title: Co-expression of α9β1 integrin and VEGF-D confers lymphatic metastatic ability to a human breast cancer cell line MDA-MB-468LN.
abstract: OBJECTIVE
  Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice.
  
  
  RESULTS
  A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells.
  
  
  CONCLUSIONS
  Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.
authors.affiliation: Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Mousumi
authors.initials: M
authors.lastname: Majumder
authors.forename: Elena
authors.initials: E
authors.lastname: Tutunea-Fatan
authors.forename: Xiping
authors.initials: X
authors.lastname: Xin
authors.forename: Mauricio
authors.initials: M
authors.lastname: Rodriguez-Torres
authors.forename: Jose
authors.initials: J
authors.lastname: Torres-Garcia
authors.forename: Ryan
authors.initials: R
authors.lastname: Wiebe
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Timoshenko
authors.forename: Rabindra N
authors.initials: RN
authors.lastname: Bhattacharjee
authors.forename: Ann F
authors.initials: AF
authors.lastname: Chambers
authors.forename: Peeyush K
authors.initials: PK
authors.lastname: Lala
mesh_terms: D000818:Animals; D001940:Breast / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016023:Integrins / Q000235:genetics*; D008207:Lymphatic Metastasis; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness / Q000235:genetics; D042643:Vascular Endothelial Growth Factor D / Q000235:genetics*
chemical_list: D016023:Integrins; D042643:Vascular Endothelial Growth Factor D; C113256:integrin alpha 9 beta 1