Therapeutics, Targets, and Chemical Biology Small-Molecule Inhibitors of Phosphatidylinositol 3-Kinase/ Akt Signaling Inhibit Wnt/β-Catenin Pathway Cross-Talk and Suppress Medulloblastoma Growth Ninib Baryawno 1 , Baldur Sveinbjörnsson 1,2 , Staffan Eksborg 1 , Ching-Shih Chen 3 , Per Kogner 1 , and John Inge Johnsen 1 Abstract Activation of the β-catenin and receptor kinase pathways occurs often in medulloblastoma, the most common pediatric malignant brain tumor. In this study, we show that molecular cross-talk between the β-catenin and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways is crucial to sustain medulloblas- toma pathophysiology. Constitutive activation of phosphoinositide-dependent protein kinase 1 (PDK1), Akt, and glycogen synthase kinase 3β (GSK-3β) was detected by immunohistochemistry in all primary medul- loblastomas examined (n = 41). Small-molecule inhibitors targeting the PI3K/Akt signaling pathway affected β-catenin signaling by inhibition of GSK-3β activity, resulting in cytoplasmic retention of β-catenin and reduced expression of its target genes cyclin D1 and c-Myc. The PDK1 inhibitor OSU03012 induced mitochondrial- dependent apoptosis of medulloblastoma cells and enhanced the cytotoxic effects of chemotherapeutic drugs in a synergistic or additive manner. In vivo, OSU03012 inhibited the growth of established medulloblastoma xenograft tumors in a dose-dependent manner and augmented the antitumor effects of mammalian target of rapamycin inhibitor CCI-779. These findings demonstrate the importance of cross-talk between the PI3K/ Akt and β-catenin pathways in medulloblastoma and rationalize the PI3K/Akt signaling pathway as a thera- peutic target in treatment of this disease. Cancer Res; 70(1); 266–76. ©2010 AACR. Introduction Medulloblastoma, a primitive neuroectodermal tumor, is the most common malignant pediatric brain tumor. Medul- loblastoma can originate from cerebellar granule neural pre- cursor cells located in the external granule layer (1, 2) and typically develop in the cerebellum. Medulloblastoma cells retain many features that resemble the progenitor cells of the embryonic brain (2), and more than half of these tu- mors display abnormal activation of the Hedgehog or Wnt signaling pathways (3), supporting a model of embryonal tumorigenesis. Wnt signaling plays a central role in modulating the bal- ance between proliferation and differentiation of progenitor cells during embryonic central nervous system development (4). The canonical Wnt signaling pathway operates by stabi- lizing β-catenin. In the absence of Wnt/Wingless ligand activation, β-catenin is sequestered in the cytoplasm by a multiprotein complex, which encompasses the adenomatous polyposis coli protein, axin1, axin2/conductin, casein kinase 1, and glycogen synthase kinase-3β (GSK-3β). In this state, β-catenin is phosphorylated at the NH 2 -terminal serine and threonine residues by GSK-3β, which targets it for ubiquiti- nation and proteolytic degradation (5). Activation of Wnt sig- naling by binding of Wnt ligands to a Frizzled receptor inhibits the formation of the multiprotein complex and GSK-3β–mediated phosphorylation of β-catenin, resulting in an accumulation of hypophosphorylated β-catenin in the cytosol. Stabilized hypophosphorylated β-catenin eventually translocates to the nucleus where it interacts with members of the T-cell factor/lymphoid enhancer binding factor (TCF/ LEF) family of transcription factors, leading to modulated expression of a broad range of genes, such as MYC and CCND1 (6, 7). Proteins regulating signaling through the phosphatidyli- nositol 3-kinase (PI3K)/Akt pathway are frequently altered in human cancer, including medulloblastoma (8, 9). Acti- vated Akt significantly augments Sonic-Hedgehog–induced medulloblastoma formation in mice (10). Activation of the PI3K/Akt signaling pathway is important for prolifera- tion of human medulloblastoma cells and cancer stem cells residing in the perivascular niche following irradiation (11, 12). Moreover, medulloblastomas frequently display re- duced expression of the Akt inhibitor PTEN (the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10) caused by promoter hypermethylation Cancer Research Authors' Affiliations: 1 Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden; 2 Division of Immunology, IMB, University of Tromsö, Tromsö, Norway; and 3 Division of Medicinal Chemistry and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Ninib Baryawno, Childhood Cancer Research Unit, Q6:05, Astrid Lindgrens Children's Hospital, S-17176 Stockholm, Sweden. Phone: 46-851-777515; Fax: 46-851-773475; E-mail: ninib. baryawno@ki.se. doi: 10.1158/0008-5472.CAN-09-0578 ©2010 American Association for Cancer Research. Cancer Res; 70(1) January 1, 2010 266 Research. on September 14, 2015. © 2010 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst December 22, 2009; DOI: 10.1158/0008-5472.CAN-09-0578 Research. on September 14, 2015. © 2010 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst December 22, 2009; DOI: 10.1158/0008-5472.CAN-09-0578 Research. on September 14, 2015. © 2010 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst December 22, 2009; DOI: 10.1158/0008-5472.CAN-09-0578 Research. on September 14, 2015. © 2010 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst December 22, 2009; DOI: 10.1158/0008-5472.CAN-09-0578