Utilization of Liquid Chromatography Mass Spectrometry Analyses to Identify LKB1–APC Interaction in Modulating Wnt/β-Catenin Pathway of Lung Cancer Cells

Abstract

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 GSK3b was dispensable for the association of LKB1 and APC. Importantly, LKB1 binds to APC to suppress the Wnt/b-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 inhibits the proliferation of gastric cancer cells by suppressing the nuclear translocation of Yap and β-catenin

“…LKB1 acts as a critical regulator in the AMPK/mTOR, Wnt/β-catenin, and Hippo signaling pathways in various types of cancers (10,11,14). However, the underlying mechanisms linking LKB1 to its diverse functions in GC remain unclear, and they are further complicated by the cross-talk between different signaling pathways.…”

“…10 in the study by Nguyen et al (11)]. In lung cancer cells, LKB1 binds directly to the adenomatous polyposis coli (APC)/glycogen synthase kinase-3β (GSK3β) destruction complex, inducing the degradation of β-catenin (14), and thus preventing Wnt/β-catenin signaling. Yap was also reported to protect β-catenin from degradation in the cytoplasm, by recruiting β-transducin repeat containing E3 ubiquitin protein ligase (β-TrCP) ubiquitin ligase away from the Axin/GSK3β destruction complex (18) in cancer cells.…”

“…Although LKB1 has been reported to suppress the proliferation and survival of many types of cancer cells (11,14,23), its effects on GC cells required further research. Therefore, AGS GC cells were transfected with a full-length LKB1 vector (LKB1 cells) or an empty vector (Vec cells).…”

“…AMPK suppresses the mammalian target of rapamycin (mTOR), and thereby inhibits proliferation, polarity and energy metabolism of cancer cells (10)(11)(12)(13). LKBI is also an essential tumor suppressor in the Wnt/β-catenin (14), Hedgehog (15) and Hippo (10) signaling pathways, where it blocks the growth, migration, and epithelial-mesenchymal transition of cancer cells.…”

NF90 is a novel influenza A virus NS1‐interacting protein that antagonizes the inhibitory role of NS1 on PKR phosphorylation

“…Each protein band was excised, destained, reduced, alkylated, and digested with trypsin. The polypeptides were extracted, purified, and then analyzed using a MicrOTOF‐QII Nanoflow LC‐MS/MS system (Bruker Daltonics, Bremen, Germany) as described previously . Peaklist files were generated using data analysis 4.0 software (Bruker Daltonics), and database searches against human and IAV sequences were performed using the mascot search engine version 2.3.01 (Matrix Science, Boston, MA, USA).…”