2. 安庆医药高等专科学校, 安庆 246052;
3. 安徽医科大学第一附属医院肿瘤内科, 合肥 230032
2. Anqing Medical College, Anqing 246052, Anhui, China;
3. Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei 230032, Anhui, China
结肠癌是常见的消化道恶性肿瘤,存在于癌组织中的结肠癌干细胞(colon cancer stem cells,CCSCs)在肿瘤发生、增殖、转移以及化学抵抗过程中发挥重要作用。目前,大量研究主要集中在CCSCs标记物的筛选和确认方面,本文就近几年关于CCSCs生物学特性的相关机制作一综述,为临床结肠癌的诊断、治疗和预防提供参考。 1 CCSCs的特征
CCSCs是一小群具有无限增殖潜能的细胞,具有干细胞特性,可自我更新和形成不同分化程度的肿瘤细胞,目前已被认为是肿瘤发生、增殖、转移和药物抵抗的重要因素。了解CCSCs的特征有助于筛选分离CCSCs,从而进一步研究其与肿瘤相关的生物学行为。 1.1 CCSCs与非CCSCs之间的转化
细胞类型的可塑性在特定条件下可引起肿瘤干细胞和非干细胞之间的相互转化。Schwitalla等[ 1 ]发现在炎性肿瘤微环境中,核因子NF-κB通过活化的Wnt信号通路,诱导非干细胞去分化,从而使组织获得肿瘤发生的能力,而消除特定的肠上皮细胞RelA/p65(NF-κB亚单位)可阻断肠腺腺窝干细胞的扩增,表明体内炎症环境对CCSCs的产生和去分化具有重要作用。Yang等[ 2 ]则发现CCSCs和非CCSCs在正常和辐射环境中存在固有的相互转换和动态平衡,TGF-β1通过激活上皮间充质转化(epithelial-mesenchymal transition,EMT)在平衡中扮演重要角色。因此,通过干预特定条件下非CCSCs向CCSCs转化,可能对彻底清除癌细胞具有重要意义。 1.2 CCSCs标记物
大量研究显示结肠癌细胞株可表达CD133、CD29、CD24、CD166、Lgr5和ALDH[ 3,4,5 ] 等标记物,这些标记物的表达可能与Hes1基因表达上调有关[ 6 ]。具有这些标记物的细胞群具有干细胞特征,在体内外表现出显著的肿瘤生成、侵袭以及化学抵抗能力。进一步的研究发现,Lgr5抗体在正常组织中不被染色,在高分化肿瘤细胞中有单个或多达4个干细胞簇被染色,在低分化肿瘤中有9~81个干细胞簇被染色,提示Lgr5表达的改变与肿瘤等级的重构相关[ 7,8 ]。PHLDA1(pleckstrin homology-like domain,family A,member 1) 在结肠癌组织中表达也很广泛,在肿瘤侵袭的边缘常有增强表达和核定位。小干扰RNA(siRNA)介导的PHLDA1抑制可阻止癌细胞体外迁移、锚定非依赖性生长和体内肿瘤生成,提示PHLDA1可能也是潜在的人肠肿瘤细胞标记物[ 9 ]。另有研究发现,白介素-22(IL-22)通过激活转录因子STAT3以及促进H3K79组蛋白甲基转移酶DOT1L生成,诱导干细胞基因Nanog、Sox2、Pou5F1表达,维持肿瘤干细胞特征及致瘤潜能[ 10 ]。 2 CCSCs的增殖和转移 2.1 CCSCs增殖的相关机制
Yes相关蛋白(Yes-associated protein,Yap)作为一种转录共激活因子,其在癌组织中高表达是促使CCSCs增殖的必需因素。Zhou等[ 11 ]观察了71份结肠癌病例组织,发现有68份高表达Yap,36个结肠癌细胞株中有30个高表达Yap,结果引起干细胞群扩大,癌细胞增殖,而消除Yap可减弱β-catenin和Notch信号转导,抑制细胞增殖和生存。在 HCT116细胞株中,同样发现表皮生长因子(epidermal growth factor,EGF)对维持干细胞增殖是必需的,通过抑制EGFR自身磷酸化和下游信号通路蛋白AKT、ERK 1/2,可进而抑制CCSCs增殖,诱导CCSCs凋亡[ 12 ]。锌指转录因子GATA6是骨形成蛋白(bone morphogenetic protein,BMP)通路重要调节因子,Whissell等[ 13 ]发现其不但可促进CCSCs标记物Lgr5表达,而且还可抑制BMP信号通路,阻止肿瘤细胞分化,而敲除GATA6基因可提高BMP表达水平,阻断肿瘤干细胞自我更新,引发CCSCs终末分化、凋亡。此外,Lin等[ 14 ]发现ALDH(+)/CD133(+)CCSCs表达高磷酸化STAT3,姜黄素则可抑制这种改变,减少STAT3下游靶基因表达;Kantara等[ 15 ]则发现,通过RNA干扰抑制干细胞标记物DCLK1蛋白活性,同样可增强姜黄素抗肿瘤的特性,诱导CCSCs凋亡。
Shenoy等[ 16 ]研究了结肠炎向癌转化(colitis-to-cancer transition,CCT)的机制,从溃疡性结肠炎患者中分离了ALDH(+)CCSCs,发现Wnt/β-catenin信号通路的激活可使CCSCs表现出高度的克隆和致瘤潜能,促使CCT的发生。另有研究显示在肿瘤微环境中,炎性细胞因子IFN-γ和TNF-α可刺激小鼠C26 CCSCs群,通过缺氧诱导因子-1α(hypoxia-inducible factor 1α,HIF-1α)信号通路高表达血管内皮生长因子(vascular endothelial growth factor,VEGF),促进肿瘤血管生成,导致肿瘤生长[ 17 ]。而IL-6可通过影响Notch-1信号通路,增强HCT116和HT-29细胞株CCSCs的增殖能力[ 18 ]。这些研究提示炎症改变了肿瘤微环境,通过多种炎性因子与信号通路的交互作用,导致肿瘤细胞逃避免疫监视,最终引起肿瘤细胞增殖、转移和复发。 2.2 CCSCs转移的相关机制
高迁移率蛋白A1(high mobility group A1,HMGA1)是结肠癌细胞转移分子机制中关键的转录因子。Belton等[ 19 ]发现在转基因小鼠中HMGA1可诱导CCSCs表达Twist1蛋白,抑制E-cadherin的表达,促进肿瘤转移发生,而HMGA1的敲除可阻断肿瘤细胞的锚定非依赖性生长,抑制体内肿瘤肝转移的发生。Pasto等[ 20 ]发现Delta样配体4(DLL4)激活Notch信号通路,可导致干细胞标记物MUSASHI(MSI-1)表达增加,而使用Notch2/3抗体可降低MSI-1水平,增加Notch通路抑制因子NUMB的表达,进而抑制肿瘤的转移。Zubeldia等[ 21 ]将Mc38-luc(TGF-β1)细胞(colon adenocarcinoma cells expressing luciferase pretreated with TGF-β1)注射到小鼠脾脏,发现TGF-β1除了可促进原发肿瘤的生长,也促使了癌细胞的肝转移。这可能与TGF-β1引起Mc38细胞Smad2、Smad3和Smad1/5/8活化,增强细胞侵袭和跨内皮迁移能力有关。 3 CCSCs与化学治疗抵抗相关的机制
化学抵抗是结肠癌药物治疗效果低下的主要原因。Colak等[ 22 ]研究显示CCSCs发生选择性化学治疗抵抗与线粒体诱导效应降低有关,通过使用BH3类似物ABT-737和WEHI-539可抑制抗凋亡蛋白Bcl-xL活性,降低线粒体内膜去极化阈值,提高化学治疗敏感性,增强化学治疗效果。Van Houdt等[ 23 ]发现凋亡蛋白抑制剂BIRC6是结肠癌伴肝转移患者CCSCs对奥沙利铂和顺铂抵抗的重要调节因子,针对BIRC6的治疗有助于消除CCSCs。另有研究显示,表达同源异型盒基因cdx1的SW1222细胞株对紫杉醇诱导的细胞毒性有显著抵抗,这与cdx1、Bcl-2的表达、caspase-3活力、LC3-Ⅱ/LC3-Ⅰ 的比例上调有关,cdx1的静默和溶酶体抑制剂bafilomycinA的治疗能提高紫杉醇诱导的细胞毒性,瘤内注射cdx1 siRNA能显著抑制接种了cdx1癌细胞的移植瘤生长[ 24 ]。Inoda等[ 25 ]将表达特异肿瘤抗原蛋白Cep55/c10orf3_193 的细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)克隆41过继转移,可显著抑制对化学治疗药物伊立替康、依托泊甙抵抗的SW480细胞株CCSCs的增殖,提示以Cep55/c10orf3_193蛋白为基础的癌症疫苗治疗或者CTL过继转移是治疗有化学抵抗CCSCs的一种可行途径。
4 MicroRNAs(miRNAs)对CCSCs的调控
MiRNAs是一种内源性非编码RNA,通过结合靶基因mRNA的3′-UTR在转录后水平调节基因表达。Fang等[ 26 ]测定了SW1116细胞株 CD133(+)/CD44(+)CCSCs中31种上调和31种下调的miRNAs,如miR29a、miR29b、miR449b、miR4524等,基因本体和通路分析显示这些表达差异与CCSCs的细胞周期功能、细胞分化、信号通路、细胞骨架蛋白、细胞基质黏附紧密相关,提示miRNAs在调控CCSCs增殖、分化和转移等方面扮演重要角色。
Ascl2 是一种bHLH(basic helix-loop-helix)转录因子,在结肠癌组织中显著表达,Zhu等[ 27 ]在HT-29细胞株CCSCs中使用shRNA干扰导致Ascl2表达下降,减弱了癌细胞集落形成、侵袭迁移能力,表达干细胞特征的相关基因CD133、Sox2、Oct4、Lgr5、Bmi1和C-myc水平也显著降低。而通过miR-302b mimic可恢复shRNA-Asc12/HT-29肿瘤球形成、侵袭等细胞行为以及干细胞特征相关基因的表达,提示Ascl2可能是抑制CCSCs的潜在靶点,miR-302b在这个过程中发挥重要作用。在HCT-15CD44(+)CCSCs中,Ju等[ 33 ]发现通过激活透明质酸 (hyaluronan,HA)/CD44信号通路,可促使转录因子Snail与miR-203启动子区E-盒(E-box)结合,引起miR-203表达下调,导致CD44(+)CCSCs呈现明显的干细胞特征以及侵袭、转移、分化能力。Xu等[ 29 ]通过联机miRNA靶点预测数据库分析发现,肿瘤抑制因子RhoBTB1是miR-31的一个新靶点,在人结肠癌HT-29 CCSCs中RhoBTB1被miR-31直接抑制而显著降低表达,提示miR-31与结肠肿瘤发生相关。miR-21在不同条件下可发挥致癌基因或抑癌基因双重功能,但在CCSCs中的作用却并不明确。 Yu等[ 30 ]通过稳定转染使富含CCSCs的细胞株HCT116过度表达miR-21,发现可引起TGF-β2(transforming growth factor beta 2)受体(TGF-βR2)下调,降低了与miR-21结合的TGF-βR2 3′-UTR活性,导致肿瘤球形成能力增强,提示miR-21可通过TGF-βR2发挥致瘤作用。同样在SW116CCSCs中,Yu等[ 31 ]发现miR-93类似物可通过Wnt、NF-κB、Notch信号通路的相互作用显著降低组蛋白去乙酰化酶8(histone deacetylase8,HDAC8)和TLE4 mRNA和蛋白水平,从而抑制CCSCs增殖。
葡萄糖调节蛋白78(GRP78)是一种重要的内质网分子伴侣和信号调节因子,在肿瘤中常过度表达,导致肿瘤发生和化学抵抗。Su等[ 32 ]通过慢病毒转染技术,使结肠癌HCT116细胞株中miR-30d、 miR-181a、 miR-199a-5p表达增高,发现它们可共同作用于GRP78的3′-UTR,导致GRP78表达下调,诱导肿瘤细胞凋亡,增强肿瘤细胞对化学治疗药物的敏感性。Bitarte等[ 32 ]观察了具有干细胞特征的结肠癌球,发现miR-451表达下调不但引起靶基因COX-2 和巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)表达增高,激活Wnt信号通路,促使CCSCs增殖,同时还上调ATP 结合盒 B 亚家族成员1(ABCB1)基因表达,导致癌细胞对伊立替康不敏感,提示miR-451也是抑制结肠癌发生和药物抵抗的一个重要因素。 5 结 语
CCSCs在结肠癌发生发展的各个阶段均发挥重要作用。虽然目前对于CCSCs标记物的研究取得较大进展,但对导致肿瘤干细胞增殖、转移、化学抵抗等生物学行为的相关机制还不甚清楚,可能涉及到肿瘤微环境、细胞器、信号通路的交互影响以及基因突变等诸多因素。进一步深入研究影响CCSCs生物学行为的细胞分子机制将有助于临床提高肿瘤诊断、治疗效果,为预测肿瘤预后提供新的标记物。而关于miRNAs对CCSCs生物学行为调控机制的研究则为阐明肿瘤发病机制提供了新途径。
6 利益冲突
所有作者声明本文不涉及任何利益冲突。
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