炎性因子OSM诱导乳腺癌恶性演进的分子机制研究
摘要
炎症在肿瘤的发生、发展中发挥重要的作用，被称为“肿瘤的第七大特征”。炎性微环境中发生的一系列生物化学反应推动肿瘤的恶性转化，上皮-间质转化（epithelial-mesenchymal transition, EMT）是肿瘤恶性转化过程中的重要事件。在EMT转化过程中，上皮细胞极性丧失，细胞-细胞及细胞-基质间的粘附减弱，上皮细胞的标志分子E-cadherin下调，间质细胞的标志分子Vimentin、Fibronectin及EMT的关键调控分子ZEB1、Snail、Twist等表达上调。同时，一系列促转移、促侵袭相关基因被诱导，使肿瘤细胞的迁移、侵袭能力增强。
炎性微环境中存在大量的炎性因子，已知炎性因子TNF-α、TGF-β、IL-6等参与了EMT过程。抑瘤素（Oncostatin M, OSM）是IL-6家族的重要成员。前期研究提示，在肿瘤细胞培养上清中存在的OSM可能与EMT发生相关。在本研究中，我们通过对乳腺癌组织芯片（n=75）的免疫组化分析，观察到约80%的乳腺癌组织高表达OSM，而乳腺良性疾病及正常乳腺组织中OSM的表达常呈阴性。OSM在乳腺癌组织中以自分泌和旁分泌的形式表达，特别在乳腺癌侵袭前缘，OSM表达呈强阳性，提示OSM可能参与乳腺癌的转移调控。此外，对7种人乳腺癌细胞系的分析亦证实，具有高侵袭能力的MDA-231和MDA-435S细胞表达较高水平的OSM。体外实验结果显示，OSM刺激可诱导上皮型的MCF-7和T47D乳腺癌细胞向间质样细胞转化，E-cadherin下调，Vimentin、Fibronectin以及ZEB1、Snail表达上调，细胞的迁移、侵袭能力显著增强，表明OSM是一个强有力的EMT诱导因子。
miRNA在EMT过程中执行重要的功能，其中let-7及miR-200家族成员在EMT中的作用最受关注。在Src诱导的MCF-10A细胞转化过程中，let-7参与NF-κB介导的表观遗传学调控，miR-200可直接抑制E-cadherin的转录抑制子（ZEB1和ZEB2）的表达，ZEB1/miR-200正反馈调控环路在EMT中发挥重要的作用。我们的进一步研究发现，OSM可诱导let-7及miR-200表达下调，但二者呈现独特的变化特征，即let-7表现为瞬时下调，而miR-200的下调则较为滞后而持久。外源性过表达let-7或miR-200mimics，可逆转OSM诱导的EMT发生，使E-cadherin表达得到恢复，并抑制Fibronectin和ZEB1表达，导致细胞的迁移、侵袭能力降低，提示let-7瞬时性下调及miR-200持续性下调在OSM诱导的EMT过程中具有重要的意义。
OSM主要激活MEK、JAK/Stat、JNK等信号通路。我们利用MEK通路抑制剂PD98059、JNK通路抑制剂SP600125、JAK/Stat通路抑制剂WP1066预处理MCF-7细胞，发现这些抑制剂可不同程度地抑制OSM诱导的EMT。已知MEK、JNK、JAK2信号通路的激活均可导致Stat3活化。我们的研究证实，OSM刺激可诱导Stat3持续性活化，而敲低Stat3可显著抑制OSM诱导的EMT，并可逆转OSM对let-7和miR-200的下调效应，而过表达组成型激活的Stat3C则可明显下调let-7和miR-200的表达。miRNA主要通过与其靶基因的3’非翻译区（3’UTR）结合而调控靶基因的表达。为了进一步证实Stat3对let-7及miR-200的调控作用，我们分别构建了含let-7和miR-200靶基因HMGA23’UTR及ZEB13’UTR的荧光素酶报告载体，分别用这些载体与Stat3C表达质粒共转染MCF-7细胞。结果显示，过表达Stat3C可明显增强HMGA23’UTR及ZEB13’UTR的活性，而对HMGA25’UTR无影响。敲低Stat3或抑制Stat3信号通路活化均可抑制let-7靶基因HMGA2的表达，进一步表明Stat3对let-7和miR-200的表达具有特异性的调控作用，提示Stat3可能是OSM诱导EMT的关键调控分子，通过调控miRNA表达而影响EMT程序。
我们发现，与OSM诱导的let-7瞬时性下调相呼应的是，let-7靶基因HMGA2、c-Myc、Ras均出现瞬时性表达增强，过表达let-7mimics能抑制OSM对HMGA2、c-Myc、Ras的诱导作用。值得注意的是，HMGA2是一个胚胎早期基因，在胚胎发育期高表达于未分化的增殖性细胞，而在成体组织中不表达；let-7的表达恰与之相反，在胚胎阶段难以检测到，而在胚胎发育末期开始上调，提示let-7-HMGA2轴的瞬时变化在OSM诱导的EMT中可能是一个始动效应。我们发现，敲低HMGA2可显著逆转EMT表型，抑制乳腺癌细胞的侵袭能力，并可使OSM诱导的miR-200下调得到恢复，表明let-7和HMGA2的表达动力学变化不仅对于OSM诱导的表观调控具有启动作用，而且对于miR-200的下调亦产生重要的影响。
通过对let-7转录本（pri-let-7）及成熟体（mat-let-7）表达的分析，我们发现OSM影响了let-7成熟体的表达，提示Stat3激活可能影响let-7加工成熟。已知RNA结合蛋白Lin-28A和Lin-28B是let-7家族成员加工成熟的关键负调控分子。我们观察到，OSM可诱导Lin-28B的瞬时性表达增强，此表达变化特征与let-7下调的动力学变化一致。敲低Lin-28B表达可显著削弱OSM诱导的EMT效应，揭示了Lin-28B-let-7-HMGA2轴的瞬时激活在OSM诱导的EMT过程中的关键作用。对Lin-28B启动子序列分析显示，Lin-28B启动子序列中含有6个Stat3的潜在结合位点。我们通过ChIP实验证实，外源性及内源性Stat3均能与Lin-28B启动子-2405和-369bp区的Stat3反应元件结合。更为重要的是，在OSM诱导下，内源性Stat3与Lin-28B启动子的结合具有与Lin-28B表达、let-7下调及HMGA2上调相似的时空特征。以上证据证明，OSM诱导的Lin-28B一过性表达增强决定了let-7-HMGA2轴的瞬时开启。
为了验证OSM是否可在体内诱导EMT，我们建立了稳定表达GFP的MCF-7/GFP乳腺癌细胞移植瘤模型。当瘤体可触及时，进行瘤体内注射OSM，每周一次，连续6周。60天后处死动物，分离瘤体、肺、肝组织，对瘤组织称重，并作H&E染色和抗GFP抗体免疫组化染色。结果发现，OSM处理明显诱导了乳腺癌自发性肺转移。对瘤组织分析结果显示，OSM处理组的肿瘤组织中E-cadherin表达下调，Vimentin和ZEB1表达上调，Stat3磷酸化水平升高。同时，一系列与转移侵袭相关的基因（MMP-2、MMP-7、MMP-9、COX-2、CXCR-4）均被诱导表达。对瘤组织miRNA分析的结果显示，OSM处理组let-7表达与对照组似无明显差别。但是，OSM注射组的瘤组织中miR-200显著下调。这些结果充分表明，OSM在体内亦具有促EMT效应，并提示OSM诱导的miR-200-ZEB1轴在肿瘤恶性表型的维持过程中发挥重要作用。
上述研究结果证实， OSM诱导的EMT受转录因子及miRNA共同构成的复杂网络调控，而Stat3协调、控制并决定了Lin-28B-let-7-HMGA2及miR-200-ZEB1轴的动力学变化，开启并维持乳腺癌EMT过程，赋予乳腺癌细胞明显的间质样表型，促进乳腺癌的恶性进展。我们的研究首次报道了OSM的体内促EMT效应及OSM在乳腺癌恶性演进中的作用；首次揭示了OSM诱导乳腺癌细胞发生EMT的“开关维持”机制；首次报道了Stat3对Lin-28B的转录调控作用。我们的研究成果为炎性微环境诱导肿瘤恶性转化的分子调控网络提供了新信息，为深入探索炎性信号通路在肿瘤发生、发展及转移中的分子机制打下了基础。
Inflammation, which plays a critical role in tumor initiation and progression, isconsidered as “the seventh hallmark of cancer”. Inflammatory microenvironmentpromotes tumor malignant transformation via a cascade of biochemical reactions.Epithelial-mesenchymal transition (EMT) is an important event in the progression andmetastasis of tumors. During EMT, the polarization of epithelial cells is lost; cell-cellor cell-stroma adhesion is decreased; the epithelial marker E-cadherin isdownregulated and mesenchymal markers vimentin and fibronectin and EMT keyregulators ZEB1, Snai1and Twist are upregulated. Additionally, a series ofpro-metastasis and pro-invasion genes are induced, which endows epithelial cells withstrong migratory and invasive potentials.
There is a large amount of cytokines in the inflammatory microenvironment. It hasbeen known that inflammatory cytokines, such as TNF-α, TGF-β and IL-6, areinvolved in the regulation of EMT. Oncostatin M (OSM) is an important member ofIL-6family. A previous study suggested that OSM in the cultural supernatant of tumorcells was associated with EMT of tumor cells. In the present study, byimmunohistochemical analysis using a breast cancer tissue array (n=75), we show thatOSM was overexpressed in approximately80%breast cancer tissues, while almostabsent in the majority of benign and normal breast tissues. OSM is expressed in anautocrine/paracrine fashion in breast cancer tissues, especially at the invasive front oftumors, implicating that OSM may play a role in breast carcinoma metastasis.Examination of OSM expression in seven breast cancer cell lines demonstrates thatthe level of OSM is high in highly invasive MDA-231and MDA-435S cells. In vitrostudies showed that OSM induced a mesenchymal phenotype in two luminal cell linesMCF-7and T47D. After OSM stimulation, E-cadherin was downregulated andvimentin, fibronectin, ZEB1and Snail were upregulated. In addition, the migratoryand invasive abilities of the cells were enhanced greatly, suggesting that OSM is apotent inducer of EMT.
miRNAs exert important functions during EMT. The roles of let-7and miR-200family members in EMT have attracted more attentions. Let-7is involved in theNF-κB-mediated epigenetic regulation during Src-induced transformation ofMCF-10A cells. miR-200directly suppresses the expression of ZEB1and ZEB2,which are the transcriptional inhibitors of E-cadherin. Our further study showed that OSM reduced the expression of let-7and miR-200remarkably. However,downregulation of let-7and miR-200exhibited different temporal patterns. Let-7wasdecreased rapidly but transiently, whereas downregulation of miR-200occurred laterbut kept persistent. OSM-induced EMT could be reversed by ectopic expression oflet-7or miR-200mimics, with a restoration in E-cadherin expression, reduction infibronectin, ZEB1and inhibition of migratory and invasive potentials in tumor cells.These results indicate that both transient and sustained downregulation of thesemiRNAs are engaged in OSM-induced EMT.
The effect of OSM is mainly mediated by MEK, JAK/Stat and JNK signalingpathways. Pre-treatment of MCF-7cells with the MEK inhibitor PD98059, JNKinhibitor SP600125or JAK/Stat inhibitor WP1066could inhibit OSM-induced EMTindividually. All these signaling pathways converge on the activation of Stat3.Knockdown of Stat3dramatically inhibited OSM-induced EMT and restored theexpression of let-7and miR-200. Overexpression of constitutively activated Stat3Crepressed the expression of let-7and miR-200. miRNAs exert the effects primarilythrough binding to the3’ UTR of their target genes. To further confirm the role ofStat3in the regulation of let-7and miR-200expression, we constructed the luciferasereporter plasmids containing the3’UTR of HMGA2and ZEB1, which are the targetsof let-7and miR-200, respectively and transfected the plasmids into MCF-7cellstogether with the Stat3C expression plasmid. The data showed that overexpression ofStat3C remarkably enhanced the activities of HMGA2and ZEB13’UTR, but noeffect was observed on the HMGA25’UTR. Moreover, the expression of HMGA2could be inhibited by either silencing Stat3or blocking Stat3-mediated signalingpathways, demonstrating that the expression of let-7and miR-200is modulated byStat3. The data implicates that Stat3is a key regulator in OSM-induced EMT byinfluencing the expression of miRNAs.
We noticed that a transient downregulation in let-7expression induced by OSMwas echoed by a transient upregulation in its target genes HMGA2, c-Myc and Ras.Ecotopic expression of let-7mimics fully suppressed the effects of OSM. Notably,HMGA2as an early embryonic gene is highly expressed in undifferentiatedproliferating cells during embryogenesis and becomes silent in adult tissues.Conversely, the expression of let-7is barely detectable in embryonic stages butupregulated at the end of embryonic development. We speculate that the transientactivation of let-7-HMGA2axis may act as an epigenetic switch to triggerOSM-induced EMT. Knockdown of HMGA2markedly reversed the EMT phenotype,repressed invasive capability of breast cancer cells and restored the expression ofmiR-200, which was downregulated later under OSM stimulation. These data suggestthat the dynamic alterations of let-7and HMGA2not only elicit OSM-induced EMT,but also impact the expression of miR-200.
Analysis of the pri-let-7and mature let-7levels indicated that OSM inhibited theexpression of mature let-7, suggesting that OSM interferes with processing andmaturation of let-7. It has been reported that the RNA binding proteins Lin-28andLin-28B are negative regulators of let-7processing. Our results show that OSMinduced a transient upregulation in the Lin-28B expression, which is coincident withthe dynamic alteration of the let-7downregulation. Knockdown of Lin-28Bremarkably impaired OSM-induced EMT, demonstrating that transient activation ofLin-28B-let-7-HMGA2axis plays a critical role in OSM-induced EMT. Sequenceanalysis reveals that the Lin-28B promoter harbors six potential Stat3bindingelements. Using ChIP assays, we demonstrated that both exogenous and endogenousStat3could bind to the Stat3responsive elements at-2405and-369bp regions in theLin-28B promoter. Importantly, endogenous Stat3interacted with the Lin-28Bpromoter in a similar temporal pattern of the Lin-28B expression, let-7downregulation and HMGA2upregulation. These data reveals that transient inductionof Lin-28B by OSM determines the transient activation of the let-7-HMGA2axis.
To validate the effect of OSM on EMT in vivo, we established the tumor-bearingmouse model by inoculating MCF-7/GFP cells into BALB/c nude mice. When thexenografts were palpable, OSM were injected into the tumors once a week forconsecutive6weeks. After60days, animals were killed, and tumors, livers and lungswere dissected and subjected to H&E staining and immunohistochemistry with theanti-OSM antibody. The data showed that OSM stimulation greatly promotedspontaneous lung metastasis of the tumors. Examination of the tumor tissuesdemonstrated that the expression of E-cadherin was reduced in the tumor tissuesinjected with OSM, concomitant with a upregulation in the expression of vimentin,ZEB1and enhancement of Stat3phosphorylation. In addition, a series ofpro-metastasis genes (MMP-2, MMP-7, MMP-9, COX-2and CXCR4) weredramatically induced. No striking difference in the let-7expression was observedbetween control and OSM-injected tumors. However, the expression of miR-200wasdefinitely reduced in OSM-treated tumor tissues. The data confirms that OSM caninduce EMT in vivo and the activation of miR-200-ZEB1axis is involved in themaintenance of tumor malignant phenotype.
Taken together, OSM-induced EMT is regulated by a complex network consistingof the transcription factors and miRNAs. The activation of Stat3controls the dynamicalterations of Lin-28B-let-7-HMGA2and miR-200-ZEB1axises which initiate andmaintain EMT, endowing breast cancer cells with mesenchymal phenotype andpromoting breast cancer progression. Our results reveal for the first time the effects ofOSM on EMT in vivo and breast cancer metastasis, and a novel “switching andmaintaining” mechanism in OSM-induced EMT. We also demonstrate for the firsttime that Lin-28B is transcriptionally regulated by Stat3. Our study provides new information for the regulatory network of inflammatory microenviroment in tumortransformation. They will be helpful for further exploration into the molecularmechanism, by which inflammatory signaling pathways modulate tumor formation,progression and metastasis.
炎性微环境中存在大量的炎性因子，已知炎性因子TNF-α、TGF-β、IL-6等参与了EMT过程。抑瘤素（Oncostatin M, OSM）是IL-6家族的重要成员。前期研究提示，在肿瘤细胞培养上清中存在的OSM可能与EMT发生相关。在本研究中，我们通过对乳腺癌组织芯片（n=75）的免疫组化分析，观察到约80%的乳腺癌组织高表达OSM，而乳腺良性疾病及正常乳腺组织中OSM的表达常呈阴性。OSM在乳腺癌组织中以自分泌和旁分泌的形式表达，特别在乳腺癌侵袭前缘，OSM表达呈强阳性，提示OSM可能参与乳腺癌的转移调控。此外，对7种人乳腺癌细胞系的分析亦证实，具有高侵袭能力的MDA-231和MDA-435S细胞表达较高水平的OSM。体外实验结果显示，OSM刺激可诱导上皮型的MCF-7和T47D乳腺癌细胞向间质样细胞转化，E-cadherin下调，Vimentin、Fibronectin以及ZEB1、Snail表达上调，细胞的迁移、侵袭能力显著增强，表明OSM是一个强有力的EMT诱导因子。
miRNA在EMT过程中执行重要的功能，其中let-7及miR-200家族成员在EMT中的作用最受关注。在Src诱导的MCF-10A细胞转化过程中，let-7参与NF-κB介导的表观遗传学调控，miR-200可直接抑制E-cadherin的转录抑制子（ZEB1和ZEB2）的表达，ZEB1/miR-200正反馈调控环路在EMT中发挥重要的作用。我们的进一步研究发现，OSM可诱导let-7及miR-200表达下调，但二者呈现独特的变化特征，即let-7表现为瞬时下调，而miR-200的下调则较为滞后而持久。外源性过表达let-7或miR-200mimics，可逆转OSM诱导的EMT发生，使E-cadherin表达得到恢复，并抑制Fibronectin和ZEB1表达，导致细胞的迁移、侵袭能力降低，提示let-7瞬时性下调及miR-200持续性下调在OSM诱导的EMT过程中具有重要的意义。
OSM主要激活MEK、JAK/Stat、JNK等信号通路。我们利用MEK通路抑制剂PD98059、JNK通路抑制剂SP600125、JAK/Stat通路抑制剂WP1066预处理MCF-7细胞，发现这些抑制剂可不同程度地抑制OSM诱导的EMT。已知MEK、JNK、JAK2信号通路的激活均可导致Stat3活化。我们的研究证实，OSM刺激可诱导Stat3持续性活化，而敲低Stat3可显著抑制OSM诱导的EMT，并可逆转OSM对let-7和miR-200的下调效应，而过表达组成型激活的Stat3C则可明显下调let-7和miR-200的表达。miRNA主要通过与其靶基因的3’非翻译区（3’UTR）结合而调控靶基因的表达。为了进一步证实Stat3对let-7及miR-200的调控作用，我们分别构建了含let-7和miR-200靶基因HMGA23’UTR及ZEB13’UTR的荧光素酶报告载体，分别用这些载体与Stat3C表达质粒共转染MCF-7细胞。结果显示，过表达Stat3C可明显增强HMGA23’UTR及ZEB13’UTR的活性，而对HMGA25’UTR无影响。敲低Stat3或抑制Stat3信号通路活化均可抑制let-7靶基因HMGA2的表达，进一步表明Stat3对let-7和miR-200的表达具有特异性的调控作用，提示Stat3可能是OSM诱导EMT的关键调控分子，通过调控miRNA表达而影响EMT程序。
我们发现，与OSM诱导的let-7瞬时性下调相呼应的是，let-7靶基因HMGA2、c-Myc、Ras均出现瞬时性表达增强，过表达let-7mimics能抑制OSM对HMGA2、c-Myc、Ras的诱导作用。值得注意的是，HMGA2是一个胚胎早期基因，在胚胎发育期高表达于未分化的增殖性细胞，而在成体组织中不表达；let-7的表达恰与之相反，在胚胎阶段难以检测到，而在胚胎发育末期开始上调，提示let-7-HMGA2轴的瞬时变化在OSM诱导的EMT中可能是一个始动效应。我们发现，敲低HMGA2可显著逆转EMT表型，抑制乳腺癌细胞的侵袭能力，并可使OSM诱导的miR-200下调得到恢复，表明let-7和HMGA2的表达动力学变化不仅对于OSM诱导的表观调控具有启动作用，而且对于miR-200的下调亦产生重要的影响。
通过对let-7转录本（pri-let-7）及成熟体（mat-let-7）表达的分析，我们发现OSM影响了let-7成熟体的表达，提示Stat3激活可能影响let-7加工成熟。已知RNA结合蛋白Lin-28A和Lin-28B是let-7家族成员加工成熟的关键负调控分子。我们观察到，OSM可诱导Lin-28B的瞬时性表达增强，此表达变化特征与let-7下调的动力学变化一致。敲低Lin-28B表达可显著削弱OSM诱导的EMT效应，揭示了Lin-28B-let-7-HMGA2轴的瞬时激活在OSM诱导的EMT过程中的关键作用。对Lin-28B启动子序列分析显示，Lin-28B启动子序列中含有6个Stat3的潜在结合位点。我们通过ChIP实验证实，外源性及内源性Stat3均能与Lin-28B启动子-2405和-369bp区的Stat3反应元件结合。更为重要的是，在OSM诱导下，内源性Stat3与Lin-28B启动子的结合具有与Lin-28B表达、let-7下调及HMGA2上调相似的时空特征。以上证据证明，OSM诱导的Lin-28B一过性表达增强决定了let-7-HMGA2轴的瞬时开启。
为了验证OSM是否可在体内诱导EMT，我们建立了稳定表达GFP的MCF-7/GFP乳腺癌细胞移植瘤模型。当瘤体可触及时，进行瘤体内注射OSM，每周一次，连续6周。60天后处死动物，分离瘤体、肺、肝组织，对瘤组织称重，并作H&E染色和抗GFP抗体免疫组化染色。结果发现，OSM处理明显诱导了乳腺癌自发性肺转移。对瘤组织分析结果显示，OSM处理组的肿瘤组织中E-cadherin表达下调，Vimentin和ZEB1表达上调，Stat3磷酸化水平升高。同时，一系列与转移侵袭相关的基因（MMP-2、MMP-7、MMP-9、COX-2、CXCR-4）均被诱导表达。对瘤组织miRNA分析的结果显示，OSM处理组let-7表达与对照组似无明显差别。但是，OSM注射组的瘤组织中miR-200显著下调。这些结果充分表明，OSM在体内亦具有促EMT效应，并提示OSM诱导的miR-200-ZEB1轴在肿瘤恶性表型的维持过程中发挥重要作用。
上述研究结果证实， OSM诱导的EMT受转录因子及miRNA共同构成的复杂网络调控，而Stat3协调、控制并决定了Lin-28B-let-7-HMGA2及miR-200-ZEB1轴的动力学变化，开启并维持乳腺癌EMT过程，赋予乳腺癌细胞明显的间质样表型，促进乳腺癌的恶性进展。我们的研究首次报道了OSM的体内促EMT效应及OSM在乳腺癌恶性演进中的作用；首次揭示了OSM诱导乳腺癌细胞发生EMT的“开关维持”机制；首次报道了Stat3对Lin-28B的转录调控作用。我们的研究成果为炎性微环境诱导肿瘤恶性转化的分子调控网络提供了新信息，为深入探索炎性信号通路在肿瘤发生、发展及转移中的分子机制打下了基础。
Inflammation, which plays a critical role in tumor initiation and progression, isconsidered as “the seventh hallmark of cancer”. Inflammatory microenvironmentpromotes tumor malignant transformation via a cascade of biochemical reactions.Epithelial-mesenchymal transition (EMT) is an important event in the progression andmetastasis of tumors. During EMT, the polarization of epithelial cells is lost; cell-cellor cell-stroma adhesion is decreased; the epithelial marker E-cadherin isdownregulated and mesenchymal markers vimentin and fibronectin and EMT keyregulators ZEB1, Snai1and Twist are upregulated. Additionally, a series ofpro-metastasis and pro-invasion genes are induced, which endows epithelial cells withstrong migratory and invasive potentials.
There is a large amount of cytokines in the inflammatory microenvironment. It hasbeen known that inflammatory cytokines, such as TNF-α, TGF-β and IL-6, areinvolved in the regulation of EMT. Oncostatin M (OSM) is an important member ofIL-6family. A previous study suggested that OSM in the cultural supernatant of tumorcells was associated with EMT of tumor cells. In the present study, byimmunohistochemical analysis using a breast cancer tissue array (n=75), we show thatOSM was overexpressed in approximately80%breast cancer tissues, while almostabsent in the majority of benign and normal breast tissues. OSM is expressed in anautocrine/paracrine fashion in breast cancer tissues, especially at the invasive front oftumors, implicating that OSM may play a role in breast carcinoma metastasis.Examination of OSM expression in seven breast cancer cell lines demonstrates thatthe level of OSM is high in highly invasive MDA-231and MDA-435S cells. In vitrostudies showed that OSM induced a mesenchymal phenotype in two luminal cell linesMCF-7and T47D. After OSM stimulation, E-cadherin was downregulated andvimentin, fibronectin, ZEB1and Snail were upregulated. In addition, the migratoryand invasive abilities of the cells were enhanced greatly, suggesting that OSM is apotent inducer of EMT.
miRNAs exert important functions during EMT. The roles of let-7and miR-200family members in EMT have attracted more attentions. Let-7is involved in theNF-κB-mediated epigenetic regulation during Src-induced transformation ofMCF-10A cells. miR-200directly suppresses the expression of ZEB1and ZEB2,which are the transcriptional inhibitors of E-cadherin. Our further study showed that OSM reduced the expression of let-7and miR-200remarkably. However,downregulation of let-7and miR-200exhibited different temporal patterns. Let-7wasdecreased rapidly but transiently, whereas downregulation of miR-200occurred laterbut kept persistent. OSM-induced EMT could be reversed by ectopic expression oflet-7or miR-200mimics, with a restoration in E-cadherin expression, reduction infibronectin, ZEB1and inhibition of migratory and invasive potentials in tumor cells.These results indicate that both transient and sustained downregulation of thesemiRNAs are engaged in OSM-induced EMT.
The effect of OSM is mainly mediated by MEK, JAK/Stat and JNK signalingpathways. Pre-treatment of MCF-7cells with the MEK inhibitor PD98059, JNKinhibitor SP600125or JAK/Stat inhibitor WP1066could inhibit OSM-induced EMTindividually. All these signaling pathways converge on the activation of Stat3.Knockdown of Stat3dramatically inhibited OSM-induced EMT and restored theexpression of let-7and miR-200. Overexpression of constitutively activated Stat3Crepressed the expression of let-7and miR-200. miRNAs exert the effects primarilythrough binding to the3’ UTR of their target genes. To further confirm the role ofStat3in the regulation of let-7and miR-200expression, we constructed the luciferasereporter plasmids containing the3’UTR of HMGA2and ZEB1, which are the targetsof let-7and miR-200, respectively and transfected the plasmids into MCF-7cellstogether with the Stat3C expression plasmid. The data showed that overexpression ofStat3C remarkably enhanced the activities of HMGA2and ZEB13’UTR, but noeffect was observed on the HMGA25’UTR. Moreover, the expression of HMGA2could be inhibited by either silencing Stat3or blocking Stat3-mediated signalingpathways, demonstrating that the expression of let-7and miR-200is modulated byStat3. The data implicates that Stat3is a key regulator in OSM-induced EMT byinfluencing the expression of miRNAs.
We noticed that a transient downregulation in let-7expression induced by OSMwas echoed by a transient upregulation in its target genes HMGA2, c-Myc and Ras.Ecotopic expression of let-7mimics fully suppressed the effects of OSM. Notably,HMGA2as an early embryonic gene is highly expressed in undifferentiatedproliferating cells during embryogenesis and becomes silent in adult tissues.Conversely, the expression of let-7is barely detectable in embryonic stages butupregulated at the end of embryonic development. We speculate that the transientactivation of let-7-HMGA2axis may act as an epigenetic switch to triggerOSM-induced EMT. Knockdown of HMGA2markedly reversed the EMT phenotype,repressed invasive capability of breast cancer cells and restored the expression ofmiR-200, which was downregulated later under OSM stimulation. These data suggestthat the dynamic alterations of let-7and HMGA2not only elicit OSM-induced EMT,but also impact the expression of miR-200.
Analysis of the pri-let-7and mature let-7levels indicated that OSM inhibited theexpression of mature let-7, suggesting that OSM interferes with processing andmaturation of let-7. It has been reported that the RNA binding proteins Lin-28andLin-28B are negative regulators of let-7processing. Our results show that OSMinduced a transient upregulation in the Lin-28B expression, which is coincident withthe dynamic alteration of the let-7downregulation. Knockdown of Lin-28Bremarkably impaired OSM-induced EMT, demonstrating that transient activation ofLin-28B-let-7-HMGA2axis plays a critical role in OSM-induced EMT. Sequenceanalysis reveals that the Lin-28B promoter harbors six potential Stat3bindingelements. Using ChIP assays, we demonstrated that both exogenous and endogenousStat3could bind to the Stat3responsive elements at-2405and-369bp regions in theLin-28B promoter. Importantly, endogenous Stat3interacted with the Lin-28Bpromoter in a similar temporal pattern of the Lin-28B expression, let-7downregulation and HMGA2upregulation. These data reveals that transient inductionof Lin-28B by OSM determines the transient activation of the let-7-HMGA2axis.
To validate the effect of OSM on EMT in vivo, we established the tumor-bearingmouse model by inoculating MCF-7/GFP cells into BALB/c nude mice. When thexenografts were palpable, OSM were injected into the tumors once a week forconsecutive6weeks. After60days, animals were killed, and tumors, livers and lungswere dissected and subjected to H&E staining and immunohistochemistry with theanti-OSM antibody. The data showed that OSM stimulation greatly promotedspontaneous lung metastasis of the tumors. Examination of the tumor tissuesdemonstrated that the expression of E-cadherin was reduced in the tumor tissuesinjected with OSM, concomitant with a upregulation in the expression of vimentin,ZEB1and enhancement of Stat3phosphorylation. In addition, a series ofpro-metastasis genes (MMP-2, MMP-7, MMP-9, COX-2and CXCR4) weredramatically induced. No striking difference in the let-7expression was observedbetween control and OSM-injected tumors. However, the expression of miR-200wasdefinitely reduced in OSM-treated tumor tissues. The data confirms that OSM caninduce EMT in vivo and the activation of miR-200-ZEB1axis is involved in themaintenance of tumor malignant phenotype.
Taken together, OSM-induced EMT is regulated by a complex network consistingof the transcription factors and miRNAs. The activation of Stat3controls the dynamicalterations of Lin-28B-let-7-HMGA2and miR-200-ZEB1axises which initiate andmaintain EMT, endowing breast cancer cells with mesenchymal phenotype andpromoting breast cancer progression. Our results reveal for the first time the effects ofOSM on EMT in vivo and breast cancer metastasis, and a novel “switching andmaintaining” mechanism in OSM-induced EMT. We also demonstrate for the firsttime that Lin-28B is transcriptionally regulated by Stat3. Our study provides new information for the regulatory network of inflammatory microenviroment in tumortransformation. They will be helpful for further exploration into the molecularmechanism, by which inflammatory signaling pathways modulate tumor formation,progression and metastasis.
引文
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