Although DNA 5-hydroxymethylcytosine (5hmC) is recognized as an important epigenetic mark in cancer, its precise role in lymph node metastasis remains elusive. In this study, we investigated how 5hmC associates with lymph node metastasis in breast cancer. Accompanying with high expression of TET1 and TET2 proteins, large numbers of genes in the metastasis-positive primary tumors exhibit higher 5hmC levels than those in the metastasis-negative primary tumors. In contrast, the TET protein expression and DNA 5hmC decrease significantly within the metastatic lesions in the lymph nodes compared to those in their matched primary tumors. Through genome-wide analysis of 8 sets of primary tumors, we identified 100 high-confidence metastasis-associated 5hmC signatures, and it is found that increased levels of DNA 5hmC and gene expression of MAP7D1 associate with high risk of lymph node metastasis. Furthermore, we demonstrate that MAP7D1, regulated by TET1, promotes tumor growth and metastasis. In conclusion, the dynamic 5hmC profiles during lymph node metastasis suggest a link between DNA 5hmC and lymph node metastasis. Meanwhile, the role of MAP7D1 in breast cancer progression suggests that the metastasis-associated 5hmC signatures are potential biomarkers to predict the risk for lymph node metastasis, which may serve as diagnostic and therapeutic targets for metastatic breast cancer.
淋巴结转移是影响乳腺癌患者生存预后的重要因素，而DNA 5-羟甲基胞嘧啶（5hmC）作为肿瘤中重要的表观修饰，其在乳腺癌淋巴结转移过程中的改变与调控机制尚未可知。为此，该研究分别从整体水平与基因水平探索了5hmC与淋巴结转移之间的相关性。作者发现相比于无淋巴结转移的乳腺癌（PT），DNA羟甲基化酶TET1、TET2在有淋巴结转移的乳腺癌（MT）组织中显著高表达，且大量基因的5hmC水平显著升高。另一方面，与乳腺癌原发灶（MT）相比，与之配对的淋巴结转移灶（MLN）中肿瘤细胞的DNA 5hmC与TET蛋白表达水平均呈现显著性降低。上述结果提示DNA 5hmC的动态改变与淋巴结转移密切相关。基于此，作者选取了8对PT与MT肿瘤样本，通过手动显微切割去除间质组织、利用其中的肿瘤组织进行了全基因组的DNA 5hmC测序。通过数据分析，该研究筛选到100个与乳腺癌淋巴结转移高度相关的差异羟甲基化基因（DhMGs），这些基因有望应用于临床早期乳腺癌淋巴结转移能力的评估。在此基础上，作者收集了更多肿瘤样本中对这些基因的5hmC修饰与转录表达水平进行检测，最终鉴定出 5hmC修饰与RNA表达水平与淋巴结转移密切相关的数个基因。其中MT样本中MAP7D1 外显子区域5hmC修饰与RNA表达水平均显著高于PT样本，由此推测 MAP7D1基因可能与乳腺癌淋巴结转移的生物学过程相关。为探其究竟，作者分别进行了体内、体外实验，最终发现MAP7D1表达受TET1调控，且可以促进乳腺癌细胞的增殖、侵袭与转移。综上，该研究利用临床标本的全基因组5hmC测序分析揭示了乳腺癌淋巴结转移过程中5hmC的动态变化规律，鉴定出一批有望用于评估早期乳腺癌淋巴结转移能力的5hmC修饰基因，同时发现MAP7D1基因具有促进乳腺癌淋巴结转移的生物学功能以及作为乳腺癌患者潜在治疗靶点的应用前景。





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