JMJD2B对大肠癌代谢应激的影响及其与ERK-MAPK的关系

Metabolic stress is a common phenomenon in varieties of solid tumors. Compensatory mechanisms to overcome metabolic stress (glucose deprivation) are vital to protect tumor cells from metabolic stress induced death and prolong cell survival. Histone demethylase JMJD2B gene is necessary for tumor survival. We also confirmed JMJD2B can promote colorectal cancer cell survival through regulation of proliferation, apoptosis, and cell cycle transition; and found significant upregulation of JMJD2B during glucose deprivation, along with activation of ERK-MAPK. Accordingly, we assume that ERK-MAPK and JMJD2B are closely related with adaptive response to metabolic stress in colorectal cancer cells. However, researches on their effects during metabolic stress and molecular mechanism of ERK-MAPK regulating JMJD2B are rare. Therefore, on the basis of former study, we aim to (1) clarify JMJD2B expression and its relationship with ERK-MAPK during glucose deprivation, and its effect on colorectal cancer cell survival; (2) explore the molecular mechanism of ERK-MAPK regulating JMJD2B during glucose deprivation; (3) analyze the correlation between JMJD2B, glucose metabolism-related protein, and ERK-MAPK, and their relationship with clinical pathological features and prognosis. The implementation of this task will provide new ideas for clinical treatment of colorectal cancer.

代谢应激是实体瘤的常见现象。对代谢应激（葡萄糖缺乏）的代偿机制对于肿瘤细胞克服其诱发的死亡并维持生存至关重要。组蛋白去甲基化酶JMJD2B是肿瘤生存所必需的基因。我们亦证实JMJD2B通过调控增殖、凋亡、周期等促进大肠癌细胞生存；并发现JMJD2B在葡萄糖缺乏时显著上调，且伴随ERK-MAPK的活化。据此我们推测ERK-MAPK和JMJD2B与大肠癌细胞适应代谢应激密切相关。然而，目前罕有研究代谢应激下两者的具体作用机理及相互调控的分子机制。因此我们拟在前期基础上，(1)明确葡萄糖缺乏时JMJD2B的表达及其与ERK-MAPK的关系，以及对大肠癌细胞生存的影响；(2)探讨葡萄糖缺乏时ERK-MAPK调控JMJD2B的分子机制；(3)分析大肠癌组织中JMJD2B、葡萄糖代谢相关蛋白、ERK-MAPK之间的相关性，及其与临床病理和预后的关系。本课题的实施将为临床治疗提供新思路。

代谢应激是实体瘤的常见现象。对代谢应激（葡萄糖缺乏）的代偿机制对于肿瘤细胞克服其诱发的死亡并维持生存至关重要。组蛋白去甲基化酶JMJD2B是肿瘤生存所必需的基因。在前期研究中，我们证实JMJD2B通过调控增殖、凋亡、周期等促进大肠癌细胞的生存，并发现JMJD2B在葡萄糖缺乏时显著上调，且伴随ERK-MAPK的活化，因此我们开展了如下研究。首先，我们通过无糖饥饿培养干预抑制细胞对葡萄糖的摄取时，发现大肠癌细胞JMJD2B蛋白水平显著上调，而同时RNA水平未见明显变化，葡萄糖缺乏时JMJD2B表达上调，同时伴随ERK1/2磷酸化水平上升。生存实验证实磷酸化ERK1/2在葡萄糖缺乏下依赖JMJD2B从而发挥作用。co-IP方法证实JMJD2B和磷酸化ERK1/2之间存在蛋白-蛋白相互作用；伴随磷酸化ERK1/2表达增加，JMJD2B和磷酸化ERK1/2在细胞内存在明显的共定位，抑制ERK-MAPK信号通路的活化可并破坏JMJD2B和磷酸化ERK1/2在细胞内相互作用和共定位。 其次，我们进一步探讨磷酸化ERK1/2能否磷酸化JMJD2B蛋白。免疫沉淀方法证实了JMJD2B丝氨酸／苏氨酸位点磷酸化水平在葡萄糖缺乏下显著升高，将JMJD2B蛋白中可能的ERK1/2磷酸化位点分别突变成丙氨酸，构建质粒转染发现其磷酸化水平较野生型显著降低，提示上述位点可以被磷酸化；同时JMJD2B T305磷酸化特异性抗体证实葡萄糖缺乏时JMJD2B T305位点磷酸化水平增加，并受到p-ERK的调控。 第三，我们随后验证发现Thr305, Ser352, Ser566 and Thr1065位点磷酸化对于JMJD2B稳定性发挥重要作用。CHX抑制实验提示即使抑制了蛋白质的合成，JMJD2B蛋白降解在葡萄糖缺乏时也是受到抑制的。我们同时发现JMJD2B对葡萄糖的摄取能力以及乳酸的产生也有着重要的影响。JMJD2B通过结合至GLUT1基因启动子区发挥对GLUT1的转录激活作用从而促进大肠癌细胞对葡萄糖的摄取，大肠癌组织芯片中亦证实JMJD2B与Pp-ERK1/2与GLUT1之间存在正相关关系。