Molecular and Epigenetic Control of Wnt/b-catenin-mediated oncogenesis by KDM4B

项目概要/摘要 异常Wnt/β-catenin信号传导与各种类型的人类癌症相关，最值得注意的是 结直肠癌（CRC）由于APC和CTNNB 1（β-连环蛋白）突变。CRC是第三常见的 癌症是全球第四大常见死因。Wnt/β-catenin信号通路显示其致癌性 通过诱导Wnt靶基因如CCND 1（细胞周期蛋白D1）和MYC（c-Myc）的表达， β-连环蛋白/T细胞因子（Tcf）介导的转录。尽管APC和CTNNB 1突变在肿瘤发生中起关键作用， 人类CRC的发生和发展，新出现的证据表明表观遗传修饰起作用， 在人CRC起始和发展中协同作用。一小部分癌症干细胞（CSC）或癌症 具有自我更新和维持肿瘤能力的起始细胞已经从人CRC中分离出来。我们和 其他人发现β-连环蛋白/Tcf介导的转录在人结肠直肠CSC中被过度激活 与非CSC肿瘤细胞相比。在本申请中，我们鉴定了赖氨酸特异性脱甲基酶4 B（KDM 4 B）， 清除H3 K9 me 3标记的基因在人CRC中高度表达。击倒或药理学 在人CRC细胞中抑制KDM 4 B显著抑制Wnt靶基因的表达。探讨 KDM 4 B在体内的功能作用，我们产生了携带条件性KDM 4 b的Kdm 4 bflox/flox（Kdm 4 bf/f）小鼠， 等位基因尽管小鼠中Kdm 4 b的缺失会导致肠道异常和隐窝干细胞减少， 与β-catenin的缺失不同，我们发现小鼠可以耐受KDM 4 B缺陷，这表明靶向 KDM 4 B可能为人类CRC的治疗提供一个安全的治疗窗口。我们发现， 在ApcMin/+小鼠中，Kdm 4 b肠缺失完全抑制肠肿瘤发生， 由于β-连环蛋白/Tcf介导的转录增加导致小肠多发性腺瘤。另外我们 发现KDM 4 B与肠干细胞转录因子ASCL 2相互作用， 反馈以增强β-连环蛋白/Tcf介导的转录。基于这些令人兴奋的发现，我们假设 KDM 4 B可能在表观遗传学上控制Wnt/β-catenin介导的肿瘤发生， 人结肠直肠CSC的致瘤潜力。利用分子、细胞、遗传和表观遗传的方法， 我们将确定：1）KDM 4 B的敲除或药理学抑制是否抑制CRC肿瘤发生 2）KDM 4 B是否在表观遗传学上控制人CSC的致瘤潜力和自我更新 CRCs; 3）KDM 4 B如何通过表观遗传学控制Wnt/β-连环蛋白介导的转录和肿瘤发生 删除H3 K9 me 3。由于组蛋白去甲基化酶是一种很容易被小分子靶向的酶， 抑制剂，我们研究的新发现可能会导致开发治疗人类CRC的新策略 和其他癌症。

Project Summary/Abstract Abnormal Wnt/β-catenin signaling is associated with various types of human cancers, most notably colorectal cancers (CRCs) due to APC and CTNNB1 (β-catenin) mutations. CRCs are the third most common cancer worldwide and the fourth most common cause of death. Wnt/β-catenin signaling exhibits its oncogenic activities by inducing the expression of Wnt target genes such as CCND1 (Cyclin D1) and MYC (c-Myc) through β-catenin/T cell factor (Tcf)-mediated transcription. Although APC and CTNNB1 mutations play a critical role in human CRC initiation and development, emerging evidence suggests that epigenetic modifications act synergistically in human CRC initiation and development. A small subset of cancer stem cells (CSCs) or cancer initiating cells with the ability to self-renew and maintain the tumor has been isolated from human CRCs. We and others have found that β-catenin/Tcf-mediated transcription is hyperactivated in human colorectal CSCs compared to non-CSC tumor cells. In this application, we identified that lysine-specific demethylase 4B (KDM4B) that erases H3K9me3 marks was highly expressed in human CRCs. The knockdown or pharmacological inhibition of KDM4B in human CRC cells significantly inhibited the expression of Wnt target genes. To explore the functional roles of KDM4B in vivo, we generated Kdm4bflox/flox (Kdm4bf/f) mice that carry conditional Kdm4b alleles. Although the deletion of Kdm4b in mice causes intestinal abnormalities and reduced crypt stem cells, unlike the deletion of β-catenin, we found that mice can tolerate KDM4B deficiency, indicating that targeting KDM4B might provide a safe therapeutic window for the treatment of human CRCs. Consistently, we found that the intestinal deletion of Kdm4b completely inhibited intestinal tumorigenesis in ApcMin/+ mice which develop multiple adenomas in the small intestine due to increased β-catenin/Tcf-mediated transcription. Additionally, we found that KDM4B interacts with the intestinal stem cell transcription factor ASCL2 which serve as a positive feedback to enhance β-catenin/Tcf-mediated transcription. Based on these exciting discoveries, we hypothesize that KDM4B might epigenetically control Wnt/β-catenin-mediated oncogenesis and is required for the tumorigenic potentials of human colorectal CSCs. Using molecular, cellular, genetic and epigenetic approaches, we will determine: 1) Whether the knockout or pharmacological inhibition of KDM4B inhibits CRC tumorigenesis in vivo; 2) Whether KDM4B epigenetically controls tumorigenic potentials and self-renewal of CSCs in human CRCs; 3) How KDM4B epigenetically controls Wnt/β-catenin-mediated transcription and tumorigenesis by erasing H3K9me3. Since histone demethylases are enzymes which can be readily targeted by small molecules inhibitors, novel findings from our studies may lead to develop novel strategies for the treatment of human CRCs and other cancers.