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Contrasting its role in breast cancer (BCa) initiation, estrogen signaling has a protective effect in later stages, where estrogen receptor (ER)alpha loss associates with aggressive metastatic disease. We asked whether the beneficial effect of estrogen signaling in late-stage BCa is attributable to the recently reported estrogen-mediated antagonism of the pro-metastatic transcription factor Runx2.

MCF7/Rx2dox breast cancer cells were engineered with a lentivirus expressing Runx2 in response to doxycycline (dox). Cells treated with dox and/or estradiol (E2) were subjected to genome-wide expression profiling, RT-qPCR analysis of specific genes, and MatrigelTM invasion assays. Knockdown of genes of interest was performed using lentiviruses expressing appropriate shRNAs, either constitutively or in response to dox. Gene expression in BCa tumors was investigated using a cohort of 557 patients compiled from publicly available datasets. Association of gene expression with clinical metastasis was assessed by dichotomizing patients into those expressing genes of interest at either high or low levels, and comparing the respective Kaplan-Meier curves of metastasis-free survival.

Runx2 induced epithelial-mesenchymal transition (EMT) evidenced by acquisition of a fibroblastic morphology, decreased expression of E-cadherin, increased expression of vimentin and invasiveness. Runx2 also stimulated SNAI2 expression in a WNT- and TGFbeta-dependent manner, and knockdown of SNAI2 abrogated the pro-metastatic activities of Runx2. E2 antagonized Runx2's pro-metastatic activities, including SNAI2 upregulation. In primary BCa tumors, Runx2 activity, SNAI2 expression, and metastasis were positively correlated, and SNAI2 expression was negatively correlated with ERalpha. However, the negative correlation between SNAI2 and ERalpha in bone-seeking BCa cells was weaker than the respective negative correlation in tumors seeking lung. Furthermore, the absence of ERalpha in primary tumors was associated with lung- and brain- but not with bone-seeking metastasis, and tumor biopsies from bone metastastatic sites displayed the unusual combination of high Runx2/SNAI2 and high ERalpha expression.

E2 antagonizes Runx2-induced EMT and invasiveness of BCa cells, partly through attenuating expression of SNAI2, a Runx2 target required for mediating its pro-metastatic property. That ERalpha loss promotes non-osseous metastasis by unleashing Runx2/SNAI2 is supported by negative correlation observed in corresponding tumors. Unknown mechanisms in bone-seeking BCa allow high Runx2/SNAI2 expression despite high ERalpha levels.

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