Role of muscarinic receptors expression in non-tumorigenic breast cells

OROÑO MANUEL; MARTÍNEZ PULIDO PAOLA; LOMBARDI GABRIELA; GOMEZ GIAMBALVO DILEYVIC; SALES MARIA ELENA

Congreso; LXI Reunión Anual de la SAIC; 2016

Angiogenesis is one of the most important steps in tumor progression. The development of new capillaries involves the participation of pro-angiogenic factors like vascular endothelial growth factor-A (VEGF-A) and extracellular matrix remodelation by metalloproteinases (MMP). Our previous works demonstrated that muscarinic acetylcholine receptors (mAChR) are over-expressed in human breast cancer tumors in comparison to normal breast tissue. We reported that non-tumorigenic human breast cells, MCF-10A neither show immunolabelling for mAChR, VEGF-A and MMP-9 nor stimulate angiogenic response in NUDE mice. Here, we developed stable MCF-10A cell lines that express different mAChR subtypes (mAChR-lines) and we investigated the effect of mAChR activation on VEGF-A levels, MMP-9 activity and tumor neovascular response induced in vivo. We demonstrated by Western blot that the transfection of mAChR induced de novo VEGF-A and MMP9 synthesis in comparison to non-transfected MCF-10A cells. Both effects were potentiated by the addition of the cholinergic agonist carbachol (CARB) and reverted by the preincubation with muscarinic antagonists or by silencing mAChRs with specific interference RNA (iRNA). CARB also increased MMP9 activity in the supernatants of mAChR-lines. We also observed that mAChR-lines inoculated in the skin of NUDE mice induced a strong neovascular response (vessel mean density±SD) (mAChR3:5.0±1.1; mAChR4:6.4±0.7; mAChR3R4:4.8±0.5) and CARB significantly enhanced angiogenic response (mAChR3:6.7±0.8; mAChR4: 8,6±0.9; mAChR3R4: 6.4±0.6) (p