Abstract
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.
Highlights
Axillary lymph node metastasis is the single most important prognostic factor for patient survival [1,2] and represents the transition from local to systemic disease in breast cancer
We found increased expression of polycomb repression complex 2 (PRC2) genes in lymph node metastatic tumor cells compared to primary tumor cells
To capture subtle yet biologically important changes, the Gene eXpression Network Analysis (GXNA) program [8] was used to determine significantly changed networks/subnetworks between lymph node metastasis and primary tumor. This produced a network of thirty-one genes with a significant score of 15 (Table 1). This network was significantly up-regulated in lymph node metastasis compared to primary tumor and will be referred to as lymph nodes (LNs) Met Set
Summary
Axillary lymph node metastasis is the single most important prognostic factor for patient survival [1,2] and represents the transition from local to systemic disease in breast cancer. In an effort to solve this issue, Vecchi and his colleagues used microarray profiling in combination with tissue microarray to further validate the cellular origins of genes identified in microarray studies With this approach, they showed four genes with epithelial origin to be down-regulated in lymph node metastasis [5]. Ellsworth and colleagues performed gene expression analyses on laser capture microdissected tumor cells and found 13 up-regulated genes and 38 down-regulated genes in lymph node metastases [6]. Their data improved current understanding of lymph node metastatic process and provided important insights on potential genetic targets for the treatment of breast cancer metastases. It is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression
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