SIRT7 通过去琥珀酰化作用激活TET2在乳腺癌发生发展中作用机制探索

Breast cancer is the most common female malignancies, the related mechanism investigations have drawn much attention. There are reports showing SIRT7 overexpressed in breast cancer. However, the molecular mechanism of this effect is unclear. Our preliminary data showed: 1. SIRT7 knockdown reduced cell migration potential of breast cancer cells; 2. Evidences of SIRT7 interacted with TET2; 3. SIRT7 KD led to 5hmC level decreasing, and counteracted 5hmC increasing by TET2 overexpression ;4. Clues that SIRT7 might act as desuccinylase for TET2. May SIRT7 regulate the function of TET2 by its desuccinylase activity, and hence affecting breast cancer invasion and metastasis? Our project is going to utilize affinity purification coupled with mass-spectrometric technique to analyze and identify the landscape of the TET2 modification changes under the condition of SIRT7 overexpression or knockdown in breast cancer cell lines; combined with the in vivo and in vitro enzyme activity assay of SIRT7 to confirm its desuccinylase activity and map the modification sites of TET2; analyze the converged downstream target genes of SIRT7 and TET2 by RNAseq, determine the effect of TET2 desuccinylation by SIRT7 on downstream target genes and metastasis of cellular, animal breast cancer models.

乳腺癌是女性最常见的恶性肿瘤，其相关的机制研究一直备受关注。有研究显示SIRT7在乳腺癌中高表达，其表达水平与肿瘤分级相关，而具体的分子机制尚不清楚。本课题前期的预实验结果显示：1、SIRT7敲低使得乳腺癌细胞迁移能力下降；2、亲和纯化加质谱分析及内源性和外源性的CoIP验证显示SIRT7与TET2有相互作用；3、在MCF-7细胞中敲低SIRT7导致5hmC水平下降、并可对抗TET2升高5hmC作用；4、SIRT7可能通过去琥珀酰化影响TET2活性。本项目拟利用亲和纯化联合质谱分析鉴定乳腺癌细胞系中SIRT7敲低或过表达后TET2修饰谱的改变情况；结合体外、体内酶活性测定明确TET2为SIRT7去琥珀酰化酶活性的底物，并确定修饰位点；利用RNAseq技术分析SIRT7和TET2可能共同影响的下游靶基因,干扰SIRT7对修饰位点的催化活性观察其对下游靶基因及细胞、动物模型中乳腺癌转移的影响。

胰腺癌为全世界生存率最差、死亡率最高的肿瘤之一，越来越多的证据表明，代谢重编程在胰腺癌的发生发展、治疗及预后中发挥了重要作用。在长期炎症、缺氧、营养剥夺等不良环境中，胰腺癌通常依靠广泛而独特的代谢改变来满足自身无限生长的需求。作为NAD+依赖的蛋白质去酰化酶，SIRT7可通过NAD+感受代谢变化，并将代谢变化与表观遗传联系起来，从而进一步影响代谢、基因组稳定性、衰老过程以及肿瘤的发生发展。SIRT7在胰腺癌代谢中发挥的作用至今仍不清楚。我们通过本项目资助证明了：（1）在环境低葡萄糖压力下SIRT7胞质易位减轻其对ACOX2的转录抑制，使得胰腺癌细胞可以转而利用过氧化酶体脂肪酸氧化维持增殖，赋予胰腺癌细胞更强的低糖耐受力；（2）SIRT7缺失可通过改变GLUT3第二内含子上组蛋白的H3K122su水平上调GLUT3的表达，并进一步通过影响胰腺癌细胞的葡萄糖摄取能力促进胰腺癌的增殖、转移以及癌细胞干性。SRIT7调控GLUT3的新机制有望为胰腺癌的靶向治疗提供新的线索。