CANCER GENOMICS & PROTEOMICS 18 : 261-272 (2021) doi:10.21873/cgp.20257 Analysis of the Protein–Protein Interaction Network Identifying c-Met as a Target of Gigantol in the Suppression of Lung Cancer Metastasis NITHIKOON AKSORN 1, NATTANAN LOSUWANNARAK 2, SUCHARAT TUNGSUKRUTHAI 3,4 , SITTIRUK ROYTRAKUL 5 and PITHI CHANVORACHOTE 3,4 1Department of Clinical Pathology, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok, Thailand; 2Department of Pharmacology and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Huachiew Chalermprakiet University, Samutprakarn, Thailand; 3Cell-based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand; 4Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand; 5Functional Ingredients and Food Innovation Research Group, National Center for Genetic Engineering and Biotechnology, Pathumthani, Thailand Abstract. Background/Aim: c-Met (mesenchymal-epithelial adhesion enriched in 40 nodes with 25 edges, 39 proteins of transition factor) facilitates cancer progression and is cell migration enriched in 39 nodes with 76 edges in down- recognized as a promising drug target. The molecular target regulated proteins, 30 proteins of cell adhesion enriched in of gigantol from Dendrobium draconis in suppressing cancer 30 nodes with 21 edges, and 22 proteins of cell migration metastasis is largely unknown. Materials and Methods: enriched in 22 nodes with 22 edges in up-regulated protein. Proteins affected by gigantol treatment were subjected to c-Met was identified as a central protein of the PPI network proteomic and bioinformatic analysis. Protein–Protein in the largest degree. KEGG mapper further suggested that interaction (PPI) networks were constructed by the Search c-Met, PI3K, and AKT were the regulatory proteins affected Tool for the Retrieval of Interacting Genes (STRING). The by gigantol. To confirm, the effects of gigantol on c-Met, the Kyoto Encyclopedia of Genes and Genomes (KEGG) p-PI3K, PI3K, p-AKT, and AKT proteins were investigated database and hub gene were used to enrich the dominant by western blotting and the results showed a consistent effect pathways. Western blot analysis and immunofluorescence of gigantol in the suppression of the c-Met/PI3K/AKT signal. were used to validate the effect of gigantol on the target Next, immunofluorescence showed a dramatic decrease in protein and signaling. Results: Gigantol down-regulates 41 c-Met, PI3K and AKT activation in response to gigantol. adhesion proteins and 39-migratory proteins, while it up- Conclusion: c-Met is an important target of gigantol regulates 30 adhesion-related proteins and 22 proteins treatment in lung cancer cells. Gigantol suppresses controlling cell migration. The key components of our metastasis-related cell motility through decreasing c-Met constructed PPI network comprised 41 proteins of cell resulting in PI3K/AKT signaling disruption. Lung cancer is one of the leading causes of worldwide mortality and accounts for approximately 27% of all cancer- This article is freely accessible online. related deaths (1). Metastasis is responsible for the majority of lung cancer deaths and most lung cancer patients are found Correspondence to: Professor Dr. Pithi Chanvorachote, Department with metastasis at the time of first diagnosis. Cancer of Pharmacology and Physiology, Faculty of Pharmaceutical metastasis involves a multi-step process, including cell Sciences, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. Tel: +66 22188344, e-mail: detachment, cell migration, cell invasion, survival in the [email protected]; [email protected] circulation, and establishment of new tumors (2). Cell adhesion plays a crucial role in cell to cell and cell to Key Words: Gigantol, c-Met, metastasis, lung cancer, PI3K, proteomics. extracellular matrix (ECM) interactions through a group of 261 CANCER GENOMICS & PROTEOMICS 18 : 261-272 (2021) cell adhesion molecules (CAMs), such as cadherins, integrins fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/ml or selectins and lymphocyte homing receptors (CD44). penicillin, and streptomycin. Therefore, reduction of these cell-cell adhesions and cell-ECM Chemicals and reagents. Gigantol was purchased from Sigma adhesions is an early step in cancer cell dissemination (3). In chemical, Inc. (Chemical Express, Bangkok, Thailand). RPMI-1640 addition, cell migration plays an important prerequisite medium, phosphate buffer saline, glutamine, penicillin, and process in a loose ECM attachment and loss of polarity, which streptomycin were purchased from Gibco Company (Grand Island, facilitates cancer cell invasion and metastasis (4). Regarding NY, USA). Fetal bovine serum (FBS), and agarose were obtained the upstream signaling of migratory control, c-Met, also from Bio-Rad Laboratories (Hercules, CA, USA). Primary antibodies known as MET (mesenchymal-epithelial transition factor), a against c-Met, p-AKT, AKT, p-PI3K, PI3K, and β- actin and specific receptor tyrosine kinase encoded by the MET gene, has been horseradish peroxidase (HRP)-link secondary antibody were obtained from Cell Signaling Technology, Inc. (Danver, MA, USA). shown to facilitate several metastatic behaviors. The binding of hepatocyte growth factor (HGF) to c-Met triggers the Proteomics sample preparation. The H460 cells treated with 20 receptor activation and transmits the HGF/c-Met axis through μM of gigantol for 24 h were dissolved with 0.5% SDS and the interaction and activation of other tyrosine kinases, protein concentration was measured by the Lowry’s method. An resulting in the induction of PI3K/AKT, Ras/MAPK, equal amount of pooled protein from control or gigantol-treated JAK/STAT, SRC, and Wnt/ β- catenin (5-8). The activation of cells (50 μg each) were subjected to in-solution digestion. Samples signals downstream of c-Met drive several cellular processes were completely dissolved in 10 mM ammonium bicarbonate (NH 4HCO 3) containing 10 mM dithiothreitol (DTT) for 1 hour at that stimulate cell proliferation, migration, invasion, and 60˚C and alkylated with 15 mM iodoacetamide (IAA) in 10 mM angiogenesis. In lung cancer, overexpression of the c-Met NH 4HCO 3 for 45 min in the dark, and then and digested with 1 ng protein has been reported and found to be linked with a high protein per 20 ng sequencing grade trypsin (Promega, Walldorf, metastasis potential, increased tumor growth, and drug Germany) at 37˚C overnight. The trypsiniszed peptides were resistance (9, 10). Recently, c-Met has been considered an protonated with 0.1% formic acid before injection into LC- important target for therapeutic approaches (11). MS/MS. Gigantol, a bibenzyl compound from Dendrobium LC/MS-MS analysis. LC/MS-MS for protein quantitation and draconis , has attracted increasing attention in recent years as identification from the digested samples was conducted using the it exhibits several potential effects on cancer cells. One study Ultimate 3000 Nano/Capillary LC System (Thermo Scientific, reported that gigantol inhibits lung cancer proliferation Gloucester, UK) coupled to a Hybrid quadrupole Q-Tof impact II™ through induction of GSK3 β- mediated MYC ubiquitin- (Bruker Daltonics, Coventry, UK) equipped with a Nano-captive proteasome degradation (12). In addition, gigantol was spray ion source with a nanocolumn (PepMap100, C18, 300 μm shown to inhibit the expression of Slug, the regulator of i.d.×5 mm). Solvent A contained 0.1% formic acid and solvent B epithelial–to–mesenchymal transition (EMT), and diminished contained 80% ACN in water containing 0.1% formic acid were used to elute peptides. Peptide separation was achieved with a linear cancer stem cell properties through ATP-dependent tyrosine gradient from 5% to 55% of solvent B at a constant flow rate of kinase (AKT) inhibition (13, 14). However, the complete 0.30 μL/min for 30 minutes including a regeneration and an profile of how gigantol can control cell migration and equilibration step at 90% and 10% eluent B, respectively. Next, the adhesion is currently unknown. Consequently, the present LC-MS/MS raw data were converted into a software file format study aimed to unravel the cellular pathways affected by mzXML (Compass 1.9 software, Bruker Daltonics). The MS and gigantol treatment in lung cancer cells, focusing on cell MS/MS spectra which over the range 150-2200 m/z were generated migration and cell adhesion, as these behaviors are related for further protein identification against database search. to the metastasis potential of cancer. We performed Proteomics data and bioinformatics analysis. The MS/MS spectra proteomic analysis on gigantol-treated lung cancer cells and were searched using MaxQuant 1.6.6.0 to identify proteins and used bioinformatics to examine the affected pathways. These peptides. MS/MS spectra were searched by the Andromeda search findings might be useful for the development of this engine against the Uniprot Homo sapiens database as following compound for lung cancer treatment as well as might benefit parameters: maximum of two miss cleavages, mass tolerance of 20 the further prediction of its potential pharmacological ppm for main search, enzyme (trypsin), variable modifications activities based on its cellular mechanism of action. (carbamidomethylation of cysteine as fixed modification, oxidation of methionine and acetylation of the protein N-terminus). For protein identification,