NBA1调节PHGDH酶活的分子机制及其对乳腺癌发展的影响

The de novo synthesis pathway of serine is one of the most significant pathways branching from glycolysis. It provides the essential precursors of macromolecules and helps cells to maintain redox status which is crucial to tumor cell growth. PHGDH is the rate-limiting enzyme of serine synthesis pathway. Genetic and functional evidence indicates that PHGDH is essential for breast cancer tumorigenesis. However, the underlying mechanism of regulating PHGDH enzyme activity is poorly understood. We have previously identified that NBA1, a TNKS binding protein, interacts with PHGDH, and that glucose deprivation enhanced their interaction. Knockdown of NBA1 significantly decreased PHGDH activity and serine synthesis; vice versa, overexpression of NBA1 stimulated PHGDH activity, which strongly indicates that NBA1 is a major regulatory protein on PHGDH activity. By crossing NBA-/- and Wnt1 transgenic mice, we found that deletion of NBA1 seriously decreased the growth of primary breast cancer. We hereby propose to further explore the regulatory mechanism of NBA1 on PHGDH activity and its effect on breast cancer development using a combined approaches of biochemistry, molecular biology and cell biology. The accomplishment of this project will elucidate a new regulatory mechanism of PHGDH activity and provides important information for clinical therapy of breast cancer treatment.

丝氨酸合成途径是糖酵解的重要分支，能够为细胞生长提供大分子合成的原料，并在维持细胞氧化还原平衡方面发挥重要功能。PHGDH是丝氨酸合成途径的限速酶，功能基因组学表明其与乳腺癌发生密切相关，但目前对PHGDH活性的调控机制知之甚少。前期工作发现PHGDH能够与TNKS的结合蛋白NBA1相互作用，而且在葡萄糖饥饿的情况下相互作用显著增强。在细胞内敲低NBA1显著降低了PHGDH的活性和丝氨酸的合成，而过量表达NBA1则能增加PHGDH的活性，说明NBA1是PHGDH活性的重要调控因子。此外，通过NBA1-/-与Wnt1转基因杂交小鼠的研究发现NBA1缺失显著抑制了原发性乳腺癌的生长速度。因此，我们拟采用生化、分子和细胞生物学的方法进一步研究NBA1对PHGDH活性的调控机制及其对乳腺癌细胞生长的影响。本项目的顺利完成有望揭示一种新的调控PHGDH活性的机制，并为乳腺癌的治疗提供重要的理论依据。

丝氨酸合成通路为生物大分子的合成提供丝氨酸和甘氨酸，为一些癌细胞的快速生长提供重要的支持。PHGDH是丝氨酸合成通路的限速酶。但是PHGDH的活性调节仍然不清楚。我们发现NBA1与PHGDH存在相互作用， NBA1通过201-298 氨基酸结构域与PHGDH相互作用增强PHGDH的酶活性。在PHGDH 高表达的乳腺癌细胞MDA-MB-468中，敲低NBA1使细胞中 a-KG水平降低。U-13C-葡萄糖标记实验显示，NBA1敲低显著内源合成的丝氨酸、甘氨酸以及AMP水平。在MDA-MB-468乳腺原位移植瘤实验中，NBA1敲低显著抑制肿瘤的生长。我们还发现在葡萄糖饥饿刺激时，NBA1与PHGDH的相互作用会增强，回补3PG会解除这种增强作用。敲低NBA1会抑制 MDA-MB-468细胞在葡萄糖饥饿刺激下的细胞存活。这些结果显示NBA1调节PHGDH的酶活促进乳腺癌的生长，为PHGDH高表达的乳腺癌治疗提供了新的靶点。