Ibrutinib, a Bruton’s tyrosine kinase inhibitor, exhibits antitumoral activity and induces autophagy in glioblastoma

BackgroundGlioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, is a novel anticancer drug used for treating several types of cancers. In this study, we aimed to determine the role of ibrutinib on GBM.MethodsCell proliferation was determined by using cell viability, colony formation, and 5-ethynyl-2′-deoxyuridine (EdU) assays. Cell cycle and cell apoptosis were analyzed by flow cytometry. Cell migratory ability was evaluated by wound healing assays and trans-well migration assays. ATG7 expression was knocked-down by transfection with Atg7-specific small interfering RNA. Overexpression of active Akt protein was achieved by transfecting the cells with a plasmid expressing constitutively active Akt (CA-Akt). Transmission electron microscopy was performed to examine the formation of autophagosomes in cells. Immunofluorescence and western blot analyses were used to analyze protein expression. Tumor xenografts in nude mice and immunohistochemistry were performed to evaluate the effect of ibrutinib on tumor growth in vivo.ResultsIbrutinib inhibited cellular proliferation and migration, and induced apoptosis and autophagy in LN229 and U87 cells. Overexpression of the active Akt protein decreased ibrutinib-induced autophagy, while inhibiting Akt by LY294002 treatment enhanced ibrutinib-induced autophagy. Specific inhibition of autophagy by 3-methyladenine (3MA) or Atg7 targeting with small interfering RNA (si-Atg7) enhanced the anti-GBM effect of ibrutinib in vitro and in vivo.ConclusionsOur results indicate that ibrutinib exerts a profound antitumor effect and induces autophagy through Akt/mTOR signaling pathway in GBM cells. Autophagy inhibition promotes the antitumor activity of ibrutinib in GBM. Our findings provide important insights into the action of an anticancer agent combining with autophagy inhibitor for malignant glioma.

[1]  Stephen Yip,et al.  Molecular pathology in adult gliomas: diagnostic, prognostic, and predictive markers , 2010, The Lancet Neurology.

[2]  M. Hsiao,et al.  Preclinical investigation of ibrutinib, a Bruton's kinase tyrosine (Btk) inhibitor, in suppressing glioma tumorigenesis and stem cell phenotypes , 2016, Oncotarget.

[3]  N. Li,et al.  IL10 inhibits starvation-induced autophagy in hypertrophic scar fibroblasts via cross talk between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways , 2016, Cell Death and Disease.

[4]  C. James,et al.  Akt and Autophagy Cooperate to Promote Survival of Drug-Resistant Glioma , 2010, Science Signaling.

[5]  A. Kimchi,et al.  Life and death partners: apoptosis, autophagy and the cross-talk between them , 2009, Cell Death and Differentiation.

[6]  G. Mills,et al.  Monitoring autophagy in glioblastoma with antibody against isoform B of human microtubule-associated protein 1 light chain 3 , 2008, Autophagy.

[7]  F. Braet,et al.  KCa3.1 mediates dysfunction of tubular autophagy in diabetic kidneys via PI3k/Akt/mTOR signaling pathways , 2016, Scientific Reports.

[8]  F. Tao,et al.  Targeting Btk with ibrutinib inhibit gastric carcinoma cells growth. , 2016, American journal of translational research.

[9]  A. Thorburn,et al.  Recent insights into cell death and autophagy , 2015, The FEBS journal.

[10]  J. Leonard,et al.  BTK inhibition targets in vivo CLL proliferation through its effects on B-cell receptor signaling activity , 2014, Leukemia.

[11]  K. Heider,et al.  Inhibition of Epidermal Growth Factor Receptor Activity by Two Pyrimidopyrimidine Derivatives , 2004, Journal of Pharmacology and Experimental Therapeutics.

[12]  D. Moras,et al.  Cysteine mapping in conformationally distinct kinase nucleotide binding sites: application to the design of selective covalent inhibitors. , 2011, Journal of medicinal chemistry.

[13]  D. Klionsky,et al.  The machinery of macroautophagy , 2013, Cell Research.

[14]  E. Sen,et al.  Ebselen sensitizes glioblastoma cells to Tumor Necrosis Factor (TNFα)‐induced apoptosis through two distinct pathways involving NF‐κB downregulation and Fas‐mediated formation of death inducing signaling complex , 2008, International journal of cancer.

[15]  S. Kondo,et al.  Inhibition of autophagy at a late stage enhances imatinib‐induced cytotoxicity in human malignant glioma cells , 2009, International journal of cancer.

[16]  M. Lotze,et al.  Autophagy inhibition in combination cancer treatment. , 2009, Current opinion in investigational drugs.

[17]  Michael L. Wang,et al.  Selective antitumor activity of ibrutinib in EGFR-mutant non-small cell lung cancer cells. , 2014, Journal of the National Cancer Institute.

[18]  L. Ricci-Vitiani,et al.  Chemotherapy resistance of glioblastoma stem cells , 2006, Cell Death and Differentiation.

[19]  W. Weiss,et al.  PI3K Signaling in Glioma—Animal Models and Therapeutic Challenges , 2009, Brain pathology.

[20]  Atique U. Ahmed,et al.  The role of glioma stem cells in chemotherapy resistance and glioblastoma multiforme recurrence , 2015, Expert review of neurotherapeutics.

[21]  Nicole Grabinski,et al.  Ibrutinib (ImbruvicaTM) potently inhibits ErbB receptor phosphorylation and cell viability of ErbB2-positive breast cancer cells , 2014, Investigational New Drugs.

[22]  Seamus J. Martin,et al.  Autophagy in malignant transformation and cancer progression , 2015, The EMBO journal.

[23]  T. Ueno,et al.  LC3 and Autophagy. , 2008, Methods in molecular biology.

[24]  S. Pittaluga,et al.  Ibrutinib inhibits BCR and NF-κB signaling and reduces tumor proliferation in tissue-resident cells of patients with CLL. , 2014, Blood.

[25]  E. White,et al.  The Double-Edged Sword of Autophagy Modulation in Cancer , 2009, Clinical Cancer Research.

[26]  K. Guan,et al.  mTOR: a pharmacologic target for autophagy regulation. , 2015, The Journal of clinical investigation.

[27]  G. Cooper,et al.  Role of Glycogen Synthase Kinase-3 in the Phosphatidylinositol 3-Kinase/Akt Cell Survival Pathway* , 1998, The Journal of Biological Chemistry.

[28]  C. Yuan,et al.  Bruton's tyrosine kinase (Btk) inhibitor ibrutinib suppresses stem-like traits in ovarian cancer , 2015, Oncotarget.

[29]  Lunquan Sun,et al.  Targeting autophagy to sensitive glioma to temozolomide treatment , 2016, Journal of Experimental & Clinical Cancer Research.

[30]  Sharad Kumar,et al.  Cell death by autophagy: facts and apparent artefacts , 2011, Cell Death and Differentiation.

[31]  D. Klionsky,et al.  Autophagosome formation: core machinery and adaptations , 2007, Nature Cell Biology.

[32]  Ajjai Alva,et al.  Regulation of an ATG7-beclin 1 Program of Autophagic Cell Death by Caspase-8 , 2004, Science.

[33]  B. Manning,et al.  Signal integration by mTORC1 coordinates nutrient input with biosynthetic output , 2013, Nature Cell Biology.

[34]  Yamei Chen,et al.  Ibrutinib for B cell malignancies , 2014, Experimental Hematology & Oncology.

[35]  G. Reifenberger,et al.  Molecular Neuropathology of Gliomas , 2009, International journal of molecular sciences.

[36]  Michael S. Cohen,et al.  Structural Bioinformatics-Based Design of Selective, Irreversible Kinase Inhibitors , 2005, Science.

[37]  David P. Davis,et al.  Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents , 2008, The Journal of cell biology.

[38]  E. Sen,et al.  Kaempferol induces apoptosis in glioblastoma cells through oxidative stress , 2007, Molecular Cancer Therapeutics.

[39]  K. Dalby,et al.  Targeting the pro-death and pro-survival functions of autophagy as novel therapeutic strategies in cancer , 2010, Autophagy.

[40]  J. Menéndez,et al.  Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab , 2009, PloS one.

[41]  J. Merlio,et al.  Autophagy inhibition cooperates with erlotinib to induce glioblastoma cell death , 2011, Cancer biology & therapy.

[42]  Zsuzsanna Szatmári,et al.  Autophagy—from molecular mechanisms to clinical relevance , 2016, Cell Biology and Toxicology.

[43]  Craig B. Thompson,et al.  Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes , 2004, Nature Cell Biology.

[44]  N. Saba,et al.  Ibrutinib downregulates a subset of miRNA leading to upregulation of tumor suppressors and inhibition of cell proliferation in chronic lymphocytic leukemia , 2017, Leukemia.