International Journal of Molecular Sciences Article Tepotinib Inhibits the Epithelial–Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3β, E-Cadherin, and Mucin 5AC and 6 Levels 1, 1, 1 2 2 Sung-Hwa Sohn y, Hee Jung Sul y, Bohyun Kim , Bum Jun Kim , Hyeong Su Kim and Dae Young Zang 1,2,* 1 Hallym Translational Research Institute, Hallym University Sacred Heart Hospital, Anyang 14066, Korea; [email protected] (S.-H.S.); [email protected] (H.J.S.); [email protected] (B.K.) 2 Department of Internal Medicine, Hallym University Medical Center, Hallym University College of Medicine, Anyang-si, Gyeonggi-do 14068, Korea; [email protected] (B.J.K.); [email protected] (H.S.K.) * Correspondence: [email protected]; Tel.: +82-31-380-4167 These authors contributed equally to this work. y Received: 22 July 2020; Accepted: 19 August 2020; Published: 21 August 2020 Abstract: Aberrant expression of mucins (MUCs) can promote the epithelial–mesenchymal transition (EMT), which leads to enhanced tumorigenesis. Carcinogenesis-related pathways involving c-MET and β-catenin are associated with MUCs. In this study, we characterized the expression of EMT-relevant proteins including MET, β-catenin, and E-cadherin in human gastric cancer (GC) cell lines, and further characterized the differential susceptibility of these cell lines compared with the c-MET inhibitor tepotinib. We assessed the antitumor activity of tepotinib in GC cell lines. The effects of tepotinib on cell viability, apoptotic cell death, EMT, and c-MET and β-catenin signaling were evaluated by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS), flow cytometry, Western blotting, and qRT-PCR. The antitumor efficacy was assessed in MKN45 xenograft mice. Tepotinib treatment induced apoptosis in c-MET-amplified SNU620, MKN45, and KATO III cells, but had no effect on c-MET-reduced MKN28 or AGS cells. Tepotinib treatment also significantly reduced the protein levels of phosphorylated and total c-MET, phosphorylated and total ERK, β-catenin, and c-MYC in SNU620 and MKN45 cells. In contrast, this drug was only slightly active against KATO III cells. Notably, tepotinib significantly reduced the expression of EMT-promoting genes such as MMP7, COX-2, WNT1, MUC5B, and c-MYC in c-MET-amplified GC cells and increased the expression of EMT-suppressing genes such as MUC5AC, MUC6, GSK3β, and E-cadherin. In a mouse model, tepotinib exhibited good antitumor growth activity along with increased E-cadherin and decreased phosphorylated c-MET (phospho-c-MET) protein levels. Collectively, these results suggest that tepotinib suppresses tumor growth and migration by negatively regulating c-MET-induced EMT. These findings provide new insights into the mechanism by which MUC5AC and MUC6 contribute to GC progression. Keywords: c-MET; EMT; gastric cancer; MUC5AC; MUC5B; MUC6; tepotinib 1. Introduction Mucins (MUCs) are part of carcinogenesis-related pathways, involving β-catenin, HGF/c-MET, NF-κB, MAPK, Ras/Erk, VEGF, c-JNK, and TGF-β [1–4]. MUCs are responsible for the formation of a protective mucous membrane barrier [5,6]. Aberrant expression of MUCs can promote the Int. J. Mol. Sci. 2020, 21, 6027; doi:10.3390/ijms21176027 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 13 aInt. protective J. Mol. Sci. 2020 mucous, 21, 6027 membrane barrier [5,6]. Aberrant expression of MUCs can promote2 ofthe 12 epithelial–mesenchymal transition (EMT) [7,8]. Among these MUCs, MUC5AC and MUC6 are specific to stomach mucus [9]. The stomach has a two-layered mucus system, consisting of an inner, attachedepithelial–mesenchymal mucus and an outer, transition unattached (EMT) [mucus7,8]. Among layer, theseboth MUCs,comprised MUC5AC of MUC5AC, and MUC6 which are acts specific as a diffusionto stomach barrier mucus for [9 hydrochloric]. The stomach acid has [10 a– two-layered13] produced mucus in stomach system, glands. consisting These of glands an inner, also attached secrete gel-formingmucus and anMUC6 outer, and unattached pepsin [14]. mucus layer, both comprised of MUC5AC, which acts as a diffusion barrierThe for Wnt/ hydrochloricβ-catenin, acid HGF/c-MET, [10–13] produced and Ra ins/Erk stomach pathways glands. have These been glands associated also secrete with gel-forming numerous cancersMUC6 and[15– pepsin17] and [14 ].mediate EMT in gastric cancer (GC) [18–20]. The EMT process includes adherenceThe Wnt junction/β-catenin, components HGF/c-MET, (e.g., E-cadherin and Ras/Erk (ECAD)), pathways matrix have metalloproteinase been associated with(MMP) numerous 7 and 9, tumorcancers microenvironment [15–17] and mediate components EMT in gastric(e.g., cancercyclooxygenase-2 (GC) [18– 20(COX-2)),]. The EMTand processc-MET includes[21–23]. Aberrantlyadherence junctionactivated components c-MET can (e.g.,drive E-cadherin tumorigenesis, (ECAD)), leading matrix to aggressive metalloproteinase cancer phenotypes (MMP) 7 and and 9, poortumor prognosis microenvironment by promoting components tumor (e.g.,cell survival, cyclooxygenase-2 migration, (COX-2)), EMT, and and c-METtreatment [21– resistance23]. Aberrantly [23]. Therefore,activated c-MET c-MET can has drive been tumorigenesis, highlighted as leading a promising to aggressive target in cancer GC. In phenotypes particular, andidentifying poor prognosis proper GCby promotingsubgroups tumoris important cell survival, for the selection migration, of EMT,c-MET and inhibitors. treatment There resistance is a need [23 to]. Therefore,define the c-MET levelshas been using highlighted various GC as acells promising with different target in c-MET GC. In expression particular, statuses. identifying The proper inhibitory GC subgroups activities of is 1449important FDA-approved for the selection drugs of were c-MET evaluated inhibitors. in SNU There620 is acells need during to define the thescreening c-MET levelsof new using therapeutic various agentsGC cells for with the ditreatmentfferent c-MET of c-MET-related expression statuses. GC. Am Theong inhibitorythese drugs, activities tepotinib of 1449showed FDA-approved the highest inhibitorydrugs were activity; evaluated therefore, in SNU620 this cells drug during requires the screening furtherof examination. new therapeutic In the agents present for the study, treatment we evaluatedof c-MET-related the effects GC. of Among tepotinib these on drugs,the suppression tepotinib showedof GC proliferation, the highest inhibitory apoptosis, activity; EMT, and therefore, tumor progression.this drug requires further examination. In the present study, we evaluated the effects of tepotinib on the suppression of GC proliferation, apoptosis, EMT, and tumor progression. 2. Results 2. Results 2.1. Effective Effective Dose Dose of Tepotinib in c-MET-Positive CellsCells We tested the dose-dependent inhibitory effect effectss of tepotinib in c-MET-amplifiedc-MET-amplified SNU620 and MKN45 cells cells (Figure (Figure 1).1). The The cells cells were were treated treated with with different di fferent concentrations concentrations of tepotinib of tepotinib for 48 for h, 48and h, theand optimal the optimal dose was dose determined was determined by evaluating by evaluating cell viability cell viability using an using MTS assay. an MTS Tepotinib assay. Tepotinibinhibited cellinhibited viability, cell viability,with an average with an average50% inhibitory 50% inhibitory concentration concentration (IC50) of (IC50) 7 nM of in 7 MKN45 nM in MKN45 cells and cells 9 andnM in9nM SNU620 in SNU620 cells. It cells. can Itbe can seen be that seen the that inhibiti the inhibitionon rate of rate tepotinib of tepotinib against against SNU620 SNU620 and andMKN45 MKN45 cell linescell lines increased increased with with increasing increasing concentration. concentration. Tepotinib Tepotinib inhibited inhibited the the growth growth of of the the SNU620 SNU620 and MKN45 cells in a concentration-dependent manner. (a) (b) Figure 1. The effect effect of tepotinib in c-MET-amplifiedc-MET-amplified gastric cancer (GC) cells. SNU620 and MKN45 cells were treated with various concentrations of tepotinib for 48 h. ( (aa)) SNU620 SNU620 and and MKN45 MKN45 cells cells were were treatedtreated with tepotinib and viability was measured after 4848 h.h. The ICIC50 forfor tepotinib tepotinib and and condition is shown in the figure. figure. ( (bb)) Relationship Relationship between between activity activity and and conc concentrationentration of tepotinib tepotinib against against SNU620 and MKN45 cells. Data Data are are means ± standardstandard deviation. deviation. IC IC50: average: average 50% 50% inhibitory inhibitory concentration. concentration. ± 50 2.2. Decreased Migration of c-MET-Amplified GC Cells by Tepotinib 2.2. Decreased Migration of c-MET-Amplified GC Cells by Tepotinib c-MET inducesinduces cancer cancer cell cell migration migration via thevia stimulationthe stimulation of cancer of cellcancer motility cell [motility23]. The migration[23]. The migrationrate of c-MET-amplified rate of c-MET-amplified MKN45 cells MKN45 was significantly cells was significantly decreased decreased by tepotinib by treatment tepotinib comparedtreatment with that of MKN28 cells (Figure2). These findings indicate that tepotinib decreases the motility of c-MET-amplified GC cells. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 13 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 13 compared with that of MKN28 cells (Figure 2). These findings indicate that tepotinib decreases the compared with that of MKN28 cells