Tepotinib Inhibits the Epithelial–Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3β, E-Cadherin, and Mucin 5AC and 6 Levels
Abstract
:1. Introduction
2. Results
2.1. Effective Dose of Tepotinib in c-MET-Positive Cells
2.2. Decreased Migration of c-MET-Amplified GC Cells by Tepotinib
2.3. Effect of Tepotinib on Cell Apoptosis
2.4. Tepotinib Inhibition of c-MET Activation and EMT in c-MET-Amplified GC Cells
2.5. Tepotinib Inhibits the Growth of Subcutaneous Xenograft Tumors in Nude Mice
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Reagents
4.2. Growth Inhibition Assays
4.3. Cell Migration Analysis
4.4. Apoptosis Analysis
4.5. Quantitative Real-Time PCR (qRT-PCR) Analysis
4.6. Immunofluorescence Microscopy
4.7. Western Analysis
4.8. Mice Xenograft Study
4.9. Histology and Immunohistochemistry
4.10. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
EMT | Epithelial–mesenchymal transition |
GC | Gastric cancer |
MUCs | Mucins |
ECAD | E-cadherin |
MMP7 | Matrix metalloproteinase 7 |
COX-2 | Cyclooxygenase-2 |
References
- Singh, P.K.; Hollingsworth, M.A. Cell surface-associated mucins in signal transduction. Trends Cell Biol. 2006, 16, 467–476. [Google Scholar] [CrossRef] [PubMed]
- Singh, P.K.; Behrens, M.E.; Eggers, J.P.; Cerny, R.L.; Bailey, J.M.; Shanmugam, K.; Gendler, S.J.; Bennett, E.P.; Hollingsworth, M.A. Phosphorylation of MUC1 by Met modulates interaction with p53 and MMP1 expression. J. Biol. Chem. 2008, 283, 26985–26995. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bozkaya, G.; Korhan, P.; Cokakli, M.; Erdal, E.; Sagol, O.; Karademir, S.; Korch, C.; Atabey, N. Cooperative interaction of MUC1 with the HGF/c-Met pathway during hepatocarcinogenesis. Mol. Cancer 2012, 11, 64. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Liu, Q.; Cheng, Z.; Luo, L.; Yang, Y.; Zhang, Z.; Ma, H.; Chen, T.; Huang, X.; Lin, S.Y.; Jin, M.; et al. C-terminus of MUC16 activates Wnt signaling pathway through its interaction with beta-catenin to promote tumorigenesis and metastasis. Oncotarget 2016, 7, 36800–36813. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hanisch, F.G. O-glycosylation of the mucin type. Biol. Chem. 2001, 382, 143–149. [Google Scholar] [CrossRef] [PubMed]
- Corfield, A.P. Mucins: A biologically relevant glycan barrier in mucosal protection. Biochim. Et Biophys. Acta 2015, 1850, 236–252. [Google Scholar] [CrossRef]
- Hollingsworth, M.A.; Swanson, B.J. Mucins in cancer: Protection and control of the cell surface. Nat. Rev. Cancer 2004, 4, 45–60. [Google Scholar] [CrossRef]
- Rajabi, H.; Kufe, D. MUC1-C Oncoprotein Integrates a Program of EMT, Epigenetic Reprogramming and Immune Evasion in Human Carcinomas. Biochim. Et Biophys. Acta Rev. Cancer 2017, 1868, 117–122. [Google Scholar] [CrossRef]
- Johansson, M.E.; Sjovall, H.; Hansson, G.C. The gastrointestinal mucus system in health and disease. Nat. Rev. Gastroenterol. Hepatol. 2013, 10, 352–361. [Google Scholar] [CrossRef] [Green Version]
- Atuma, C.; Strugala, V.; Allen, A.; Holm, L. The adherent gastrointestinal mucus gel layer: Thickness and physical state in vivo. Am. J. Physiol. Gastrointest. Liver Physiol. 2001, 280, G922–G929. [Google Scholar] [CrossRef] [Green Version]
- Ho, S.B.; Roberton, A.M.; Shekels, L.L.; Lyftogt, C.T.; Niehans, G.A.; Toribara, N.W. Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression. Gastroenterology 1995, 109, 735–747. [Google Scholar] [CrossRef]
- Nordman, H.; Davies, J.R.; Lindell, G.; de Bolos, C.; Real, F.; Carlstedt, I. Gastric MUC5AC and MUC6 are large oligomeric mucins that differ in size, glycosylation and tissue distribution. Biochem. J. 2002, 364, 191–200. [Google Scholar] [CrossRef]
- Schade, C.; Flemstrom, G.; Holm, L. Hydrogen ion concentration in the mucus layer on top of acid-stimulated and inhibited rat gastric mucosa. Gastroenterology 1994, 107, 180–188. [Google Scholar] [CrossRef]
- Bartman, A.E.; Buisine, M.P.; Aubert, J.P.; Niehans, G.A.; Toribara, N.W.; Kim, Y.S.; Kelly, E.J.; Crabtree, J.E.; Ho, S.B. The MUC6 secretory mucin gene is expressed in a wide variety of epithelial tissues. J. Pathol. 1998, 186, 398–405. [Google Scholar] [CrossRef]
- Zhang, M.; Zhu, G.Y.; Gao, H.Y.; Zhao, S.P.; Xue, Y. Expression of tissue levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in gastric adenocarcinoma. J. Surg. Oncol. 2011, 103, 243–247. [Google Scholar] [CrossRef] [PubMed]
- Javle, M.M.; Gibbs, J.F.; Iwata, K.K.; Pak, Y.; Rutledge, P.; Yu, J.; Black, J.D.; Tan, D.; Khoury, T. Epithelial-mesenchymal transition (EMT) and activated extracellular signal-regulated kinase (p-Erk) in surgically resected pancreatic cancer. Ann. Surg. Oncol. 2007, 14, 3527–3533. [Google Scholar] [CrossRef] [PubMed]
- Zhang, Y.; Xia, M.; Jin, K.; Wang, S.; Wei, H.; Fan, C.; Wu, Y.; Li, X.; Li, X.; Li, G.; et al. Function of the c-Met receptor tyrosine kinase in carcinogenesis and associated therapeutic opportunities. Mol. Cancer 2018, 17, 45. [Google Scholar] [CrossRef] [PubMed]
- Humar, B.; Blair, V.; Charlton, A.; More, H.; Martin, I.; Guilford, P. E-cadherin deficiency initiates gastric signet-ring cell carcinoma in mice and man. Cancer Res. 2009, 69, 2050–2056. [Google Scholar] [CrossRef] [Green Version]
- Song, Y.; Li, Z.X.; Liu, X.; Wang, R.; Li, L.W.; Zhang, Q.Y. The Wnt/beta-catenin and PI3K/Akt signaling pathways promote EMT in gastric cancer by epigenetic regulation via H3 lysine 27 acetylation. Tumor Biol. 2017, 39. [Google Scholar] [CrossRef] [Green Version]
- Leung, E.; Xue, A.; Wang, Y.; Rougerie, P.; Sharma, V.P.; Eddy, R.; Cox, D.; Condeelis, J. Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway. Oncogene 2017, 36, 2680–2692. [Google Scholar] [CrossRef]
- Yasui, W.; Oue, N.; Aung, P.P.; Matsumura, S.; Shutoh, M.; Nakayama, H. Molecular-pathological prognostic factors of gastric cancer: A review. Gastric Cancer J. Int. Gastric Cancer Assoc. Jpn. Gastric Cancer Assoc. 2005, 8, 86–94. [Google Scholar] [CrossRef] [PubMed]
- Fanelli, M.F.; Chinen, L.T.; Begnami, M.D.; Costa, W.L.J.; Fregnami, J.H.; Soares, F.A.; Montagnini, A.L. The influence of transforming growth factor-alpha, cyclooxygenase-2, matrix metalloproteinase (MMP)-7, MMP-9 and CXCR4 proteins involved in epithelial-mesenchymal transition on overall survival of patients with gastric cancer. Histopathology 2012, 61, 153–161. [Google Scholar] [CrossRef] [PubMed]
- Jeon, H.M.; Lee, J. MET: Roles in epithelial-mesenchymal transition and cancer stemness. Ann. Transl. Med. 2017, 5, 1–5. [Google Scholar] [CrossRef] [Green Version]
- Jonckheere, N.; Skrypek, N.; Van Seuningen, I. Mucins and tumor resistance to chemotherapeutic drugs. Biochim. Biophys. Acta 2014, 1846, 142–151. [Google Scholar] [CrossRef] [Green Version]
- Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018, 68, 394–424. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Takahashi, T.; Saikawa, Y.; Kitagawa, Y. Gastric cancer: Current status of diagnosis and treatment. Cancers 2013, 5, 48–63. [Google Scholar] [CrossRef] [Green Version]
- Dicken, B.J.; Bigam, D.L.; Cass, C.; Mackey, J.R.; Joy, A.A.; Hamilton, S.M. Gastric adenocarcinoma: Review and considerations for future directions. Ann. Surg. 2005, 241, 27–39. [Google Scholar] [CrossRef] [PubMed]
- Hack, S.P.; Bruey, J.M.; Koeppen, H. HGF/MET-directed therapeutics in gastroesophageal cancer: A review of clinical and biomarker development. Oncotarget 2014, 5, 2866–2880. [Google Scholar] [CrossRef] [Green Version]
- Thiery, J.P. Epithelial-mesenchymal transitions in tumour progression. Nat. Rev. Cancer 2002, 2, 442–454. [Google Scholar] [CrossRef]
- Shook, D.; Keller, R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech. Dev. 2003, 120, 1351–1383. [Google Scholar] [CrossRef]
- Gnemmi, V.; Bouillez, A.; Gaudelot, K.; Hemon, B.; Ringot, B.; Pottier, N.; Glowacki, F.; Villers, A.; Vindrieux, D.; Cauffiez, C.; et al. MUC1 drives epithelial-mesenchymal transition in renal carcinoma through Wnt/beta-catenin pathway and interaction with SNAIL promoter. Cancer Lett. 2014, 346, 225–236. [Google Scholar] [CrossRef] [PubMed]
- Ponnusamy, M.P.; Lakshmanan, I.; Jain, M.; Das, S.; Chakraborty, S.; Dey, P.; Batra, S.K. MUC4 mucin-induced epithelial to mesenchymal transition: A novel mechanism for metastasis of human ovarian cancer cells. Oncogene 2010, 29, 5741–5754. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Comamala, M.; Pinard, M.; Theriault, C.; Matte, I.; Albert, A.; Boivin, M.; Beaudin, J.; Piche, A.; Rancourt, C. Downregulation of cell surface CA125/MUC16 induces epithelial-to-mesenchymal transition and restores EGFR signalling in NIH:OVCAR3 ovarian carcinoma cells. Br. J. Cancer 2011, 104, 989–999. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Baldus, S.E.; Hanisch, F.G. Biochemistry and pathological importance of mucin-associated antigens in gastrointestinal neoplasia. Adv. Cancer Res. 2000, 79, 201–248. [Google Scholar]
- Lahdaoui, F.; Messager, M.; Vincent, A.; Hec, F.; Gandon, A.; Warlaumont, M.; Renaud, F.; Leteurtre, E.; Piessen, G.; Jonckheere, N.; et al. Depletion of MUC5B mucin in gastrointestinal cancer cells alters their tumorigenic properties: Implication of the Wnt/beta-catenin pathway. Biochem. J. 2017, 474, 3733–3746. [Google Scholar] [CrossRef]
- Perrais, M.; Pigny, P.; Buisine, M.P.; Porchet, N.; Aubert, J.P.; Van Seuningen-Lempire, I. Aberrant expression of human mucin gene MUC5B in gastric carcinoma and cancer cells. Identification and regulation of a distal promoter. J. Biol. Chem. 2001, 276, 15386–15396. [Google Scholar] [CrossRef] [Green Version]
- Pinto-de-Sousa, J.; Reis, C.A.; David, L.; Pimenta, A.; Cardoso-de-Oliveira, M. MUC5B expression in gastric carcinoma: Relationship with clinico-pathological parameters and with expression of mucins MUC1, MUC2, MUC5AC and MUC6. Virchows Arch. Int. J. Pathol. 2004, 444, 224–230. [Google Scholar] [CrossRef]
- Buisine, M.P.; Devisme, L.; Maunoury, V.; Deschodt, E.; Gosselin, B.; Copin, M.C.; Aubert, J.P.; Porchet, N. Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. I. Stomach. A relationship to gastric carcinoma. J. Histochem. Cytochem. Off. J. Histochem. Soc. 2000, 48, 1657–1666. [Google Scholar] [CrossRef] [Green Version]
- Giannoni, E.; Bianchini, F.; Masieri, L.; Serni, S.; Torre, E.; Calorini, L.; Chiarugi, P. Reciprocal activation of prostate cancer cells and cancer-associated fibroblasts stimulates epithelial-mesenchymal transition and cancer stemness. Cancer Res. 2010, 70, 6945–6956. [Google Scholar] [CrossRef] [Green Version]
- Kabashima, A.; Maehara, Y.; Kakeji, Y.; Baba, H.; Koga, T.; Sugimachi, K. Clinicopathological features and overexpression of matrix metalloproteinases in intramucosal gastric carcinoma with lymph node metastasis. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2000, 6, 3581–3584. [Google Scholar]
- Zheng, G.; Lyons, J.G.; Tan, T.K.; Wang, Y.; Hsu, T.T.; Min, D.; Succar, L.; Rangan, G.K.; Hu, M.; Henderson, B.R.; et al. Disruption of E-cadherin by matrix metalloproteinase directly mediates epithelial-mesenchymal transition downstream of transforming growth factor-beta1 in renal tubular epithelial cells. Am. J. Pathol. 2009, 175, 580–591. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Docetaxel: New indication. Metastatic gastric cancer: Keep using fluorouracil-based chemotherapy. No tangible progress. Prescr. Int. 2008, 17, 107. [PubMed]
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Sohn, S.-H.; Sul, H.J.; Kim, B.; Kim, B.J.; Kim, H.S.; Zang, D.Y. Tepotinib Inhibits the Epithelial–Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3β, E-Cadherin, and Mucin 5AC and 6 Levels. Int. J. Mol. Sci. 2020, 21, 6027. https://doi.org/10.3390/ijms21176027
Sohn S-H, Sul HJ, Kim B, Kim BJ, Kim HS, Zang DY. Tepotinib Inhibits the Epithelial–Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3β, E-Cadherin, and Mucin 5AC and 6 Levels. International Journal of Molecular Sciences. 2020; 21(17):6027. https://doi.org/10.3390/ijms21176027
Chicago/Turabian StyleSohn, Sung-Hwa, Hee Jung Sul, Bohyun Kim, Bum Jun Kim, Hyeong Su Kim, and Dae Young Zang. 2020. "Tepotinib Inhibits the Epithelial–Mesenchymal Transition and Tumor Growth of Gastric Cancers by Increasing GSK3β, E-Cadherin, and Mucin 5AC and 6 Levels" International Journal of Molecular Sciences 21, no. 17: 6027. https://doi.org/10.3390/ijms21176027