Oxidative pentose phosphate pathway regulates AMPK homeostasis by balancing opposing LKB1 and PP2A

项目摘要/摘要： 新陈代谢途径和细胞信号网络之间的相互作用 在癌细胞中的“重新编程”在很大程度上仍然是未知的。磷酸戊糖氧化途径(OxIPPP) 在细胞糖酵解、生物合成和氧化还原动态平衡的代谢协调中起着至关重要的作用 生产核苷酸和脂肪生物合成的前体，以及抗氧化剂NADPH 癌细胞快速增殖过程中产生的活性氧(ROS)。有三种关键的酶 沿着oxIPPP。葡萄糖-6-磷酸脱氢酶(G6PD)是糖酵解的第一种酶 中间葡萄糖-6-磷酸(G6P)到6-磷酸葡萄糖内酯(6PG1)，并产生NADPH。这个 第二种酶6-磷酸葡萄糖内酯酶(PGLS)将6PG1转化为6-磷酸葡萄糖酸(6PG)。第三 酶6-磷酸葡萄糖酸脱氢酶(6PGD)将6PG转化为核酮糖-5-磷酸(Ru-5-P)，还 产生NADPH。我们最近报道，在癌细胞中，6PGD通常被赖氨酸乙酰化激活 并通过控制其产物Ru-5-P激活脂肪生成，从而抑制LKB1-AMPK途径 破坏活性LKB1复合体(Shane等人，2014 Mol Cell；Lin等人，2015年，NAT Cell Biol.)。有趣的是，我们 研究发现，尽管Ru-5-P和随后的LKB1减少，G6PD的敲除并没有改变AMPK的激活 激活，这是由于AMPK的上游磷酸酶PP2A的活性增强。相比之下，击倒 6PGD或PGLS使PP2A活性降低。从机制上讲，G6PD或PGLS的基因敲除减少或增加 6PG1水平，增强了Src对PP2A的抑制磷酸化。有两种形式的 6PG1，γ-6-磷酸葡萄糖内酯(γ-6PGl)是一种产生的功能未知的oxIPPP副产物 通过δ-6-磷酸葡萄糖内酯(δ-6GL)的分子内重排，而δ-6PG1是唯一的 PGLS的底物，并能快速自发水解。因此，γ-6PG1相对稳定。 加入δ-6GL，但不参加oxIPPP。进一步的研究表明，γ-6PG1而不是δ-6GL促进 SRC-PP2A结合，可能是通过与Src结合而不是PP2A，并促进PP2A的招募。 因此，我们推测G6PD、PGLS和6PGD在AMPK的调节中起着不同的作用 通过oxipPP中间体Ru-5-P和An平衡对立的LKB1和PP2A的动态平衡 分别是oxIPPP的副产品γ-6PGl和γ-6PGl，以前被认为是 OxIPPP具有未知的生理功能，是连接代谢oxIPPP的信号分子 与Src-PP2A-AMPK信号通路有关。具体目标是：(1)阐明分子生物学和分子生物学 γ-6PG1依赖的信号基础通过抑制PP2A参与AMPK的激活； (2)确定G6PD和PGLS对AMPK激活、氧化还原动态平衡和肿瘤的不同作用 (3)评价oxIPPP抑制剂和AMPK激活剂联合治疗儿童骨肉瘤的临床疗效。 人白血病和肺癌细胞的体外和体内实验。

Project Summary/Abstract: The interplay between metabolic pathways and cell signaling networks that contribute to the “metabolic reprogramming” in cancer cells remains largely unknown. The oxidative pentose phosphate pathway (oxiPPP) plays a crucial role in the metabolic coordination of glycolysis, biosynthesis and redox homeostasis in cells by producing precursors for nucleotide and lipid biosynthesis, as well as antioxidant NADPH that quenches the reactive oxygen species (ROS) produced during rapid proliferation of cancer cells. There are three key enzymes along the oxiPPP. The first enzyme glucose-6-phosphate dehydrogenase (G6PD) converts glycolytic intermediate glucose-6-phosphate (G6P) to 6-phosphogluconolactone (6PGL) and produces NADPH. The second enzyme 6-phosphogluconolactonase (PGLS) converts 6PGL to 6-phosphogluconate (6PG). The third enzyme 6-phosphogluconate dehydrogenase (6PGD) converts 6PG to ribulose-5-phosphate (Ru-5-P) and also produces NADPH. We recently reported that 6PGD is commonly activated by lysine acetylation in cancer cells and activates lipogenesis through controlling its product Ru-5-P, which inhibits the LKB1-AMPK pathway by disrupting the active LKB1 complex (Shan et al., 2014 Mol Cell; Lin et al., 2015, Nat Cell Biol.). Interestingly, we found that knockdown of G6PD did not alter AMPK activation despite decreased Ru-5-P and subsequent LKB1 activation, due to enhanced activity of PP2A, the upstream phosphatase of AMPK. In contrast, knockdown of 6PGD or PGLS reduced PP2A activity. Mechanistically, knockdown of G6PD or PGLS decreased or increased 6PGL level, respectively, which enhanced the inhibitory phosphorylation of PP2A by Src. There are two forms of 6PGL, γ-6-phosphogluconolactone (γ-6PGL) is an oxiPPP byproduct with unknown function that is generated through intramolecular rearrangement of δ-6-phosphogluconolactone (δ-6GL), while δ-6PGL is the only substrate of PGLS and can undergo quick spontaneous hydrolysis. Thus, γ-6PGL is relatively stable compared to δ-6GL but does not participate in oxiPPP. Further studies revealed that γ-6PGL, but not δ-6GL, promotes Src-PP2A association, probably by binding to Src but not PP2A and enhancing PP2A recruitment. Thus, we hypothesize that G6PD, PGLS and 6PGD play differential roles in regulation of AMPK homeostasis by balancing the opposing LKB1 and PP2A, through the oxiPPP intermediate Ru-5-P and an oxiPPP “byproduct” γ-6PGL, respectively; and γ-6PGL, previously considered as a “dead end” byproduct of the oxiPPP with unknown physiological function, functions as a signaling molecule that links the metabolic oxiPPP with the Src-PP2A-AMPK signaling pathway. The specific aims are proposed: (1) To elucidate the molecular and signaling basis underlying γ-6PGL-dependent contribution to AMPK activation through inhibition of PP2A by Src; (2) To determine the differential effects of G6PD and PGLS on AMPK activation, redox homeostasis and tumor growth; and (3) To evaluate combined therapy with oxiPPP inhibitors and AMPK activator in the treatment of human leukemia and lung cancer cells in vitro and in vivo.