Department of Physiological Sciences, School of Medicine, University of Barcelona-IDIBELL, L'Hospitalet de Llobregat, Spain.
Programs of Molecular Mechanisms and Experimental Therapeutics in Oncology (Oncobell), and Cancer Therapeutics Resistance (ProCURE), Catalan Institute of Oncology, Bellvitge Institute for Biomedical Research (IDIBELL), L'Hospitalet del Llobregat, Spain.
Cell Death Dis. 2022 Aug 24;13(8):730. doi: 10.1038/s41419-022-05177-x.
On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from C-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies.
在葡萄糖限制下，上皮细胞可以经历细胞吞噬作用，即一种细胞吞噬自身细胞的自噬过程，从而使细胞能够在这种代谢应激下大量存活。因此，我们假设减少蛋白质糖基化可能参与了这种细胞存活途径的激活。通过在显微镜下直接观察或用 Rho-GTPase 激酶抑制剂（ROCKi）处理经历细胞吞噬作用的细胞，观察到细胞向凋亡+坏死转变，发现葡萄糖剥夺可促进 MCF7 乳腺癌模型中的细胞吞噬作用。在这种情况下，用 N-乙酰葡萄糖胺抑制蛋白糖基化缺陷部分挽救了细胞吞噬作用，而在用衣霉素或 NGI-1（而非其他非相关的内质网应激诱导剂，如 thapsigargin 或氨基酸限制）限制葡萄糖存在下的糖基化则刺激了细胞吞噬作用。葡萄糖剥夺会上调线粒体磷酸烯醇丙酮酸羧激酶（PEPCK-M；PCK2），从而增强细胞存活。因此，我们推测 PEPCK-M 可以通过使用替代底物将关键代谢物转移到糖基部分来在这个过程中发挥作用。在没有葡萄糖的情况下，使用 iPEPCK-2 抑制 PEPCK-M 促进了细胞吞噬作用，而其过表达则抑制了细胞吞噬作用。PEPCK-M 抑制作用对 Concanavalin A 结合所确定的总蛋白糖基化以及完全糖基化的 LAMP1 或 E-钙粘蛋白的特定比例有直接作用。通过 GC/MS 测量，PEPCK-M 含量依赖于从 C-谷氨酰胺标记物到糖酵解中间产物直至葡萄糖-6-磷酸、核糖和核酮糖-5-磷酸的通量，以及代谢物的含量。总而言之，我们首次证明了蛋白质糖基化缺陷先于并启动了细胞吞噬作用，并表明 PEPCK-M 参与了这种存活程序，以减轻葡萄糖剥夺的后果。这些结果对我们理解肿瘤代谢和治疗策略具有广泛的意义。
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