.1 CCR7 Up-Regulated VEGF-D in NSCLC Cell Lines:treatment of A549 cells with human CCL21 increase the expression' of VEGF-D mRNA and protein.Treatment of A549 cells with CCR7 antibody significantly attenuated the endogenous VEGF-D protein level.
Of the three cell lines tested,we found that CCL21 increased the expression of VEGF-D,and blocking CCR7 decreased the expression of VEGF-D,indicating that CCL21 could regulate VEGF-D via CCR7.
2.2 Involvement of ERK_(1/2) and Akt in CCR7-Mediated VEGF-D Up-Regulation: we detected p-ERK_(1/2),ERK_(1/2),p-Akt,Akt protein expressions in A549 cells.Western blotting showed that incubation of A549 cells with CCL21 increased the expressions of p-ERK_(1/2) and p-Akt,while exhibiting no effect on the total protein levels of ERK_(1/2) or Akt.Furthermore,Specific blocking CCR7 expression by anti-CCR7 Ab inhibited the expression of p-ERK_(1/2) and p- Akt.Furthermore,the VEGF-D mRNA and protein expression in A549 cells were decreased significantly after incubation with PD98059 or LY294002,respectively.However,in A549 cells,LY294002 did not inhibit ERK_(1/2) phosphorylation,and PD98059 did not affect phosphorylation of Akt under basal condition.
2.3 CCR7 Expression Correlate with VEGF-D Level,Lymphatic Vessel Density, Clinical Stages,Lymph Node Metastasis,and Patient Survival in NSCLC: Immunohistochemical analysis of 90 NSCLC specimens revealed that the VEGF-D expression was significantly associated with the expression of CCR7(r =0.455,P＜0.001).Quantification of the D2-40-positive vessels in the tumors indicated that tumor with high expression of CCR7 had a higher LVD than in low CCR7 expressing tumors (28.09±10.57 for CCR7 high expression tumors,n=63,and 20.58±7.6 for CCR7 low expression tumors,n=27,P=0.001).Tumors with VEGF-D high expression had a higher LVD than in low VEGF-D expressing tumors 30.14±9.87 for VEGF-D high expression tumors,n=54,and 19.38±7.22 for VEGF-D low expression tumors,n=36, P＜0.001).Patients with CCR7 high expression were more likely than low expression patients to have advanced disease(P=0.003) and lymph node metastasis(P=0.004). VEGF-D high expression patients were more likely than low expression patients to have advanced disease(P=0.005) and lymph node metastasis(P=0.001).For CCR7 low expression patients had a statistically significantly longer survival than CCR7 high expression patients(P=0.036).The median survival time for patients with tumors with high VEGF-D was 16±3.8 months;95%confidence interval 8.6-23.4 months, significantly shorter than that for patients with tumors with low VEGF-D expression (P=0.009).
Conclusions
1、CCR7 expression was correlated positively with the expressions of HIF-1αand HIF-2αin 94 cases NSCLC tissues.Each of them was significantly associated with lymph node metastasis and clinical pathological stage.
2、Hypoxia- HIF-1α,2α-CCR7-ERK1/2 pathway is likely to regulate the invasion and migration of lung cancer cells under hypoxic tissue microenvironments.HIF-1α, HIF-2αand CCR7 activation might play a role in promoting a more malignant phenotype of NSCLC and represents novel therapeutic strategies.
3、Acause-effect link between CCR7 over-expression and tumor lymphangiogenesis through VEGF-D activity was demonstrated in lung cancer.CCR7 could regulate VEGF-D expression through ERK1/2 and Akt signaling pathway.Our findings open newer therapeutic modalities against lymphangiogenesis and lymphatic borne。
引文
1 Kunz M,Ibrahim SM.Molecular responses to hypoxia in tumor cells.Mol Cancer 2003;2:23.
2 Pennacchietti S,Michieli P,Galluzzo M,Mazzone M,Giordano S,Comoglio PM.Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene.Cancer Cell 2003;3(4):347-61.
3 Semenza GL.Targeting HIF-1 for cancer therapy.Nat Rev Cancer 2003;3(10):721-32.
4 Hanahan D,Folkman J.Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell 1996;86(3):353-64.
5 Wood SM,Wiesener MS,Yeates KM,et al.Selection and analysis of a mutant cell line defective in the hypoxia-inducible factor-1 alpha-subunit (HIF-1 alpha).Characterization of hif-1 alpha-dependent and-independent hypoxia-inducible gene expression.J Biol Chem 1998;273(14):8360-8.
6 Zlotnik A,Yoshie O.Chemokines:a new classification system and their role in immunity.Immunity 2000;12(2):121-7.
7 Dieu MC,Vanbervliet B,Vicari A,et al.Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites.J Exp Med 1998;188(2):373-86.
8 Hirao M,Onai N,Hiroishi K,et al.CC chemokine receptor-7 on dendritic cells is induced after interaction with apoptotic tumor cells:critical role in migration from the tumor site to draining lymph nodes.Cancer Res 2000;60(8):2209-17.
9 Muller A,Homey B,Soto H,et al.Involvement of chemokine receptors in breast cancer metastasis.Nature 2001;410(6824):50-6.
10 Ding Y,Shimada Y,Maeda M,et al.Association of CC chemokine receptor 7 with lymph node metastasis of esophageal squamous cell carcinoma.Clin Cancer Res 2003;9(9):3406-12.
11 Wang J,Xi L,Hunt JL,et al.Expression pattern of chemokine receptor 6 (CCR6) and CCR7 in squamous cell carcinoma of the head and neck identifies a novel metastatic phenotype.Cancer Res 2004;64(5):1861-6.
12 Takeuchi H,Fujimoto A,Tanaka M,Yamano T,Hsueh E,Hoon DS.CCL21 chemokine regulates chemokine receptor CCR7 bearing malignant melanoma cells.Clin Cancer Res 2004;10(7):2351-8.
13 Shuyi Y,Juping D,Zhiqun Z,et al.A Critical Role of CCR7 in Invasiveness and metastasis of SW620 Colon Cancer Cell in Vitro and in Vivo.Cancer Biol Ther 2008;7(7).
14 Ishigami S,Natsugoe S,Nakajo A,et al.Prognostic value of CCR7 expression in gastric cancer.Hepatogastroenterology 2007;54(76):1025-8.
15 Kodama J,Hasengaowa,Kusumoto T,et al.Association of CXCR4 and CCR7 chemokine receptor expression and lymph node metastasis in human cervical cancer.Ann Oncol 2007;18(1):70-6.
16 Wilson JL,Burchell J,Grimshaw MJ.Endothelins induce CCR7 expression by breast tumor cells via endothelin receptor A and hypoxia-inducible factor-1.Cancer Res 2006;66(24):11802-7.
17 Giatromanolaki A,Koukourakis MI,Sivridis E,et al.Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival.Br J Cancer 2001;85(6):881-90.
18 Talks KL,Turley H,Garter KC,et al.The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2alpha in normal human tissues,cancers,and tumor-associated macrophages.Am J Pathol 2000;157(2):411-21.
19 Takanami I.Overexpression of CCR7 mRNA in nonsmall cell lung cancer:correlation with lymph node metastasis.Int J Cancer 2003;105(2):186-9.
20 Fan LF,Diao LM,Chen DJ,et al.[Expression of HIF-1 alpha and its-relationship to apoptosis and proliferation in lung cancer].Ai Zheng 2002;21(3):254-8.
21 Lau SK,Boutros PC,Pintilie M,et al.Three-gene prognostic classifier for early-stage non small-cell lung cancer.J Clin Oncol 2007;25(35):5562-9.
22 Liu YL,Yu JM,Song XR,Wang XW,Xing LG,Gao BB.Regulation of the chemokine receptor CXCR4 and metastasis by hypoxia-inducible factor in non small cell lung cancer cell lines.Cancer Biol Ther 2006;5(10):1320-6.
23 Gort EH,Groot AJ,van der Wall E,van Diest PJ,Vooijs MA.Hypoxic regulation of metastasis via hypoxia-inducible factors.Curr Mol Med 2008;8(1):60-7.
24 Redondo-Munoz J,Jose Terol M,Garcia-Marco JA,Garcia-Pardo A.Matrix metalloproteinase-9 is up-regulated by CCL21/CCR7 interaction via extracellular signal-regulated kinase-1/2 signaling and is involved in CCL21-driven B-cell chronic lymphocytic leukemia cell invasion and migration.Blood 2008;111(1):383-6.
25 Wang J,Zhang X,Thomas SM,et al.Chemokine receptor 7 activates phosphoinositide-3 kinase-mediated invasive and prosurvival pathways in head and neck cancer cells independent of EGFR.Oncogene 2005;24(38):5897-904.
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27 Bajetto A,Barbero S,Bonavia R,et al.Stromal cell-derived factor-1 alpha induces astrocyte proliferation through the activation of extracellular signal-regulated kinases 1/2 pathway.J Neurochem 2001;77(5):1226-36.
28 Billadeau DD,Chatterjee S,Bramati P,et al.Characterization of the CXCR4 signaling in pancreatic cancer cells.Int J Gastrointest Cancer 2006;37(4):110-9.
29 Jain RK.,Fenton BT.Intratumoral lymphatic vessels:a case of mistaken identity or malfunction? J Natl Cancer Inst 2002;94:417-421.
30 Mashino K,Sadanaga N,Yamaguchi H,et al.Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma.Cancer Res 2002;15:2937-41.
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34 Skobe M,Hawighorst T,Jackson DG,et al.Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med 2001;7:192-198.
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36 Herrmann E,Eltze E,Bierer S,et al.VEGF-C,VEGF-D and Flt-4 in transitional bladder cancer:relationships to clinicopathological parameters and long-term survival.Anticancer Res 2007;27:3127-33.
37 Maekawa S,Iwasaki A,Shirakusa T,et al.Correlation between lymph node metastasis and the expression of VEGF-C,VEGF-D and VEGFR-3 in Tl lung adenocarcinoma.Anticancer Res 2007;27:3735-41.
38 Van Iterson V,Leidenius M,von Smitten K,et al.VEGF-D in association with VEGFR-3 promotes nodal metastasis in human invasive lobular breast cancer.Am J Clin Pathol 2007;128:759-66.
39 Yonemura Y,Endo Y,Tabata K,et al.Role of VEGF-C and VEGF-D in lymphangiogenesis in gastric cancer.Int J Clin Oncol 2005;10:318-27.
40 Stearns ME,Wang M,Hu Y,et al.Expression of a flt-4 (VEGFR3) splicing variant in primary human prostate tumors.VEGF D and flt-4t (Delta773-1081) overexpression is diagnostic for sentinel lymph node metastasis.Lab Invest 2004;84:785-95.
41 Yasuoka H,Nakamura Y,Zuo H,et al.VEGF-D expression and lymph vessels play an important role for lymph node metastasis in papillary thyroid carcinoma.Mod Pathol 2005;18:1127-33.
42 Onogawa S,Kitadai Y,Tanaka S,et al.Expression of VEGF-C and VEGF-D at the invasive edge correlates with lymph node metastasis and prognosis of patients with colorectal carcinoma.Cancer Sci 2004;95:32-9.
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2 Pennacchietti S,Michieli P,Galluzzo M,Mazzone M,Giordano S,Comoglio PM.Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene.Cancer Cell 2003;3(4):347-61.
3 Semenza GL.Targeting HIF-1 for cancer therapy.Nat Rev Cancer 2003;3(10):721-32.
4 Hanahan D,Folkman J.Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell 1996;86(3):353-64.
5 Wood SM,Wiesener MS,Yeates KM,et al.Selection and analysis of a mutant cell line defective in the hypoxia-inducible factor-1 alpha-subunit (HIF-1 alpha).Characterization of hif-1 alpha-dependent and-independent hypoxia-inducible gene expression.J Biol Chem 1998;273(14):8360-8.
6 Zlotnik A,Yoshie O.Chemokines:a new classification system and their role in immunity.Immunity 2000;12(2):121-7.
7 Dieu MC,Vanbervliet B,Vicari A,et al.Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites.J Exp Med 1998;188(2):373-86.
8 Hirao M,Onai N,Hiroishi K,et al.CC chemokine receptor-7 on dendritic cells is induced after interaction with apoptotic tumor cells:critical role in migration from the tumor site to draining lymph nodes.Cancer Res 2000;60(8):2209-17.
9 Muller A,Homey B,Soto H,et al.Involvement of chemokine receptors in breast cancer metastasis.Nature 2001;410(6824):50-6.
10 Ding Y,Shimada Y,Maeda M,et al.Association of CC chemokine receptor 7 with lymph node metastasis of esophageal squamous cell carcinoma.Clin Cancer Res 2003;9(9):3406-12.
11 Wang J,Xi L,Hunt JL,et al.Expression pattern of chemokine receptor 6 (CCR6) and CCR7 in squamous cell carcinoma of the head and neck identifies a novel metastatic phenotype.Cancer Res 2004;64(5):1861-6.
12 Takeuchi H,Fujimoto A,Tanaka M,Yamano T,Hsueh E,Hoon DS.CCL21 chemokine regulates chemokine receptor CCR7 bearing malignant melanoma cells.Clin Cancer Res 2004;10(7):2351-8.
13 Shuyi Y,Juping D,Zhiqun Z,et al.A Critical Role of CCR7 in Invasiveness and metastasis of SW620 Colon Cancer Cell in Vitro and in Vivo.Cancer Biol Ther 2008;7(7).
14 Ishigami S,Natsugoe S,Nakajo A,et al.Prognostic value of CCR7 expression in gastric cancer.Hepatogastroenterology 2007;54(76):1025-8.
15 Kodama J,Hasengaowa,Kusumoto T,et al.Association of CXCR4 and CCR7 chemokine receptor expression and lymph node metastasis in human cervical cancer.Ann Oncol 2007;18(1):70-6.
16 Wilson JL,Burchell J,Grimshaw MJ.Endothelins induce CCR7 expression by breast tumor cells via endothelin receptor A and hypoxia-inducible factor-1.Cancer Res 2006;66(24):11802-7.
17 Giatromanolaki A,Koukourakis MI,Sivridis E,et al.Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival.Br J Cancer 2001;85(6):881-90.
18 Talks KL,Turley H,Garter KC,et al.The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2alpha in normal human tissues,cancers,and tumor-associated macrophages.Am J Pathol 2000;157(2):411-21.
19 Takanami I.Overexpression of CCR7 mRNA in nonsmall cell lung cancer:correlation with lymph node metastasis.Int J Cancer 2003;105(2):186-9.
20 Fan LF,Diao LM,Chen DJ,et al.[Expression of HIF-1 alpha and its-relationship to apoptosis and proliferation in lung cancer].Ai Zheng 2002;21(3):254-8.
21 Lau SK,Boutros PC,Pintilie M,et al.Three-gene prognostic classifier for early-stage non small-cell lung cancer.J Clin Oncol 2007;25(35):5562-9.
22 Liu YL,Yu JM,Song XR,Wang XW,Xing LG,Gao BB.Regulation of the chemokine receptor CXCR4 and metastasis by hypoxia-inducible factor in non small cell lung cancer cell lines.Cancer Biol Ther 2006;5(10):1320-6.
23 Gort EH,Groot AJ,van der Wall E,van Diest PJ,Vooijs MA.Hypoxic regulation of metastasis via hypoxia-inducible factors.Curr Mol Med 2008;8(1):60-7.
24 Redondo-Munoz J,Jose Terol M,Garcia-Marco JA,Garcia-Pardo A.Matrix metalloproteinase-9 is up-regulated by CCL21/CCR7 interaction via extracellular signal-regulated kinase-1/2 signaling and is involved in CCL21-driven B-cell chronic lymphocytic leukemia cell invasion and migration.Blood 2008;111(1):383-6.
25 Wang J,Zhang X,Thomas SM,et al.Chemokine receptor 7 activates phosphoinositide-3 kinase-mediated invasive and prosurvival pathways in head and neck cancer cells independent of EGFR.Oncogene 2005;24(38):5897-904.
26 Leelawat K,Leelawat S,Narong S,Hongeng S.Roles of the MEK1/2 and AKT pathways in CXCL12/CXCR4 induced cholangiocarcinoma cell invasion.World J Gastroenterol 2007;13(10):1561-8.
27 Bajetto A,Barbero S,Bonavia R,et al.Stromal cell-derived factor-1 alpha induces astrocyte proliferation through the activation of extracellular signal-regulated kinases 1/2 pathway.J Neurochem 2001;77(5):1226-36.
28 Billadeau DD,Chatterjee S,Bramati P,et al.Characterization of the CXCR4 signaling in pancreatic cancer cells.Int J Gastrointest Cancer 2006;37(4):110-9.
29 Jain RK.,Fenton BT.Intratumoral lymphatic vessels:a case of mistaken identity or malfunction? J Natl Cancer Inst 2002;94:417-421.
30 Mashino K,Sadanaga N,Yamaguchi H,et al.Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma.Cancer Res 2002;15:2937-41.
31 Schimanski CC,Schwald S,Simiantonaki N et al.Effect of Chemokine Receptors CXCR4 and CCR7 on the Metastatic Behavior of Human Colorectal Cancer.Clin Cancer Res 2005;11:1743-50.
32 Gao F,Lu YM,Cao ML,et al.Expression and quantification of LYVE-1 in human colorectal cancer.Clin Exp Med 2006;6:65-71.
33 Stacker SA,Caesar C,Baldwin ME,et al.VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.Nat Med 2001;7:186-191.
34 Skobe M,Hawighorst T,Jackson DG,et al.Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med 2001;7:192-198.
35 Makinen T,Veikkola T,Mustjoki S,et al.Isolated lymphatic endothelial cells transduce growth,survival and migratory signals via the VEGF-C/D receptor VEGFR-3.EMBO J 2001;20:4762-4773.
36 Herrmann E,Eltze E,Bierer S,et al.VEGF-C,VEGF-D and Flt-4 in transitional bladder cancer:relationships to clinicopathological parameters and long-term survival.Anticancer Res 2007;27:3127-33.
37 Maekawa S,Iwasaki A,Shirakusa T,et al.Correlation between lymph node metastasis and the expression of VEGF-C,VEGF-D and VEGFR-3 in Tl lung adenocarcinoma.Anticancer Res 2007;27:3735-41.
38 Van Iterson V,Leidenius M,von Smitten K,et al.VEGF-D in association with VEGFR-3 promotes nodal metastasis in human invasive lobular breast cancer.Am J Clin Pathol 2007;128:759-66.
39 Yonemura Y,Endo Y,Tabata K,et al.Role of VEGF-C and VEGF-D in lymphangiogenesis in gastric cancer.Int J Clin Oncol 2005;10:318-27.
40 Stearns ME,Wang M,Hu Y,et al.Expression of a flt-4 (VEGFR3) splicing variant in primary human prostate tumors.VEGF D and flt-4t (Delta773-1081) overexpression is diagnostic for sentinel lymph node metastasis.Lab Invest 2004;84:785-95.
41 Yasuoka H,Nakamura Y,Zuo H,et al.VEGF-D expression and lymph vessels play an important role for lymph node metastasis in papillary thyroid carcinoma.Mod Pathol 2005;18:1127-33.
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