Neuropeptide Y Y5-receptor activation on breast cancer cells acts as a paracrine system that stimulates VEGF expression and secretion to promote angiogenesis.
Accumulating data implicate a pathological role for sympathetic neurotransmitters like neuropeptide Y (NPY) in breast cancer progression. Our group and others reported that NPY promotes proliferation and migration in breast cancer cells, however the angiogenic potential of NPY in breast cancer is unknown. Herein we sought to determine if NPY promotes angiogenesis in vitro by increasing vascular endothelial growth factor (VEGF) expression and release from 4T1 breast cancer cells. Western blot analysis revealed that NPY treatment caused a 52 ± 14% increase in VEGF expression in the 4T1 cells compared to non-treated controls. Using selective NPY Y-receptor agonists (Y1R, Y2R and Y5R) we observed an increase in VEGF expression only when cells were treated with Y5R agonist. Congruently, using selective Y1R, Y2R, or Y5R antagonists, NPY-induced increases in VEGF expression in 4T1 cells were attenuated only under Y5R antagonism. Endothelial tube formation assays were conducted using conditioned media (CM) from NPY treated 4T1 cells. Concentration-dependent increases in number of branch points and complete endothelial networks were observed in HUVEC exposed to NPY CM. CM from Y5R agonist treated 4T1 cells caused similar increases in number of branch points and complete endothelial networks. VEGF concentration was quantified in CM (ELISA) from agonist experiments; we observed a 2-fold and 2.5-fold increase in VEGF release from NPY and Y5R agonist treated 4T1 cells respectively. Overall these data highlight a novel mechanism by which NPY may promote breast cancer progression, and further implicate a pathological role of the NPY Y5R.
Department of Medical Biophysics, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada.
Philip J
PJ
Medeiros
Dwayne N
DN
Jackson
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D009478:Neuropeptide Y / Q000819:agonists / Q000378:metabolism*; D019899:Paracrine Communication; D017476:Receptors, Neuropeptide Y / Q000819:agonists / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000378:metabolism
D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C100568:neuropeptide Y5 receptor
4T1, Mammary carcinoma; Ang1; Ang2; Breast cancer; CM; DMEM; Dulbecco's minimal essential medium; ELISA; FBS; FGF; Fetal bovine serum; HUVEC; IL-6; MMP; NE; NPY; Neuropeptide Y; PDGF; TTBS; VEGF; Vascularization; Y1R; Y1agonist,; Y2R; Y2agoinst,; Y5R; Y5agonist; [Leu31, Pro34]-Neuropeptide Y; [cPP1-7, NPY19-23, Ala31, Aib32, Gln34]-hPancreatic polypeptide; angiopoietin 1; angiopoietin 2; conditioned media; enzyme-linked immunosorbent assay; fibroblast growth factor; human umbilical endothelial vein cells; interleukin 6; matrix metalloproteinase; neuropeptide Y; norepinephrine; peptide YY (3–36); platelet-derived growth factor; tris-buffered saline+Tween20; vascular endothelial growth factor