Co-Expression of [alpha]9[beta]1 Integrin and VEGF-D Confers Lymphatic Metastatic Ability to a Human Breast Cancer Cell Line MDA-MB-468LN

• Published in: PloS one Vol. 7; no. 4; p. e35094
• Main Authors: Majumder, Mousumi, Tutunea-Fatan, Elena, Xin, Xiping, Rodriguez-Torres, Mauricio, Torres-Garcia, Jose, Wiebe, Ryan, Timoshenko, Alexander V, Bhattacharjee, Rabindra N, Chambers, Ann F, Lala, Peeyush K
• Format: Journal Article
• Language: English
• Published: Public Library of Science 24.04.2012
• Subjects: Analysis; Antibodies; Breast cancer; Cancer metastasis; Endothelium; Integrins; Macrophages; Vascular endothelial growth factor
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0035094

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 [alpha]9[beta]1 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 [alpha]9[beta]1 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 [alpha]9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or [alpha]9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. 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 [alpha]9[beta]1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of [alpha]9[beta]1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with [alpha]9[beta]1 blocking antibody or knock-down of [alpha]9. 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 [alpha]9-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 [alpha]9 in 468LN cells. Differential capacity for VEGF-D production and [alpha]9[beta]1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.