Johnson Mathieu, Nowlan Sarah, Sahin Gülsüm, Barnett David A, Joy Andrew P, Touaibia Mohamed, Cuperlovic-Culf Miroslava, Zofija Avizonis Daina, Turcotte Sandra
Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB, Canada.
Atlantic Cancer Research Institute, Moncton, NB, Canada.
Front Oncol. 2022 Feb 9;12:841054. doi: 10.3389/fonc.2022.841054. eCollection 2022.
Kidney cancer is one of the top ten cancer diagnosed worldwide and its incidence has increased the last 20 years. Clear Cell Renal Cell Carcinoma (ccRCC) are characterized by mutations that inactivate the von Hippel-Lindau (VHL) tumor suppressor gene and evidence indicated alterations in metabolic pathways, particularly in glutamine metabolism. We previously identified a small molecule, STF-62247, which target VHL-deficient renal tumors by affecting late-stages of autophagy and lysosomal signaling. In this study, we investigated ccRCC metabolism in VHL-deficient and proficient cells exposed to the small molecule. Metabolomics profiling using 1H NMR demonstrated that STF-62247 increases levels of glucose, pyruvate, glycerol 3-phosphate while glutamate, asparagine, and glutathione significantly decreased. Diminution of glutamate and glutamine was further investigated using mass spectrometry, western blot analyses, enzymatic activities, and viability assays. We found that expression of SLC1A5 increases in VHL-deficient cells treated with STF-62247, possibly to stimulate glutamine uptake intracellularly to counteract the diminution of this amino acid. However, exogenous addition of glutamine was not able to rescue cell viability induced by the small molecule. Instead, our results showed that VHL-deficient cells utilize glutamine to produce fatty acid in response to STF-62247. Surprisingly, this occurs through oxidative phosphorylation in STF-treated cells while control cells use reductive carboxylation to sustain lipogenesis. We also demonstrated that STF-62247 stimulated expression of stearoyl-CoA desaturase (SCD1) and peripilin2 (PLIN2) to generate accumulation of lipid droplets in VHL-deficient cells. Moreover, the carnitine palmitoyltransferase 1A (CPT1A), which control the entry of fatty acid into mitochondria for β-oxidation, also increased in response to STF-62247. CPT1A overexpression in ccRCC is known to limit tumor growth. Together, our results demonstrated that STF-62247 modulates cellular metabolism of glutamine, an amino acid involved in the autophagy-lysosome process, to support lipogenesis, which could be implicated in the signaling driving to cell death.
肾癌是全球确诊的十大癌症之一，其发病率在过去20年中有所上升。透明细胞肾细胞癌（ccRCC）的特征是使冯·希佩尔-林道（VHL）肿瘤抑制基因失活的突变，并且有证据表明代谢途径发生改变，尤其是谷氨酰胺代谢。我们之前鉴定出一种小分子STF-62247，它通过影响自噬和溶酶体信号传导的后期阶段来靶向VHL缺陷型肾肿瘤。在本研究中，我们调查了暴露于该小分子的VHL缺陷型和正常细胞中的ccRCC代谢。使用1H NMR进行代谢组学分析表明，STF-62247可提高葡萄糖、丙酮酸、3-磷酸甘油的水平，而谷氨酸、天冬酰胺和谷胱甘肽则显著降低。使用质谱、蛋白质免疫印迹分析、酶活性和活力测定进一步研究了谷氨酸和谷氨酰胺的减少情况。我们发现，用STF-62247处理的VHL缺陷型细胞中SLC1A5的表达增加，这可能是为了刺激细胞内谷氨酰胺的摄取，以抵消这种氨基酸的减少。然而，外源添加谷氨酰胺并不能挽救该小分子诱导的细胞活力。相反，我们的结果表明，VHL缺陷型细胞利用谷氨酰胺来响应STF-62247产生脂肪酸。令人惊讶的是，这在经STF处理的细胞中是通过氧化磷酸化发生的，而对照细胞则利用还原羧化来维持脂肪生成。我们还证明，STF-62247刺激硬脂酰辅酶A去饱和酶（SCD1）和周脂素2（PLIN2）的表达，从而在VHL缺陷型细胞中产生脂滴积累。此外，控制脂肪酸进入线粒体进行β氧化的肉碱棕榈酰转移酶1A（CPT1A）也响应STF-62247而增加。已知ccRCC中CPT1A的过表达会限制肿瘤生长。总之，我们的结果表明，STF-62247调节参与自噬-溶酶体过程的氨基酸谷氨酰胺的细胞代谢，以支持脂肪生成，这可能与驱动细胞死亡的信号传导有关。
