Functional Analysis of Histone Demethylase Activity in Hypoxic Cancer Cells

项目总结（见说明）： 肿瘤内不受控制的细胞生长导致缺氧，因为细胞的代谢需求超过了肿瘤脉管系统提供氧气和营养物的能力。为了应对缺氧微环境，细胞进行大规模的转录重编程以促进存活。缺氧诱导转录因子（HIF）是缺氧反应的主要调节因子，并诱导糖酵解基因、细胞迁移因子和血管生成因子的表达。HIF还诱导几种转录调节因子的表达，包括几种组蛋白脱甲基酶，为缺氧细胞提供延长或固定促存活基因表达的机制。这些组蛋白去甲基化酶之一JMJD2B使三甲基化组蛋白H3赖氨酸9（H3K9me3）去甲基化，这是被抑制的染色质结构的关键标志物。JMJD2B的低分化诱导可能在激活基因表达以促进肿瘤生长中起重要作用。与该假设一致，JMJD2B表达的强制敲低降低了肿瘤异种移植物的生长，并且在卵巢癌中过表达。在该提议中，将使用肿瘤异种移植实验在体内测试JMJD2B调节肿瘤生长的程度，并通过测定JMJD2B表达导致的细胞增殖、侵袭和血管生成在体外测试JMJD2B调节肿瘤生长的程度（特异性目的1）。随后的生化实验将确定缺氧中特定靶基因调控的机制，鉴定缺氧中JMJD2B调控的新基因，并表征对致瘤表型重要的基因的关键途径的调控（特异性目的2）。本提案中描述的实验将建立JMJD2B用于调节肿瘤发生的机制，而 为增强肿瘤治疗寻找新的靶向途径。

PROJECT SUMMARY (See instructions): Uncontrolled cell growth within a tumor results in hypoxia as the metabolic needs of the cells exceed the ability of the tumor vasculature to provide oxygen and nutrients. In response to the hypoxic microenvironment, cells undergo a massive reprogramming of transcription to promote survival. The Hypoxia Inducible Transcription Factors (HIFs) are primary regulators of the hypoxic response and induce the expression of glycolytic genes, cell migration factors, and angiogenic factors. HIFs also induce expression of several transcriptional regulators, including several histone demethylases, providing a mechanism for the hypoxic cell to extend or fix the expression of pro-survival genes. One of these histone demethylases, JMJD2B, demethylates tri-methylated histone H3 lysine 9 (H3K9me3), a key marker of repressed chromatin structure. Hypoxic induction of JMJD2B may play an important role in activating gene expression to promote tumor growth. Consistent with this hypothesis, forced knock-down of JMJD2B expression reduces growth of tumor xenografts, and is overexpressed in ovarian cancers. In this proposal, the extent to which JMJD2B regulates tumor growth will be tested in vivo using tumor xenograft experiments and in vitro by assaying for cell proliferation, invasion, and angiogenesis as a result of JMJD2B expression (Specific Aim 1). Subsequent biochemical experiments will determine the mechanism of specific target gene regulation in hypoxia, identify new genes regulated by JMJD2B in hypoxia, and characterize the regulation of key pathways of genes important for the tumorigenic phenotype (Specific Aim 2). The experiments described in this proposal will establish the mechanisms utilized by JMJD2B to regulate tumorigenesis, while identifying new pathways to target for enhanced tumor therapies.