Repurposing Potential of Riluzole as an ITAF Inhibitor in mTOR Therapy Resistant Glioblastoma
Abstract
:1. Introduction
2. Results
2.1. Molecular Docking Screening Identifies Riluzole as a Potential hnRNP A1 Inhibitor
2.2. Riluzole Blocks IRES Activity and hnRNP A1-IRES mRNA Binding in Glioblastoma
2.3. Riluzole Blocks Association of UP1 with IRES RNAs
2.4. Riluzole Directly Binds to hnRNP A1 within the Predicted IRES-J007 Binding Pocket
2.5. Riluzole and mTOR Inhibitors Display Synergistic Anti-GBM Properties In Vitro
2.6. In Vivo Effects of PP242 and Riluzole Cotherapy
3. Discussion
4. Materials and Methods
4.1. Cell Lines, Constructs and Transfections
4.2. In Silico Docking Screening
4.3. Recombinant Proteins, Antibodies and Reagents
4.4. IRES Reporter Assays, In Vitro RNA Pull-Down Assays, Filter Binding Assays and Polysome Analyses
4.5. Immunoblotting and Quantitative Real-Time PCR
4.6. Cell Proliferation, Cell-Cycle Distribution and TUNEL Assays
4.7. Xenograft Studies
4.8. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
IRES | Internal ribosome entry site |
ITAF | IRES trans-acting factor |
ALS | Amyotrophic lateral sclerosis |
hnRNP A1 | Heterogeneous nuclear ribonucleoprotein A1 |
mTOR | Mechanistic target of rapamycin |
mTORC | Mechanistic target of rapamycin complex |
CNS | Central nervous system |
PI3K | Phosphatidylinositol 3-kinase |
EGFR | Epidermal growth factor receptor |
PTEN | Phosphatase and tensin homolog |
AKT/PKB | Protein kinase B |
SPR | Surface plasmon resonance |
FDA | Food and drug administration, United States |
PDX | Patient derived xenograft |
RNP | Ribonucleoprotein |
RRM | RNA recognition motif |
GST | Glutathione S-transferase |
FITC | Fluorescein isothiocyanate |
SCID | Severe combined immunodeficient |
TUNEL | Terminal deoxynucleotidyl transferase dUTP nick end labeling |
qrt-PCR | Quantitative reverse transcription polymerase chain reaction |
References
- Gupta, S.C.; Sung, B.; Prasad, S.; Webb, L.J.; Aggarwal, B.B. Cancer drug discovery by repurposing: Teaching new tricks to old dogs. Trends Pharmacol. Sci. 2013, 34, 508–517. [Google Scholar] [CrossRef] [PubMed]
- Shim, J.S.; Liu, J.O. Recent advances in drug repositioning for the discovery of new anticancer drugs. Int. J. Biol. Sci. 2014, 10, 654–663. [Google Scholar] [CrossRef] [Green Version]
- Dunn, G.P.; Rinne, M.L.; Wykosky, J.; Genovese, G.; Quayle, S.N.; Dunn, I.F.; Agarwalla, P.K.; Chheda, M.G.; Campos, B.; Wang, A.; et al. Emerging insights into the molecular and cellular basis of glioblastoma. Genes Dev. 2012, 26, 756–784. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ostrom, Q.T.; Gittleman, H.; Xu, J.; Kromer, C.; Wolinsky, Y.; Kruchko, C.; Barnholtz-Sloan, J.S. CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2009-2013. Neuro Oncol. 2016, 18, v1–v75. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fan, Q.W.; Weiss, W.A. Targeting the RTK-PI3K-mTOR axis in malignant glioma: Overcoming resistance. Curr. Top. Microbiol. Immunol. 2010, 347, 279–296. [Google Scholar]
- Cloughesy, T.F.; Cavenee, W.K.; Mischel, P.S. Glioblastoma: From molecular pathology to targeted treatment. Annu. Rev. Pathol. 2014, 9, 1–25. [Google Scholar] [CrossRef] [Green Version]
- Prados, M.D.; Byron, S.A.; Tran, N.L.; Phillips, J.J.; Molinaro, A.M.; Ligon, K.L.; Wen, P.Y.; Kuhn, J.G.; Mellinghoff, I.K.; de Groot, J.F.; et al. Toward precision medicine in glioblastoma: The promise and the challenges. Neuro Oncol. 2015, 17, 1051–1063. [Google Scholar] [CrossRef] [Green Version]
- Miller, J.J.; Wen, P.Y. Emerging targeted therapies for glioma. Expert Opin. Emerg. Drugs 2016, 21, 441–452. [Google Scholar] [CrossRef]
- Gini, B.; Zanca, C.; Guo, D.; Matsutani, T.; Masui, K.; Ikegami, S.; Yang, H.; Nathanson, D.; Villa, G.R.; Shackelford, D.; et al. The mTOR kinase inhibitors, CC214-1 and CC214-2, preferentially block the growth of EGFRvIII-activated glioblastomas. Clinical Cancer Res. 2013, 19, 5722–5732. [Google Scholar] [CrossRef] [Green Version]
- Saxton, R.A.; Sabatini, D.M. mTOR Signaling in Growth, Metabolism, and Disease. Cell 2017, 168, 960–976. [Google Scholar] [CrossRef] [Green Version]
- Fan, Q.W.; Nicolaides, T.P.; Weiss, W.A. Inhibiting 4EBP1 in Glioblastoma. Clinical Cancer Res. 2018, 24, 14–21. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Masui, K.; Shibata, N.; Cavenee, W.K.; Mischel, P.S. mTORC2 activity in brain cancer: Extracellular nutrients are required to maintain oncogenic signaling. BioEssays 2016, 38, 839–844. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Benavides-Serrato, A.; Lee, J.; Holmes, B.; Landon, K.A.; Bashir, T.; Jung, M.E.; Lichtenstein, A.; Gera, J. Specific blockade of Rictor-mTOR association inhibits mTORC2 activity and is cytotoxic in glioblastoma. PLoS ONE 2017, 12, e0176599. [Google Scholar] [CrossRef] [PubMed]
- Mecca, C.; Giambanco, I.; Donato, R.; Arcuri, C. Targeting mTOR in Glioblastoma: Rationale and Preclinical/Clinical Evidence. Dis. Markers 2018, 2018, 9230479. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Iwanami, A.; Gini, B.; Zanca, C.; Matsutani, T.; Assuncao, A.; Nael, A.; Dang, J.; Yang, H.; Zhu, S.; Kohyama, J.; et al. PML mediates glioblastoma resistance to mammalian target of rapamycin (mTOR)-targeted therapies. Proc. Natl. Acad. Sci. USA 2013, 110, 4339–4344. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Benavides-Serrato, A.; Anderson, L.; Holmes, B.; Cloninger, C.; Artinian, N.; Bashir, T.; Gera, J. mTORC2 modulates feedback regulation of p38 MAPK activity via DUSP10/MKP5 to confer differential responses to PP242 in glioblastoma. Genes Cancer 2014, 5, 393–406. [Google Scholar] [PubMed] [Green Version]
- Tanaka, K.; Babic, I.; Nathanson, D.; Akhavan, D.; Guo, D.; Gini, B.; Dang, J.; Zhu, S.; Yang, H.; De Jesus, J.; et al. Oncogenic EGFR signaling activates an mTORC2-NF-kappaB pathway that promotes chemotherapy resistance. Cancer Discov. 2011, 1, 524–538. [Google Scholar] [CrossRef] [Green Version]
- Shi, Y.; Sharma, A.; Wu, H.; Lichtenstein, A.; Gera, J. Cyclin D1 and c-myc internal ribosome entry site (IRES)-dependent translation is regulated by AKT activity and enhanced by rapamycin through a p38 MAPK- and ERK-dependent pathway. J. Biol. Chem. 2005, 280, 10964–10973. [Google Scholar] [CrossRef] [Green Version]
- Martin, J.; Masri, J.; Cloninger, C.; Holmes, B.; Artinian, N.; Funk, A.; Ruegg, T.; Anderson, L.; Bashir, T.; Bernath, A.; et al. Phosphomimetic substitution of heterogeneous nuclear ribonucleoprotein A1 at serine 199 abolishes AKT-dependent internal ribosome entry site-transacting factor (ITAF) function via effects on strand annealing and results in mammalian target of rapamycin complex 1 (mTORC1) inhibitor sensitivity. J. Biol. Chem. 2011, 286, 16402–16413. [Google Scholar]
- Jo, O.D.; Martin, J.; Bernath, A.; Masri, J.; Lichtenstein, A.; Gera, J. Heterogeneous nuclear ribonucleoprotein A1 regulates cyclin D1 and c-myc internal ribosome entry site function through Akt signaling. J. Biol. Chem. 2008, 283, 23274–23287. [Google Scholar] [CrossRef] [Green Version]
- Holcik, M. Could the eIF2alpha-Independent Translation Be the Achilles Heel of Cancer? Front. Oncol. 2015, 5, 264. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Komar, A.A.; Hatzoglou, M. Exploring Internal Ribosome Entry Sites as Therapeutic Targets. Front. Oncol. 2015, 5, 233. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Holcik, M. Targeting translation for treatment of cancer--a novel role for IRES? Curr. Cancer Drug Targets 2004, 4, 299–311. [Google Scholar] [CrossRef] [PubMed]
- Holmes, B.; Lee, J.; Landon, K.A.; Benavides-Serrato, A.; Bashir, T.; Jung, M.E.; Lichtenstein, A.; Gera, J. Mechanistic Target of Rapamycin (mTOR) Inhibition Synergizes with Reduced Internal Ribosome Entry Site (IRES)-mediated Translation of Cyclin D1 and c-MYC mRNAs to Treat Glioblastoma. J. Biol. Chem. 2016, 291, 14146–14159. [Google Scholar] [CrossRef] [Green Version]
- Trott, O.; Olson, A.J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31, 455–461. [Google Scholar] [CrossRef] [Green Version]
- Cloninger, C.; Bernath, A.; Bashir, T.; Holmes, B.; Artinian, N.; Ruegg, T.; Anderson, L.; Masri, J.; Lichtenstein, A.; Gera, J. Inhibition of SAPK2/p38 enhances sensitivity to mTORC1 inhibition by blocking IRES-mediated translation initiation in glioblastoma. Mol. Cancer Ther. 2011, 10, 2244–2256. [Google Scholar] [CrossRef] [Green Version]
- Thoreen, C.C.; Chantranupong, L.; Keys, H.R.; Wang, T.; Gray, N.S.; Sabatini, D.M. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 2012, 485, 109–113. [Google Scholar] [CrossRef]
- Shi, Y.; Frost, P.; Hoang, B.; Yang, Y.; Fukunaga, R.; Gera, J.; Lichtenstein, A. MNK kinases facilitate c-myc IRES activity in rapamycin-treated multiple myeloma cells. Oncogene 2013, 32, 190–197. [Google Scholar] [CrossRef] [Green Version]
- Zarate, C.A.; Manji, H.K. Riluzole in psychiatry: A systematic review of the literature. Expert Opin. Drug Metab. Toxicol. 2008, 4, 1223–1234. [Google Scholar] [CrossRef]
- Samano, C.; Nistri, A. Mechanism of Neuroprotection Against Experimental Spinal Cord Injury by Riluzole or Methylprednisolone. Neurochem. Res. 2019, 44, 200–213. [Google Scholar] [CrossRef]
- Cifra, A.; Mazzone, G.L.; Nistri, A. Riluzole: What it does to spinal and brainstem neurons and how it does it. Neuroscientist 2013, 19, 137–144. [Google Scholar] [CrossRef] [PubMed]
- Rosas, H.D.; Koroshetz, W.J.; Jenkins, B.G.; Chen, Y.I.; Hayden, D.L.; Beal, M.F.; Cudkowicz, M.E. Riluzole therapy in Huntington’s disease (HD). Mov. Disord. 1999, 14, 326–330. [Google Scholar] [CrossRef]
- Kim, H.J.; Kim, N.C.; Wang, Y.D.; Scarborough, E.A.; Moore, J.; Diaz, Z.; MacLea, K.S.; Freibaum, B.; Li, S.; Molliex, A.; et al. Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature 2013, 495, 467–473. [Google Scholar] [CrossRef] [PubMed]
- Khan, A.J.; LaCava, S.; Mehta, M.; Schiff, D.; Thandoni, A.; Jhawar, S.; Danish, S.; Haffty, B.G.; Chen, S. The glutamate release inhibitor riluzole increases DNA damage and enhances cytotoxicity in human glioma cells, in vitro and in vivo. Oncotarget 2019, 10, 2824–2834. [Google Scholar] [CrossRef]
- Shah, R.; Singh, S.J.; Eddy, K.; Filipp, F.V.; Chen, S. Concurrent Targeting of Glutaminolysis and Metabotropic Glutamate Receptor 1 (GRM1) Reduces Glutamate Bioavailability in GRM1(+) Melanoma. Cancer Res. 2019, 79, 1799–1809. [Google Scholar] [CrossRef] [Green Version]
- Wadosky, K.M.; Shourideh, M.; Goodrich, D.W.; Koochekpour, S. Riluzole induces AR degradation via endoplasmic reticulum stress pathway in androgen-dependent and castration-resistant prostate cancer cells. Prostate 2019, 79, 140–150. [Google Scholar] [CrossRef]
- Sachkova, A.; Sperling, S.; Mielke, D.; Schatlo, B.; Rohde, V.; Ninkovic, M. Combined Applications of Repurposed Drugs and Their Detrimental Effects on Glioblastoma Cells. Anticancer. Res. 2019, 39, 207–214. [Google Scholar] [CrossRef]
- Lemieszek, M.K.; Stepulak, A.; Sawa-Wejksza, K.; Czerwonka, A.; Ikonomidou, C.; Rzeski, W. Riluzole Inhibits Proliferation, Migration and Cell Cycle Progression and Induces Apoptosis in Tumor Cells of Various Origins. Anticancer Agents Med. Chem. 2018, 18, 565–572. [Google Scholar] [CrossRef]
- Doble, A. The pharmacology and mechanism of action of riluzole. Neurology 1996, 47, S233–S241. [Google Scholar] [CrossRef]
- McGeer, E.G.; McGeer, P.L. Pharmacologic approaches to the treatment of amyotrophic lateral sclerosis. Biol. Drugs 2005, 19, 31–37. [Google Scholar] [CrossRef]
- Miller, R. Riluzole for ALS: What is the evidence? Amyotroph. lateral Scler. Mot. Neuron Disord. 2003, 4, 135. [Google Scholar] [CrossRef]
- Speyer, C.L.; Nassar, M.A.; Hachem, A.H.; Bukhsh, M.A.; Jafry, W.S.; Khansa, R.M.; Gorski, D.H. Riluzole mediates anti-tumor properties in breast cancer cells independent of metabotropic glutamate receptor-1. Breast Cancer Res. Treat. 2016, 157, 217–228. [Google Scholar] [CrossRef] [PubMed]
- Dolfi, S.C.; Medina, D.J.; Kareddula, A.; Paratala, B.; Rose, A.; Dhami, J.; Chen, S.; Ganesan, S.; Mackay, G.; Vazquez, A.; et al. Riluzole exerts distinct antitumor effects from a metabotropic glutamate receptor 1-specific inhibitor on breast cancer cells. Oncotarget 2017, 8, 44639–44653. [Google Scholar] [CrossRef] [PubMed]
- Milane, A.; Tortolano, L.; Fernandez, C.; Bensimon, G.; Meininger, V.; Farinotti, R. Brain and plasma riluzole pharmacokinetics: Effect of minocycline combination. J. Pharm. Pharm. Sci. 2009, 12, 209–217. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Milane, A.; Fernandez, C.; Vautier, S.; Bensimon, G.; Meininger, V.; Farinotti, R. Minocycline and riluzole brain disposition: Interactions with p-glycoprotein at the blood-brain barrier. J. Neurochem. 2007, 103, 164–173. [Google Scholar] [CrossRef] [PubMed]
- Gao, M.; Skolnick, J. A comprehensive survey of small-molecule binding pockets in proteins. PLoS Comput. Biol. 2013, 9, e1003302. [Google Scholar] [CrossRef] [Green Version]
- Ko, C.C.; Chen, Y.J.; Chen, C.T.; Liu, Y.C.; Cheng, F.C.; Hsu, K.C.; Chow, L.P. Chemical proteomics identifies heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as the molecular target of quercetin in its anti-cancer effects in PC-3 cells. J. Biol. Chem. 2014, 289, 22078–22089. [Google Scholar] [CrossRef] [Green Version]
- Fridell, R.A.; Truant, R.; Thorne, L.; Benson, R.E.; Cullen, B.R. Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta. J. Cell Sci. 1997, 110, 1325–1331. [Google Scholar]
- Rebane, A.; Aab, A.; Steitz, J.A. Transportins 1 and 2 are redundant nuclear import factors for hnRNP A1 and HuR. RNA 2004, 10, 590–599. [Google Scholar] [CrossRef] [Green Version]
- Didiot, M.C.; Hewett, J.; Varin, T.; Freuler, F.; Selinger, D.; Nick, H.; Reinhardt, J.; Buckler, A.; Myer, V.; Schuffenhauer, A.; et al. Identification of cardiac glycoside molecules as inhibitors of c-Myc IRES-mediated translation. J. Biomol. Screen. 2013, 18, 407–419. [Google Scholar] [CrossRef] [Green Version]
- Vaklavas, C.; Meng, Z.; Choi, H.; Grizzle, W.E.; Zinn, K.R.; Blume, S.W. Small molecule inhibitors of IRES-mediated translation. Cancer Biol. Ther. 2015, 16, 1471–1485. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Malina, A.; Khan, S.; Carlson, C.B.; Svitkin, Y.; Harvey, I.; Sonenberg, N.; Beal, P.A.; Pelletier, J. Inhibitory properties of nucleic acid-binding ligands on protein synthesis. FEBS Lett. 2005, 579, 79–89. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Speyer, C.L.; Bukhsh, M.A.; Jafry, W.S.; Sexton, R.E.; Bandyopadhyay, S.; Gorski, D.H. Riluzole synergizes with paclitaxel to inhibit cell growth and induce apoptosis in triple-negative breast cancer. Breast Cancer Res. Treat. 2017, 166, 407–419. [Google Scholar] [CrossRef] [PubMed]
- Gao, W.Q.; Ma, J.; Sun, L.L.; Li, Q.; Zhu, R.Y.; Jin, J. Paclitaxel-mediated human aryl hydrocarbon receptor mRNA translation by an internal ribosomal entry site-dependent mechanism. Oncol. Rep. 2017, 38, 3211–3219. [Google Scholar] [CrossRef] [PubMed]
- Li, Q.; Gao, W.Q.; Dai, W.Y.; Yu, C.; Zhu, R.Y.; Jin, J. ATF2 translation is induced under chemotherapeutic drug-mediated cellular stress via an IRES-dependent mechanism in human hepatic cancer Bel7402 cells. Oncol. Lett. 2016, 12, 4795–4802. [Google Scholar] [CrossRef] [Green Version]
- Gao, W.; Li, Q.; Zhu, R.; Jin, J. La Autoantigen Induces Ribosome Binding Protein 1 (RRBP1) Expression through Internal Ribosome Entry Site (IRES)-Mediated Translation during Cellular Stress Condition. Int. J. Mol. Sci. 2016, 17, 1174. [Google Scholar] [CrossRef] [Green Version]
- Fu, Q.; Chen, Z.; Gong, X.; Cai, Y.; Chen, Y.; Ma, X.; Zhu, R.; Jin, J. beta-Catenin expression is regulated by an IRES-dependent mechanism and stimulated by paclitaxel in human ovarian cancer cells. Biochem. Biophys. Res. Commun. 2015, 461, 21–27. [Google Scholar] [CrossRef]
- Pineiro, D.; Gonzalez, V.M.; Salinas, M.; Elena Martin, M. Analysis of the protein expression changes during taxol-induced apoptosis under translation inhibition conditions. Mol. Cell. Biochem. 2010, 345, 131–144. [Google Scholar] [CrossRef]
- Law, V.; Knox, C.; Djoumbou, Y.; Jewison, T.; Guo, A.C.; Liu, Y.; Maciejewski, A.; Arndt, D.; Wilson, M.; Neveu, V.; et al. DrugBank 4.0: Shedding new light on drug metabolism. Nucleic Acids Res. 2014, 42, D1091–D1097. [Google Scholar] [CrossRef] [Green Version]
- Bonnal, S.; Pileur, F.; Orsini, C.; Parker, F.; Pujol, F.; Prats, A.C.; Vagner, S. Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA. J. Biol. Chem. 2005, 280, 4144–4153. [Google Scholar] [CrossRef] [Green Version]
- Holmes, B.; Benavides-Serrato, A.; Saunders, J.T.; Landon, K.A.; Schreck, A.J.; Nishimura, R.N.; Gera, J. The protein arginine methyltransferase PRMT5 confers therapeutic resistance to mTOR inhibition in glioblastoma. J. Neurooncol. 2019, 145, 11–22. [Google Scholar] [CrossRef] [PubMed]
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Benavides-Serrato, A.; Saunders, J.T.; Holmes, B.; Nishimura, R.N.; Lichtenstein, A.; Gera, J. Repurposing Potential of Riluzole as an ITAF Inhibitor in mTOR Therapy Resistant Glioblastoma. Int. J. Mol. Sci. 2020, 21, 344. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21010344
Benavides-Serrato A, Saunders JT, Holmes B, Nishimura RN, Lichtenstein A, Gera J. Repurposing Potential of Riluzole as an ITAF Inhibitor in mTOR Therapy Resistant Glioblastoma. International Journal of Molecular Sciences. 2020; 21(1):344. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21010344
Chicago/Turabian StyleBenavides-Serrato, Angelica, Jacquelyn T. Saunders, Brent Holmes, Robert N. Nishimura, Alan Lichtenstein, and Joseph Gera. 2020. "Repurposing Potential of Riluzole as an ITAF Inhibitor in mTOR Therapy Resistant Glioblastoma" International Journal of Molecular Sciences 21, no. 1: 344. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21010344