Analysis of a mechanism of Girdin-mediated regulation of cancer cell metabolism

我们实验室以前的工作将肌动蛋白结合蛋白Girdin描述为内皮细胞、癌细胞和成神经细胞迁移的关键调节因子，其依赖于细胞外信号，包括生长因子刺激。我们以前也发现Girdin也与氨基酸转运蛋白相互作用，以调节氨基酸诱导的mTORC 1激活。为了进一步研究Girdin在癌症代谢中的作用，我们采用亲和柱色谱法分离Girdin相互作用蛋白，并鉴定了丙酮酸激酶M2（PKM 2），这是一种调节肿瘤生长所必需的瓦尔堡效应的关键酶。通过免疫共沉淀（co-IP）、蛋白质印迹分析和免疫荧光证实PKM 2与Girdin的相互作用。接下来，为了检查Girdin在癌症代谢中的作用，我们通过市售的生化试剂盒评估了Girdin耗竭对ATP和乳酸产生以及葡萄糖消耗的影响。我们还研究了Girdin在PKM 2 Y105磷酸化中的作用，这与PKM 2酶活性高度相关。我们还通过IHC分析了Girdin在肺癌进展中的作用，其中我们发现Girdin的表达与肺癌组织中PKM 2的表达相关。综上所述，我们从研究中获得的数据揭示了Girdin在癌症代谢和癌症进展中的作用，并显示了Girdin通过影响有氧糖酵解参与调节化疗效果的可能性。

The previous work from our laboratory characterized the actin-binding protein Girdin as a critical regulator of migration of endothelial cells, cancer cells, and neuroblasts, which depends on extracellular cues including growth factor stimulation. We also previously found that Girdin also interacts with amino acids transporters to regulate amino acids-induced mTORC1 activation. To further investigate the role of Girdin in cancer metabolism, we employed affinity column chromatography to isolate Girdin-interacting proteins, and identified Pyruvate Kinase M2 (PKM2), a key enzyme that regulates the Warburg effect that is necessary for tumor growth, as a candidate. The interaction of PKM2 with Girdin was confirmed by co-immunoprecipitation (co-IP), western blot analysis and immunofluorescence. Next, to check on the role of Girdin in cancer metabolism, we evaluated the effect of Girdin depletion on ATP and lactate production, and glucose consumption by commercially available biochemical kits. We also investigated the role of Girdin in PKM2 Y105 phosphorylation, which is highly related to PKM2 enzyme activity. We also analyzed the role of Girdin in lung cancer progression through IHC, where we found that the expression of Girdin is correlated with the expression of PKM2 in lung cancer tissue. In summary, the data we got from the study revealed the role of Girdin in cancer metabolism and cancer progression, and showed the possibility that Girdin is involved in the modulation of chemotherapy effect through affecting aerobic glycolysis.