lde225 and Glioblastoma

Since PI3K/Akt/mTOR and sonic hedgehog (SHH) signaling pathways are highly activated in glioblastoma-initiating cells (GICs), we examined the effects of inhibiting these pathways on GIC characteristics and tumor growth in mice. NVP-LDE-225 (inhibitor of Smoothened) inhibited the expression of Gli1, Gli2, Smoothened, Patched1, and Patched2, and induced the expression of SuFu, whereas NVP-BEZ-235 (dual inhibitor of PI3K and mTOR) inhibited the expression of p-PI3K, p-Akt, p-mTOR, and p-p70S6K. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting the self-renewal capacity of GICs, expression of pluripotency maintaining factors (Nanog, c-Myc, Oct4, and Sox2), Musashi1, cyclin D1, and Bcl-2, and transcription and expression of Gli, and in inducing the expression of cleaved caspase-3, cleaved PARP and Bim. Additionally, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting epithelial-mesenchymal transition. Finally, the combination of NVP-LDE-225 and NVP-BEZ-235 was superior in inhibiting tumor growth, regulating the expression of pluripotency promoting factors, stem cell markers, cell cycle, and cell proliferation, and modulating EMT compared to single agent alone. In conclusion, the combined inhibition of PI3K/Akt/mTOR and SHH pathways was superior to single pathway inhibition in suppressing glioblastoma growth by targeting GICs.

Topics: Animals; Antineoplastic Agents; Biphenyl Compounds; Cell Proliferation; Glioblastoma; Hedgehog Proteins; Humans; Imidazoles; Mice; Mice, SCID; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridines; Quinolines; Signal Transduction; Smoothened Receptor; TOR Serine-Threonine Kinases

Glioblastoma multiforme is the most common form of primary brain tumor, often characterized by poor survival. Glioblastoma initiating cells (GICs) regulate self-renewal, differentiation, and tumor initiation properties and are involved in tumor growth, recurrence, and resistance to conventional treatments. The sonic hedgehog (SHH) signaling pathway is essential for normal development and embryonic morphogenesis. The objectives of this study were to examine the molecular mechanisms by which GIC characteristics are regulated by NPV-LDE-225 (Smoothened inhibitor; (2,2'-[[dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N-dimethylbenzenamine).. Cell viability and apoptosis were measured by XTT and annexin V-propidium iodide assay, respectively. Gli translocation and transcriptional activities were measured by immunofluorescence and luciferase assay, respectively. Gene and protein expressions were measured by quantitative real-time PCR and Western blot analyses, respectively.. NPV-LDE-225 inhibited cell viability, neurosphere formation, and Gli transcriptional activity and induced apoptosis by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. NPV-LDE-225 increased the expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-R1/DR4, TRAIL-R2/DR5, and Fas and decreased the expression of platelet derived growth factor receptor-α and Bcl2, and these effects were abrogated by Gli1 plus Gli2 short hairpin RNAs. NPV-LDE-225 enhanced the therapeutic potential of FasL and TRAIL by upregulating Fas and DR4/5, respectively. Interestingly, NPV-LDE-225 induced expression of programmed cell death 4 and apoptosis and inhibited cell viability by suppressing micro RNA (miR)-21. Furthermore, NPV-LDE-225 inhibited pluripotency-maintaining factors Nanog, Oct4, Sox2, and cMyc. The inhibition of Bmi1 by NPV-LDE-225 was regulated by induction of miR-128. Finally, NPV-LDE-225 suppressed epithelial-mesenchymal transition by upregulating E-cadherin and inhibiting N-cadherin, Snail, Slug, and Zeb1 through modulating the miR-200 family. Our data highlight the importance of the SHH pathway for self-renewal and early metastasis of GICs.

Topics: Apoptosis; Biphenyl Compounds; Blotting, Western; Brain Neoplasms; Cadherins; Caspase 3; Cell Adhesion; Cell Movement; Cell Proliferation; Electrophoretic Mobility Shift Assay; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Glioblastoma; Hedgehog Proteins; Humans; Immunoenzyme Techniques; MicroRNAs; Neoplastic Stem Cells; Polycomb Repressive Complex 1; Pyridines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Spheroids, Cellular; TNF-Related Apoptosis-Inducing Ligand; Transcription Factors; Tumor Cells, Cultured; Zinc Finger Protein GLI1

In glioblastoma, phosphatidylinositol 3-kinase (PI3K) signaling is frequently activated by loss of the tumor suppressor phosphatase and tensin homolog (PTEN). However, it is not known whether inhibiting PI3K represents a selective and effective approach for treatment. We interrogated large databases and found that sonic hedgehog (SHH) signaling is activated in PTEN-deficient glioblastoma. We demonstrate that the SHH and PI3K pathways synergize to promote tumor growth and viability in human PTEN-deficient glioblastomas. A combination of PI3K and SHH signaling inhibitors not only suppressed the activation of both pathways but also abrogated S6 kinase (S6K) signaling. Accordingly, targeting both pathways simultaneously resulted in mitotic catastrophe and tumor apoptosis and markedly reduced the growth of PTEN-deficient glioblastomas in vitro and in vivo. The drugs tested here appear to be safe in humans; therefore, this combination may provide a new targeted treatment for glioblastoma.

Topics: Aminopyridines; Animals; Biphenyl Compounds; Brain Neoplasms; Cell Line, Tumor; Enzyme Inhibitors; Glioblastoma; Hedgehog Proteins; Humans; Mice; Mice, Nude; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; PTEN Phosphohydrolase; Pyridines; Ribosomal Protein S6 Kinases; Signal Transduction; Xenograft Model Antitumor Assays