尼克酰胺-N-甲基转移酶作为乳腺癌分子分型和药物疗效新标志物的可行性研究

Nicotinamide N-methyltransferase (NNMT), an enzyme involved in the biotransformation and detoxification of many drugs and xenobiotic compounds, was found over-expressed in a wide range of tumor tissues. Our previous study demonstrated that high NNMT expression is significantly associated with unfavorable prognosis in renal cell cancer. Our preliminary studies on breast cancer revealed that NNMT enhances cell proliferation and may have a potential role in tumor development and progression and drug resistance. However, the related mechanism remains unknown. The goal of this project was to validate NNMT as a biomarker of breast cancer which indicating molecular subtype and therapy effects.. Cell proliferation assay, plate colony formation assay, apoptosis analysis, cell cycle analysis, nude mice tumorigenesis experiments and drug resistance tests were designed to study the biological function changes in breast cancer cells by constructing NNMT up-regulated and NNMT knockdown cells models. The effect of NNMT on energy metabolism, Nicotinamide balance, SIRT1, PARP, cholesterol synthesis, PI3K/AKT and MAPK/ERK signal pathways were set to explore the mechanisms related to proliferation and drug resistance. Expression of NNMT and other markers such as ER, PR, HER-2 in a large cohort of breast cancer tissues will be applied to analyze the clinical usage of NNMT as a tumor marker. Correlation analysis was performed to understand the associations between NNMT expression and clinico-pathological parameters such as grade, subtype, drug effect and survival in breast cancer..The results may help to explain the mechanisms related to NNMT effect on breast cancers and suggest that subtype specific responses should be evaluated in future clinical treatments for breast cancer. The application of NNMT with other markers in the clinical setting could improve the targeting of adjuvant therapies to those women most likely to benefit.

肿瘤标志物的检测是肿瘤分型和制定肿瘤个体化治疗方案的基础。尼克酰胺-N-甲基转移酶（NNMT）是近年发现的新的肿瘤蛋白质酶，在多种肿瘤组织中呈高表达，本课题组前期研究发现NNMT促进乳腺癌细胞的增殖，极可能与肿瘤的发生和放、化疗敏感性有重要关系，但其主要作用机制及调控通路仍不清楚。本研究拟在前期研究基础上采用NNMT基因导入和RNA干扰技术，研究该蛋白表达变化对乳腺癌细胞的增殖、药敏、凋亡、细胞周期及裸鼠成瘤的影响；重点研究NNMT与乳腺癌临床病理分期、分型、化疗效果及预后的关系和不同NNMT表达水平对乳腺癌细胞能量代谢、尼克酰胺平衡、细胞胆固醇合成以及PI3K/AKT、MAPK/ERK分子信号通路等的影响，期望通过本研究阐明NNMT促进乳腺癌细胞增殖的主要机制及通路；筛选出该通路中的关键分子，为个体化治疗方案的制定及新型分子靶向特异性阻断剂的研发提供基础。

尼克酰胺-N-甲基转移酶（NNMT）是近年发现的新肿瘤相关蛋白酶，在多种肿瘤组织中高表达，本课题组前期研究发现NNMT促进乳腺癌细胞的增殖，极可能与肿瘤的发生和化疗敏感性有重要关系，但其主要作用机制及调控通路仍不清楚。本研究在前期研究基础上研究该蛋白对乳腺癌细胞的增殖、化疗敏感性、凋亡、细胞周期及裸鼠成瘤的影响；明确了该蛋白对乳腺癌细胞增殖和耐药的作用机制、信号通路及通路中的关键分子；也分析了NNMT与乳腺癌临床病理分期、分型、化疗效果及预后的关系。生物学功能研究结果显示：NNMT的过表达可以抑制乳腺癌细胞凋亡，促进肿瘤细胞增殖及裸鼠移植瘤的形成和生长；在化疗敏感性方面，NNMT的过表达可以增强乳腺癌细胞对化疗药物的抗性；NNMT蛋白对乳腺癌细胞增殖和耐药的作用机制研究结果显示：NNMT的过表达可提高肿瘤细胞内尼克酰胺平衡的关键分子1-MNA含量，从而降低细胞内ROS的水平，同时激活PI3K/AKT和MAPK/ERK信号通路，增加下游抗凋亡蛋白Bcl-2、Bcl-xL表达，抑制细胞色素c从线粒体释放到细胞质中，从而降低Caspase-9、Caspase-3剪切体含量，进一步证明NNMT通过降低乳腺癌细胞线粒体途径的凋亡，促进细胞增殖；通过检测能量代谢中几个关键分子，发现NNMT的过表达增加了乳腺癌细胞内ATP的含量，降低NAD+和NADH的含量，表明NNMT在乳腺癌细胞内能量代谢中起重要作用，推测可能是NNMT促进肿瘤细胞增殖的另一种作用机制；建立了NNMT表达检测的免疫组化，对乳腺癌标本库中265例病人组织进行NNMT表达检测，显示NNMT在乳腺癌病人组织的阳性率为58.92%，结合临床资料发现NNMT的过表达与三阴性乳腺癌和蒽环类耐药成正相关性。通过本项目的研究，我们认为NNMT促进乳腺癌细胞增殖和抗蒽环类药物，PI3K/AKT和MAPK/ERK信号通路的激活以及细胞凋亡相关分子改变是其主要机制，进一步明确了NNMT作为乳腺癌靶向治疗标志物的潜在价值，也为研发其下游关键分子新型靶向特异性阻断剂提供基础；开展NNMT与乳腺癌临床病理分期、分型、化疗效果及预后关系的研究，建立了一套较为完整的对NNMT研究和临床检测的技术，也为细化乳腺癌的病理分型提供新思路，有助于肿瘤个体化治疗方案的制定。