The angiogenic role of the alpha 9-nicotinic acetylcholine receptor in triple-negative breast cancers
Sonjid Ochirbat (a), Tzu-Chun Kan (b, c), Chun-Chun Hsu (b, d, e), Tzu-Hsuan Huang (b, f), Kuo-Hsiang Chuang (g), Michael Chen (g), Chun-Chia Cheng (h), Chun-Chao Chang (i, j, k), Sri Rahayu (l), Jungshan Chang (a, b, m*)

a) International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan
b) Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan
c) Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
d) School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
e) Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei 11031, Taiwan.
f) Department of Medical Research, Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan
g) Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 11031, Taiwan
h) Research Center of Radiation Medicine, Chang Gung University, Taoyuan 33302, Taiwan
i) Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei 11031, Taiwan
j) Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan
k) TMU Research Center for Digestive Medicine, Taipei Medical University, Taipei 11031, Taiwan
l) Department of Biology, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
m) International Ph.D. Program for Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan

Abstract

Nicotine acts as an angiogenic factor by stimulating endogenous cholinergic pathways. Several subtypes of nicotinic acetylcholine receptors (nAChRs) have been demonstrated to be closely correlated to the formation and progression of different types of cancers. Recently, several studies have found that nicotinic acetylcholine receptors &#945-9 (&#945-9-nAChRs) are highly expressed in breast tumors, especially in tumors derived from patients diagnosed at advanced stages. In vitro studies have demonstrated that activation of &#945-9-nAChRs is associated with increased proliferation and migration of breast cancer. To study the tumor-promoting role of &#945-9-nAChRs in breast cancers, we generated a novel anti-&#945-9-nAChR and methoxy-polyethylene glycol (mPEG) bispecific antibody (&#945-9 BsAb) for dissecting the molecular mechanism on &#945-9-nAChR-mediated tumor progression. Unexpectedly, we discovered the angiogenic role of &#945-9-nAChR in nicotine-induced neovascularization of tumors. It revealed &#945-9 BsAbs reduced nicotine-induced endothelial cell tube formation, blood vessel development in matrigel plug assay and angiogenesis in microtube array membrane murine model (MTAMs). To unbraid the molecular mechanism of &#945-9-nAChR in nicotine-mediated angiogenesis, the &#945-9 BsAbs were applied and revealed the inhibitory roles in nicotine-induced production of hypoxia-inducible factor-2 alpha (HIF-2&#945-), vascular endothelial growth factor A (VEGF-A), phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2), vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase-9 (MMP9) from triple-negative breast cancer cells (MDA-MB-231), suggesting &#945-9-nAChRs played an important role in nicotine-induced angiogenesis. To confirm our results, the shRNA targeting &#945-9-nAChRs was designed and used to silence &#945-9-nAChR expression and then evaluated the angiogenic role of &#945-9-nAChRs. The results showed &#945-9 shRNA also played an inhibitory effect in blocking the nicotine-induced angiogenic signaling. Taken together, &#945-9-nAChR played a critical role in nicotine-induced angiogenesis and this bispecific antibody (&#945-9 BsAb) may serve as a potential therapeutic candidate for treatments of the &#945-9 positive cancers.

Keywords: Nicotine, Nicotinic acetylcholine receptors &#945-9, Bispecific antibody, Angiogenesis and Microtube array Membrane murine model.

Topic: Biology