Some recent progress on breast cancer stem cells

doi:10.3969/j.issn.2095-4344.2017.21.022

Abstract

Abstract:

BACKGROUND: With the in-depth exploration of the mechanism of breast cancer occurrence and increasing attention has been paid to breast cancer stem cell development.
OBJECTIVE: To review the main progress in the characteristics, related signaling pathways, epithelial- mesenchymal transition, breast cancer metastasis and tumor microenvironment about breast cancer stem cells.
METHODS: A computer-based retrieval of PubMed database was performed in order to search relevant articles published from 2001 to 2017, using the keywords of “breast cancer, stem cells, signaling pathway, epithelial- mesenchymal transition, tumor microenvironment”. After removal of repetitive or irrelevant articles, 60 articles were finally reviewed.
RESULTS AND CONCLUSION: The tumor stem cell theory explains the root cause of tumor metastasis and recurrence, and provides a new insight into the tumor treatment. With the further study on the molecular pathogenesis, characteristics and related molecular regulation mechanisms of breast cancer stem cells, the role of breast cancer stem cells is explicated in the development and progression of breast cancer, which is of great significance in the tumor treatment targeting breast cancer stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Breast Neoplasms, Neoplastic Stem Cells, Tissue Engineering

CLC Number:

R318

Luo Zhi-ping, Lau Clara Bik-san, Tan Ning-hua. Some recent progress on breast cancer stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3414-3419.

Figures/Tables 1

2.1 乳腺癌干细胞的基本特征 2006年，美国癌症研究协会定义肿瘤干细胞为存在于实体肿瘤组织中具有无限自我更新能力、多向分化潜能以及高度致瘤性等特性的细胞亚群[5]。肿瘤干细胞的来源主要有4个方面：①正常成体干细胞、祖细胞或体细胞表型改变或基因突变所致；②病毒的某些蛋白影响了某些癌基因及抑癌基因；③受刺激后的静止的胚胎干细胞；④致癌突变细胞与干细胞的融合[6]。肿瘤干细胞学说的提出具有重要意义：①有利于肿瘤预防及治疗方法的转变；②有利于肿瘤的风险评估、早期检测及肿瘤预防；③有助于开发靶向肿瘤干细胞的药物，为癌症治疗提供新的思路[7]。 2003年，Al-Hajj等[8]提出乳腺癌干细胞是乳腺癌的起源，并且首次分离得到实体乳腺癌干细胞；从乳腺癌组织中分离到的CD44+/CD24-乳腺癌干细胞能在重症非肥胖型糖尿病联合免疫缺陷小鼠(NOD/SCID)体内形成肿瘤，并且在小鼠瘤体中发现同种CD44+/CD24-细胞亚群，该细胞也可在NOD/SCID小鼠体内成瘤，此研究证实了乳腺癌组织中乳腺癌干细胞的存在，引发了肿瘤干细胞研究的热潮。 2.2 乳腺癌干细胞的分选和鉴定 2.2.1 表面标志物分选法不同类型的肿瘤细胞表面表达有不同的肿瘤干细胞分子标志物，该标志物被广泛应用于鉴定和分选肿瘤干细胞[9]。表面标志物分选法的原理是利用抗原抗体的相互结合，使用流式细胞仪或者免疫磁珠将带有干细胞表面分子标志物的细胞与其他细胞分离，从而得到肿瘤干细胞亚群[10]。Al-Hajj等[8]将从乳腺癌实体肿瘤组织中分选出的CD44+/CD24-细胞接种到NOD/SCID小鼠体内，发现仅需200个CD44+/CD24-细胞就能使小鼠成瘤。该研究成为使用细胞表面标志物分选乳腺癌干细胞的基础，CD44+/CD24-为乳腺癌干细胞最常用的表面标志物。 2.2.2 侧群细胞分选法侧群细胞是指能够将脂溶性DNA结合染料排出细胞外的一小群细胞，在使用流式细胞仪分选的散点图中，这些细胞位于大多数细胞的一侧；研究发现，肿瘤中的侧群细胞具有干细胞样特性[11]。Patrawala等[12]从人实体肿瘤组织中使用侧群细胞分选技术分离得到肿瘤干细胞；其中乳腺癌细胞系MCF-7肿瘤干细胞侧群数约占总细胞数的0.2%，侧群细胞在小鼠体内成瘤所需细胞数比非侧群细胞更少，成瘤性更强，成瘤周期短。Luminal型乳腺癌中的侧群细胞具有致瘤性，Luminal型MCF-7细胞中的侧群细胞能够在NOD/SCID小鼠体内成瘤[13]。 2.2.3 醛脱氢酶(aldehyde dehydrogenase，ALDH)活性测定分选法该方法鉴定肿瘤干细胞的依据是细胞中醛脱氢酶的活性[14]。活化的醛脱氢酶能够将不带电荷的醛脱氢酶底物BAAA转化为荧光产物BAA，高表达醛脱氢酶的细胞会呈现明亮荧光，此细胞可以使用流式细胞仪进行鉴定和分离[15]。Ginestier等[14]首次发现ALDH-1阳性肿瘤细胞与肿瘤不良预后有密切关系。Croker等[16]研究发现ALDH-1活性高的乳腺癌具有更强的侵袭和转移能力。ALDH异构体ALDH1A3的表达影响乳腺癌患者肿瘤干细胞中ALDH的活性，从而影响肿瘤的复发转移[17]。ALDH-1阳性细胞与恶性肿瘤的转移、复发以及耐药性的产生密切相关[18]。 2.3 调控乳腺癌干细胞的信号转导通路调控乳腺癌干细胞的信号转导通路主要有Notch、Wnt和Hedgehog信号通路，乳腺癌干细胞的自我更新和分化过程由这3条主要的信号通路调控[19]。乳腺癌干细胞信号通路的正常，可以保证乳腺癌干细胞的自我更新和分化[20]。靶向信号通路来控制肿瘤干细胞的增殖、分化，已经成为研究抗癌药物的新热点。 2.3.1 Notch信号通路 Notch可以与邻近细胞表面Notch配体发生相互作用，从而调节细胞的生长和变化[21]。Notch信号通路能够调控脊椎动物和无脊椎动物的细胞增殖、凋亡，对于神经系统的发育和器官形成起着重要的作用[22]。同时Notch信号通路与肿瘤干细胞的自我更新能力密切相关，在乳腺癌的发生发展中发挥关键作用[23]。Harrison等[24]研究发现，Notch信号通路的激活会促进正常乳腺干细胞和乳腺肿瘤干细胞的增殖。检测不同乳腺癌细胞系中Notch4的信号活性，在乳腺癌干细胞中的表达水平比正常未分化的乳腺细胞中的表达水平高8倍，抑制Notch1和Notch4的活性，会降低乳腺肿瘤干细胞的活性。Notch信号通路活化的细胞亚群表现出较强的自我更新能力、多向分化潜能，以及增强体内致瘤性等肿瘤干细胞的特性。 2.3.2 Wnt信号通路 Wnt信号通路调控着乳腺癌干细胞以及其他各种干细胞的分化，同时在调控脊椎动物和无脊椎动物的胚胎发育过程中也发挥重要作用[25]。在多数组织中，Wnt信号通路激活能够引起癌症的发生[26]。Wnt配体是一种调节组织稳态的胞外分泌蛋白，Cai等[27]研究发现，Wnt信号通路中的转录因子LEF1/TCF可以通过黏附蛋白β-catenin进入细胞核发生转录，从而维持正常组织和肿瘤组织中干细胞自我更新和未分化的特性。Jang等[28]研究发现，在乳腺癌肿瘤干细胞中Wnt/β-catenin的分子表达水平高于实体肿瘤组织中的表达水平，Wnt/β-catenin小分子抑制剂CWP232228能够通过减弱IGF-I的活性抑制β-catenin调控的转录，从而减少乳腺癌干细胞的数目。 2.3.3 Hedgehog信号通路 Hedgehog信号通路包括HH配体、Shh、Ihh、Dhh以及Patched-1和Patched-2等跨膜受体[29]。Hedgehog信号通路中PTCH1、Gli1、Gli2在人乳腺干细胞中高度表达，Hedgehog信号通路的激活能增强乳腺癌干细胞自我更新能力[30]。Ramaswamy等[31]研究表明PI3K/AKT信号通路通过保护蛋白酶体降解在调控Hedgehog中发挥重要作用，靶向Hedgehog通路或者联合PI3K/AKT信号通路可能成为治疗耐药性肿瘤的新的治疗方法。长链非编码RNA(lncRNAs)通过激活Hedgehog信号通路能够增强Twist阳性乳腺癌细胞中乳腺癌干细胞的产生；活化的Hedgehog信号通路增加GLI1、SOX2和OCT4的表达从而维持肿瘤干细胞的特性，增强Twist阳性乳腺癌细胞的体外乳腺微球体形成能力以及体内致瘤性；沉默Twist阳性乳腺癌细胞中lncRNA-Hh基因则减弱Hedgehog信号通路的激活从而降低肿瘤干细胞相关的SOX和OCT4的表达水平，减弱微球体形成能力以及致瘤性[32]。 2.4 乳腺癌干细胞与上皮间质转化有研究发现，恶性肿瘤细胞在发生上皮间质转化后可以转化为肿瘤干细胞[33]。在上皮间质转化进程中，上皮特性的细胞会失去细胞间黏附转化为具有间质表型细胞[34]。上皮间质转化是恶性肿瘤细胞获得迁移和侵袭能力的重要生物学过程[35]。 Mani等[36]研究发现，通过转染Twist和Snail基因或使用TGF-β诱导乳腺上皮细胞发生上皮间质转化，均能够增加乳腺上皮细胞微球体形成能力及自我更新能力。发生上皮间质转化后的人乳腺上皮细胞获得了间质细胞样特性，细胞游离性增加，检测发现细胞表达有干细胞标志物，这些细胞的乳腺微球体形成能力增强；从人乳腺上皮中分离出的乳腺癌干细胞也表达与上皮间质转化相关的分子标志物，由此说明肿瘤干细胞特性与上皮间质转化的发生密切相关。Aktas等[37]研究发现乳腺癌患者中循环肿瘤细胞关系到肿瘤干细胞的存在，而肿瘤循环细胞发生上皮间质转化，从而获得转移能力。Dimeo等[38]研究发现，上皮间质转化与肿瘤细胞自我更新能力密切相关，从而使其获得转移能力。Hollier等[39]研究发现，转录因子FOXC2存在于间质细胞及干细胞中，在诱导发生上皮间质转化和肿瘤干细胞中发挥作用；降低FOXC2的表达能够抑制间充质细胞的侵袭能力和肿瘤干细胞特性，同时减弱微球体形成及肿瘤形成能力；过表达FOXC2则能够诱导肿瘤干细胞标志物的表达并能够促进人乳腺上皮细胞的自发性转移；因此，FOXC2及其相关基因对于抗上皮间质转化的肿瘤治疗提供一个很好的靶点。Sun等[40]研究发现，乳腺癌干细胞表达神经节苷脂GD2，上皮间质转化能够诱导GD2和GD3S的表达；GD2阳性细胞相比于GD2阴性细胞高表达间质细胞标志物(N-cadherin，vimentin，twist，snail)，低表达上皮细胞标志物(E-cadherin)。Asiedu等[41]研究发现，通过TGF-β/TNF-α诱导乳腺癌细胞发生上皮间质转化后，细胞呈现稳定的干细胞特性，细胞自我更新能力和致瘤性提高，并且增强了对奥沙利铂、依托泊苷、紫杉醇等化疗药物的耐药性；检测TGF-β/TNF-α诱导产生的乳腺癌干细胞的基因表达，发现claudin 3、4、7和cytokeratin 18的基因表达降低，这些变化表明间质细胞和肿瘤干细胞的分子标志物的升高以及预示了肿瘤的不良预后。泛素连接酶CUL4A在肿瘤发生过程中作用关键，Wang等[42]研究了CUL4A在乳腺癌等肿瘤中的表达水平，肿瘤中CUL4A的表达水平和在远隔器官内的表达呈正相关；在正常和癌变人乳腺上皮细胞中过表达CUL4A会增强它们体内和体外的肿瘤特性；相反在侵袭性乳腺癌中敲除CUL4A基因则会抑制这些过程；CUL4A调控上皮间质转化的诱导因子ZEB1，沉默ZEB1基因会阻断CUL4A诱导的细胞增殖、上皮间质转化、肿瘤形成和转移。Sarrio等[43]研究人员用28种不同的标志物在479种侵袭性乳腺癌和12种癌肉瘤组织样本中对免疫组化做了组织微阵列分析发现：基底细胞样型乳腺癌中上皮间质转化标志物(vimentin、smooth-muscle- actin、N-cadherin、cadherin-11)表达升高，细胞外基质重塑和侵袭相关蛋白表达升高，而上皮细胞标志物(E-cadherin和cytokeratins)表达下调；大多数乳腺癌具有基底细胞样型，同时间质细胞标志物增多。乳腺癌患者体内的肿瘤循环细胞中包含很多的间质表型的细胞，连续监测11例患者的循环肿瘤细胞，发现间质表型的循环肿瘤细胞和肿瘤发展关系密切[44]。这些研究结果揭示了上皮间质转化的发生和肿瘤干细胞之间的密切联系，以上皮间质转化为切入点，寻找靶向肿瘤干细胞的药物，为临床治疗乳腺癌的研究提供了一个新的思路。 2.5 肿瘤干细胞与乳腺癌转移乳腺癌干细胞、胚胎干细胞信号通路及上皮间质转化之间相互作用结果促进了乳腺癌的发展和转移[45]，上皮间质转化和肿瘤干细胞促进乳腺癌的发展和转移[46]。肿瘤转移涉及多个步骤，而肿瘤转移灶形成的关键步骤是转移性集聚，乳腺癌干细胞在转移性集聚过程中发挥重要作用[47]。侵袭性的肿瘤细胞需要在远隔转移部位诱导骨膜蛋白的表达，才能发生转移性集聚；而骨膜蛋白在维系肿瘤干细胞特性中的作用非常关键，骨膜蛋白功能的阻断会造成肿瘤转移的抑制[48]。乳腺癌患者体内的肿瘤干细胞具有致瘤性[49]，血液中的循环肿瘤细胞能够在移植瘤小鼠模型体内造成肿瘤转移[50]。 Sheridan等[51]研究发现，CD44+/CD24-乳腺癌细胞具有很强的侵袭能力，能够在远端器官侵袭并增殖形成肿瘤转移灶，分析的13种乳腺癌细胞系中有5种都含有大于30%的CD44+/CD24-细胞亚群，这些细胞系中侵袭性基因表达很高，比其他细胞系具有更强的转移能力。ALDH-1阳性的乳腺癌细胞具有高度转移能力：ALDEFLUOR实验分析来自正常和癌变乳腺组织的33种不同乳腺癌细胞系，其中有23种ALDH-1阳性细胞在体外和体内移植瘤模型中均表现出肿瘤干细胞特性，具有高度侵袭性；ALDH基因能够维系乳腺癌干细胞的特性，增强微球体形成能力及自我更新能力[52]。ALDEFLUOR实验分析SUM149、MARY-X等不同的炎性乳腺癌细胞系以及原代移植瘤中的肿瘤干细胞，体内体外实验均表明高ALDH-1活性的肿瘤干细胞调控炎性乳腺癌的侵袭和转移能力；而且，炎性乳腺癌中ALDH-1的表达水平是患者早期转移和不良预后的一个独立的指示性因素[53]。 2.6 乳腺癌干细胞与肿瘤微环境肿瘤微环境在肿瘤的局部耐药性、免疫逃脱和远端转移等多个肿瘤发生、发展的关键步骤中发挥作用[54]。肿瘤微环境主要包括间质干细胞、细胞外基质、肿瘤相关成纤维细胞、脂肪细胞、内皮细胞、免疫细胞等，是肿瘤细胞和邻近组织之间的部分[55]。肿瘤干细胞的特性会受到肿瘤微环境组成成分的影响，研究肿瘤微环境可以为癌症提供一个新的治疗思路[56]。白细胞介素6能够将不同乳腺癌细胞系中的非肿瘤干细胞转化为乳腺癌干细胞；Korkaya等[57]研究发现，IL-6/NF-κB信号通路诱导EMT表型的细胞产生干细胞特性；经过白细胞介素6诱导的乳腺癌干细胞能够对HER2的治疗产生耐受。曲妥单抗耐药可能由白细胞介素6循环介导，阻断白细胞介素6循环也许能够克服曲妥单抗的耐药性。Singh等[58]研究发现白细胞介素8在乳腺癌中的高表达与乳腺癌不良预后有关。研究表明，白细胞介素8的表达水平与乳腺微球体形成能力有关，可以通过其同源性受体CXCR1、CXCR2影响乳腺癌干细胞的自我更新能力，调控其活性；白细胞介素8诱导EGFR/HER2及其下游信号通路的激活，抑制SRC、EGFR/HER2、PI3K磷酸化则会消除这种效果，IL-8/CXCR1/2信号通路由新的SRC和EGFR/HER2调控；此研究结果揭示了白细胞介素8在调控患者来源的乳腺癌干细胞活性时所发挥的作用，在临床治疗中联合使用CXCR1/2抑制剂和抗HER2治疗能够抑制患者体内乳腺癌干细胞的活性，提高HER2阳性乳腺癌患者的生存率。 2.7 乳腺癌干细胞与治疗通常认为，乳腺癌复发的根本原因在于乳腺癌干细胞对传统化疗药物产生了耐药性[59]。从化疗后的原发乳腺癌患者肿瘤组织中取出肿瘤细胞，检测肿瘤组织中的CD44+/CD24-乳腺癌干细胞比例，化疗后干细胞亚群的比例与化疗前相比明显增加，乳腺癌细胞微球体形成能力也提高；而在经过拉帕替尼治疗后的HER-2阳性乳腺癌患者取出的肿瘤细胞中，CD44+/CD24-表型细胞没有明显增高。在乳腺癌的治疗方案中，应该联合使用传统化疗药物和靶向肿瘤干细胞的药物，来增加乳腺癌的治愈率，减少乳腺癌复发的概率，提高患者的生存率[60]。"

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