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

项目概要/摘要： 代谢途径和细胞信号网络之间的相互作用，有助于“代谢” 癌细胞中的“重编程”在很大程度上仍是未知的。氧化戊糖磷酸途径（oxiPPP） 在细胞糖酵解、生物合成和氧化还原稳态的代谢协调中起着至关重要的作用， 产生核苷酸和脂质生物合成的前体，以及抗氧化剂NADPH， 癌细胞快速增殖过程中产生的活性氧（ROS）。有三种关键酶 沿着oxiPPP。第一种酶葡萄糖-6-磷酸脱氢酶（G6 PD）将糖酵解转化为葡萄糖。 葡萄糖-6-磷酸（G6 P）转化为6-磷酸葡萄糖内酯（6PGL）并产生NADPH。的 第二种酶6-磷酸葡糖酸内酯酶（PGLS）将6PGL转化为6-磷酸葡糖酸盐（6PG）。第三 酶6-磷酸葡糖酸脱氢酶（6PGD）将6PG转化为核酮糖-5-磷酸（Ru-5-P），并且还 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，Ru-5-P通过以下途径抑制LKB 1-AMPK途径： 破坏活性LKB 1复合物（Shan等，2014 Mol Cell; Lin等人，2015，Nat Cell Biol.）。有意思的是，我们 发现敲低G6 PD并不改变AMPK的激活，尽管Ru-5-P和随后的LKB 1 激活，由于PP 2A的活性增强，AMPK的上游磷酸酶。相反， 6PGD或PGLS降低PP 2A活性。在机制上，G6 PD或PGLS的敲低降低或增加 6PGL水平，增强Src对PP 2A磷酸化的抑制作用。有两种形式 6PGL，γ-6-磷酸己内酯（γ-6PGL）是一种oxiPPP副产物，功能未知， 通过δ-6-磷酸内酯（δ-6GL）的分子内重排，而δ-6PGL是唯一的 PGLS的底物，并可进行快速自发水解。因此，γ-6PGL相对稳定， 至δ-6GL，但不参与oxiPPP。进一步的研究表明，γ-6PGL，而不是δ-6GL，促进 Src-PP 2A结合，可能是通过结合Src而不是PP 2A并增强PP 2A募集。 因此，我们推测G6 PD、PGLS和6PGD在AMPK的调节中发挥不同的作用 通过平衡相对的LKB 1和PP 2A，通过oxiPPP中间体Ru-5-P和一个 oxiPPP“副产物”γ-6PGL;和γ-6PGL，先前被认为是oxiPPP“死端”副产物。 oxiPPP具有未知的生理功能，作为一种信号分子，连接代谢oxiPPP Src-PP 2A-AMPK信号通路。具体目标是：（1）阐明分子和分子生物学机制， Src通过抑制PP 2A对AMPK活化的γ-6PGL依赖性作用的信号基础; (2)为了确定G6 PD和PGLS对AMPK活化、氧化还原稳态和肿瘤的不同作用， 评价oxiPPP抑制剂和AMPK激活剂联合治疗对生长的影响。 人白血病和肺癌细胞的体外和体内研究。

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.