2015, Vol. 50
据统计,乳腺癌已成为全球女性恶性肿瘤之首,发病率和致死率均高居首位。乳腺癌的转移和侵袭 是其致死的主要原因。随着对乳腺癌的深入研究,人们逐渐认识到上皮-间质转化 (epithelial-mesenchymal transition,EMT) 在乳腺癌发生及转移中的重要作用。为了侵袭正常组织和迁移至其他器官,肿瘤细胞
往往需要通过EMT丢失极性和细胞间接触等上皮细胞样表型特征,从而获得迁移和侵袭能力[1]。而在乳腺癌中,超过95% 的癌细胞来源于上皮细胞。乳腺癌上皮细胞的转移、乳腺导管原位癌发展为浸润性乳腺癌、乳腺癌治疗效果差及患者对化疗药物产生耐药性等方面与EMT过程密切相关[2]。此外,临床研究也发现临床样本中乳腺癌细胞发生了明显的EMT样 变化。
TGF-β信号通路是广泛认可的介导调控EMT过程的信号通路。所以,研究TGF-β信号通路在EMT中的调控机制对于了解乳腺癌的转移侵袭机制及确定治疗方案意义重大。该信号通路主要通过Smad依赖型和非依赖型信号通路调控下游转录因子,参与EMT过程的调控。本文就Smad依赖型信号通路的信号分子TGF-βs、Smad蛋白和转录因子 (表 1) 以及Smad非依赖性信号通路参与调控乳腺癌上皮-间质转化的研究进展予以综述。
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表 1 参与调控乳腺癌EMT的主要信号分子 |
若干生长因子被证实与EMT过程的发生有关,而TGF-β是最早被发现能够在小鼠正常乳腺上皮细胞NMuMG细胞中诱导EMT过程发生的生长因子[3, 4],随后又证实其在肿瘤细胞EMT的发生过程中也发挥重要作用[5]。TGF-β超家族包括将近30多种蛋白,如TGF-βs、activins、inhibins、nodal、myostatin和骨形成蛋白 (bone morphogenetic protein,BMPs) 等[6]。本文中的TGF-β生长因子主要包括TGF-β1、TGF-β2和TGF-β3。TGF-β1在乳腺癌EMT过程中的作用研究得最为广泛,仅有少量研究乳腺上皮细胞EMT的文献涉及TGF-β2和TGF-β3。TGF-β具有多功能生物学活性[7],除调控发育、增殖及免疫应答的作用外,在肿瘤发生过程中有着双重作用: 在早期通过诱导凋亡和细胞周期阻滞发挥肿瘤抑制作用,而在后期能促进肿瘤细胞的转移[8]。TGF-β的促癌作用通常与其诱导EMT过程同步发生,促进肿瘤细胞的迁移和侵袭。在乳腺癌的体外实验中,TGF-β I型受体持续 活化,即TGF-β信号通路持续活化,转基因小鼠模型中血管外肺转移能力增强; 当敲除TGFβ II型受体 后,即TGF-β信号通路持续抑制,原位瘤的生长和转移瘤的侵袭受到抑制[9]; 另外,TGF-β还能够促进其他小鼠模型中乳腺癌细胞的骨转移[10]。在乳腺癌细胞中,TGF-β能通过Smad信号通路诱导Angptl4促 进肿瘤细胞转移至肺部; 当该信号通路活性受到抑制时,ER-乳腺癌细胞系的肺转移能力下降[11]。除体外实验外,EMT发生的相关蛋白上皮细胞样标志蛋白E-cadherin、Cytokeratins,间质细胞样标志蛋白N-cadherin、Vimentin,调控细胞紧密连接的ZO、Claudins、Occludins,桥粒斑蛋白Desmoplakin以及基质金属蛋白酶MMP2、MMP9等蛋白均为TGF-β调控的下游靶蛋白[12]。因此,TGF-βs被认为在EMT发生过程中起着主导作用。
2 Smad蛋白与EMTTGF-β通过Smad依赖型信号通路,即Smad信号通路参与调控EMT过程,该信号通路的传导需要多种蛋白共同完成。细胞膜表面存在有TGF-βs特有的受体四聚体复合物,包括两个I型受体ALK-5,即TβR-I和两个II型受体TβRII。II型受体是持续活化状态,但当受体与配体结合时,其磷酸化I型受体 GS结构域丝/苏氨酸残基,使I型受体活化,继而识别并磷酸化下游Smads蛋白末端SSXS序列。Smads可进一步分为3种: 受体调节型Smads (R-Smads)、共同通用型Smads (Co-Smads) 以及抑制型Smads (I-Smads)[6]。R-Smads包括Smad2和Smad3,二者均由两个高度保守的多肽片段MH1 (the Mad homology 1) 和MH2片段以及连接多肽组成[13]。在受体失活状态下,非磷酸化Smad2和Smad3以单体形式存在; 当C-末端丝氨酸被TβR-I磷酸化后,磷酸化的Smad2和Smad3形成同源寡聚体,然后迅速与Smad4结合形成异源寡聚体[14, 15],该寡聚体转入核内后,通过与转录因子相互结合活化或抑制相关基因的转录。另 外在该信号通路中,还有抑制型的Smad7参与,与Smad2和Smad3竞争性结合ALK-5,起抑制TGF-β信号通路的作用[16]。
传统意义上,Smad2/3是通过C-末端丝氨酸的磷酸化,与Smad4结合并转入细胞核,传递生长抑制 信号,被认为是常规Smads途径; 而最新的研究表明Smad2/3连接多肽的某些丝、苏氨酸位点能被ERK (extracellular signal-regulated kinase)、JNK (c-Jun N- terminal kinase)、p38、CDK (cyclin-dependent kinase) 及GSK (glycogen synthase kinase) 等激酶磷酸化,与Smad4结合转入细胞核,协同或者拮抗Smad2/3 C-末端丝氨酸的磷酸化,传递促癌信号,被认为是非常规Smads途径[17]。研究指出,Ras过度活化能组成性活化小鼠胃上皮细胞中JNK信号通路,促进Smad2/3 Linker的磷酸化,通过上调AP1和MMPs,促进肿瘤侵袭。非常规Smads途径能传递迁移和侵袭信号,这提示pSmad2/3L途径与EMT过程有关[18]。在肺癌细胞中,JNK1能促进TGF-β1诱导的EMT作用,当受ERK激酶调控的Smad3连接多肽磷酸化位点突变失活时,JNK1的作用受到抑制; 反过来,磷酸化位点持续激活,能增强JNK1促进EMT相关基因转录的作用[19]。虽然已有详尽的研究指出Smad2/3 Linker磷酸化异构体对该信号通路传递信号的影响,但是关于Smad2/3磷酸化异构体与乳腺癌细胞中EMT过程之间关系的报道较少,有待深入研究。
Smad2和Smad3蛋白在TGF-β调控乳腺癌EMT过程中作用显著。Smad2和Smad3过表达都能够在乳腺上皮细胞中诱导EMT过程的发生[4, 20]。由于Smad2和Smad3蛋白高度同源,其突变株均能不同程度地干扰另一种蛋白的活性,所以无法比较Smad2或Smad3两种蛋白的重要性。Valcourt等[20]发现单独过表达Smad2或Smad3并不足以引起乳腺上皮细胞发生EMT过程,这可能说明这两种蛋白对Smad信号通路诱导EMT过程来说都是必需的。在皮肤癌细胞中,Smad2敲除显著促使细胞发生EMT过程[21]。而在乳腺癌细胞中,Smad2敲除的MDA-MB-231细胞的侵袭性显著增强; Smad3的敲除阻碍肿瘤细胞侵袭转移[22]。因此,Smad2很有可能在乳腺癌中起肿瘤抑制因子的作用。另外,在乳腺癌MCF10A细胞中,抑制内源性Smad3的功能明显抑制小鼠肺转移斑的形成; 而过表达Smad3 C端片段能够增强TGF-β诱导的转移[22, 23]。Dzwonek等[24]进一步提出Smad3对于TGF-β诱导的EMT来说,比Smad2更重要。在小鼠乳腺Nme上皮细胞中,Smad3下敲能干扰TGF-β引起的细胞形态、细胞连接、细胞骨架以及MMPs的表达变化; 而Smad2下敲并不影响TGF-β引起的一系列改变。在不加TGF-β诱导的情况下,Smad2或Smad3的下敲均不能影响细胞形态、细胞连接和细胞骨架。这些报道提示,Smad3在乳腺肿瘤的生长和转移过程中起关键作用。尽管有大量研究显示Smad2与Smad3在乳腺癌中发挥作用,这二者在TGF-β诱导乳腺癌EMT过程中的明确地位和具体机制还有待进一步研究。
Smad4对于TGF-β调控的EMT过程,也是不可或缺的。在NMuMG细胞系NM18和NM14细胞中敲除Smad4后,TGF-β不能诱导EMT过程的发生; 在体内实验中,下敲Smad4能够抑制MDA-MB-231细胞向骨转移[25]。共同通用型的Smad4不仅参与了TGF-βs诱导的EMT调控,也参与了BMPs诱导的EMT调控过程,所以无法单独研究Smad4对TGF-βs信号通路的调控作用。与之类似,Smad7既是TGF-β信号通路也是BMP信号通路的拮抗剂。高表达Smad7后,TGF-βs不能诱导乳腺上皮细胞发生EMT过程[26]; Smad7高表达还能抑制乳腺癌细胞转移至肺或肝[27]。Papageorgis等[28]发现Smad7过表达能诱导乳腺癌MIII细胞表达表观遗传沉默的上皮基因,获得上皮样细胞表型并且抑制间质样MCF10CA1h细胞的迁移和侵袭。另有研究表明,miR-106b-25基因簇在乳腺癌细胞中,以Smad7为靶点提高I型受体的表达水平,促进TGF-β信号通路的活化,从而引起EMT过程[29]。这些研究均表明Smad7能作为乳腺癌细胞EMT过程的抑制因子。
3 转录因子与EMTSmads蛋白是通过与DNA结合活化或抑制下游转录因子的转录而发挥对乳腺癌EMT的调控作用。这些TGF-β信号通路下游的转录因子主要包括三大转录因子家族: Snail、ZEB和碱性螺旋-环-螺旋(basic helix-loop-helix,bHLH) 家族。
Snail家族是研究最为广泛的与EMT有关的效应蛋白。Snail家族中,Snail1 (Snail) 和Snail2 (Slug) 均在乳腺癌中高表达[30, 31, 32],且与TGF-β调控的EMT有关。不同的是Snail1几乎涉及到所有EMT过程[33, 34],在乳腺上皮细胞中与Smad3和Smad4形成复合物,抑制TGF-β诱导的EMT过程中Occludin、E-cadherin等蛋白的表达[35]; Snail2虽然比Snail1表达更广泛,但被证实在某些情况下与EMT过程无关[34, 36]。Snail1和Snail2均能抑制编码上皮标志蛋白E-cadherin的 基因CDH1的表达,但E-cadherin的下调并不总是与Snail1相关,这表明可能存在Snail1的辅助抑制因子。Dong等[37]发现Snail-G9a-Dnmt1复合物抑制果糖-1,6-双磷酸酯酶 (fructose-1,6-biphosphatase,FBP1) 的表达,而后者的缺失是发生EMT并引起管腔样向基底样乳腺癌细胞转变所必需的一个环节,与患者的低存活率相关。这些提示,Snail复合物可作为治疗基底样乳腺癌的潜在靶点。Snail除了能抑制上皮标志蛋白E-cadherin、Claudin-1、Occludin和Cytokeratin的表达外,还能促进Fibronectin、Vitronectin和N-cadherin等间质标志蛋白的表达[12],因此能作为参与TGF-β调控EMT过程中极为重要的转录因子。
ZEB家族是TGF-β通路下游的另一转录因子,包括ZEB1 (又称为δ EF1或AREB6) 和ZEB2 [又称为Smad相互作用蛋白1 (Smad-interacting protein 1,SIP1)]。ZEB1和ZEB2在乳腺癌细胞中均过表达[31],二者与Smad3/Smad4复合物相互作用,共同调控下游基因的表达[38]。TGF-β能够促进ZEB1和ZEB2的表达,抑制E-cadherin的表达。同时沉默这两种转录因子能完全抵消TGF-β对E-cadherin的抑制作用,而间质标志蛋白,如Fibronectin、N-cadherin和Vimentin则不受ZEB1和ZEB2沉默的影响。另外,Ets1蛋白下调时抑制TGF-β诱导的ZEB1和ZEB2表达,提示Ets1可能作为ZEB1和ZEB2上游转录调控因子发挥作用[39]。在鼠乳腺上皮细胞中,ZEB不依赖于Snail转录因子,直接抑制E-cadherin的表达,这也进一步表明ZEB家族是E-cadherin的直接抑制因子[39, 40]。ZEB1与非编码小RNA microRNA-200之间存在反 馈回路: ZEB2直接抑制miR-200家族miR-141和miR-200c的表达,强烈激活乳腺癌等肿瘤上皮细胞的上皮分化; 而ZEB1和EMT激动剂TGF-β2是这些microRNAs调控的主要下游靶点[41]。
bHLH家族也能参与调控EMT过程。HLH家族分子的基本结构包括由一个环连接的两个平行的α螺旋形成的二聚体。基于组织分布、二聚化特性以及DNA结合特异性,分成七大类[42]。类型I蛋白E12和E47,类型II蛋白Twists和类型V蛋白Ids参与EMT过程的调控。Twists是转移癌和浸润性癌症的重要调控因子[43],在人乳腺癌中过表达[44]。Twists的活性形式为二聚体形式。在乳腺癌细胞系中,Twist能与E-cadherin启动子结合,抑制E-cadherin的表达[45]。除了E-cadherin外,Twist1和Twist2还能抑制Occludin和Claudin-7的表达,促进Vimentin和N-cadherin的表达,增强迁移和侵袭[43, 46]。另外,Twist1和Twist2 可与H-Ras协同,共同调控Ras转化的MCF10A细胞的EMT过程[46]。Ids是bHLH家族的负性转录因子,能调控Twist蛋白的表达; 当其表达被TGF-β抑制时,其对Twist的抑制作用解除,从而诱导EMT过程[47, 48]。在乳腺癌的肺转移过程中,ID基因的参与被认为是不可或缺的。由于Twist1是Id1的靶蛋白,Id1能够促使间质形态依赖于Twist1的乳腺癌细胞发生间质上皮转化 (mesenchymal to epithelial transition,MET); 而Id1下调能抑制MET过程并且显著抑制乳腺癌细胞的肺转移[49]。
除了上述三大转录因子家族外,还发现许多调控EMT的新转录因子。HMGA2 (high mobility group A2) 是参与TGF-β调控EMT过程中的下游效应元件。TGF-β能通过Smad3/Smad4依赖型通路促进HMGA2的表达上调,而HMGA2的异常表达又能引起Snail1/2和Twist1的表达,从而参与调控EMT过程[50]。SOX转录因子中SOX4被认为是TGF-β调控的另一转录因子,参与调控TGF-β诱导的人乳腺上皮细胞EMT过程[51]。在人乳腺癌临床样本中,SOX4异常高表达,可作为三阴性乳腺癌的潜在生物标志[52]。其他一些转录因子包括FOX转录因子和GATA家族等,也与EMT的调控过程有关,但他们与TGF-β信号通路下游Snail、Twist和Zeb转录因子之间的关系和参与EMT调控的具体作用机制尚不明确[53]。
4 Smad非依赖型信号通路与EMTTGF-β除了通过Smad信号通路参与调控EMT外,还能通过其他Smad非依赖型信号通路参与调控EMT过程。
丝裂原活化蛋白激酶 (mitogen activated protein kinase,MAPK) 和TGF-β信号通路之间存在相互作用。TGF-β能通过p38/MAPK和ERK/MAPK信号通路促进H-Ras介导的细胞迁移和侵袭[54]。在NMuMG细胞中,TGF-β1提高ERK的磷酸化水平和ERK激酶的活性; 当加入MEK抑制剂抑制ERK的磷酸化和激酶活性后,TGF-β1丧失诱导EMT的作用。结果说明TGF-β1在体外诱导EMT过程需要ERK信号通路的活化[55]。随着对作用机制的深入研究,Lee等[56]发现TGF-β通过ShcA调控ERK/MAPK信号通路。I型受体与ShcA结合,磷酸化其酪氨酸和丝氨酸位点。磷酸化的酪氨酸为GRB2和SOS提供对接位点,从而启动ERK/MAPK信号通路。除此之外,构建缺失ShcA酪氨酸磷酸化位点的突变体后,发现3个已知的酪氨酸磷酸化位点的缺失能完全抵消TGF-β对乳腺癌细胞运动和侵袭的促进作用[57],这提示ShcA有可能参与TGF-β调控EMT过程。
除MAPK信号通路外,乳腺癌中NF-κB信号通路也能够与TGF-β信号通路共同调节乳腺癌EMT过程的发生发展[58]。在Ras转化的乳腺癌细胞系EpRas细胞中,TGF-β诱导EMT过程依赖于NF-κB信号通路: TGF-β联合NF-κB使EpRas细胞更容易发生EMT; 抑制NF-κB信号通路活性能阻断TGF-β诱导的EMT作用; 体内实验证实抑制NF-κB信号通路的活性能抑制TGF-β诱导的EMT引起的肺转移[59]。这说明NF-κB信号通路与TGF-β、Ras信号通路之间存在某种合作。借助TAK1/IKKβ复合物,乳腺癌的发生能将TGF-β从NF-κB活性的抑制剂转化成激动剂。在正常的乳腺上皮细胞中,TGF-β诱导EMT,促使TAB1并入TAK1:IKKβ复合物。只有在TGF-β诱导下发生EMT的乳腺上皮细胞或恶性的乳腺癌上皮细胞中,该复合物才能介导TGF-β诱导NF-κB活性[60]。这些提示NF-κB信号通路有可能在乳腺癌后期参与TGF-β的调控活动。
TGF-β与PI3K/Akt信号通路之间存在交叉作用: TGF-β1能磷酸化Akt/PKB,活化Akt信号通路; 而PI3K/Akt信号通路参与TGF-β信号通路介导的乳腺上皮细胞EMT过程。当PI3K/Akt信号通路受抑制时,TGFβ-1诱导的Smad2磷酸化和促乳腺癌细胞迁移作用均受到抑制[61]。Xue等[62]发现Akt/PKB介导Twist1磷酸化,通过调控TGF-β2转录,活化TGF-βR/Smad2信号通路,诱导EMT并促进乳腺癌转移,继而通过反馈回路维持PI3K/Akt信号通路的高度活化。最近的研究指出Akt2是TGF-β诱导EMT过程中重要的调控因子[63, 64]。
雷帕霉素靶蛋白 (mammalian target of rapamycin,mTOR) 信号通路是新发现的参与TGF-β调控EMT过程的Smad非依赖型信号通路。在小鼠乳腺上皮细胞中,mTORC1 (mTOR complex 1) 抑制剂雷帕霉素,在不改变EMT表型的情况下,能够抑制TGF-β诱导的EMT过程中的迁移、黏附和侵袭[65]。与mTORC1类似,mTORC2 (mTOR complex 2) 调控TGF-β诱导的EMT; 抑制mTORC2活性并不能改变上皮细胞 表型,但能够减弱TGF-β诱导的EMT相关基因表达、细胞迁移、侵袭和细胞骨架重建等方面的变化[66]。mTORC2有可能通过调控RhoA的活性参与TGF-β诱导的EMT过程中的骨架重建。所以说,mTOR信号通路的活化在TGF-β诱导的EMT过程中发挥重要的作用。
5 小结正如前面所述,TGF-β的确能诱导乳腺上皮细胞以及乳腺癌上皮样细胞发生EMT过程,并且通过Smad依赖型信号通路或者Smad非依赖型信号通路调控该过程的发生发展 (图 1)。TGF-β对乳腺癌的促癌作用可能体现在: TGF-β具有通过EMT过程产生肿瘤干细胞从而启动癌变的能力[67]; TGF-β介导的EMT过程促进乳腺癌转移和侵袭,而转移和侵袭是乳腺癌致死及复发的最主要原因[68]; EMT与乳腺癌耐药相关,特别是TGF-β诱导的EMT的发生与乳腺癌细胞获得对化疗药物的耐药性密切相关[69, 70, 71]。因此,可以将拮抗TGF-β信号通路作为潜在的治疗方法来阻止乳腺癌EMT过程,从而控制其癌变、转移、侵袭以及复发。目前,TGF-β疗法有多种给药策略,包括: 联合给药、大分子抑制剂、小分子TGF-β RI激酶抑制剂、反义化合物以及TGF-β特异性中和抗体等[72]。随着诊断手法的进步和乳腺癌模型的建立,对乳腺癌中TGF-β信号通路及其参与EMT调控机制的深入研究,TGF-β疗法必将作为乳腺癌治疗手段中的一种且发挥重要作用。
|
图 1 TGF-β诱导乳腺癌EMT的主要信号通路 |
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