CELL BIOLOGY AND SIGNALING

Abstract

Ikaros, a DNA-binding protein also known as Ikaros family zinc finger protein 1 (IKZF1), was initially shown to function critically in hematopoietic differentiation. Deletion or mutation of Ikaros has been associated with lymphoblastic, as well as acute and chronic myelogenous leukemias demonstrating that Ikaros can act as a tumor suppressor. Ikaros is predominantly expressed in a variety of tissue including the brain, suggesting that Ikaros may be involved in the physiological functions of glial growth. Other recent studies have shown that Ikaros can regulate the transition of neural progenitor cells to postmitotic neurons which eventually differentiate into glial cells. Deregulation of glial development and/or growth has been shown to initiate glioblastoma (GBM) growth suggesting that Ikaros may be involved in GBM tumorigenesis. The goal of this study was to determine if Ikaros plays an important role in glial tumorigenesis. Analysis using the TCGA and Rembrandt databases demonstrated that decreased expression of Ikaros leads to worse prognosis in GBM patients (P < 0.01). In addition, the Ikaros promoter was found to be hypermethylated (85% of samples) in GBM samples suggesting that inhibition of Ikaros may contribute to glial tumorigenesis. To determine if Ikaros is involved in glial tumorigenesis we generated stable GBM cell lines that were transduced with HA-Ikaros. Enhanced expression of Ikaros resulted in a 3- to 4-fold reduction in proliferation and a 2- to 3-fold reduction in colony formation. In addition, exogenous expression of Ikaros inhibited the migration of GBM cells suggesting that Ikaros may also be involved in the invasiveness of glial cancers. Together our data suggests that Ikaros may play an important role in suppressing glial tumor formation and may be a novel biomarker for GBMs. Gliomas are the most frequently occurring primary malignancies in the central nervous system, and glioblastoma (GBM) is the most common and aggressive of these tumors. Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (alpha and/or alpha') and two beta regulatory subunits, which phosphorylates over 300 substrates. CK2 is a key suppressor of apoptosis, promotes angiogenesis, and enhances activation of the NF-kappaB, PI3K/AKT, Wnt and Notch signaling pathways. Elevated CK2 expression/activity has been reported in many tumors, including GBM, and it has been suggested that CK2 is essential for cancer cell survival. We have recently discovered that CK2 is a novel interaction partner of the Janus Kinases JAK1 and JAK2, and is required for activation of the JAK/STAT-3 pathway. Aberrant activation of signaling pathways including NF-kappaB, PI3K/AKT and JAK/STAT-3, has been implicated in tumor progression in GBM, and since CK2 is involved in their activation, we evaluated the expression and function of CK2 in the context of this cancer. Analysis of 537 GBMs from The Cancer Genome Atlas Project indicated the CSNK2A1 gene, encoding for CK2alpha, is frequently amplified in GBM (33.7%), which is significantly associated with the classical subtype of GBM. Inhibition of CK2 activity by pharmacological inhibitors (CX-4945) or knockdown of CK2 expression suppresses activation of the JAK/STAT, NF-kappaB and AKT pathways in primary human GBM xenograft cells. CK2 inhibitors decrease the adhesion and migration of GBM cells, in part through inhibition of integrin betal and integrin alpha4 expression. CK2 inhibitors also suppress growth, colony formation and cell cycle progression of GBM cells, and induce apoptosis of these cells. In vivo, CX-4945 significantly inhibits tumor growth and promotes survival in mice with intracranial human GBM xenografts by suppressing numerous signaling pathways. Therefore, CK2 inhibitors may be considered for treatment of patients with GBM. p53 dysfunction plays a major role in the pathobiology of malignant brain tumors, and numerous post-translational modifications can regulate p53 activity. Nitric oxide, a gaseous signaling molecule, contributes to both physiologic and pathologic states through S-nitrosylation of protein cysteine residues, thereby altering protein function. Signaling associated with nitric oxide has increasingly been implicated in the biology of malignant brain tumors, and so we hypothesized that p53 might be regulated by S-nitrosylation. p53 contains ten cysteine residues, several of which are essential for zinc coordination and DNA binding. Using the biotin switch method, we detect S-nitrosylation of p53 in cells exposed to nitric oxide donors, as well as under physiologic conditions in mouse brain. We demonstrate p53 S-nitrosylation in ONS76 medulloblastoma cells which express endogenous neuronal nitric oxide synthase (nNOS). Manipulation of nitric oxide levels in cell lines suggests nitric oxide-dependent regulation of p53-mediated transcriptional activity and cell death. S-nitrosylation is a novel post-translational modification of p53 that may regulate p53 response to cell stressors and contribute to the pathogenesis of medulloblastoma and other malignant brain tumors in which nitric oxide is generated. Glioblastoma is a diverse disease that can be divided into distinct subtypes on the basis of molecular and genetic profiling. The Proneural subtype has an expression profile, which resembles that of oligodendrocyte progenitor cells (OPCs), and is associated with a specific set of genetic alterations. However the functional relationships between the Proneural phenotype and the associated genetic alterations have not been resolved. To address this issue, we used a mouse model of Proneural glioma and longitudinally tracked alterations in gene copy number (by array CGH) and expression profiles (by RNA-Seq) at multiple time points during tumor progression. We showed that murine gliomas induced by retrovirus delivery of PDGF and Cre-mediated deletion of Pten, acquired a specific set of genetic deletions with remarkable consistency as they progressed from low to high grade tumors. Cross-species analyses revealed that subsets of these genes are also selectively deleted in human Proneural glioblastoma. Expression analysis at early time points showed that mouse tumors had a Proneural expression pattern, characterized by high levels of OPC genes, prior to the acquisition of the recurrent gene deletions. Further analysis identified transcription factors that act as master regulators of the Proneural phenotype, revealing a transcriptional network that was highly connected to p53 as one of the key master regulators at early and late stages of glioma progression. Experimental alteration of this regulatory network by upfront deletion of Trp53, led to accelerated glioma formation and obviated the acquisition of subsequent deletions. These results show the interplay between the pre-existing regulatory network, which defines cellular phenotype, and the genetic alterations that accumulate during progression of Proneural glioma.