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

项目摘要/摘要 Wnt/β-Catenin信号异常与多种人类癌症有关，最显著的是 APC和CTNNB1(β-catenin)突变导致的结直肠癌。CRC是第三种最常见的 癌症是全球第四大最常见的死亡原因。Wnt/β-连环蛋白信号转导系统显示其致癌作用 通过诱导Wnt靶基因CCND1(Cyclin D1)和Myc(c-Myc)的表达而发挥活性 β-连环蛋白/T细胞因子(Tcf)介导的转录。尽管APC和CTNNB1突变在APC和CTNNB1基因突变中起关键作用 人类结直肠癌的启动和发展，新的证据表明表观遗传修饰起作用 在人类结直肠癌的启动和发展中协同作用。一小部分癌症干细胞(CSCs)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的癌细胞中分离出来。我们和 另有研究发现，β-连环蛋白/Tcf介导的转录在人大肠肿瘤干细胞中高度激活。 与非CSC肿瘤细胞相比。在本应用中，我们鉴定了赖氨酸特异性去甲基酶4B(KDM4B) 擦除H3K9me3标记的基因在人癌组织中高度表达。击倒或药理作用 抑制KDM4B对人结直肠癌细胞Wnt靶基因的表达有显著抑制作用。去探索 KDM4B在体内的功能作用，我们建立了携带条件性Kdm4b的Kdm4bFlox/Flox(Kdm4bf/f)小鼠 等位基因。虽然Kdm4b基因在小鼠体内的缺失会导致肠道异常和隐窝干细胞减少， 与β-连环蛋白的缺失不同，我们发现小鼠可以耐受KDM4B缺乏，这表明靶向 KDM4B可能为人结直肠癌的治疗提供一个安全的治疗窗口。一如既往，我们发现 肠道Kdm4b基因缺失可完全抑制ApcMin/小鼠肠道肿瘤的发生 由于β-连环蛋白/Tcf介导的转录增加而导致的小肠多发性腺瘤。此外，我们 发现KDM4B与肠道干细胞转录因子ASCL2相互作用，ASCL2为阳性 反馈增强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 KDM4B可能在表观遗传学上控制Wnt/β-连环蛋白介导的肿瘤发生，这是肿瘤发生所必需的 人结直肠肿瘤干细胞的致瘤潜能。利用分子、细胞、遗传和表观遗传学方法， 我们将确定：1)KDM4B的基因敲除或药物抑制是否能抑制结直肠癌的发生 2)KDM4B是否在表观遗传学上控制CSCs的致瘤潜能和自我更新 3)KDM4B如何通过表观遗传学调控Wnt/β-catenin介导的转录和肿瘤发生 正在擦除H3K9me3。由于组蛋白去甲基酶是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癌的新策略 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.