title: Molecular Pathways Mediating Metastases to the Brain via Epithelial-to-Mesenchymal Transition: Genes, Proteins, and Functional Analysis.
abstract: BACKGROUND
  Brain metastases are the leading cause of morbidity and mortality among patients with disseminated cancer. The development of metastatic disease involves an orderly sequence of steps enabling tumor cells to migrate from the primary tumor and colonize at secondary locations. In order to achieve this complex metastatic potential, a cancer cell is believed to undergo a cellular reprogramming process involving the development of a degree of stemness, via a proposed process termed epithelial-to-mesenchymal transition (EMT). Upon reaching its secondary site, these reprogrammed cancer stem cells submit to a reversal process designated mesenchymal-to-epithelial transition (MET), enabling establishment of metastases. Here, we examined the expression of markers of EMT, MET, and stem cells in metastatic brain tumor samples.
  
  
  METHODS
  Immunohistochemical analyses were performed to establish the markers of EMT and MET. Co-expression of these markers was determined by immunofluorescence analysis. Gene-expression analysis was conducted using tissues from brain metastases of primary adenocarcinoma of the lung compared to non-metastatic tissue. Cell proliferation was carried out using 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide S-phase entry analysis, by determining the 5-ethynyl-2'-deoxyuridine incorporation. Scratch wound and chemotactic migration assays were performed in an astrocytic setting.
  
  
  RESULTS
  Metastatic brain tumor samples displayed expression of epithelial markers (zinc finger protein SNAI1 and Twist-related protein-1), as well as the mesenchymal marker vimentin. The stem cell marker CD44 was also highly expressed. Moreover, co-expression of the epithelial marker E-cadherin with the mesenchymal marker vimentin was evident, suggesting a state of transition. Expression analysis of transcription factor genes in metastatic brain tumor samples demonstrated an alteration in genes associated with neurogenesis, differentiation, and reprogramming. Furthermore, tumor cells grown in astrocytic medium displayed increased cell proliferation and enhanced S-phase cell-cycle entry. Additionally, chemotactic signaling from the astrocytic environment promoted tumor cell migration. Primary tumor cells and astrocytes were also shown to grow amicably together, forming cell-to-cell interactions.
  
  
  CONCLUSIONS
  These findings suggest that cellular reprogramming via EMT/MET plays a critical step in the formation of brain metastases, where the cerebral milieu provides a microenvironment suitable for the development of metastatic disease.
authors: Department of Neurosurgery, New York Medical College, Valhalla, NY, U.S.A.
authors: Dhruve S
authors: DS
authors: Jeevan
authors: Department of Neurosurgery, New York Medical College, Valhalla, NY, U.S.A.
authors: Jared B
authors: JB
authors: Cooper
authors: Department of Pathology, New York Medical College, Valhalla, NY, U.S.A.
authors: Alex
authors: A
authors: Braun
authors: Department of Neurosurgery, New York Medical College, Valhalla, NY, U.S.A.
authors: Raj
authors: R
authors: Murali
authors: Department of Neurosurgery, New York Medical College, Valhalla, NY, U.S.A. meena_jhanwar@nymc.edu.
authors: Meena
authors: M
authors: Jhanwar-Uniyal
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism*; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism / Q000556:secondary*; D002450:Cell Communication; D002453:Cell Cycle; D002465:Cell Movement; D049109:Cell Proliferation; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007124:Immunoenzyme Techniques; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor
keywords: Metastatic tumors; brain metastasis; epithelial-to-mesenchymal transition; homing; invasion; stem cells