PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation <em>In Situ</em>

BackgroundLymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression. Methodology/Principal FindingsWe performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors. Conclusions/SignificanceThis study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.背景 淋巴结转移是乳腺癌进展进程中的关键事件，因此阐明介导区域淋巴结转移进展的潜在分子机制具有重要研究价值。 方法与主要发现 我们对来自人类乳腺原发肿瘤与淋巴结转移灶的纯化肿瘤细胞开展了基因表达谱分析。通过微阵列（microarray）网络分析，我们发现相较于原发肿瘤细胞，淋巴结转移肿瘤细胞中多梳抑制复合体2（polycomb repression complex 2, PRC2）的核心亚基EED与EZH2的表达水平显著上调，该结果经实时定量PCR（real-time PCR）验证。此外，对全组织切片进行免疫组织化学（immunohistochemical, IHC）染色与定量图像分析后发现，淋巴结转移灶中表达EZH2的肿瘤细胞比例显著高于配对的原发乳腺肿瘤，且该现象与原位肿瘤细胞增殖呈显著正相关。我们进一步探究了转移性肿瘤细胞中PRC2基因上调的潜在机制，通过基因集富集分析（Gene Set Enrichment Analysis, GSEA）发现，淋巴结转移灶中存在E2F基因、MYC靶基因的表达上调，以及抑癌基因E-钙粘蛋白（E-cadherin）靶基因的表达下调。我们进一步通过免疫组织化学检测了潜在EZH2靶蛋白E-钙粘蛋白在配对原发灶/淋巴结转移样本中的表达情况，结果显示相较于配对原发肿瘤，其在淋巴结转移灶中的表达量显著降低。 结论与意义 本研究证实，相较于乳腺原发肿瘤，乳腺癌淋巴结转移灶中表观遗传沉默复合体PRC2/EED-EZH2的表达显著上调，且其与原位肿瘤细胞增殖呈正相关。与此同时，PRC2的靶蛋白E-钙粘蛋白在淋巴结转移灶中显著下调，这表明PRC2可通过抑制E-钙粘蛋白的表达，在淋巴结转移进程中促进上皮间质转化（epithelial mesenchymal transition, EMT）。上述结果提示，由PRC2蛋白介导的表观遗传调控或许可赋予肿瘤细胞在淋巴结中定植生长的额外优势，这为开发治疗与预防乳腺癌淋巴结转移的表观遗传药物提供了新的可能性。