title: Regulation of breast cancer metastasis by Runx2 and estrogen signaling: the role of SNAI2.
abstract: BACKGROUND
  In contrast to its role in breast cancer (BCa) initiation, estrogen signaling has a protective effect in later stages, where estrogen receptor (ER)α 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.
  
  
  METHODS
  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 Matrigel™ 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.
  
  
  RESULTS
  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 stimulated SNAI2 expression in a WNT- and transforming growth factor (TGF)β-dependent manner, and knockdown of SNAI2 abrogated the pro-metastatic activities of Runx2. E2 antagonized the pro-metastatic activities of Runx2, including SNAI2 upregulation. In primary BCa tumors, Runx2 activity, SNAI2 expression, and metastasis were positively correlated, and SNAI2 expression was negatively correlated with ERα. However, the negative correlation between SNAI2 and ERα in bone-seeking BCa cells was weaker than the respective negative correlation in tumors seeking lung. Furthermore, the absence of ERα in primary tumors was associated with lung- and brain- but not with bone metastasis, and tumor biopsies from bone metastatic sites displayed the unusual combination of high Runx2/SNAI2 and high ERα expression.
  
  
  CONCLUSIONS
  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 ERα loss promotes non-osseous metastasis by unleashing Runx2/SNAI2 is supported by the negative correlation observed in corresponding tumors. Unknown mechanisms in bone-seeking BCa allow high Runx2/SNAI2 expression despite high ERα level.
authors.affiliation: Department of Biochemistry & Molecular Biology, Keck School of Medicine of the University of Southern California, 2250 Alcazar Street, Los Angeles, CA 90033, USA. chimgee@usc.edu
authors.forename: Nyam-Osor
authors.initials: NO
authors.lastname: Chimge
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Baniwal
authors.forename: Gillian H
authors.initials: GH
authors.lastname: Little
authors.forename: Yi-bu
authors.initials: YB
authors.lastname: Chen
authors.forename: Michael
authors.initials: M
authors.lastname: Kahn
authors.forename: Debu
authors.initials: D
authors.lastname: Tripathy
authors.forename: Zea
authors.initials: Z
authors.lastname: Borok
authors.forename: Baruch
authors.initials: B
authors.lastname: Frenkel
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000401:mortality / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D004967:Estrogens / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009362:Neoplasm Metastasis; D015398:Signal Transduction; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism; D051153:Wnt Proteins / Q000378:metabolism
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D004967:Estrogens; C000604408:SNAI2 protein, human; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D051153:Wnt Proteins