通过 TAT-Cx43 特异性抑制 c-Src 靶向体外和体内神经胶质瘤干细胞的代谢可塑性

Targeting metabolic plasticity in glioma stem cells in vitro and in vivo through specific inhibition of c-Src by TAT-Cx43.

机构信息

Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Calle Pintor Fernando Gallego 1, Salamanca 37007, Spain; Departamento de Bioquímica y Biología Celular, Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, Salamanca 37007, Spain; Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Virgen de la Vega, 10ª planta, Paseo de San Vicente, 58-182, Salamanca 37007, Spain.

Precision Nutrition and Cancer Program, Molecular Oncology and Nutritional Genomics of Cancer Group, IMDEA Food Institute, CEI UAM + CSIC, Carretera de Canto Blanco 8 E, Madrid 28049, Spain.

出版信息

EBioMedicine. 2020 Dec;62:103134. doi: 10.1016/j.ebiom.2020.103134. Epub 2020 Nov 27.

DOI:10.1016/j.ebiom.2020.103134

PMID:33254027

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7708820/

Abstract

BACKGROUND

Glioblastoma is the most aggressive primary brain tumour and has a very poor prognosis. Inhibition of c-Src activity in glioblastoma stem cells (GSCs, responsible for glioblastoma lethality) and primary glioblastoma cells by the peptide TAT-Cx43 reduces tumorigenicity, and boosts survival in preclinical models. Because c-Src can modulate cell metabolism and several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells, here we explored the inhibition of advantageous GSC metabolic plasticity by the c-Src inhibitor TAT-Cx43.

METHODS

Metabolic impairment induced by the c-Src inhibitor TAT-Cx43 in vitro was assessed by fluorometry, western blotting, immunofluorescence, qPCR, enzyme activity assays, electron microscopy, Seahorse analysis, time-lapse imaging, siRNA, and MTT assays. Protein expression in tumours from a xenograft orthotopic glioblastoma mouse model was evaluated by immunofluorescence.

FINDINGS

TAT-Cx43 decreased glucose uptake in human GSCs and reduced oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on brain cell metabolism, including rat neurons, human and rat astrocytes, and human neural stem cells. TAT-Cx43 impaired metabolic plasticity, reducing GSC growth and survival under different nutrient environments. Finally, GSCs intracranially implanted with TAT-Cx43 showed decreased levels of important metabolic targets for cancer therapy, such as hexokinase-2 and GLUT-3.

INTERPRETATION

The reduced ability of TAT-Cx43-treated GSCs to survive in metabolically challenging settings, such as those with restricted nutrient availability or the ever-changing in vivo environment, allows us to conclude that the advantageous metabolic plasticity of GSCs can be therapeutically exploited through the specific and cell-selective inhibition of c-Src by TAT-Cx43.

FUNDING

Spanish Ministerio de Economía y Competitividad (FEDER BFU2015-70040-R and FEDER RTI2018-099873-B-I00), Fundación Ramón Areces. Fellowships from the Junta de Castilla y León, European Social Fund, Ministerio de Ciencia and Asociación Española Contra el Cáncer (AECC).

摘要

背景

胶质母细胞瘤是最具侵袭性的原发性脑肿瘤，预后极差。肽 TAT-Cx43 抑制胶质母细胞瘤干细胞（负责胶质母细胞瘤致死性的细胞）和原代胶质母细胞瘤中的 c-Src 活性，可降低肿瘤发生能力，并提高临床前模型中的存活率。由于 c-Src 可以调节细胞代谢，并且有几项报道显示由于癌细胞代谢重排，各种抗肿瘤药物的临床疗效不佳，因此我们在此探索 c-Src 抑制剂 TAT-Cx43 抑制有利的 GSC 代谢可塑性。

方法

通过荧光法、western blot、免疫荧光、qPCR、酶活性测定、电子显微镜、 Seahorse 分析、延时成像、siRNA 和 MTT 测定评估 c-Src 抑制剂 TAT-Cx43 在体外对代谢的影响。通过免疫荧光评估异种移植原位胶质母细胞瘤小鼠模型肿瘤中的蛋白表达。

结果

TAT-Cx43 降低了人 GSCs 中的葡萄糖摄取，并减少了氧化磷酸化，而没有代偿性增加糖酵解，对包括大鼠神经元、人源和大鼠星形胶质细胞以及人神经干细胞在内的脑细胞代谢没有影响。TAT-Cx43 损害了代谢可塑性，降低了不同营养环境下 GSC 的生长和存活能力。最后，用 TAT-Cx43 颅内植入的 GSCs 显示出癌症治疗的重要代谢靶标水平降低，如己糖激酶-2 和 GLUT-3。