Cancer Res：蛋白质组学和代谢组学联合分析揭示肾癌中分级依赖的代谢重编程

现如今，蛋白质组学和代谢组学越来越多地运用于疾病领域的研究，如何把这两个组学的数据有效结合用于科学研究，仍然是一个研究的难点。本篇文献会利用蛋白质组学和代谢组学两种技术研究处于不同分级的肾癌组织，并着重从KEGG通路分析着手，将蛋白质组学和代谢组学的数据进行整合分析，很好地揭示了多条参与调控肾癌分级的通路。

文献来源：Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis. Cancer Res. 2015 Jun 15; 75(12): 2541-52.

文章摘要

肾癌（RCC）通常伴随多发的系统损害，常被称为“内科医生的肿瘤”，提示其存在着复杂的、非生理的代谢通路，目前缺乏有效的治疗靶点。本研究依据Fuhrman分级标准，对不同分级的肾癌组织进行了蛋白质组学和非靶向代谢组学的联合分析，揭示了厌氧糖酵解、氧化代谢、谷氨酰胺代谢通路以及色氨酸分解代谢途径参与调控的分级依赖的代谢重编程，有助于临床针对不同分级肾癌患者的个性化治疗，并为新药研发提供潜在的靶点。

实验设计

• 蛋白质组学和代谢组学的核心应用思路 ---- 差异比较（Fuhrman Grade）

Table 1. Human RCC samples analyzed by metabolomics and proteomics

*Matched control was provided for each patient.

• 利用KEGG信号通路对蛋白质组学和代谢组学的数据进行整合分析，揭示肾癌中分级依赖的信号通路及代谢重编程；结合体外功能实验对每一条信号通路进行验证。

研究结果

• 厌氧糖酵解/乳酸发酵在高分级的肾癌组织中显著上调（Fig.1）

厌氧糖酵解/乳酸发酵相关的代谢产物（葡萄糖、葡糖-6-磷酸、果糖-6-磷酸以及乳酸）在高分级的组织中显著上调；同时，与正常组织相比，参与该过程的酶（GAPDH、PKM2及LDHA等）在肿瘤组织中也显著上调；此外，调控丙酮酸进入TCA循环的丙酮酸羧化酶（PC）在肿瘤组织中显著下调，进一步证实肿瘤细胞中Warburg effect是能量的主要来源，而非TCA循环。

Figure 1. Aerobic glycolysis is grade-dependently upregulated in RCC. Combined proteomics and metabolomics（蛋白质组和代谢组整合）data of human RCC tissue were overlaid onto a stylized KEGG-based pathway（KEGG通路）diagram. Green, metabolite; orange, enzyme; black dotted arrow, metabolism; red arrow, upregulated pathway; blue arrow, downregulated pathway; G6P, glucose-6-phosphate; PEP, F6P, fructose 6-phosphate, phosphoenolpyruvate; OAA, oxaloacetate; GPI, glucose-6-phosphate isomerase; ALDOB, aldolase B; TPI1, triosephosphate isomerae 1; PGK1, phosphoglycerate kinase 1; ENO, enolase; PKM, pyruvate kinase; AKR1A1, aldo-keto reductase family 1, member A1; LDH, lactate dehydrogenase.

• 脂肪酸β-氧化在高分级的肾癌组织中显著下调（Fig.3）

短链游离脂肪酸的水平随肾癌组织分级的增加而下降，而肉毒碱和酰基肉碱在所有肿瘤组织中上调表达；同时，β-氧化过程中的大部分酶在肿瘤组织中显著下调，提示肾癌组织中乙酰辅酶A氧化下调，从而导致酰基肉碱的积累。

Figure 3. Acylcarnitines are increased and b-oxidation is downregulated in RCC. Combined proteomics and metabolomics（蛋白质组和代谢组整合）data of human RCC tissue were overlaid onto a stylized KEGG-based pathway（KEGG通路）diagram. Green, metabolite; orange, enzyme; black dotted arrow, metabolism; red, upregulated pathway; blue arrow, downregulated pathway. ACSL, acyl-CoA synthetase long-chain; CPT, carnitine palmitoyltransferase; HADH, carnitine palmitoyltransferase alpha subunit; HADHA, hydroxyacyl-CoA dehydrogenase; EHHADH, enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase; SCEH, short-chain enoyl-CoA hydratase; MCAD, medium-chain specific acyl-CoA dehydrogenase; VLCAD, very long-chain specific acyl-CoA dehydrogenase; ACAT, acetyl-CoA acetyltransferase.

• 谷氨酰胺通路在高分级肾癌中重编程进入GSH/GSSG抗氧化系统（Fig.4）

谷氨酰胺可以调控下游的TCA循环、尿素循环以及GSH通路；研究发现酰胺转化成为GSH和GSSG通路中的代谢产物水平与肾癌分级正相关，而催化GSH转化成为谷氨酰胺的酶下调表达，提示在高分级肾癌中，谷氨酰胺代谢重编程进入GSH途径，从而减弱氧化压力，有助于肿瘤细胞存活。

Figure 4. The glutamine pathway bolstered the glutathione system. Combined proteomics and metabolomics（蛋白质组和代谢组整合）data of human RCC tissue were overlaid onto a stylized KEGG-based pathway（KEGG通路）diagram. Levels of metabolites and enzymes were graphed grade dependently. Green, metabolite; orange, enzyme; black dotted arrow, metabolism; red arrow, upregulated pathway; blue arrow, downregulated pathway; ROS, reactive oxygen species; GPX1, glutathione peroxidase 1; GSTT1, glutathione S-transferase theta 1; GSTO1, glutathione S-transferase omega 1; GSTM3, glutathione S-transferase mu 3; GSTP1, glutathione S-transferase pi 1; GSTA2, glutathione S-transferase alpha 2; GSTK1, glutathione S-transferase kappa 1; GGT5, gamma-glutamyltransferase 5; GABA, gamma-aminobutyric acid; a-KG, a-ketoglutarate; GLS, glutaminase; ACY1, aminoacylase 1; ASS1, argininosuccinate synthase 1.

• TCA循环在肾癌组织中显著下调（Fig.5）

TCA循环上游的3个主要通路，即糖酵解、脂肪酸代谢和谷氨酰胺途径在肾癌组织中均发生了重编程，导致TCA循环显著下调。

Figure 5. The TCA cycle is not fed by glycolysis, the fatty acid pathway, or the glutamine pathway in RCC. A, combined proteomics and metabolomics data（蛋白质组和代谢组整合）of human RCC tissue were overlaid onto a stylized KEGG-based pathway（KEGG通路）diagram. Green, metabolite; orange, enzyme; black dotted arrow, metabolism; red arrow, upregulated pathway; blue arrow, downregulated pathway; ROS, reactive oxygen species; IDH, isocitrate dehydrogenase; GABA, gamma-aminobutyric acid; SDHA, succinate dehydrogenase complex subunit A; SDHB, succinate dehydrogenase complex subunit B. B, OCR was measured with a Seahorse XF24 Analyzer in 786-O (RCC; top plots), Caki-1 (RCC; bottom plots), and NHK (right plot) cells maintained in their growth media. Cells were treated 30 minutes before OCR measurement with glucose-depleted media (Glc-; left plot), glutamine-depleted media (Gln-; right plot), etomoxir (Eto) 50 mmol/L, or 2-DG (middle plot) 5 mmol/L unless stated otherwise. DMSO was used as the vehicle solution for etomoxir and 2-DG treatments. Data are the average OCR from six wells per group. Error bars, SEM. The data are representative of three repeats.

Ø 色氨酸代谢通路在高分级肾癌中上调，抗炎代谢产物增加（Fig.6）

色氨酸代谢通路中免疫抑制代谢物犬尿氨酸和羟基喹啉铜在肾癌组织中显著上调，而催化色氨酸进行其它代谢通路的酶活性呈现分级依赖的下调。

Figure 6. Tryptophan metabolism favors a grade-dependent increase in immune-suppressive metabolites. A, combined proteomics and metabolomics data（蛋白质组和代谢组整合）of human RCC tissue were overlaid onto a stylized KEGG-based pathway（KEGG通路）diagram. Green, metabolite; orange, enzyme; black dotted arrow, metabolism; red arrow, upregulated pathway; blue arrow, downregulated pathway; MAO, monoamine oxidase; DDC, dopa decarboxylase; ALDH, aldehyde dehydrogenase; IDO, indoleamine 2,3-dioxygenase; TDO, tryptophan 2,3-dioxygenase. B, 786-O (VHL-mut) and Caki-1 (VHL-wt) cells were plated in 6-well plates the day before treating with either human IFN-g (50 ng/mL) and/or MTH-trp (100 mmol/L) for 4 days and immunoblotted（免疫印迹）with the antibodies indicated. C, 786-O (VHL-mut) and Caki-1 (VHL-wt) cells were plated in 6-well plates the day before treating with either human IFN-g (50 ng/mL) and/or MTH-trp (100 mmol/L) for 3 days. The conditioned media were harvested and tryptophan (TRP) and kynurenine (KN) were measured（检测色氨酸和犬尿氨酸）by HPLC and normalized for cell number counted using the cell viability assay kit （归一化校正）(EMD Millipore) on a MUSE (EMD Millipore). Data shown are mean (n ? 3) and SD. _, P < 0.05. mM/1M cell, mM/1 million cells.

通过蛋白质组学和代谢组学的联合分析，揭示了肾癌组织中分级依赖的代谢通路的变化(Fig.7)

Figure 7. Summary of grade-dependent metabolic pathway alterations in RCC. With higher grade, glycolysis was directed toward lactate metabolism at the expense of TCA cycle intermediaries. Fatty acid b-oxidation was decreased, whereas the glutamine pathway served to attenuate oxidative stress, thereby increasing cancer cell survival. Tryptophan was metabolized preferentially to immunosuppressive compounds. Red, increased; blue, decreased.

亮点

• 蛋白质组学和代谢组学的联合分析系统地揭示了肾癌中能量和及免疫相关代谢的重编程，避免了单一组学技术的局限性；

• 对基于组学数据提出的假说进行严谨的体外功能实验确证；

• 基于不同分级肾癌的研究更有助于临床个体化治疗，并为药物研发提供了潜在的靶点。

• 通过蛋白质组学的筛选和后续验证，该研究证实LOXL2的表达水平与结肠癌的预后有关，并可通过区分II期和III期阶段为后续的治疗决策提供帮助。

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