Utilization of liquid chromatography mass spectrometry analyses to identify LKB1-APC interaction in modulating Wnt/β-catenin pathway of lung cancer cells

• Published in: Molecular cancer research Vol. 12; no. 4; pp. 622 - 635
• Main Authors: Jian, Shu-Fang, Hsiao, Chang-Chun, Chen, Shin-Yi, Weng, Ching-Chieh, Kuo, Tzu-Lei, Wu, Deng-Chyang, Hung, Wen-Chun, Cheng, Kuang-Hung
• Format: Journal Article
• Language: English
• Published: United States 01.04.2014
• Subjects: Adenomatous Polyposis Coli Protein - analysis; Adenomatous Polyposis Coli Protein - metabolism; Animals; beta Catenin - metabolism; Cell Proliferation; Chromatography, High Pressure Liquid - methods; HEK293 Cells; Heterografts; Humans; Lung Neoplasms - chemistry; Lung Neoplasms - genetics; Lung Neoplasms - metabolism; Male; Mice; Mice, Inbred C57BL; Mice, SCID; Phosphorylation; Protein-Serine-Threonine Kinases - analysis; Protein-Serine-Threonine Kinases - metabolism; RNA, Small Interfering - analysis; RNA, Small Interfering - metabolism; Signal Transduction; Tandem Mass Spectrometry - methods; Wnt Signaling Pathway
• ISSN: 1541-7786; 1557-3125
• DOI: 10.1158/1541-7786.MCR-13-0487

STK11/LKB1, a serine/threonine protein kinase and tumor suppressor, is a key upstream kinase of adenine monophosphate-activated protein kinase, which is a kinase involved in controlling cell polarity and maintaining cellular energy homeostasis. LKB1 is mutated in a significant number of Peutz-Jeghers syndrome (PJS) cases and sporadic cancers, and is most frequently mutated in lung adenocarcinomas; however, little is known about how LKB1 is involved in lung cancer progression. In this study, immunoprecipitation-HPLC tandem mass spectrometry (IP-LC-MS/MS) was performed to identify novel proteins interacting with LKB1 in lung cancer. Interestingly, many LKB1-interacting proteins acquired from the LC-MS/MS approach were mapped, using MetaCore pathway analysis, to the cystic fibrosis transmembrane conductance regulator activation pathway. Moreover, it was determined that LKB1 directly interacts with APC, and this LKB1-APC interaction was further confirmed by reverse immunoprecipitation assays, but GSK3β was dispensable for the association of LKB1 and APC. Importantly, LKB1 binds to APC to suppress the Wnt/β-catenin signaling pathway, which is known to be involved in cell proliferation and migration. Subsequent analysis of the downstream targets of the Wnt/TCF pathway led to the identification of several Wnt-regulated genes, such as CD44, COX-2, survivin, and c-Myc, whose expression levels are downregulated by LKB1. In summary, these results demonstrate that LKB1 regulates the Wnt pathway through a direct interaction with APC to suppress the tumorigenic/metastatic potential of lung tumors. LKB1 status influences the molecular circuitry (Wnt/β-catenin pathway), cellular biology, and may serve as a potential therapeutic node in genetically defined subsets of lung cancer.