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

项目概要/摘要 异常的 Wnt/β-连环蛋白信号传导与多种类型的人类癌症相关，尤其是 APC 和 CTNNB1（β-连环蛋白）突变引起的结直肠癌 (CRC)。 CRC 是第三常见的 癌症是全世界第四大常见死因。 Wnt/β-连环蛋白信号传导显示其致癌性 通过诱导 Wnt 靶基因的表达来发挥活性，例如 CCND1 (Cyclin D1) 和 MYC (c-Myc) β-连环蛋白/T 细胞因子 (Tcf) 介导的转录。尽管 APC 和 CTNNB1 突变在 人类结直肠癌的发生和发展，新出现的证据表明表观遗传修饰起作用 在人类 CRC 的发生和发展中具有协同作用。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从人类结直肠癌中分离出来。我们和 其他人发现 β-catenin/Tcf 介导的转录在人类结直肠 CSC 中过度激活 与非 CSC 肿瘤细胞相比。在此应用中，我们鉴定了赖氨酸特异性脱甲基酶 4B (KDM4B) 消除 H3K9me3 标记的蛋白在人类 CRC 中高度表达。击倒或药理学 抑制人CRC细胞中的KDM4B可显着抑制Wnt靶基因的表达。探索 为了研究 KDM4B 在体内的功能作用，我们生成了携带条件 Kdm4b 的 Kdm4bflox/flox (Kdm4bf/f) 小鼠 等位基因。虽然小鼠中Kdm4b的缺失会导致肠道异常和隐窝干细胞减少， 与删除 β-连环蛋白不同，我们发现小鼠可以耐受 KDM4B 缺陷，这表明靶向 KDM4B 可能为人类 CRC 的治疗提供安全的治疗窗口。一致地，我们发现 Kdm4b 肠道缺失完全抑制了 ApcMin/ 小鼠肠道肿瘤的发生 由于 β-catenin/Tcf 介导的转录增加，导致小肠多发性腺瘤。此外，我们 发现 KDM4B 与肠干细胞转录因子 ASCL2 相互作用，后者起到积极的作用 反馈以增强 β-catenin/Tcf 介导的转录。基于这些令人兴奋的发现，我们假设 KDM4B 可能通过表观遗传方式控制 Wnt/β-catenin 介导的肿瘤发生，并且是 人类结直肠癌干细胞的致瘤潜力。使用分子、细胞、遗传和表观遗传方法， 我们将确定： 1) KDM4B 的敲除或药物抑制是否会抑制 CRC 肿瘤发生 体内； 2) KDM4B是否通过表观遗传学控制人类CSC的致瘤潜力和自我更新 CRC； 3) KDM4B如何通过表观遗传控制Wnt/β-catenin介导的转录和肿瘤发生 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结直肠癌的新策略 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.