糖酵解关键酶己糖激酶及其O-GlcNAc糖基化修饰对肿瘤细胞Warburg效应的功能作用

O-GlcNAc is a sugar attachment to serine or threonine hydroxyl moieties on nuclear and cytoplasmic proteins. O-GlcNAc modified proteins are involved in transcription, translation, cytoskeletal assembly, signal transduction, and many other cellular functions. Emerging research indicates that O-GlcNAcylation is involved in many diseases, such as diabetes and cancer. Compared with normal cells, cancer cells show abnormal metabolism. Cancer cells consume glucose avidly and produce elevated levels of lactic acid through glycolytic pathway even in the presence of oxygen. This phenomenon is known as aerobic glycolysis or the "Warburg effect". However, the mechanism by which the Warburg effect occurs is not well known. Hexokinase (HK) is the first key enzyme to regulate glycolysis and HK2, an isoform of HK, is almost overexpressed in cancer cells. Our preliminary studies indicate that HK2 is modified by O-GlcNAc. In this research, we will study the effect of O-GlcNAcylation of HK2 on Warburg effect. Firstly, we will identify the actual O-GlcNAc sites on HK2; Secondly, we will construct vectors with mutation of HK2 and obtain stable transfected cancer cells overexpressing HK2 without the O-GlcNAc sites; Thirdly, we will examine the effect on cellular metabolism and cell proliferation by using these cell lines under the circumstance of different oxygen or glucose concentrations. Meanwhile, we will investigate the mechanism by which the GlcNAcylation affect the unusual metabolism in cancer cells. Fourthly, we will detect the tumor growth in xenograft nude mice bearing these cell lines to examine whether modulation of O-GlcNAc levels can affect the ability of cancer cells to grow in mice. Finally, due to our findings that HK2 overexpressing enhanced O-GlcNAcylation levels for many proteins in cells, we will analysis the proteins with changed O-GlcNAc levels and investigate the possible mechanisms. The outcome of the proposed research will provide a novel mechanism underlying the Warburg effect.

肿瘤细胞具有在氧气充足的条件下大量摄取葡萄糖进行有氧糖酵解的Warburg效应，但其产生机制还没有统一的认识。己糖激酶（HK）是糖酵解途径中的第一个调控关键酶，在肿瘤细胞中HK的2型变异体HK2蛋白过度表达。我们的前期工作证实HK2是O-GlcNAc修饰蛋白，蛋白质的O-GlcNAc糖基化修饰广泛参与多种细胞生命活动的调控，并与肿瘤、糖尿病等多种疾病密切相关。本项目首先鉴定HK2的O-GlcNAc糖基化位点，并通过构建载体获得糖基化位点缺失的HK2高表达细胞株，研究HK2 的O-GlcNAc糖基化修饰对Warburg效应的功能作用。另外我们发现HK2过度表达促进了细胞中其它蛋白质的O-GlcNAc糖基化修饰，本项目拟鉴定这些蛋白质，并初步探索HK2过度表达是如何通过改变其它蛋白质的O-GlcNAc糖基化修饰水平进而影响Warburg效应的。本项目将为肿瘤细胞独特的能量代谢方式提供新的观点。

细胞质和细胞核内存在O-GlcNAc糖基化修饰的蛋白质，O-GlcNAc糖基化修饰和磷酸化修饰一样是动态过程，修饰水平随着细胞环境的变化而改变。O-GlcNAc糖基化影响蛋白质的稳定性和生物学活性。己糖激酶（HK2）是糖酵解过程中3个限速酶中的第一个限速酶，影响细胞能量的代谢，肿瘤细胞中HK2过度表达并促进细胞的增殖。本项目研究发现HK2是O-GlcNAc糖基化修饰的蛋白质，经过质谱鉴定糖基化位点是S32。我们构建S32位点氨基酸突变的HK2表达载体，进而获得糖基化位点缺失的HK2高表达的肿瘤细胞MDA-MB-231/S32A，然后与具有糖基化位点的HK2高表达细胞MDA-MB-231/HK2进行生物学方面的比较。在正常培养条件以及在低浓度葡萄糖培养条件下，与细胞MDA-MB-231/HK2相比较，细胞MDA-MB-231/S32A的增殖速度没有变化，糖酵解各个指标比如葡萄糖、乳酸、丙酮酸和耗氧量都没有显著性差异。然而，在抗肿瘤药物紫杉醇处理时，没有糖基化的MDA-MB-231/S32A细胞凋亡数量显著高于MDA-MB-231/HK2细胞。MDA-MB-231/S32A和MDA-MB-231/HK2细胞的荷瘤裸鼠试验表明，两种荷瘤裸鼠的肿瘤生长速度没有差异，但是两者分别给以紫杉醇治疗，没有糖基化的MDA-MB-231/S32A荷瘤组的肿瘤生长缓慢，紫杉醇的抗肿瘤活性高于MDA-MB-231/HK2组；并且，MDA-MB-231/S32A荷瘤组肿瘤的细胞凋亡数量显著高于MDA-MB-231/HK2组。进一步研究发现，细胞MDA-MB-231/S32A中HK2与VDAC1的蛋白相互作用低于MDA-MB-231/HK2细胞；和MDA-MB-231/HK2细胞相比较，在紫杉醇诱导下，MDA-MB-231/S32A细胞caspase 3和PARP蛋白的活性升高，细胞色素C从线粒体释放到细胞质中增多。以上结果表明，HK2的O-GlcNAc糖基化增强了HK2与VDAC1的相互作用，导致促凋亡蛋白Bax不能和线粒体膜结合，线粒体内的细胞色素C不能释放到细胞质中，抑制了凋亡的产生，从而促进细胞的增殖。本研究表明HK2糖基化对糖酵解没有影响，但对细胞凋亡产生影响。