循环肿瘤细胞Stat3/Twist双信号通路交互作用对EMT编程的乳腺癌转移的调控与干预

Metastasis is responsible for the majority of breast cancer-related deaths. The presence of CTCs is a robust independent prognostic indicator of progression-free survival and overall survival in patients with metastatic breast cancer. Our recent preliminary clinical data also shows that CTCs changes in breast cancer patients are associated with metastasis. Emerging evidence indicates that the epithelial-to-mesenchymal transition (EMT) and breast cancer stem cells (CSCs) cooperate to produce circulating tumor cells (CTCs) that are highly competent for metastasis; whereas, the reverse process-MET is thought to allow the outgrowth of these cells at distant sites.Both EMT transcriptional factors (EMT-TFs) and Stat3 signal pathway have been shown to affect EMT process. However, it is unclear whether the crosstalk between Stat3 and EMT-TFs affects CTCs in vivo during the process of metastasis. In previous studies, we have reported that the role of Stat3 interaction with nuclear receptors in gene transcription and tumor cell growth. We hypothesize that Stat3 may interacts with Twist1, a major EMT transcription factor, in CTCs, which may regulate the plasticity in EMT in metastasis of breast cancer.We proposed the present study with three specific aims: 1) to investigate the effects of Stat3/Twist crosstalk in dynamic alterations ofCTC numbers, EMT/MET program and CSCphenotypeand their association with the generation, survival, apoptosis, immunoescape and colonization of CTCs,by using mouse primary breast tumor model, mouse metastatic models including spontaneous and experimental lung metastatic model, and patients CTC xenograftmouse model; 2) to identify Stat3/Twist-targeted genes that are associated with invasion and metastasisby comparing gene expression patterns associated with individual steps of mammary tumor metastasis; and 3) to explore the feasibility of targeted intervention of different types of Stat3 inhibitors on breast cancer metastasis. These studies will significantly promote our understanding of the role of CTCs and provide a novel strategy to the treatment in breast cancer metastasis.

文献和我们的临床观察均表明乳腺癌转移与循环肿瘤细胞(CTCs)变化密切相关。上皮-间充质表型转换(EMT)及逆转换MET在体内可塑性编程赋予CTCs侵袭性和肿瘤干细胞性，EMT 转录因子如Twist和Stat3 信号通路均可调节EMT。但Stat3与EMT转录因子交互作用对乳腺癌循环肿瘤细胞影响迄今尚无报道。在前期研究中我们发现Stat3与核受体交互作用影响其靶基因转录和癌细胞生长。我们推测Stat3/Twist的交互作用可能亦参与EMT编程调控的癌转移。因此本项目将应用小鼠乳癌原位和转移模型、人CTC移植裸鼠临床前模型，通过动态观察CTCs其EMT/MET转换和CSC表型，研究Stat3/Twist交互作用对CTC形成、生存、凋亡，逃逸和定植的影响；寻找受Stat3/Twist调控的转移相关基因；探讨不同Stat3抑制剂靶向干预癌转移的可行性。这对于癌转移机制阐明与治疗具有重要意义。

乳腺癌是一种分子异质性的常见恶性肿瘤，以循环肿瘤细胞、循环 cfDNA 和循环cf-miRNA 为核心的“血源性液体活检 ”逐渐受到人们重视. 为检测早期肿瘤、监测早期转移、药物疗效观察、指导个体化治疗、评价生存预后提供了新的思路和策略。但是， 该领域的研究目前主要集中在提高检测循环肿瘤生物标志物的技术及其临床应用方面， 而对于肿瘤相关信号转导分子与转录因子对循环肿瘤生物标志物水平以及这些循环肿瘤生物标志物之间的相互联系的调节尚不清楚。.基于上皮-间充质表型转换(EMT)及逆转换MET在肿瘤细胞侵袭性和干细胞性的重要性， 本项目在建立小鼠循环肿瘤细胞分选和检测体系的基础上，系统研究了Stat3与EMT转录因子及其交互作用对CTCs其EMT/MET转换和CSC表型的影响，并且进一步应用乳腺癌病人样品验证了 Stat3作为循环肿瘤生物标志物的临床意义。 所获得的主要研究结果如下：1） 4T1-GFP过表达乳腺癌稳定细胞株的构建与乳腺癌原位移植荷瘤及自发肺转移小鼠模型的建立；2）应用免疫磁珠分选结合GFP荧光检测动物模型外周血中的循环肿瘤细胞方法的建立；3）Stat3或Twist稳定过表达的三阴高侵袭性乳腺癌单克隆细胞株的构建及其EMT表型鉴定; 4) 乳腺癌细胞EMT表型和侵袭能力的异质性与STAT3的表达及活化的相关性；5）EMT转录因子Twist1表达与STAT3活化而不是STAT3表达呈正相关；6）Stat3与Twist 对乳腺癌细胞EMT 调节机制的比较；7）EMT关键表型蛋白E-cadherin：评价乳腺癌阿霉素化疗敏感性的生物标志物；8） Stat3 作为肿瘤转移早期诊断的生物标志物以及治疗药物新靶的评价; 9）WP1066抑制STAT3磷酸化可增强阿霉素的抗肿瘤活性； 10) Stat3/Oct-4/c-Myc信号通路调节肿瘤干细胞介导的三阴性乳腺癌细胞对阿霉素的耐药性以及WP1066的抑制作用。 .上述结果不仅对阐明Stat3表达与活化对EMT转录因子编程驱动的CTC动态调控与干细胞化的分子机制，鉴定E-cadherin和Stat3 作为化疗药物评价与肿瘤转移早期诊断的生物标志物均具有重要意义，而且为 WP1066合用阿霉素可能成为一种新的化疗药物组合通过克服阿霉素耐药性应用于难治性三阴性乳腺癌提供了新思路。