CUL4A通过WDR5介导的表观遗传修饰调控乳腺和乳腺癌干细胞扩增和干性

CUL4A, a member of an E3 ligase family, is significantly associated with prognosis of breast cancers. Our previous data showed that over-expression of CUL4A in breast and breast cancer cell lines can induce epithelial-mesenchymal transition of normal breast and breast cancer cell lines. Further preliminary experiments indicated that CUL4A induces NANOG, OCT4, SOX2 expression in normal breast and breast cancer cell lines, increases the proportion and stemness of cancer stem cells. Higher level of tri-methylation of H3K4 and enhanced bindings of H3K4me3 with promoter sequences if the above stem-related genes were also detected in breast cancer cell line. Taken together with previous reports, we postulated that CUL4A may induce self-renewal and differentiation potentials breast stem cells and breast cancer stem cells though WDR5 mediated H3K4 tri-methylation. To validate our preliminary experiments and test our hypothesis we planned to use serials of cellular, and molecular assays such as self-renewal and differentiation potential, flow cytometry, ChIP-sequencing, ChIP-qPCR etc in culture cells, transgenic mouse models to analysis the effects of CUL4A on stemness of normal breast and cancer stem cells and try to dissect the underlying epigenetic mechanisms. Finally we will examine the correlation of CUL4A with cancer stem cells from human breast cancer tissues and clinical characteristics of patients. The successful accomplishments of this project will further our understanding of generation, stemness and other properties of breast cancer stem cells and will shed lights on mechanisms of tumorigenesis, relapse and metastasis of breast cancers.

肿瘤干细胞被认为是肿瘤耐药、复发、转移的根源，但目前对肿瘤干细胞的调控机制了解不足。CUL4A是与乳腺癌预后密切相关的癌基因，我们初步实验显示CUL4A能诱导乳腺和乳腺癌细胞EMT及其干细胞特性，使其具有多胚层分化潜能。根据前期实验结果和文献报道，我们推测CUL4A可能通过WDR5介导的组蛋白甲基化修饰调节乳腺和乳腺癌干细胞的扩增和分化潜能。本项目拟在前期研究基础上，综合运用流式细胞术、干细胞诱导分化、基因芯片、ChIP-seq、ChIP-qPCR等技术手段在分子、细胞、转基因鼠及临床标本等层面深入探讨CUL4A调控乳腺和乳腺癌干细胞增殖和分化潜能的作用及其表观遗传学机制，并分析CUL4A表达与乳腺癌病人肿瘤干细胞、临床特征的相关性。旨在通过上述研究验证我们的假设，深化对乳腺及乳腺癌干细胞的认识，揭示乳腺癌发生及耐药、复发、转移的新机制，为进一步研究乳腺癌生物治疗方案和药物提供新思路。

乳腺癌系女性常见恶性肿瘤，是世界范围癌症死亡的主要病因之一。目前其机制仍存在很多未知之处。随着对乳腺癌研究的深入，逐渐认识到乳腺干细胞的异常增殖分化以及乳腺肿瘤干细胞的形成和扩增在乳腺癌发生及复发转移中具有重要作用。但目前对乳腺癌肿瘤干细胞的调控机制了解不足。CUL4A是与乳腺癌预后密切相关的癌基因，其在乳腺癌肿瘤干细胞调控中的作用和机制未名，我们通过本课题的研究证实正常乳腺上皮细胞MCF10A和乳腺癌上皮细胞MDA-MB-468高表达CUL4A后能够抑制上皮细胞标志蛋白E-cadherin表达，而促进N-cadherin、Fibronectin等间质细胞标志蛋白表达，细胞划痕及Transwell实验表明CUL4A能增强细胞运动迁移能力，这些特征性改变表明CUL4A能诱导乳腺上皮细胞向间质细胞转变。而大量的研究表明乳腺上皮细胞经历EMT后会表现干细胞的特征。我们的后续研究发现，长期培养的CUL4A高表达细胞能够形成“干细胞样”克隆球，免疫荧光染色显示胚胎干细胞标志分子TRA-1-60和SSEA4阳性。证明CUL4A可能在乳腺和乳腺癌干细胞增殖中具有重要作用。鉴于此，我们进行了干细胞相关特性的研究，经Western blot和RT-PCR实验证实，在CUL4A高表达的MCF10A和MDA-MB-468细胞中干细胞相关基因OCT4、NANOG、SOX2等表达均较对照组升高，体外球体形成实验也明确CUL4A高表达的MDA-MB-468细胞有更多的干细胞。此外，裸鼠体内成瘤实验也证实CUL4A能够促进肿瘤的发生。进一步发现CUL4A高表达诱导的乳腺癌干细胞具有多胚层分化潜能。因此我们检测了CUL4A与H3K4甲基化水平的关系。我们的实验发现CUL4A高表达的细胞中H3K4的三重甲基化（H3K4me3）水平显著升高，并且，染色质免疫共沉淀结合定量PCR实验重复表明，H3K4me3与上述干细胞诱导基因启动子的结合增强。本课题通过上述系统深入的研究，明确CUL4A在调节乳腺癌肿瘤干细胞中的作用于功能，深化对乳腺及乳腺癌干细胞的认识，揭示乳腺癌发生及耐药、复发、转移的新机制，为进一步研究乳腺癌生物治疗方案和药物提供新思路。