Date Published: May 31, 2019
Publisher: Public Library of Science
Author(s): Maxim Bychkov, Zakhar Shenkarev, Mikhail Shulepko, Olga Shlepova, Mikhail Kirpichnikov, Ekaterina Lyukmanova, Joseph Najbauer.
Lynx1 is the first three-finger prototoxin found in the mammalian central nervous system. It is a GPI-anchored protein modulating nicotinic acetylcholine receptors (nAChRs) in the brain. Besides the brain, the Lynx1 protein was found in the lung and kidney. Endogenous Lynx1 controls the nicotine-induced up-regulation of the expression of α7 type nAChRs in lung adenocarcinoma A549 cells as well as the cell growth. Here, we analyzed the Lynx1 expression in the set of human epithelial cells. The Lynx1 expression both at the mRNA and protein level was detected in normal oral keratinocytes, and lung, colon, epidermal, and breast cancer cells, but not in embryonic kidney cells. Co-localization of Lynx1 with α7-nAChRs was revealed in a cell membrane for lung adenocarcinoma A549 and colon carcinoma HT-29 cells, but not for breast adenocarcinoma MCF-7 and epidermoid carcinoma A431 cells. The recombinant water-soluble variant of Lynx1 without a GPI-anchor (ws-Lynx1) inhibited the growth of A549 cells causing cell cycle arrest via modulation of α7-nAChRs and activation of different intracellular signaling cascades, including PKC/IP3, MAP/ERK, p38, and JNK pathways. A549 cells treatment with ws-Lynx1 resulted in phosphorylation of the proapoptotic tumor suppressor protein p53 and different kinases participated in the regulation of gene transcription, cell growth, adhesion, and differentiation. Externalization of phosphatidylserine, an early apoptosis marker, observed by flow cytometry, confirmed the induction of apoptosis in A549 cells upon the ws-Lynx1 treatment. Our data revealed the ability of ws-Lynx1 to regulate homeostasis of epithelial cancer cells.
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels responsible for signal transduction in the central and peripheral nervous systems and in the neuromuscular junctions . nAChRs are homo- or hetero-pentamers, composed of α and non-α subunits. nAChRs were also found in non-excitable cells, like immune and epithelial cells . These non-neuronal receptors participate in proliferation, differentiation, migration and apoptosis of epithelial cells, in control of inflammation, as well as in regulation of gene transcription [3–5]. The expression of different nAChR subunits was described in epithelial human cancers, e.g. in lung cancer, mesothelioma and colon carcinoma .
nAChRs play an important role in many essential processes and are involved in nicotine-induced carcinogenesis . Previously we demonstrated that the human secreted proteins SLURPs could inhibit the growth of cancer cells of epithelial origin [18,19]. Human Lynx1 was also found to participate in the regulation of the lung cancer cells growth [26,44]. Here, for the first time we described the Lynx1 protein expression in epidermal, colon and breast epithelial cells. Colocalization of Lynx1 with α7-nAChRs in lung and colon cancer cells was observed. We also found that the water-soluble variant of human Lynx1 controls the growth and apoptosis of non-small lung cancer cells. Based on the data obtained, we propose the two-stage mechanism of the ws-Lynx1 action (Fig 7). Ws-Lynx1 interaction with α7-nAChRs activates different intracellular kinase cascades with the subsequent phosphorylation of the proapototic factor p53 and other factors participated in the control of the gene transcription as well as cell growth and apoptosis. Thus, the endogenous Ly6/uPAR proteins like Lynx1 are the promising prototypes for development of new drugs for treatment of epithelial cancers.