COORDINATE REGULATION BY MAP3K4 OF EPIGENETIC MODIFIERS CONTROLLING EMT

 描述(申请人提供)：上皮向间充质转化(EMT)是一种发育过程，具有紧密细胞-细胞粘连的非运动性上皮细胞转化为单独的运动性间充质细胞。重要的是，EMT是可逆的，通过间充质到上皮的转变(MET)将间充质细胞转换回上皮状态。EMT计划对于发育过程中的正常过程和组织再生是必不可少的。EMT在人类疾病中被重新激活，包括肾、肝和肺的组织纤维化以及癌症转移，使EMT成为药物治疗的关键靶点。小鼠(KI4)中MAP3K4激酶活性部位赖氨酸的定点突变会由于EMT中断而导致严重的发育缺陷。来自MAP3K4(KI4)小鼠的上皮滋养细胞干细胞(TSKI4细胞)在EMT的中期被独特地暂停，在保持干细胞特性的同时同时表达上皮和间充质特征。在TSKI4细胞中诱导EMT的部分原因是MAP3K4/CBP在控制上皮表型的基因启动子上失去了MAP3K4/CBP介导的组蛋白H2B乙酰化。我们最近发现，MAP3K4活性的丧失会增加另一种染色质重构体HDAC6的表达和活性。本项目的目标是明确MAP3K4调控EMT的分子网络。这个网络包括导致EMT重要基因表观遗传调控的信号通路。我们的方法是基于我们发表的工作和新的发现，表明MAP3K4通过激活CBP和抑制对EMT重要的基因启动子上的HDAC6来协调EMT。我们预测，这个MAP3K4控制的网络中的基因在EMT相关病理中起关键作用。我们的理论基础是基于我们的TSKI4干细胞和Claudin低乳腺癌细胞之间重叠的基因表达签名的发现，这两个细胞都表现出干性和EMT的特征。具有这些特征的癌症通常既有转移性，又表现出对治疗的抵抗力。调节EMT转变的蛋白质网络是潜在的治疗靶点，其理论基础是逆转EMT将恢复对治疗的敏感性。我们使用这一创新系统的初步工作已经成功地在乳腺癌中发现了控制EMT/MET的新基因。AIM1使用RNA-SEQ和CHIP-SEQ来识别控制EMT的MAP3K4/CBP/HDAC6/H2BK5Ac依赖网络。目的2是关于MAP3K4和HDAC6在EMT中的作用及其对KI4细胞和组织表型的影响的机制研究。第三个目的是针对GALNT3，一个MAP3K4/CBP/HDAC6/H2BK5Ac共同调节的基因，定义了GALNT3控制EMT的机制。总之，该提案的三个目标将定义MAP3K4通过染色质修饰物CBP和HDAC6的共同调节来协调细胞表型的信号机制。此外，这项提议将识别和表征调控上皮/间充质状态的新基因，导致发现新的生物标记物和药物靶点，用于识别和治疗与EMT相关的病理，如纤维化和转移。

 DESCRIPTION (provided by applicant): Epithelial to mesenchymal transition (EMT) is a developmental program in which non-motile epithelial cells with tight cell-cell adherence convert into individual, motile mesenchymal cells. Importantly, EMT is reversible through a mesenchymal to epithelial transition (MET) converting mesenchymal cells back to an epithelial state. The EMT program is essential for normal processes during development and for tissue regeneration. EMT is reactivated in human diseases including tissue fibrosis in the kidney, liver, and lung and in cancer metastasis, making EMT a key target for drug therapy. Targeted mutation of the active site lysine of the kinase MAP3K4 in the mouse (KI4) results in severe developmental defects due to disrupted EMT. Epithelial trophoblast stem (TS) cells from kinase-inactive MAP3K4 (KI4) mice (TSKI4 cells) are uniquely paused in the intermediate stages of EMT, expressing both epithelial and mesenchymal characteristics while maintaining stemness. Induction of EMT in TSKI4 cells is due in part to the loss of MAP3K4/CBP mediated acetylation of histone H2B on the promoters of genes controlling the epithelial phenotype. We have recently discovered that loss of MAP3K4 activity increases the expression and activity of another chromatin remodeler, HDAC6. The goal of this project is to define the molecular network regulated by MAP3K4 controlling EMT. This network includes signaling pathways leading to the epigenetic regulation of genes important for EMT. Our approach is based on both our published work and new findings showing that MAP3K4 coordinates EMT by activating CBP and inhibiting HDAC6 on the promoters of genes important for EMT. We predict that genes in this MAP3K4 controlled network are critical to EMT-related pathologies. Our rationale is based on our findings of overlapping gene expression signatures between our TSKI4 stem cells and claudin low breast cancer cells that both display characteristics of stemness and EMT. Cancers with these characteristics frequently are both metastatic and display resistance to therapy. Protein networks that regulate the EMT transition represent potential targets for therapy with the rationale that reversal of EMT would restore sensitivity towards therapy. Our preliminary work using this innovative system has successfully identified new genes in breast cancer that control EMT/MET. Aim1 uses RNA-seq and ChIP-seq to identify a MAP3K4/CBP/HDAC6/H2BK5Ac dependent network that controls EMT. Aim 2 is a mechanistic study of the role of MAP3K4 and HDAC6 in EMT and the impact on the KI4 cellular and organismal phenotypes. The third aim targets GALNT3, a MAP3K4/CBP/HDAC6/H2BK5Ac co- regulated gene, defining the mechanisms by which GALNT3 controls EMT. Together, the three aims of this proposal will define the signaling mechanisms by which MAP3K4 coordinates the cellular phenotype through the co-regulation of the chromatin modifiers CBP and HDAC6. Further, this proposal will identify and characterize novel genes regulating epithelial/mesenchymal states, leading to the discovery of new biomarkers and drug targets for the identification and treatment of EMT related pathologies like fibrosis and metastasis.