Periodic Reporting for period 1 - nAChRs-CRC-PCa-IF-RI (Nicotinic Acetylcholine Receptors nAChRs in Prostate and Colon Cell Cancer: pharmacology, mechanism, cellular (mal)function)
Okres sprawozdawczy: 2022-04-01 do 2024-03-31
• Tobacco exposure is a very well-known risk factor in cancer. One of its proposed mechanisms is through the activation of various nicotinic acetylcholine receptors nAChR, and consequently inducing and promoting cancer through different mechanisms. In lung cancer nicotine and its metabolites increase cell proliferation, invasion, migration, epithelial to mesenchymal transition (EMT) and activate specific signaling cascades principally through α7 and α9 nAChR subunits. And although current data associates cigarette smoking with prostate (PCa) and colon cancer (CRC), no study had addressed the question of whether nAChR contributed to this effect and the underlying signaling events.
• In this context, the research projects principal aim was to investigate nAChRs in the physiology/pathophysiology of CRC and PCa, where I explored the contribution of specific nAChRs to cancers hallmark functions, investigate the mechanism(s), and the respective signaling cascade(s) involved. Thus, first I set to (i) identify and characterize the role of nAChR in PCa and CRC, and (ii) investigate the role of nAChRs in migration, proliferation and invasion in CRC and PCa cancer cell lines. Here it was found that a specific subunit, the α5 nAChR subunit, was overexpressed amongst other subunits in different types of cancers, and that it was responsible for nicotine’s promotion of proliferation and migration in cell lines of breast, colon, and prostate cancer origins. Furthermore, the α5 subunit was also key for nicotine’s-promoted expression of cancer molecular markers and immune regulatory proteins. Moreover, a specific polymorphism in this subunit, which is known to be related to nicotine administration, dependence, and lung cancer, caused an inherent increase in proliferation and migration in colon cancer cells. Through a mechanism involving G-protein signaling. Taken together, our results indicate that nicotine-induced pro-oncogenic effects in PCa and CRC are mediated via the α5 subunit, adding to its characterization as a possible druggable target to target cancer.
• In this context, the research projects principal aim was to investigate nAChRs in the physiology/pathophysiology of CRC and PCa, where I explored the contribution of specific nAChRs to cancers hallmark functions, investigate the mechanism(s), and the respective signaling cascade(s) involved. Thus, first I set to (i) identify and characterize the role of nAChR in PCa and CRC, and (ii) investigate the role of nAChRs in migration, proliferation and invasion in CRC and PCa cancer cell lines. Here it was found that a specific subunit, the α5 nAChR subunit, was overexpressed amongst other subunits in different types of cancers, and that it was responsible for nicotine’s promotion of proliferation and migration in cell lines of breast, colon, and prostate cancer origins. Furthermore, the α5 subunit was also key for nicotine’s-promoted expression of cancer molecular markers and immune regulatory proteins. Moreover, a specific polymorphism in this subunit, which is known to be related to nicotine administration, dependence, and lung cancer, caused an inherent increase in proliferation and migration in colon cancer cells. Through a mechanism involving G-protein signaling. Taken together, our results indicate that nicotine-induced pro-oncogenic effects in PCa and CRC are mediated via the α5 subunit, adding to its characterization as a possible druggable target to target cancer.
• The project started with Objective 1, with the study of the expression of diverse nAChR subunits in diverse cancer types. Here we analyzed data from the Human Protein Atlas, which is based on the Cancer Genome Atlas TCGA project. And we found that throughout different types of human cancers, the α5 subunit is prominently expressed. Next, we thought to evaluate in colon cancer cell lines (CRC) and prostate cancer cell lines (PCa) whether activation of nAChRs induced calcium signals, but unfortunately, we did not have any signals with the cell lines tested.
• Next, using a pharmacological approach, we evaluated whether nicotine increased proliferation and migration in through nAChRs activation in the breast cancer cell line MCF7, the CRC line SW480, and the PCa cell line DU145. For this analysis, cells were incubated with increasing concentrations of nicotine, from 0.1 μM to 10 μM, for 72 h. In the presence of 1 µM nicotine, for all cell lines tested proliferation was significantly increased. Next, by using diverse nAChRs inhibitors we could conclude that nicotine exerted its actions through nAChRs not composed of α7 or β2 subunits. Additionally, we found that nicotine, through nAChRs, affected cellular migration in the DU145 cell line. As our qPCR studies indicated high expression levels of α5 in all cell lines, we sought to determine whether nicotine could modify proliferation and migration through the activation of α5-containing nAChRs in the analyzed cancer cell lines. Consequently, we made use of siRNA based silencing of the α5 subunit and evaluated whether it affected nicotine-induced proliferation and migration. In these cells, 1 μM nicotine failed to stimulate proliferation or migration. In summary, in MCF7, SW480, and DU145 cells, nicotine promoted proliferation and migration through the α5-subunit-containing nAChRs. Additionally we could determine that nAChRs and more specifically the α5 subunit affected the nicotine-elicited expression of immunoregulatory proteins and EMT markers. The results above clearly demonstrated that α5-containing nicotinic receptors are essential for nicotine-induced effects.
• Diverse studies have shown that a specific polymorphism in this subunit, which changes an aspartate D in position 398 to an asparagine N (referred to α5SNP), is strongly linked with nicotine dependence, quantity of smoking, and lung cancer. Additionally previous reports showed that the D398N mutation impairs α5′s function in neurons. With this information in mind, we next thought to investigate whether this mutation affects the nicotine-induced effects in proliferation and migration. In this set of experiments, DU145 cells were either transfected with the wild-type α5 construct or with its mutated version α5D398N. Then, proliferation in the absence or presence of 1 μM nicotine was studied in α5-WT- or α5-MUT-transfected PCa cells. Notably, basal proliferation and migration without nicotine was per se higher in cells transfected with α5-MUT than in those transfected with α5-WT. But the cells were not reactive towards nicotine. When we looked deeper into the mechanism, we could show that this mutation caused a persistent activation of G-proteins, and effect also observed when nicotine activated non-mutated cells. Taken together, our results demonstrate that nicotine induces proliferation through specific activation of the α5 subunit in a G-protein-mediated mechanism.
• The results of the project have been so far published in one internationally recognized journal, while another article resulting from the project is under review. Our result have been also disseminated to different target audiences by oral presentations in University of Bern Night of Science, scientific congress and in house final reports.
• Next, using a pharmacological approach, we evaluated whether nicotine increased proliferation and migration in through nAChRs activation in the breast cancer cell line MCF7, the CRC line SW480, and the PCa cell line DU145. For this analysis, cells were incubated with increasing concentrations of nicotine, from 0.1 μM to 10 μM, for 72 h. In the presence of 1 µM nicotine, for all cell lines tested proliferation was significantly increased. Next, by using diverse nAChRs inhibitors we could conclude that nicotine exerted its actions through nAChRs not composed of α7 or β2 subunits. Additionally, we found that nicotine, through nAChRs, affected cellular migration in the DU145 cell line. As our qPCR studies indicated high expression levels of α5 in all cell lines, we sought to determine whether nicotine could modify proliferation and migration through the activation of α5-containing nAChRs in the analyzed cancer cell lines. Consequently, we made use of siRNA based silencing of the α5 subunit and evaluated whether it affected nicotine-induced proliferation and migration. In these cells, 1 μM nicotine failed to stimulate proliferation or migration. In summary, in MCF7, SW480, and DU145 cells, nicotine promoted proliferation and migration through the α5-subunit-containing nAChRs. Additionally we could determine that nAChRs and more specifically the α5 subunit affected the nicotine-elicited expression of immunoregulatory proteins and EMT markers. The results above clearly demonstrated that α5-containing nicotinic receptors are essential for nicotine-induced effects.
• Diverse studies have shown that a specific polymorphism in this subunit, which changes an aspartate D in position 398 to an asparagine N (referred to α5SNP), is strongly linked with nicotine dependence, quantity of smoking, and lung cancer. Additionally previous reports showed that the D398N mutation impairs α5′s function in neurons. With this information in mind, we next thought to investigate whether this mutation affects the nicotine-induced effects in proliferation and migration. In this set of experiments, DU145 cells were either transfected with the wild-type α5 construct or with its mutated version α5D398N. Then, proliferation in the absence or presence of 1 μM nicotine was studied in α5-WT- or α5-MUT-transfected PCa cells. Notably, basal proliferation and migration without nicotine was per se higher in cells transfected with α5-MUT than in those transfected with α5-WT. But the cells were not reactive towards nicotine. When we looked deeper into the mechanism, we could show that this mutation caused a persistent activation of G-proteins, and effect also observed when nicotine activated non-mutated cells. Taken together, our results demonstrate that nicotine induces proliferation through specific activation of the α5 subunit in a G-protein-mediated mechanism.
• The results of the project have been so far published in one internationally recognized journal, while another article resulting from the project is under review. Our result have been also disseminated to different target audiences by oral presentations in University of Bern Night of Science, scientific congress and in house final reports.
• The results of the present project provide valuable insights into how human α5 nicotinic acetylcholine receptor subunit is key for nicotine signaling and its effect upon cancer cells migration and proliferation, underlining its role as a future therapeutical target. Our results are of high relevance for society in general, and scientist in particular as they help to better understand nicotine’s deleterious effects in all cancer types and also the molecular mechanisms involving nAChRs activation and signaling.