Nicotine promotes cholangiocarcinoma growth in xenograft mice
Authors: O'Brien April , Hall Chad , Ehrlich Laurent , White Tori , Lairmore Terry , Dostal David E , Glaser Shannon 
Published: November 2023

Nicotine, the major psychoactive component of cigarette smoke, can stimulate the proliferation of non‐neuronal cell types and promotes the growth of solid tumors. Nicotine exerts its functions through nicotinic acetylcholine receptors (nAChRs) and has been shown to stimulate the proliferation of a number of non‐neuronal cell types in an α7‐nAChR‐dependent mechanism due to the activation of MAPK signaling pathways. The AIM of our study was evaluate the effects of nicotine on cholangiocarcinoma proliferation.METHODSThe cholangiocarcinoma cell lines Mz‐ChA‐1, CCLP and HuCCT‐1 and the normal human cholangiocyte cell line HIBEpiC were utilized. α7‐nAChR expression was determined by immunofluorescence and real‐time PCR. The effects of nicotine on proliferation were determined by MTS proliferation assay in cells stimulated with nicotine (5, 10, 15, and 20 μM) for 48 hours. Nicotine stimulation of ERK1/2 signaling pathway was determined in Mz‐ChA‐1 cells stimulated with nicotine (10 μM) for 0, 3, 5, 10 and 30 minutes. Activation was observed by ELISA for p/tERK1/2. Phosphorylation and nuclear translocation of ERK1/2 was observed by immunofluorescence in Mz‐ChA‐1 cells treated with nicotine (10 μM) for 3 and 10 minutes. Nicotine‐stimulated ERK1/2 activity was also evaluated in MzChA‐1 cells transfected with shRNA for α7‐nAChR. Studies were also performed in Male BALB/c nude mice. Mz‐ChA‐1 cells (5 x 106) suspended in 0.2 ml of extracellular matrix gel were injected s.c. into the right and left rear flanks of the nude mice. After the formation of palpable tumors, the mice received regular drinking water or drinking water containing 50 or 200 μM nicotine. Tumor volume was measured and the expression of CK‐19, α7‐nAChR, S100A4, Ki‐67, and p‐ERK were evaluated by IHC. α7‐nAChR was evaluated in normal human liver tissue and cholangiocarcinoma samples.RESULTSMz‐ChA‐1, CCLP, HuCCT‐1 and HIBEpiC expressed the α7‐nAChR. Nicotine stimulated a dose‐dependent increase in cholangiocarcinoma proliferation at 48 hours. Nicotine induced a significant increase in the phosphorylation of ERK1/2 at 10 and 30 minutes, which was blocked by the knockdown of α7‐nAChR. By immunofluorescence, nicotine stimulated the phosphorylation and nuclear translocation of pERK1/2 in Mz‐ChA‐1 cells. Nicotine accelerated the growth of the cholangiocarcinoma tumors in our xenograft mouse model, and increased fibrosis and proliferation of the tumor cells, and pERK activation. Finally, α7‐nAChR was expressed at similar levels in normal human liver tissue and human cholangiocarcinoma.SUMMARY/CONCLUSIONSTaken together, the results of this study suggest that nicotine acts through α7‐nAChR and plays a novel role in pathogenesis of cholangiocarcinoma. Furthermore, nicotine may act as a mitogen in cholestatic liver disease processes facilitating malignant transformation.Support or Funding InformationVA Merit Award (5I01BX002192) to Dr. Glaser from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service.