Molecular Mechanisms of Fructose-induced Colorectal Cancer Cell Survival

项目摘要 我们所吃的食物与癌症的发展和进展之间已经建立了明确的联系 结直肠癌(CRC)例如，果糖的摄入增加了结直肠癌发展的风险 和CRC特有的死亡率。然而，这种联系背后的机制尚不清楚。我们已经展示了 每天适度接触口服高果糖玉米糖浆(HFCS，果糖和葡萄糖的混合物)会导致 小鼠更大、更具侵袭性的肠腺瘤。这些影响在具有遗传基因的小鼠中是不存在的 酮己糖激酶(KHK)缺乏症，KHK是一种将果糖转化为1-磷酸果糖(F1P)的酶。一个 对这些肿瘤的代谢分析表明，暴露于HFCS后，F1P高度丰富，这 增加与丙酮酸激酶(PK)活性的降低相关。因此，我们假设F1P， KHK的产物，通过作为PK的变构抑制剂促进合成代谢而促进肿瘤生长 新陈代谢和细胞存活。我们将用小鼠的生理和器官代谢、细胞 和人类器官培养，以及重组蛋白生物化学。在目标1中，我们将从基因和 在小鼠体内对PK(PKM2)的M2同工酶进行药理操作，以探讨其作为血管紧张素转换酶调节因子的作用 HFC诱导肿瘤生长。在目标2中，我们将定义果糖暴露和 癌细胞存活。然而，我们发现培养中的细胞在接触果糖时并不会生长得更快。 我们观察到细胞存活率显著提高，特别是在高细胞密度和 培养液中含有果糖的低氧。因此，我们假设F1P抑制PKM2促进缺氧细胞 生死存亡。我们将使用暴露在果糖和低氧中的细胞和有机物培养模型来验证这一假设。 我们将在这些模型中从遗传学和药理学上操纵KHK和PKM2的表达和活性 以确定这些蛋白质对细胞新陈代谢和存活的特定影响。在目标3中，我们将评估 F1P对重组PK亚型的影响，尤其是对PKM2的影响。我们假设果糖- 衍生的F1P结合并抑制PKM2。我们将进行生化活性和结构分析以 测定在F1P存在下PK亚型的动力学参数和寡聚态。这些 实验将揭示F1P如何结合和抑制PKM2的分子机制。总而言之，这些目标 将改变我们对果糖如何改变肿瘤细胞代谢的基本理解，定义 果糖/F1P/PKM2轴是结直肠癌的代谢易感性，并为PKM2提供临床前证据 激活剂作为对抗结直肠癌的一种新的治疗方式。

Project Summary Clear associations have been established between the food we eat and the development and progression of colorectal cancer (CRC). For example, the consumption of fructose increases the risk for CRC development and CRC-specific mortality. However, the mechanism underlying this association is unknown. We have shown that moderate daily exposure to oral high fructose corn syrup (HFCS, a mix of fructose and glucose) leads to larger and more aggressive intestinal adenomas in mice. These effects were absent in mice with genetic deficiency of ketohexokinase (KHK), the enzyme that converts fructose to fructose 1-phosphate (F1P). A metabolomic analysis of these tumors showed that F1P is highly abundant following HFCS exposure, and this increase correlates with a reduction in pyruvate kinase (PK) activity. Therefore, we hypothesize that F1P, the product of KHK, enhances tumor growth by acting as an allosteric inhibitor of PK to promote anabolic metabolism and cell survival. We will test this hypothesis using mouse physiology and organ metabolism, cell and human organoid culture, and recombinant protein biochemistry. In Aim 1, we will genetically and pharmacologically manipulate the M2 isozyme of PK (PKM2) in mice to interrogate its role as a mediator of HFCS-induced tumor growth. In Aim 2, we will define the mechanistic linkage between fructose exposure and cancer cell survival. We have found that cells in culture do not grow faster when exposed to fructose, however we observed a significant improvement in cell viability, especially under conditions of high cell density and hypoxia with fructose in the media. Therefore, we hypothesize that F1P inhibits PKM2 to promote hypoxic cell survival. We will test this hypothesis using cell and organoid culture models exposed to fructose and hypoxia. We will genetically and pharmacologically manipulate KHK and PKM2 expression and activity in these models to determine the specific effects of these proteins on cell metabolism and survival. In Aim 3, we will assess the effects of F1P on recombinant PK isoforms with a particular focus on PKM2. We hypothesize that fructose- derived F1P binds to and inhibits PKM2. We will perform biochemical activity and structural assays to determine the kinetic parameters and oligomeric state of PK isoforms in the presence of F1P. These experiments will reveal the molecular mechanisms of how F1P binds and inhibits PKM2. Together, these aims will change our fundamental understanding of how fructose alters tumor cell metabolism, define the fructose/F1P/PKM2 axis as a metabolic vulnerability of CRC, and provide pre-clinical evidence for PKM2 activators as a novel therapeutic modality to combat CRC.