title: A novel role for ezrin in breast cancer angio/lymphangiogenesis.
abstract: BACKGROUND
  Recent evidence suggests that tumour lymphangiogenesis promotes lymph node metastasis, a major prognostic factor for survival of breast cancer patients. However, signaling mechanisms involved in tumour-induced lymphangiogenesis remain poorly understood. The expression of ezrin, a membrane cytoskeletal crosslinker and Src substrate, correlates with poor outcome in a diversity of cancers including breast. Furthermore, ezrin is essential in experimental invasion and metastasis models of breast cancer. Ezrin acts cooperatively with Src in the regulation of the Src-induced malignant phenotype and metastasis. However, it remains unclear if ezrin plays a role in Src-induced tumour angio/lymphangiogenesis.
  
  
  METHODS
  The effects of ezrin knockdown and mutation on angio/lymphangiogenic potential of human MDA-MB-231 and mouse AC2M2 mammary carcinoma cell lines were examined in the presence of constitutively active or wild-type (WT) Src. In vitro assays using primary human lymphatic endothelial cells (hLEC), an ex vivo aortic ring assay, and in vivo tumour engraftment were utilized to assess angio/lymphangiogenic activity of cancer cells.
  
  
  RESULTS
  Ezrin-deficient cells expressing activated Src displayed significant reduction in endothelial cell branching in the aortic ring assay in addition to reduced hLEC migration, tube formation, and permeability compared to the controls. Intravital imaging and microvessel density (MVD) analysis of tumour xenografts revealed significant reductions in tumour-induced angio/lymphangiogenesis in ezrin-deficient cells when compared to the WT or activated Src-expressing cells. Moreover, syngeneic tumours derived from ezrin-deficient or Y477F ezrin-expressing (non-phosphorylatable by Src) AC2M2 cells further confirmed the xenograft results. Immunoblotting analysis provided a link between ezrin expression and a key angio/lymphangiogenesis signaling pathway by revealing that ezrin regulates Stat3 activation, VEGF-A/-C and IL-6 expression in breast cancer cell lines. Furthermore, high expression of ezrin in human breast tumours significantly correlated with elevated Src expression and the presence of lymphovascular invasion.
  
  
  CONCLUSIONS
  The results describe a novel function for ezrin in the regulation of tumour-induced angio/lymphangiogenesis promoted by Src in breast cancer. The combination of Src/ezrin might prove to be a beneficial prognostic/predictive biomarker for early-stage metastatic breast cancer.
authors.forename: Abdi
authors.initials: A
authors.lastname: Ghaffari
authors.forename: Victoria
authors.initials: V
authors.lastname: Hoskin
authors.forename: Alvin
authors.initials: A
authors.lastname: Szeto
authors.forename: Maaike
authors.initials: M
authors.lastname: Hum
authors.forename: Navid
authors.initials: N
authors.lastname: Liaghati
authors.forename: Kanji
authors.initials: K
authors.lastname: Nakatsu
authors.forename: David
authors.initials: D
authors.lastname: LeBrun
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Madarnas
authors.forename: Sandip
authors.initials: S
authors.lastname: Sengupta
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Elliott
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D003598:Cytoskeletal Proteins / Q000502:physiology*; D005260:Female; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D042583:Lymphangiogenesis; D008808:Mice, Inbred CBA; D018345:Mice, Knockout; D020125:Mutation, Missense; D009361:Neoplasm Invasiveness; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017207:Rats, Sprague-Dawley; D050796:STAT3 Transcription Factor / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D042582:Vascular Endothelial Growth Factor C / Q000378:metabolism; D019061:src-Family Kinases; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D015850:Interleukin-6; D050796:STAT3 Transcription Factor; D042461:Vascular Endothelial Growth Factor A; D042582:Vascular Endothelial Growth Factor C; D019061:src-Family Kinases; D000099149:Ezrin; C508600:IL6 protein, human; C494086:STAT3 protein, human; C467484:VEGFA protein, human