HK2蛋白的翻译后修饰在肿瘤糖酵解中的功能及其机制

Warburg effect (aerobic glycolysis) has now been widely accepted as a crucial hallmark of cancer and cancer therapeutic agents targeting the Warburg effect are being developed. Among the regulators involved in the Warburg effect, hexokinase 2 (HK2) is a key glycolytic enzyme that catalyzes the first essential step of glucose metabolism. HK2 is reportedly mainly regulated on the transcriptional level by many studies. But for posttranslational modification, there is little. Preliminary studies showed that there was a Tyr phosphorylation modification in HK2 and an interaction between HK2 and EGFR which can phosphorylate HK2. Therefore, we propose to study HK2 phosphorylation and its mechanism by using RNA interference, CRISPR/Cas9, immunoprecipitation and so on. Experiments such as glucose uptake detection, determination of lactate, ATP level detection, hexokinase assay, ECAR, OCR, growth curve analysis, transwell and animal experiment are to be performed to test the effect of HK2 phosphorylation on tumor glycolysis, growth and metastasis. Clinical significance of EGFR, HK2 and HK2 phosphorylation are to be determined in the clinical samples of tumor patients, which contributes to illuminating the molecular mechanisms of cancer development and progression providing new clues and potential therapeutic targets.

Warburg效应（有氧糖酵解）已成为恶性肿瘤重要特征之一，靶向糖酵解的肿瘤治疗药物正在被开发。己糖激酶HK2是糖酵解通路的关键酶，催化糖酵解代谢的第一步反应。目前对HK2调控研究主要在转录水平，而翻译后水平却少有报道，且HK2翻译后修饰是否调控肿瘤糖酵解和生长转移仍不清楚。我们初步研究发现，HK2存在酪氨酸磷酸化修饰，且与蛋白激酶EGFR相互作用；EGFR能使HK2发生磷酸化。因此，拟在此基础上，利用RNA干扰、CRISPR/Cas9、免疫沉淀、激酶活性检测等研究HK2磷酸化修饰及其机制；利用葡萄糖摄取检测、乳酸测定、ATP检测、HK酶活性检测、ECAR、OCR、生长曲线、Transwell、动物实验等检测HK2磷酸化对肿瘤糖酵解、生长转移的调控；利用临床标本检测EGFR、HK2及其磷酸化蛋白在肿瘤患者中的表达，确定其临床意义，为阐明肿瘤发生发展的分子机制提供新的线索和潜在的治疗靶标。

Warburg效应（有氧糖酵解）已成为恶性肿瘤重要特征之一，靶向糖酵解的肿瘤治疗药物正在被开发。己糖激酶HK2是糖酵解通路的关键酶，催化糖酵解代谢的第一步反应。目前对HK2调控研究主要在转录水平，而翻译后水平却少有报道，且HK2翻译后修饰是否调控肿瘤糖酵解和生长转移仍不清楚。我们通过免疫共沉淀实验结合NANOLC-MS/MS质谱筛选到HK2相互作用蛋白表皮生长因子受体(EGFR)。免疫共沉淀和GST pull-down实验显示了EGFR可以直接与HK2结合，并确定了二者的相互作用区域。此外，NANOLC-MS/MS质谱筛选到HK2磷酸化位点为Tyr219，且免疫共沉淀和western blot等实验证明了EGFR可于第219位酪氨酸上磷酸化HK2。葡萄糖摄取检测、乳酸测定、ATP检测、HK酶活性检测、细胞外产酸率(ECAR)、耗氧率(OCR)等糖酵解功能实验表明HK2 Tyr219磷酸化位点突变降低了HK酶活性和糖酵解能力，且CCK8实验、克隆形成、划痕、Transwell、裸鼠成瘤实验说明了HK2 Tyr219磷酸化位点突变在体内外都抑制乳腺癌细胞的生长和侵袭。通过免疫组化染色分析显示，在人乳腺癌中EGFR和 pTyr219-HK2蛋白的表达水平呈正相关。综上所述，我们的发现表明EGFR是HK2的一个新的调节因子，为预防及治疗乳腺癌提供了新的线索和潜在的治疗靶标。