肠道普雷沃菌及其代谢产物通过调控细胞自噬促进乳腺癌发展的机制研究

Breast cancer is a malignant neoplasm with the highest incidence in female population worldwide. A concrete relationship between breast cancer and gut microbiota (GM) has been found. Previous studies showed that breast cancer cells could develop as tumor quickly in conventional mice, resulting in an enrichement of Prevotella within GM. This is consistent with the findings from clinical invesigation about breast cancer and GM. However, our findings also showed that in germ-free mice, tumor could hardly develop from breast cancer cells or grew rather slowly, of which mTOR was activated while autophagy was suppressed. But all these were reversed after the germ-free mice was transplanted with fecal from the conventional mice, and serum of the recipient exhibited more potent stimulation on the cancer cell. All these suggested that Prevotella play a key role in the promotion of breast cancer, which mainly relies on its metabolites to maintain autophagy at high level. Hence, we will employ metabolomics to figure out the critical metabolite, and explore its underlying mechanism from the perspective of AMPK/mTOR-autophagy balance. The present project aims to improve the understanding of the intrinsic properties of breast cancer based on the interplay with GM, as well as to provide reasonable suggestion for the optimization in early diagnosis and treatment.

乳腺癌是世界女性人口中发病率最高的恶性肿瘤。临床研究报道肠道菌群与乳腺癌发展密切相关。课题组前期发现，乳腺癌细胞在正常动物中可迅速成瘤，且使肠道菌群中普雷沃菌（Prevotella）丰度明显升高，该结果与临床报道一致。然而，乳腺癌细胞在无菌动物中成瘤速度、成瘤率显著下降，且肿瘤组织的mTOR被激活，自噬功能被抑制；而无菌动物接受正常动物粪便移植后，上述变化得到逆转，且其血清对乳腺癌细胞有更强的刺激作用。据此课题组推测，Prevotella是促进乳腺癌发展的关键因素，它可能通过其代谢产物，使癌细胞维持较高的自噬水平，从而促进乳腺癌发展。为验证这一假说，本项目利用代谢组学技术，寻找Prevotella促进乳腺癌发展的关键代谢产物，并从AMPK/mTOR-自噬角度探讨其作用机制。本项目通过探讨肠道菌群与乳腺癌发展的内在关系，期望为深入理解乳腺癌的发生机制，推进其早诊早治提供理论依据。

乳腺癌是世界女性人口中发病率最高的恶性肿瘤。临床研究报道肠道菌群与乳腺癌发展密切相关，但两者的因果关系及肠道菌群的作用机制尚未明确。本项目研究结果发现，乳腺癌患者肠道菌群的α多样性和β多样性与对照患者有显著差异。其中，Prevotella 属在乳腺癌患者中显著富集，且该微生物在所有年龄段乳腺癌患者中的流行性也显著高于对照人群，说明Prevotella 属与乳腺癌的发展密切相关。由于Prevotella copri是肠道菌群中Prevotella 属的优势菌种，本研究选择P. copri，以4T1乳腺癌模型探讨Prevotella 属在乳腺癌发展过程中的潜在作用。结果表明，P. copri可在小鼠肠道中有效定植，且无论SPF条件下还是GF条件下，P. copri均可使肿瘤重量显著高于对照组，说明Prevotella 属（P. copri）对乳腺癌发展有重要促进作用。进一步，课题组发现P. copri显著改变了肿瘤组织蛋白表达谱和DNA甲基化谱，使UHRF1、DNMT 3A等DNA甲基化调控蛋白表达上调，抑制AMPK磷酸化，并影响了AMPK通路中数个相关基因的mRNA水平，提高组织内自噬水平，从而促进乳腺癌进展。通过靶向代谢组学分析，我们发现P. copri使荷瘤小鼠血液中色氨酸（Trp）及其代谢产物之一，吲哚-3-丙酮酸（IPyA）含量急剧下降；但P. copri可大量消耗Trp而不产生IPyA。我们证实IPyA可通过逆转UHRF1介导的负调节，激活AMPK，对癌细胞进行能量代谢重编程，抑制细胞能量生成，从而抑制细胞增殖。综上所述，本项目证实Prevotella 属的异常富集与乳腺癌发展密切相关，它可能通过消耗宿主摄入的Trp，阻碍IPyA生理积蓄，削弱宿主自身肿瘤控制能力，强化肿瘤组织内Uhrf1对AMPK的去磷酸化，抑制AMPK活性。因此，肠道菌群中的Prevotella可能是乳腺癌发展一个重要风险因子。