Inhibition of FOXM1 Leads to Suppression of Cell Proliferation, Migration, and Invasion Through AXL/eEF2 Kinase Signaling and Induces Apoptosis and Ferroptosis in GBM Cells
Abstract
1. Introduction
2. Results
2.1. eEF2K, AXL, and FOXM1 Are Upregulated in GBM Patient Tumors
2.2. Downregulation of FOXM1, AXL, and eEF2K Inhibits Proliferation and Colony Formation
2.3. FOXM1 Regulates AXL and eEF2K Expression and Physically Interacts with Them
2.4. Inhibition of FOXM1, AXL, and eEF2K Suppresses Spheroid Formation of GBM Cells
2.5. Knockdown of FOXM1, AXL, and eEF2K Suppresses Cell Migration and Invasion of GBM Cells
2.6. Downregulation of FOXM1, AXL, and eEF2K Induces Apoptosis and Ferroptosis
2.7. Knockdown of FOXM1, AXL, and eEF2K Enhances TMZ-Induced Cell Death via Apoptosis and Ferroptosis
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Cell Lines and Cell Culture
5.2. Patient Dataset Analysis
5.3. siRNA Transfection
5.4. Colony Formation Assay
5.5. Protein Extraction and Western Blot Analysis
- Membranes were blocked with 5% dry milk in Tris-buffered saline-Tween 20 (TBS-T) and incubated overnight at 4 °C with primary antibodies against eEF2K, GAPDH (Cell Signaling Technology, Danvers, MA, USA), AXL (R&D Systems, Minneapolis, MN, USA), and FOXM1 (Santa Cruz Biotechnology, Dallas, TX, USA).
- After washing with TBS-T, membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).
- Blots were imaged using the ChemiDoc™ Imaging System (Bio-Rad, Hercules, CA, USA) with chemiluminescent detection using the Immobilon Classico Western HRP Substrate (Millipore Sigma, Burlington, MA, USA).
5.6. Co-Immunoprecipitation (IP) Assay
5.7. Spheroid Formation Assay
5.8. Cell Migration Assay
5.9. In Vitro Matrigel Invasion Assay
5.10. Apoptosis Detection with Annexin V Assay
5.11. Lipid Peroxidation Assay
5.12. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
GBM | Glioblastoma Multiforme |
eEF2K | Eukaryotic Elongation Factor 2 Kinase |
FOXM1 | Forkhead-box protein M1 |
TMZ | Temozolomide |
References
- Ostrom, Q.T.; Price, M.; Neff, C.; Cioffi, G.; Waite, K.A.; Kruchko, C.; Barnholtz-Sloan, J.S. CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2015–2019. Neuro Oncol. 2022, 24, v1. [Google Scholar] [CrossRef]
- Koshy, M.; Villano, J.L.; Dolecek, T.A.; Howard, A.; Mahmood, U.; Chmura, S.J.; Weichselbaum, R.R.; McCarthy, B.J. Improved survival time trends for glioblastoma using the SEER 17 population-based registries. J. Neurooncol. 2011, 107, 207. [Google Scholar] [CrossRef]
- Grochans, S.; Cybulska, A.M.; Simińska, D.; Korbecki, J.; Kojder, K.; Chlubek, D.; Baranowska-Bosiacka, I. Epidemiology of Glioblastoma Multiforme—Literature Review. Cancers 2022, 14, 2412. [Google Scholar] [CrossRef]
- Chinot, O.L.; Wick, W.; Mason, W.; Henriksson, R.; Saran, F.; Nishikawa, R.; Carpentier, A.F.; Hoang-Xuan, K.; Kavan, P.; Cernea, D.; et al. Bevacizumab plus Radiotherapy–Temozolomide for Newly Diagnosed Glioblastoma. N. Engl. J. Med. 2014, 370, 709–722. [Google Scholar] [CrossRef] [PubMed]
- Stupp, R.; Taillibert, S.; Kanner, A.A.; Kesari, S.; Steinberg, D.M.; Toms, S.A.; Taylor, L.P.; Lieberman, F.; Silvani, A.; Fink, K.L.; et al. Maintenance Therapy With Tumor-Treating Fields Plus Temozolomide vs. Temozolomide Alone for Glioblastoma: A Randomized Clinical Trial. JAMA 2015, 314, 2535–2543. [Google Scholar] [CrossRef] [PubMed]
- Miller, K.D.; Ostrom, Q.T.; Kruchko, C.; Patil, N.; Tihan, T.; Cioffi, G.; Fuchs, H.E.; Waite, K.A.; Jemal, A.; Siegel, R.L.; et al. Brain and other central nervous system tumor statistics, 2021. CA Cancer J. Clin. 2021, 71, 381–406. [Google Scholar] [CrossRef] [PubMed]
- Poon, M.T.C.; Sudlow, C.L.M.; Figueroa, J.D.; Brennan, P.M. Longer-term (≥2 years) survival in patients with glioblastoma in population-based studies pre- and post-2005: A systematic review and meta-analysis. Sci. Rep. 2020, 10, 11622. [Google Scholar] [CrossRef]
- Biltekin, E.; Ozpolat, B. FOXM1: A Promising Target for Cancer Stemness. J. Cancer Biol. Res. 2024, 11, 1146. Available online: https://www.jscimedcentral.com/public/assets/articles/cancerbiology-11-1146.pdf (accessed on 1 June 2025).
- Dilmac, S.; Hamurcu, Z.; Ozpolat, B. Therapeutic Landscape of FOXM1 in Triple-Negative Breast Cancer and Aggressive Solid Cancers. Cancers 2024, 16, 3823. [Google Scholar] [CrossRef]
- Jackson, B.C.; Carpenter, C.; Nebert, D.W.; Vasiliou, V. Update of human and mouse forkhead box (FOX) gene families. Hum. Genom. 2010, 4, 345–352. [Google Scholar] [CrossRef]
- Shriwas, O.; Priyadarshini, M.; Samal, S.K.; Rath, R.; Panda, S.; Das Majumdar, S.K.; Muduly, D.K.; Botlagunta, M.; Dash, R. DDX3 modulates cisplatin resistance in OSCC through ALKBH5-mediated m6A-demethylation of FOXM1 and NANOG. Apoptosis 2020, 25, 233–246. [Google Scholar] [CrossRef] [PubMed]
- Modi, A.; Purohit, P.; Roy, D.; Vishnoi, J.R.; Pareek, P.; Elhence, P.; Singh, P.; Sharma, S.; Sharma, P.; Misra, S. FOXM1 mediates GDF-15 dependent stemness and intrinsic drug resistance in breast cancer. Mol. Biol. Rep. 2022, 49, 2877–2888. [Google Scholar] [CrossRef] [PubMed]
- Lee, Y.; Kim, K.H.; Kim, D.G.; Cho, H.J.; Kim, Y.; Rheey, J.; Shin, K.; Seo, Y.J.; Choi, Y.S.; Lee, J.I.; et al. FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2. PLoS ONE 2015, 10, e0137703. [Google Scholar] [CrossRef]
- Liao, G.B.; Li, X.Z.; Zeng, S.; Liu, C.; Yang, S.M.; Yang, L.; Hu, C.J.; Bai, J.Y. Regulation of the master regulator FOXM1 in cancer. Cell Commun. Signal. 2018, 16, 57. [Google Scholar] [CrossRef]
- Zhang, N.; Wu, X.; Yang, L.; Xiao, F.; Zhang, H.; Zhou, A.; Huang, Z.; Huang, S. FoxM1 Inhibition Sensitizes Resistant Glioblastoma Cells to Temozolomide by Downregulating the Expression of DNA Repair Gene Rad51. Clin. Cancer Res. 2012, 18, 5961. [Google Scholar] [CrossRef]
- Guler, A.; Hamurcu, Z.; Ulutabanca, H.; Cınar, V.; Nurdinov, N.; Erdem, S.; Ozpolat, B. Flavopiridol Suppresses Cell Proliferation and Migration and Induces Apoptotic Cell Death by Inhibiting Oncogenic FOXM1 Signaling in IDH Wild-Type and IDH-Mutant GBM Cells. Mol. Neurobiol. 2024, 61, 1061–1079. [Google Scholar] [CrossRef]
- Karakas, D.; Ozpolat, B. Eukaryotic elongation factor-2 kinase (eEF2K) signaling in tumor and microenvironment as a novel molecular target. J. Mol. Med. 2020, 98, 775–787. [Google Scholar] [CrossRef]
- Erdogan, M.A.; Ashour, A.; Yuca, E.; Gorgulu, K.; Ozpolat, B. Targeting eukaryotic elongation factor-2 kinase suppresses the growth and peritoneal metastasis of ovarian cancer. Cell Signal 2021, 81, 109938. [Google Scholar] [CrossRef]
- Ashour, A.A.; Abdel-Aziz, A.A.H.; Mansour, A.M.; Neslihan Alpay, S.; Huo, L.; Ozpolat, B. Targeting elongation factor-2 kinase (eEF-2K) induces apoptosis in human pancreatic cancer cells. Apoptosis 2014, 19, 241–258. [Google Scholar] [CrossRef]
- Onder, F.C.; Kahraman, N.; Atici, E.B.; Cagir, A.; Kandemir, H.; Tatar, G.; Tok, T.T.; Karliga, B.; Durdagi, S.; Ay, M.; et al. Target-driven design of a coumarinyl chalcone scaffold based novel EF2 Kinase inhibitor suppresses breast cancer growth in vivo. bioRxiv 2020. [Google Scholar] [CrossRef]
- Wang, N.; Cen, L.L.; Tian, Z.; An, M.M.; Gu, Q.; Zhou, X.H.; Zhang, Y.H.; Liu, L.; Zhang, J.; Yang, D.; et al. eEF2K as an important kinase associated with cancer survival and prognosis. Sci. Rep. 2024, 14, 29284. [Google Scholar] [CrossRef]
- Zhu, C.; Wei, Y.; Wei, X. AXL receptor tyrosine kinase as a promising anti-cancer approach: Functions, molecular mechanisms and clinical applications. Mol. Cancer 2019, 18, 153. [Google Scholar] [CrossRef] [PubMed]
- Onken, J.; Torka, R.; Korsing, S.; Radke, J.; Krementeskaia, I.; Nieminen, M.; Bai, X.; Ullrich, A.; Heppner, F.; Vajkoczy, P. Inhibiting receptor tyrosine kinase AXL with small molecule inhibitor BMS-777607 reduces glioblastoma growth, migration, and invasion in vitro and in vivo. Oncotarget 2016, 7, 9876–9889. [Google Scholar] [CrossRef] [PubMed]
- Tang, Y.; Zang, H.; Wen, Q.; Fan, S. AXL in cancer: A modulator of drug resistance and therapeutic target. J. Exp. Clin. Cancer Res. 2023, 42, 148. [Google Scholar] [CrossRef] [PubMed]
- Onken, J.; Vajkoczy, P.; Torka, R.; Hempt, C.; Patsouris, V.; Heppner, F.L.; Radke, J. Phospho-AXL is widely expressed in glioblastoma and associated with significant shorter overall survival. Oncotarget 2017, 8, 50403. [Google Scholar] [CrossRef]
- Liu, Y.; Xu, L.; Dou, Y.; He, Y. AXL: Shapers of tumor progression and immunosuppressive microenvironments. Mol. Cancer 2025, 24, 11. [Google Scholar] [CrossRef]
- Gjerdrum, C.; Tiron, C.; Høiby, T.; Stefansson, I.; Haugen, H.; Sandal, T.; Collett, K.; Li, S.; McCormack, E.; Gjertsen, B.T.; et al. Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival. Proc. Natl. Acad. Sci. USA 2010, 107, 1124–1129. [Google Scholar] [CrossRef]
- Liu, J.; Wang, K.; Yan, Z.; Xia, Y.; Li, J.; Shi, L.; Zou, Q.; Wan, X.; Jiao, B.; Wang, H.; et al. Axl expression stratifies patients with poor prognosis after hepatectomy for hepatocellular carcinoma. PLoS ONE 2016, 11, e0154767. [Google Scholar] [CrossRef]
- Cardone, C.; Blauensteiner, B.; Moreno-Viedma, V.; Martini, G.; Simeon, V.; Vitiello, P.P.; Ciardiello, D.; Belli, V.; Matrone, N.; Troiani, T.; et al. AXL is a predictor of poor survival and of resistance to anti-EGFR therapy in RAS wild-type metastatic colorectal cancer. Eur. J. Cancer 2020, 138, 1–10. [Google Scholar] [CrossRef]
- Hu, B.; Zhong, L.; Weng, Y.; Peng, L.; Huang, Y.; Zhao, Y.; Liang, X.J. Therapeutic siRNA: State of the art. Signal Transduct. Target. Ther. 2020, 5, 101. [Google Scholar] [CrossRef]
- Li, S.; Li, X.; Wang, N.; Zhang, C.; Sang, Y.; Sun, Y.; Xia, X.; Zheng, M. Brain targeted biomimetic siRNA nanoparticles for drug resistance glioblastoma treatment. J. Control. Release 2024, 376, 67–78. [Google Scholar] [CrossRef]
- Kim, Y.; You, J.H.; Ryu, Y.; Park, G.; Lee, U.; Moon, H.E.; Park, H.R.; Song, C.W.; Ku, J.L.; Park, S.H.; et al. ELAVL2 loss promotes aggressive mesenchymal transition in glioblastoma. npj Precis. Oncol. 2024, 8, 79. [Google Scholar] [CrossRef] [PubMed]
- Sehgal, I.; Eells, K.; Hudson, I. A Comparison of Currently Approved Small Interfering RNA (siRNA) Medications to Alternative Treatments by Costs, Indications, and Medicaid Coverage. Pharmacy 2024, 12, 58. [Google Scholar] [CrossRef] [PubMed]
- Lee, J.Y.; Kim, W.K.; Bae, K.H.; Lee, S.C.; Lee, E.W. Lipid Metabolism and Ferroptosis. Biology 2021, 10, 184. [Google Scholar] [CrossRef] [PubMed]
- Lee, S.Y. Temozolomide resistance in glioblastoma multiforme. Genes. Dis. 2016, 3, 198. [Google Scholar] [CrossRef]
- Han, H.; Du, A.; Li, J.; Han, H.; Feng, P.; Zhu, Y.; Li, X.; Tian, G.; Yu, H.; Zhang, B.; et al. Transitioning from molecular methods to therapeutic methods: An in-depth analysis of glioblastoma (Review). Oncol. Rep. 2025, 53, 48. [Google Scholar] [CrossRef]
- Krex, D.; Klink, B.; Hartmann, C.; Von Deimling, A.; Pietsch, T.; Simon, M.; Sabel, M.; Steinbach, J.P.; Heese, O.; Reifenberger, G.; et al. Long-term survival with glioblastoma multiforme. Brain 2007, 130, 2596–2606. [Google Scholar] [CrossRef]
- Fisher, J.P.; Adamson, D.C. Current FDA-Approved Therapies for High-Grade Malignant Gliomas. Biomedicines 2021, 9, 324. [Google Scholar] [CrossRef]
- Cen, L.; Gu, Q.; Zhou, X.; Lu, H.; Yang, Q. Eukaryotic Extension Factor 2 Kinase may Affect the Occurrence and Development of Glioblastoma Through Immune Cell Infiltration. Neurochem. Res. 2022, 47, 3670–3681. [Google Scholar] [CrossRef]
- Liu, X.Y.; Zhang, L.; Wu, J.P.; Zhou, L.; Ren, Y.J.; Yang, W.Q.; Ming, Z.J.; Chen, B.; Wang, J.; Zhang, Y.; et al. Inhibition of Elongation Factor-2 Kinase Augments the Antitumor Activity of Temozolomide against Glioma. PLoS ONE 2013, 8, e81345. [Google Scholar] [CrossRef]
- Yadav, M.; Sharma, A.; Patne, K.; Tabasum, S.; Suryavanshi, J.; Rawat, L.; Machaalani, M.; Eid, M.; Singh, R.P.; Choueiri, T.K.; et al. AXL signaling in cancer: From molecular insights to targeted therapies. Signal Transduct. Target. Ther. 2025, 10, 37. [Google Scholar] [CrossRef] [PubMed]
- Hutterer, M.; Knyazev, P.; Abate, A.; Reschke, M.; Maier, H.; Stefanova, N.; Knyazeva, T.; Barbieri, V.; Reindl, M.; Muigg, A.; et al. Axl and growth arrest-specific gene 6 are frequently overexpressed in human gliomas and predict poor prognosis in patients with glioblastoma multiforme. Clin. Cancer Res. 2008, 14, 130–138. [Google Scholar] [CrossRef] [PubMed]
- Vouri, M.; An, Q.; Birt, M.; Pilkington, G.J.; Hafizi, S. Small molecule inhibition of Axl receptor tyrosine kinase potently suppresses multiple malignant properties of glioma cells. Oncotarget 2015, 6, 16183. [Google Scholar] [CrossRef] [PubMed]
- Sadahiro, H.; Kang, K.D.; Gibson, J.T.; Minata, M.; Yu, H.; Shi, J.; Chhipa, R.; Chen, Z.; Lu, S.; Simoni, Y.; et al. Activation of the receptor tyrosine kinase AXL regulates the immune microenvironment in glioblastoma. Cancer Res. 2018, 78, 3002. [Google Scholar] [CrossRef]
- Behrmann, C.A.; Ennis, K.N.; Sarma, P.; Wetzel, C.; Clark, N.A.; Von Handorf, K.M.; Vallabhapurapu, S.; Andreani, C.; Reigle, J.; Scaglioni, P.P.; et al. Coordinated Targeting of S6K1/2 and AXL Disrupts Pyrimidine Biosynthesis in PTEN-deficient glioblastoma. Cancer Res. Commun. 2024, 4, 2215–2227. [Google Scholar] [CrossRef]
- Hong, X.; Chedid, K.; Kalkanis, S.N. Glioblastoma cell line-derived spheres in serum-containing medium versus serum-free medium: A comparison of cancer stem cell properties. Int. J. Oncol. 2012, 41, 1693–1700. [Google Scholar] [CrossRef]
- Wang, Z.; Zhang, S.; Siu, T.L.; Huang, S. Glioblastoma Multiforme Formation and EMT: Role of FoxM1 Transcription Factor. Curr. Pharm. Des. 2015, 21, 1268. [Google Scholar] [CrossRef]
- Hamurcu, Z.; Ashour, A.; Kahraman, N.; Ozpolat, B. FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation, invasion and tumorgenesis of human triple negative breast cancer cells. Oncotarget 2016, 7, 16619–16635. [Google Scholar] [CrossRef]
- Bowman, R.L.; Wang, Q.; Carro, A.; Verhaak, R.G.W.; Squatrito, M. GlioVis data portal for visualization and analysis of brain tumor expression datasets. Neuro Oncol. 2017, 19, 139–141. [Google Scholar] [CrossRef]
- Gravendeel, L.A.M.; Kouwenhoven, M.C.M.; Gevaert, O.; De Rooi, J.J.; Stubbs, A.P.; Duijm, J.E.; Daemen, A.; Bleeker, F.E.; Bralten, L.B.C.; Kloosterhof, N.K.; et al. Intrinsic gene expression profiles of gliomas are a better predictor of survival than histology. Cancer Res. 2009, 69, 9065–9072. [Google Scholar] [CrossRef]
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Biltekin, E.; Kahraman, N.; Gul, O.A.; Akay, Y.M.; Akay, M.; Ozpolat, B. Inhibition of FOXM1 Leads to Suppression of Cell Proliferation, Migration, and Invasion Through AXL/eEF2 Kinase Signaling and Induces Apoptosis and Ferroptosis in GBM Cells. Int. J. Mol. Sci. 2025, 26, 6792. https://doi.org/10.3390/ijms26146792
Biltekin E, Kahraman N, Gul OA, Akay YM, Akay M, Ozpolat B. Inhibition of FOXM1 Leads to Suppression of Cell Proliferation, Migration, and Invasion Through AXL/eEF2 Kinase Signaling and Induces Apoptosis and Ferroptosis in GBM Cells. International Journal of Molecular Sciences. 2025; 26(14):6792. https://doi.org/10.3390/ijms26146792
Chicago/Turabian StyleBiltekin, Ezgi, Nermin Kahraman, Ogun Ali Gul, Yasemin M. Akay, Metin Akay, and Bulent Ozpolat. 2025. "Inhibition of FOXM1 Leads to Suppression of Cell Proliferation, Migration, and Invasion Through AXL/eEF2 Kinase Signaling and Induces Apoptosis and Ferroptosis in GBM Cells" International Journal of Molecular Sciences 26, no. 14: 6792. https://doi.org/10.3390/ijms26146792
APA StyleBiltekin, E., Kahraman, N., Gul, O. A., Akay, Y. M., Akay, M., & Ozpolat, B. (2025). Inhibition of FOXM1 Leads to Suppression of Cell Proliferation, Migration, and Invasion Through AXL/eEF2 Kinase Signaling and Induces Apoptosis and Ferroptosis in GBM Cells. International Journal of Molecular Sciences, 26(14), 6792. https://doi.org/10.3390/ijms26146792