肿瘤相关巨噬细胞调控PKM2磷酸化促进结肠癌发生发展的研究

Aerobic glycolysis of tumors not only provides energy for rapid tumor growth, but also constructs its own intermediate storage pool. Our previous studies have found that tumor-associated macrophages (TAM) are involved in the regulation of aerobic glycolysis in tumors. PKM2 is a key enzyme in glycolysis. It has multiple functions of pyruvate kinase and non-pyruvate kinase activity. It is an important regulator of tumorigenesis and development. At present, the mechanism of PKM2 non-pyruvate kinase is not completely clear. When we studied TAM, we found that TAM can regulate aerobic glycolysis and promote the non-pyruvate kinase activity of PKM2. The mechanism may be that TAM can activate c-SRC by secreting a variety of cytokines, promote the binding of c-SRC to PKM2 and phosphorylate specific site of PKM2, so that PKM2 can play the role of non-pyruvate kinase. To confirm the above hypothesis, we intend to use metabolomics, Co-IP, ChIP and protein mass spectrometry to identify the phosphorylation sites of TAM acting on PKM2 through c-SRC and to study the mechanism of specific phosphorylation sites, so as to provide new clues for elucidating the function of TAM and the mechanism of abnormal glucose metabolism in tumors.

肿瘤有氧糖酵解不仅满足了肿瘤细胞快速供能的需求，还构建了自己的中间产物储备池。我们前期研究发现肿瘤相关巨噬细胞(TAM)参与对结肠肿瘤细胞有氧糖酵解的调节。PKM2是糖酵解过程中的关键酶，具有丙酮酸激酶和非丙酮酸激酶的多重功能，是肿瘤发生发展的重要调控分子，目前对PKM2非丙酮酸激酶功能的调控机制并不完全清楚。我们前期研究TAM时意外发现TAM能调控有氧糖酵解，促进PKM2的丙酮酸激酶功能向非丙酮酸激酶功能的转换，机制可能是TAM通过分泌多种细胞因子，激活c-SRC，促进c-SRC与PKM2结合并磷酸化PKM2的特定位点，使PKM2发挥非丙酮酸激酶的功能。为证实以上假说，本课题组拟采用代谢组学、Co-IP、ChIP、蛋白质质谱等技术明确TAM通过c-SRC作用于PKM2的磷酸化位点并研究特定磷酸化位点的作用机理，为阐明TAM的功能及肿瘤糖代谢异常的机制提供新线索。

肿瘤有氧糖酵解是肿瘤细胞快速供能的生存方式，有氧糖酵解的过程中会产生大量的代谢中间产物，这些中间产物对于肿瘤细胞快速增殖至关重要。我们前期研究发现肿瘤相关巨噬细胞(TAM)参与对结肠肿瘤细胞有氧糖酵解的调节。PKM2是糖酵解过程中的关键酶，具有丙酮酸激酶和非丙酮酸激酶的多重功能，是肿瘤发生发展的重要调控分子，目前对PKM2非丙酮酸激酶功能的调控机制并不完全清楚。因此本项目主要研究了TAM通过c-SRC作用于PKM2的磷酸化位点并研究特定磷酸化位点的作用机理极其应用价值。.首先我们发现TAM可以促进结肠肿瘤细胞的有氧糖酵解。验证了TAM可以促进结肠肿瘤的转移。接下来我们通过Co-IP证实TAM-CM可以促进c-SRC与PKM2的结合，并且c-SRC与PKM2的这种结合是依赖于TAM-CM的。接下来我们证实了TAM-CM促进结肠肿瘤细胞糖酵解是依赖于c-SRC的。我们通过蛋白质谱分析发现c-SRC可以会磷酸化PKM2的Y175位酪氨酸。而TAM-CM可以通过c-SRC来引起PKM2 Y175磷酸化的。我们进一步证实了TAM-CM可以促进PKM2从细胞质转移至细胞核中，而入核后的PKM2可以促进c-Myc和GLUT1的表达，也可以促进EMT，促进了Vimentin和N-Cadherin的表达，抑制了E-Cadherin的表达。.本项目阐明了TAM在结肠肿瘤糖代谢中的作用及机制，佐证了肿瘤微环境在肿瘤糖代谢中的重要作用，为肿瘤治疗提供了新的思路。