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Hypermethylation and Transcriptional Downregulation of the Carboxyl-Terminal Modulator Protein Gene in Glioblastomas

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Abstract

The carboxyl-terminal modulator protein (CTMP) has been identified as a negative regulator of protein kinase B/Akt. Aberrant Akt signaling is frequently observed in glioblastomas, the most common and most malignant glial brain tumors. Because loss of CTMP function and/or expression may remove the inhibitory effects on Akt and promote tumorigenesis, we studied 93 primary glioblastomas and nine glioblastoma cell lines for CTMP deletion, mutation, promoter hypermethylation, and mRNA expression. None of the tumors or cell lines had CTMP-homozygous deletions or coding sequence mutations. However, CTMP mRNA expression was lower by at least 50% relative to non-neoplastic brain tissue in 37 (40%) glioblastomas and six (67%) glioma cell lines. Reduced CTMP mRNA levels were closely associated with hypermethylation of the CTMP promoter. Furthermore, treatment of CTMP-hypermethylated A172 glioma cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A resulted in partial demethylation of the CTMP promoter and increased CTMP mRNA expression. Thus, epigenetic downregulation of CTMP transcription is a common aberration in glioblastomas.

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... Hotdog-fold thioesterases are known to utilize an active site carboxylate residue (Glu or Asp) in catalysis. Mechanistic studies directed at the hotdog-fold thioesterases from evolutionary distant organisms [10][11][12][13][14] have revealed that the catalytic carboxylate functions as a nucleophile in a two-step pathway involving the intermediacy of a mixed anhydride intermediate (Scheme 1A) or alternatively in single-step process, as a general base, responsible for the activation of the water nucleophile (Scheme 1B). However, for any given thioesterase, the task of distinguishing between these two pathways can be quite difficult owing to the limitations of the mechanistic probes currently available. ...
... Because hotdog-fold thioesterases are known to play essential roles in human health and disease [14,15] , and because effective inhibitors of these enzymes have yet to be discovered, there exists an urgent need for the development of a class of inert substrate analogs that can be used to unambiguously define the structure and mechanism of any hotdog-fold thioesterase targeted for inhibitor design. Herein, we report the synthesis of such an analog and demonstrate its successful application in defining the active site structure of a hotdogfold thioesterase, PA1618, poised for catalytic turnover. ...
Article
Thioesterase activity (hydrolysis of thioester bonds) accounts for the majority of the activities in the hotdog-fold superfamily. The structure and mechanism of catalysis for many hotdog enzymes have been elucidated by X-ray crystallography and kinetics to probe the specific substrate usage and cellular functions. However, structures of hotdog thioesterases in complex with substrate analogs reported to date utilize ligands that comprise either truncations of the substrate or include additional atoms to prevent the hydrolysis. Herein, we present the synthesis of an isosteric and isoelectronic substrate analog, benzoyl-OdCoA, and the X-ray crystal structure of a complex of the analog with P. aeruginosa hotdog thioesterase, PA1618 (at 1.72 Å resolution). The complex is compared to that of the "imperfect" substrate analog phenacyl-CoA, refined to a resolution of 1.62 Å. Kinetic and structural results are consistent with Glu64 as the catalytic residue and with Gln49 in stabilization of the transition state. Structural comparison of the two ligand-bound structures revealed a crucial ordered water molecule coordinated in the active site of the benzoyl-OdCoA structure which is not present in the phenacyl-CoA bound structure. This suggests a general base mechanism of catalysis where Glu64 activates the coordinated water nucleophile. Together, our findings reveal the importance of a more indistinguishable substrate analog to determine proper substrate binding and catalytic mechanism.
... CTMP expression phenotypically and functionally modulates the cell morphology, growth rate, and tumorigenesis of v-Akt-transformed cells, suggesting a tumor-suppressive function of CTMP. In agreement with the inhibitory role of CTMP on Akt activation, the reduction of CTMP mRNA was observed in glioma cell lines through an epigenetic regulation (21). Furthermore, the RNA-interference depletion of CTMP prevents ischemia-induced neuronal cell death (22). ...
... To clarify the CTMP function on Akt phosphorylation and tumorigenesis, it is necessary to study their correlation in specific types of human cancers. In 2004, Knobbe and colleagues reported that reduction of CTMP mRNA levels could be found in 40% (37/93) of primary glioblastomas and in 67% (6/9) of glioma cell lines, and Akt phosphorylation at S473 could be detected in all glioma cell lines, suggesting that CTMP may function as tumor suppressor by inhibiting Akt phosphorylation in human glioblastomas (21). In our study, we found that CTMP was upregulated in 136 out of 204 (66.67%) breast cancer specimens and was significantly correlated with poor patient survival. ...
Article
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Akt activation has been implicated broadly in tumorigenesis, but the basis for its dysregulation in cancer cells is incompletely understood. In this study, we sought to clarify a regulatory role for the Akt-binding carboxy-terminal modulator protein (CTMP), which has been controversial. In evaluating CTMP expression in paired normal– tumor specimens of 198 patients with breast cancer, we found that CTMP was upregulated in breast tumors, where it was associated with poor patient survival. Notably, CTMP expression also correlated positively with Akt phosphorylation in breast cancer clinical specimens and cell lines. Furthermore, ectopic expression of CTMP promoted cell proliferation and enhanced the tumorigenic properties of estrogen-dependent breast cancer cells. This effect was correlated with increased sensitivity to insulin-induced Akt phosphorylation, which is mediated primarily by the phosphoinositide 3-kinase–Akt pathway. In contrast, short hairpin RNA-mediated silencing of endogenous CTMP decreased the proliferation of estrogen-dependent or estrogen-independent breast cancer cells. Mechanistic investigations defined the N-terminal domain of CTMP at amino acids 1 to 64 as responsible for Akt binding. Taken together, our results firmly corroborate the concept that CTMP promotes Akt phosphorylation and functions as an oncogenic molecule in breast cancer.
... CTMP overexpression inactivates PKB in v-Akt-transformed cells transplanted into mice [10], in cultured cells [11] , and in a Kras-induced lung cancer model [12] . The tumor suppressor-like properties of CTMP are supported by a report demonstrating inhibition of CTMP expression by hypermethylation of its promoter in malignant glioblastomas, where PKB activity is frequently altered [13]. Mitochondria regulate cellular energy supplies, apoptosis, and signaling pathways. ...
... Evidence suggest that CTMP negatively regulates PKB activity in v-Akt transformed cells [10], ciliary ganglion neurons [11], and K-ras-induced lung cancer model [12]. This observation is further supported by a recent report showing epigenetic down-regulation of CTMP transcription in malignant glioblastomas [13] . As previously sug- gested [10], CTMP is phosphorylated in vivo in response to pervanadate stimulation (Figure 1). ...
Article
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The serine/threonine protein kinase B (PKB/Akt) is involved in insulin signaling, cellular survival, and transformation. Carboxyl-terminal modulator protein (CTMP) has been identified as a novel PKB binding partner in a yeast two-hybrid screen, and appears to be a negative PKB regulator with tumor suppressor-like properties. In the present study we investigate novel mechanisms by which CTMP plays a role in apoptosis process. CTMP is localized to mitochondria. Furthermore, CTMP becomes phosphorylated following the treatment of cells with pervanadate, an insulin-mimetic. Two serine residues (Ser37 and Ser38) were identified as novel in vivo phosphorylation sites of CTMP. Association of CTMP and heat shock protein 70 (Hsp70) inhibits the formation of complexes containing apoptotic protease activating factor 1 and Hsp70. Overexpression of CTMP increased the sensitivity of cells to apoptosis, most likely due to the inhibition of Hsp70 function. Our data suggest that phosphorylation on Ser37/Ser38 of CTMP is important for the prevention of mitochondrial localization of CTMP, eventually leading to cell death by binding to Hsp70. In addition to its role in PKB inhibition, CTMP may therefore play a key role in mitochondria-mediated apoptosis by localizing to mitochondria.
... In genome-wide methylation analyses using restriction landmark genome scanning and CpG island microarrays as well as a multi-candidate gene approach, cancer-related genes such as MGMT, CDKN2A, CDKN2B, p14ARF, CTMP, EMP3, SLC5A8, HIC-1, PCDH-γ-A11, BEX1, BEX2, LATS1, LATS2, RUNX3, and TES were found to be methylated in gliomas (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). MGMT promoter methylation has already gained major clinical significance because it is associated with positive response to alkylating chemotherapy and increased survival in glioblastoma patients (5). ...
Article
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Critical tumor suppression pathways in brain tumors have yet to be fully defined. Along with mutational analyses, genome-wide epigenetic investigations may reveal novel suppressor elements. Using differential methylation hybridization, we identified a CpG-rich region of the promoter of the dual-specificity mitogen-activated protein kinase phosphatase-2 gene (DUSP4/MKP-2) that is hypermethylated in gliomas. In 83 astrocytic gliomas and 5 glioma cell lines examined, hypermethylation of the MKP-2 promoter was found to occur relatively more frequently in diffuse or anaplastic astrocytomas and secondary glioblastomas relative to primary glioblastomas. MKP-2 hypermethylation was associated with mutations in TP53 and IDH1, exclusive of EGFR amplification, and with prolonged survival of patients with primary glioblastoma. Expression analysis established that promoter hypermethylation correlated with reduced expression of MKP-2 mRNA and protein. Consistent with a regulatory role, reversing promoter hypermethylation by treating cells with 5-aza-2'-deoxycytidine increased MKP-2 mRNA levels. Furthermore, we found that glioblastoma cell growth was inhibited by overexpression of exogenous MKP-2. Our findings reveal MKP-2 as a common epigenetically silenced gene in glioma, the inactivation of which may play a significant role in glioma development.
... PKB activity is also regulated by other proteins, like the carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB activity [28]. CTMP down regulation by epigenetic mechanisms has been identified as a common aberration in glioblastoma [29]. Together, these activating alterations in the PI3K/PKB pathway prime the cell metabolism for growth and render the cells more resistant to pro-apoptotic stimuli. ...
Article
Kinases relay incoming input to downstream proteins by covalently linking phosphate groups to their targets. This phosphorylation acts as a biological switch regulating the func-tion of the target molecules. While simple in its general design, protein kinase signaling functions in highly complex networks and cascades regulating some of the most essential cel-lular and physiological functions. Subtle changes in these networks can have devastating consequences and the deadliest among all is cancer. Primary brain tumors of glial origin show mutations and alterations in several kinase cascades leading to aberrant growth and tissue invasion. Molecular profiling has identified key pathways of gliomagenesis but due to the redundancy among glioma promoting signaling pathways, highly specific kinase inhibi-tors used in mono-therapies have not led to clear clinical benefits so far. Adding to the dis-mal prognosis of glioma patients is the strong angiogenic potential of these tumors; again, a process with a kinase as key regulator which is targeted by several molecular strategies. In this review, we summarize the kinases involved in the main features of glioblastoma, the ef-fect targeted therapies against these kinases have shown so far and critically discuss future options of targeting kinases in glioma therapy.
... In this report, we examine the in vitro properties of the human protein hTHEM4 (NP 444283), also known as "Akt C-terminal modulator protein" (CTMP). Our attention was first drawn to hTHEM4 by reports that hTHEM4 mRNA expression is weakened in primary glioblastomas and in glioblastoma cell lines (1), and that hTHEM4 can revert the phenotype (cell morphology, growth rate, and in vivo turnorigenesis) of Akt1-transformed cell lines (2). Akt1 is a serinethreonine protein kinase that plays a key role in cancer by stimulating cell proliferation and inhibiting apoptosis (3). ...
Article
Herein, we report on an in vitro kinetic activity analysis that demonstrates that the protein known as the Akt C-terminal modulator protein is a broad-range, high-activity acyl-CoA thioesterase. In vitro tests of possible activity regulation by product inhibition or by Akt1 binding gave negative results. Truncation mutants confined the thioesterase activity to the C-terminal domain, consistent with our threading model. The N-terminal domain of unknown fold and function was found to contribute to solubility.
... CTMP has been shown to inhibit PKB/Akt activation at the plasma membrane in response to various stimuli and also to have tumor suppressor-like functions. This notion was strengthened by the observation that primary glioblastomas exhibit downregulation of CTMP mRNA levels due to promoter hypermethylation [21]. We recently reported the mitochondrial localization of endogenous and exogenous CTMP [22]. ...
Article
Full-text available
Mitochondria are central to the metabolism of cells and participate in many regulatory and signaling events. They are looked upon as dynamic tubular networks. We showed recently that the Carboxy-Terminal Modulator Protein (CTMP) is a mitochondrial protein that may be released into the cytosol under apoptotic conditions. Here we report an unexpected function of CTMP in mitochondrial homeostasis. In this study, both full length CTMP, and a CTMP mutant refractory to N-terminal cleavage and leading to an immature protein promote clustering of spherical mitochondria, suggesting a role for CTMP in the fission process. Indeed, cellular depletion of CTMP led to accumulation of swollen and interconnected mitochondria, without affecting the mitochondrial fusion process. Importantly, in vivo results support the relevance of these findings, as mitochondria from livers of adult CTMP knockout mice had a similar phenotype to cells depleted of CTMP. Together, these results lead us to propose that CTMP has a major function in mitochondrial dynamics and could be involved in the regulation of mitochondrial functions.
... This observation has been confirmed by another study that described PI3K p110 mutations in 15% of glioma samples, and 21% in pediatric and 17% in adult brain tumor samples [63]. In addition, elements of the PI3K signaling pathway are also frequently mutated, such as PTEN [64, 65] and the carboxyl-terminal modulator protein (CTMB) [66, 67]. Transgenic and knockout mouse models have confirmed the role of the PI3K-PBK pathway in tumorigenesis [68]. ...
Article
Glioblastoma (GBM) is the most common primary tumor of the CNS in the adult. It is characterized by exponential growth and diffuse invasiveness. Among many different genetic alterations in GBM, e.g., mutations of PTEN, EGFR, p16/p19 and p53 and their impact on aberrant signaling have been thoroughly characterized. A major barrier to develop a common therapeutic strategy is founded on the fact that each tumor has its individual genetic fingerprint. Nonetheless, the PI3K pathway may represent a common therapeutic target to most GBM due to its central position in the signaling cascade affecting proliferation, apoptosis and migration. The read-out of blocking PI3K alone or in combination with other cancer pathways should mainly focus, besides the cytostatic effect, on cell death induction since sublethal damage may induce selection of more malignant clones. Targeting more than one pathway instead of a single agent approach may be more promising to kill GBM cells.
... Three years later, additional insights into the implication of CTMP-Akt in cancer arose, where loss of CTMP function and/or expression reduced its inhibitory effects on Akt and promoted tumorigenesis. Indeed, when primary glioblastomas and glioblastoma cell lines were studied for the consequences of CTMP deletion, mutation, promoter hypermethylation and mRNA expression, Knobbe and colleagues (Knobbe et al., 2004) found that both hypermethylation and transcriptional down-regulation of CTMP genes could be related to tumor state. In 2009, the role of CTMP in multistage lung tumorigenesis revealed that lentiviral overexpression of CTMP altered the Akt signalling pathway and inhibited DNA synthesis and cell cycle progression in lungs of 9-week-old K- ras(LA1) mice. ...
Article
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Kinases of the Akt family are integral and essential components in growth factor signaling pathways activated downstream of the membrane bound phospho-inositol-3 kinase. In light of strong homologies in the primary amino acid sequence, the three Akt kinases were long surmised to play redundant and overlapping roles in insulin signaling across the spectra of cell and tissue types. Over the last 10 years, work using mouse knockout models, cell specific inactivation, and more recently targeted gene inactivation, has brought into question the redundancy within Akt kinase isoforms and instead pointed to isoform specific functions in different cellular events and diseases. Here we concentrate on the differential roles played by Akt1 and Akt2 in a variety of cellular processes and in particular during cancer biogenesis. In this overview, we illustrate that while Akt1 and 2 are often implicated in many aspects of cellular transformation, the two isoforms frequently act in a complementary opposing manner. Furthermore, Akt1 and Akt2 kinases interact differentially with modulating proteins and are necessary in relaying roles during the evolution of cancers from deregulated growth into malignant metastatic killers. These different actions of the two isoforms point to the importance of treatments targeting isoform specific events in the development of effective approaches involving Akt kinases in human disease.
... Increasing numbers of genes associated with epigenetic alterations have been identified in human gliomas e.g., MGMT, CDKN2A, CDKN2B, p14ARF, CTMP, EMP3, SLC5A8, HIC-1, PCDH-c-A11, BEX1, BEX2, LATS1, LATS2, RUNX3, TES, DUSP4 and CASPR2. [5][6][7][8][9][10][11][12][13][14][15][16] The identification of new genes functionally involved in tumor development and progression may help to find alternative approaches for diagnostic and therapeutic evaluation. ...
Article
In a genome-wide screen using DMH (differential methylation hybridization) we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in low- and high-grade astrocytomas compared to normal brain tissue. Pyrosequencing was performed to confirm the methylation status of this CpG island in 89 astrocytic gliomas of different malignancy grades and six glioma cell lines. Hypermethylation of SGNE1/7B2 was significantly more frequent in diffuse low-grade astrocytomas as well as secondary glioblastomas and anaplastic astrocytomas as compared to primary glioblastomas. mRNA expression analysis by real-time RT-PCR indicates that SGNE1/7B2 expression is downregulated in astrocytic gliomas compared to white matter samples. Treatment of glioma cells with the demethylating agent 5-aza-2'-deoxycytidine restores the transcription of SGNE1/7B2. Overexpression of SGNE1/7B2 in T98G, A172 and U373MG glioblastoma cells significantly suppressed focus formation and led to a significant increase in apoptotic cells as determined by flow cytometric analysis in T98G cells. In summary, we have identified SGNE1/7B2 as a novel target silenced by DNA methylation in astrocytic gliomas. The high incidence of this alteration and the significant effects of SGNE1/7B2 on the growth and apoptosis of glioblastoma cells provide a first proof for a functional implication of SGNE1/7B2 inactivation in the molecular pathology of gliomas.
... wilms tumor 1 WT1 transcription factor; development [104] interferon regulatory factor 7 IRF7 apoptosis regulation [104] protocadherin gamma subfamily A, 11 PCDH-γ-A11 cell adhesion [117] solute carrier family 5 (iodide transporter), member 8 SLC5A8 ion transport; apoptosis; cell cycle regulation [96] large tumor suppressor, homolog 1 LATS1 cell cycle regulation [118] large tumor suppressor, homolog 2 LATS2 cell cycle regulation [118] testis derived transcript TES protein-protein interactions; tumor suppressor [119] runt related transcription factor 3 RUNX transcription factor; proliferation [119] ATPase, Na+/K+ transporting, beta 2 polypeptide ATP1B2/AMOG ion transport; cell size regulation [120] apolipoprotein D APOD lipid transport and metabolism [120] dmx-like 1 DMXL1 unknown; family members involved in cell regulatory processes [120] family with seqeunce similiarity 107, member A FAM107A/DRR1 cell growth regulation [120] pleckstrin and Sec 7 domain containing 3 PSD3 signal transduction regulation [120] harakiri, BCL2 interacting protein HRK apoptosis [121] caspase 8 caspase 8 apoptosis [122] Estrogen Receptor 1 ESR1 transcription [99 , 116] calcitonin related polypeptide alpha CALCA calcium regulation, signaling, regulation [116] myogenic differentiation 1 MYOD1 differentiation, muscle development [116] Ras association (RalGDS/AF-6) domain family member 5 RASSF5/NORE1A apoptosis, signal transduction, cell cycle regulation [105] tumor suppressor candidate 3 TUSC3/N33 tumor suppressor [99] hypermethylated in cancer 1 HIC1 cell cycle and transcription regulation [99] cyclin dependent kinase inhibitor 1A CDKN1A/p21 cell cycle regulation [109] cyclin dependent kinase inhibitor 2B CDKN1B/p27kip1 cell cycle regulation [109] carboxy terminal modulator protein CTMP/ THEM4 Akt inhibition [123] In this table, the names of genes that have been shown to be silenced in gliomas are listed. Moreover, the potential function of each gene and reference are also listed. ...
Article
Full-text available
Epigenetics are defined, in broad-terms, as alterations in gene expression without changes in DNA sequence. While histone modifications and DNA methylation are two classical means to regulate gene expression, miRNA has also recently been documented to govern gene expression in normal as well as cancer cells. In this review, we will first describe briefly histone modifications, DNA methylation and miRNAs and the functions of these epigenetic marks during different cellular processes involving DNA metabolism. We will then highlight some epigenetic changes in glioblastomas, a malignant form of brain tumor, and potential application of epigenetic means for diagnosis, prognosis, and treatment of gliomas. We expect that novel therapies will be developed to counter epigenetic changes in this deadly disease.
... These immunoblots are representative examples of 4 repetitions of this experiment . highly expressed in neural tissue, binds to the C-terminal regulatory domain of Akt and reduces its activity at the plasma membrane by preventing phosphorylation at serine 473 and threonine 308 (Brazil et al. 2002; Knobbe et al. 2004; Maira et al. 2001). We found that overexpression of GFP-tagged CTMP blocked stimulation of K Ca evoked by either NRG1 or TGF1, but had no effect on expression of voltage-activated Ca 2 currents (Fig. 5), consistent with observations made with using treatments that inhibit Akt signaling. ...
Article
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The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance K(Ca) channels in ciliary ganglion (CG) neurons evoked by beta-neuregulin-1 (NRG1) and transforming growth factor-beta1 (TGFbeta1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGF(beta)1. NRG1- and TGF(beta)1-evoked stimulation of K(Ca) is blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional K(Ca) channels. NRG1 and TGF(beta)1 evoked an Akt-dependent increase in cell-surface SLO alpha-subunits. These procedures have no effect on voltage-activated Ca2+ currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons.
... T-ALL cell lines (Gebhart et al., 2002) were cultured, and DNA was extracted as described previously. Glioma cells were cultured as described previously (Knobbe et al., 2004), and DNA was extracted using a Blood and Cell Culture DNA Midi Kit (Qiagen). Microsatellite marker and CDKN2A/B exon PCR as well as analysis of CDKN2A hypermethylation were performed as described in previous articles (Florl et al., 2000;Florl and Schulz, 2003). ...
Article
The CDKN2A tumor-suppressor locus on chromosome band 9p21, which encodes p16(INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutation, promoter hypermethylation, and, often, deletion. Homozygous deletions are unusually prevalent at this locus in very different human cancers. In the present study, we compared deletions in squamous cell carcinoma of the head and neck (SCCHN) cell lines to those in T-cell acute lymphatic leukemia (T-ALL), glioma, and bladder carcinoma (TCC) cell lines. Of 14 SCCHN lines, 10 showed homozygous deletions of CDKN2A, one displayed promoter hypermethylation with gene silencing, and one had a frameshift deletion in exon 2. Many deletion ends were in or proximal to the repetitive sequence clusters flanking the locus. Breakpoint junctions displayed variable microhomologies or insertions characteristic of DNA repair by nonhomologous end-joining. In general, deletions were much smaller in SCCHN than in TCC and glioma. In T-ALL, breakpoints were near consensus sites for recombination mediated by RAG (recombination activating genes) enzymes, and the structure of the junctions was consistent with this mechanism. We suggest that different mechanisms of CDKN2A deletion prevail in different human cancers. Aberrant RAG-mediated recombination may be responsible in T-ALL, and exuberant DNA repair by nonhomologous end-joining is the likely prevailing mechanism in SCCHN, but a distinct mechanism in TCC and glioma remains to be elucidated.
... Hypermethylation of the promotor for carboxyl-terminal modulator protein (CTMP) has been observed in glioblastomas and is known to prevent negative regulation of the AKT pathway, leading to uncontrolled proliferation (Knobbe et al 2004). Although these genetic differences are ...
... Instead, AKT3-174aa expression showed as a positive correlation with the patients' total survival with GBM in our study. Considering that AKT3-174aa is one of the few negative regulators of PI3K/AKT signaling (others include PTEN, PHLPP [46], and CTMP [47]), its expression level should be more intensively tested in larger cohort to confirm whether it is an independent biomarker for GBM or other types of human cancers. ...
Article
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Background: The RTK/PI3K/AKT pathway plays key roles in the development and progression of many cancers, including GBM. As a regulatory molecule and a potential drug target, the oncogenic role of AKT has been substantially studied. Three isoforms of AKT have been identified, including AKT1, AKT2 and AKT3, but their individual functions in GBM remain controversial. Moreover, it is not known if there are more AKT alternative splicing variants. Methods: High-throughput RNA sequencing and quantitative reverse transcription-PCR were used to identify the differentially expressed circRNAs in GBM samples and in paired normal tissues. High throughput RNA sequencing was used to identify circ-AKT3 regulated signaling pathways. Mass spectrometry, western blotting and immunofluorescence staining analyses were used to validate AKT3-174aa expression. The tumor suppressive role of AKT3-174aa was validated in vitro and in vivo. The competing interaction between AKT3-174aa and p-PDK1 was investigated by mass spectrometry and immunoprecipitation analyses. Results: Circ-AKT3 is a previously uncharacterized AKT transcript variant. Circ-AKT3 is expressed at low levels in GBM tissues compared with the expression in paired adjacent normal brain tissues. Circ-AKT3 encodes a 174 amino acid (aa) novel protein, which we named AKT3-174aa, by utilizing overlapping start-stop codons. AKT3-174aa overexpression decreased the cell proliferation, radiation resistance and in vivo tumorigenicity of GBM cells, while the knockdown of circ-AKT3 enhanced the malignant phenotypes of astrocytoma cells. AKT3-174aa competitively interacts with phosphorylated PDK1, reduces AKT-thr308 phosphorylation, and plays a negative regulatory role in modulating the PI3K/AKT signal intensity. Conclusions: Our data indicate that the impaired circRNA expression of the AKT3 gene contributes to GBM tumorigenesis, and our data corroborate the hypothesis that restoring AKT3-174aa while inhibiting activated AKT may provide more benefits for certain GBM patients.
... Knobbe et al. reported epigenetic silencing of carboxyl-terminal modulator protein (CTMP) gene, the products of the CTMP acts as a negative regulator of Akt, in 40% of GBMs tested, and the aberrant expression of CTMP in dysregulation of survival pathways in GBM [41]. The SLIT gene family is important in migration and axonal guidance and the the epigenetic inactivation of this genes were reported by Dallol et al. the promoter hypermethylation of the SLIT2 in 59% of glioma tissue samples tested, whereas in non-tumor brain tissue, the promoter was fully unmethylated [42]. ...
Article
Aberrations in the epigenetic machinery of the genome may results the inactivation of critical genes and the epigenetic changes are important mechanisms in the evolution of malignancies that not only contributes to tumorigenesis but may also precede genetic changes. Several epigenetic mechanisms have been observed in glioblastomas including DNA hyper-methylation of genes, histone modifications including methylation and acetylation, nucleosomal rearrangement and dysregulation of noncoding RNA expression have been shown to play a critical role in the biology of glioblastomas and to contribute to the clinical outcome. This review examines the general role of epigenetic changes in the malignant process and focuses on the known epigenetic changes and development of new therapeutic strategies against these malignancies.
... Carboxyl-terminal modulator protein (CTMP) has been reported to activate multiple signaling pathways which impinge on AKT signaling to serve roles as both as a tumor suppressor or an oncogene [16][17][18][19]. In addition to data from our lab, Ono and colleagues showed that CTMP activates AKT signaling and contributes to oncogenesis through a direct interaction in breast cancer [18,19]. ...
Article
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Trastuzumab is regarded as the primary therapy for patients with HER2-enriched breast cancer, but the pathological complete response for advanced cases is less than 30%. The underlying mechanism of trastuzumab resistance remains unclear and there are currently no conclusive biomarkers for patient response to trastuzumab. Identifying predictive biomarkers for trastuzumab response may allow treatments to be individually tailored and optimized multi-target therapies may be developed. CTMP activates AKT signaling in breast cancer and over-activation of AKT has been reported to contribute to trastuzumab resistance. In this study, we examined samples from 369 patients to investigate the correlation between CTMP expression level and patient outcome. Elevated CTMP expression was correlated with adverse outcomes in HER2-enriched patients including overall and disease-free survival as well as trastuzumab resistance. Ectopic expression of varying levels of CTMP in SkBR3 cells dose-dependently attenuated trastuzumab-mediated growth inhibition through AKT activation. In addition, inhibition of AKT signaling by AKT inhibitor IV and Rapamycin reversed CTMP-mediated trastuzumab resistance. In clinical samples, the high expression of CTMP was showed in trastuzumab non-responders and positively correlated with AKT activity. Taken together, we demonstrated that CTMP promotes AKT activation resulting in trastuzumab resistance in patients with HER2-enriched breast cancer. High CTMP expression not only predicted poor prognosis, but may also predict resistance to trastuzumab in HER2-enriched patients. Therefore, CTMP expression may be considered as a prognostic biomarker in HER2-enriched breast cancer and high expression may indicate a utility for AKT-inhibition in these patients.
... Akt activation is managed by various interacting proteins and upstream regulators. Since Maira et al. published the original study describing 27-kDA CTMP as an Akt-interacting protein in 2001 11 , accumulating evidence suggests that CTMP binds to the Akt carboxy terminus and negatively regulates its activity in lung cancer 33 , glioblastoma 34,35 , pancreatic adenocarcinoma 36 , and cervical cancer cell lines 37 , suggesting CTMP is a tumor suppressor. However, opposite results were found by Ono et al. 38 . ...
Article
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The exact regulatory mechanisms of carboxyl-terminal modulator protein (CTMP) and its downstream pathways in cancer have been controversial and are not completely understood. Here, we report a new mechanism of regulation of Akt serine/threonine kinase, one of the most important dysregulated signals in head and neck squamous cell carcinoma (HNSCC) by the CTMP pathway and its clinical implications. We find that HNSCC tumor tissues and cell lines had relatively high levels of CTMP expression. Clinical data indicate that CTMP expression was significantly associated with positive lymph node metastasis (OR = 3.8, P = 0.033) and correlated with poor prognosis in patients with HNSCC. CTMP was also positively correlated with Akt/GSK-3β phosphorylation, Snail up-regulation and E-cadherin down-regulation, which lead to increased proliferation and epithelial-to-mesenchymal transition, suggesting that CTMP expression results in enhanced tumorigenic and metastatic properties of HNSCC cells. Moreover, CTMP suppression restores sensitivity to cisplatin chemotherapy. Intriguingly, all the molecular responses to CTMP regulation are identical regardless of p53 status in HNSCC cells. We conclude that CTMP promotes Akt phosphorylation and functions as an oncogenic driver and prognostic marker in HNSCC irrespective of p53.
... Aberrant activation of Akt in GBM can occur as a result of loss of PTEN suppressive function, which leads to unchecked proliferation through an array of pro-growth and anti-apoptotic mechanisms [213,214]. In addition, the PI3K/Akt pathway can be abnormally activated as a consequence of epidermal growth factor receptor (EGFR) gene amplification and rearrangement, as well as CTMP promoter hypermethylation [215,216]. Importantly, Akt is required for the maintenance of a stem-like state in glioma [217] and Akt inhibition increases their rate of apoptosis and decreases the ability to form neurospheres [218,219]. Aberrant Akt activation has also been observed in medulloblastoma [220]. ...
Article
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Radiation therapy (RT) is frequently used as part of the standard of care treatment of the majority of brain tumors. The efficacy of RT is limited by radioresistance and by normal tissue radiation tolerance. This is highlighted in pediatric brain tumors where the use of radiation is limited by the excessive toxicity to the developing brain. For these reasons, radiosensitization of tumor cells would be beneficial. In this review, we focus on radioresistance mechanisms intrinsic to tumor cells. We also evaluate existing approaches to induce radiosensitization and explore future avenues of investigation.
... Indeed, increasing CTMP expression reduces non-small cell lung tumor progression in mice (38), exerts robust protection against ischemia-induced neuronal death (39), and restores immune function (40). It is, however, worth noting that, although the preponderance of evidence supports CTMP as a negative regulator of Akt1 phosphorylation and activation (7,39,41), a few studies suggest that CTMP can act as a positive regulator of Akt1 (6,42,43). In our work, we have elucidated a new regulatory mechanism by which the proteasomal degradation of CTMP leads to an increase in Akt1 phosphorylation. ...
Article
Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial NO synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at Thr308 and Ser473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells (PAEC) to ADMA enhanced Akt1 phosphorylation at both Thr308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog (PTEN) activity or membrane Akt1 levels. Hsp90 plays a pivotal role in maintaining Akt1 activity and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but surprisingly, overexpression of a dominant-negative Hsp90 (DN Hsp90) mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of DN Hsp90 increased Hsp70 levels and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, Carboxyl-Terminal Modulator Protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo and that increasing CTMP levels may be an avenue to treat pulmonary hypertension.
Astrocytic gliomas are the most common primary brain tumors and account for up to two thirds of all tumors of glial origin. In this review we outline the basic histological and epidemiological aspects of the different astrocytoma subtypes in adults. In addition, we summarize the key genetic alterations that have been attributed to astrocytoma patho-genesis and progression. Recent progress has been made by interpreting genetic alterations in a pathway-related context so that they can be directly targeted by the application of specific inhibitors. Also, the first steps have been taken in refining classical histopathological diagnosis by use of molecular predictive markers, for example, MGMT promoter hypermethylation in glioblastomas. Progress in this direction will be additionally accelerated by the employment of high-throughput profiling techniques, such as array-CGH and gene expression profiling. Finally, the tumor stem cell hypothesis has challenged our way of understanding astrocytoma biology by emphasizing intratumoral heterogeneity. Novel animal models will provide us with the opportunity to comprehensively study this multilayered disease and explore novel therapeutic approaches in vivo.
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Dysregulation of Akt signaling is important in a broad range of diseases that includes cancer, diabetes and heart disease. The role of Akt signaling in brain disorders is less clear. We found that global ischemia in intact rats triggered expression and activation of the Akt inhibitor CTMP (carboxyl-terminal modulator protein) in vulnerable hippocampal neurons and that CTMP bound and extinguished Akt activity and was essential to ischemia-induced neuronal death. Although ischemia induced a marked phosphorylation and nuclear translocation of Akt, phosphorylated Akt was not active in post-ischemic neurons, as assessed by kinase assays and phosphorylation of the downstream targets GSK-3beta and FOXO3A. RNA interference-mediated depletion of CTMP in a clinically relevant model of stroke restored Akt activity and rescued hippocampal neurons. Our results indicate that CTMP is important in the neurodegeneration that is associated with stroke and identify CTMP as a therapeutic target for the amelioration of hippocampal injury and cognitive deficits.
Article
The focus of this paper is the hotdog-fold thioesterase THEM2 from human (hTHEM2; Swiss-Prot entry Q9NPJ3 ). In an earlier communication (Cheng, Z., Song, F., Shan, X., Wei, Z., Wang, Y., Dunaway-Mariano, D., and Gong, W. (2006) Crystal structure of human thioesterase superfamily member 2, Biochem. Biophys. Res. Commun. 349, 172-177) we reported the apo crystal structure of hTHEM2. Herein, we report the results of an extensive hTHEM2 substrate screen, the structure determination of hTHEM2 complexed with the inert substrate analogue undecan-2-one-CoA (in which OC-CH(2)-S substitutes for OC-S) and the kinetic analysis of active site mutants. The work described in this paper represents the first reported structure-function based analysis of a human hotdog-fold thioesterase. The catalytic mechanism proposed involves the Asp65/Ser83 assisted attack of a water molecule at the Gly57/Asn50 polarized thioester CO and the Asn50 assisted departure of the thiolate leaving group. Thioesterase activity was observed with acyl-CoAs but not with the human acyl-ACP or with an acyl-Cys peptide. The medium-to-long-chain fatty acyl-CoAs displayed the smallest K(m) values. The substrate specificity profile was analyzed within the context of the liganded enzyme to define the structural determinants of substrate recognition. Based on the results of this structure-function analysis we hypothesize that the physiological role of hTHEM2 involves catalysis of the hydrolysis of cytosolic medium-to-long-chain acyl-CoA thioesters.
Article
The serine/threonine kinase, Akt, also known as PKB (Protein Kinase B) is one important signal transduction pathway that mediates the delay of neutrophil apoptosis caused by inflammatory mediators. Proteins controlled by the PKB/Akt pathway have been reported to prevent or reverse apoptotic-signaling pathways and regulate cell survival. In this review we discuss the role of PKB/Akt activation in the regulation of neutrophil activation during inflammation, and the importance of resolving the inflammatory response by inhibiting PKB/Akt activation and neutrophil survival. Furthermore, we introduce the concept of a dynamic Akt signal complex that is altered when an extracellular signal is initiated such that changes in protein-protein interactions within the Akt signal complex regulates Akt activity and cell survival. Various substrates of PKB/Akt as well as positive and negative regulators of PKB/Akt activation are discussed which in turn inhibit or enhance cellular survival.
Article
Aberrant activation of Ras signaling is a common finding in human glioblastomas. To determine the contribution of Ras gene mutations to this aberration, we screened 94 glioblastomas for mutations in the three Ras family genes NRAS, KRAS and HRAS. All tumors were additionally analyzed for mutations in BRAF, which encodes a Ras-regulated serine/threonine kinase with oncogenic properties. Mutation analysis of the entire coding regions of NRAS and KRAS, as well as the known mutation hot-spot sites in HRAS, identified somatic point mutations in two glioblastomas, both affecting codon 12 of NRAS (c.35G>A, p.G12D). Three additional tumors carried BRAF mutations altering the known hot-spot codon 599 (c.1796T>A, p.V599E). None of these five glioblastomas showed amplification of the EGFR or PDGFRA genes, while three of the tumors, including two with NRAS and one with BRAF mutation, demonstrated PTEN missense mutations or loss of PTEN mRNA expression. Taken together, our data suggest activating mutations in NRAS or BRAF as a molecular alteration that contributes to aberrant Ras signaling in a small fraction of glioblastomas.
Article
The Carboxy-Terminal Modulator Protein (CTMP) protein was identified as a PKB inhibitor that binds to its hydrophobic motif. Here, we report mitochondrial localization of endogenous and exogenous CTMP. CTMP exhibits a dual sub-mitochondrial localization as a membrane-bound pool and a free pool of mature CTMP in the inter-membrane space. CTMP is released from the mitochondria into the cytosol early upon apoptosis. CTMP overexpression is associated with an increase in mitochondrial membrane depolarization and caspase-3 and polyADP-ribose polymerase (PARP) cleavage. In contrast, CTMP knock-down results in a marked reduction in the loss of mitochondrial membrane potential as well as a decrease in caspase-3 and PARP activation. Mutant CTMP retained in the mitochondria loses its capacity to sensitize cells to apoptosis. Thus, proper maturation of CTMP is essential for its pro-apoptotic function. Finally, we demonstrate that CTMP delays PKB phosphorylation following cell death induction, suggesting that CTMP regulates apoptosis via inhibition of PKB.
Article
Carboxyl-terminal modulator protein (CTMP) is a tumor suppressor-like binding partner of Protein kinase B (PKB/Akt) that negative regulates this kinase. In the course of our recent work, we identified that CTMP is consistently associated with leucine zipper/EF-hand-containing transmembrane-1 (LETM1). Here, we report that adenovirus-LETM1 increased the sensitivity of HeLa cells to apoptosis, induced by either staurosporine or actinomycin D. As shown previously, LETM1 localized to the inner mitochondrial membrane. Electron-microscopy analysis of adenovirus-LETM1 transduced cells revealed that mitochondrial cristae were swollen in these cells, a phenotype similar to that observed in optic atrophy type-1 (OPA1)-ablated cells. OPA1 cleavage was increased in LETM1-overexpressing cells, and this phenotype was reversed by overexpression of OPA1 variant-7, a cleavage resistant form of OPA1. Taken together, these data suggest that LETM1 is a novel binding partner for CTMP that may play an important role in mitochondrial fragmentation via OPA1-cleavage.
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Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments.
Article
Glioblastomas frequently carry genetic alterations resulting in an aberrant activation of the phosphoinositol-3-kinase (Pi3k)/protein kinase B (Akt) signalling pathway, including most notably phosphatase and tensin homolog (PTEN) mutation, epidermal growth factor receptor (EGFR) amplification and rearrangement, as well as carboxyl-terminal modulator protein (CTMP) hypermethylation [Knobbe et al., (2004) Hypermethylation and transcriptional downregulation of the carboxyl-terminal modulator protein gene in glioblastomas. J Natl Cancer Institute, 96, 483-486]. Here, we investigated two further Pi3k/Akt pathway genes, namely PIK3CA (3q26.3) and phosphatidylinositol-3-kinase enhancer (PIKE) (CENTG1, 12q14), for genetic alteration and aberrant expression in a series of 97 primary glioblastomas. Single strand conformation polymorphism (SSCP) analysis of PIK3CA revealed somatic mutations in five tumours (5%). Twelve glioblastomas (12%) showed amplification of PIKE with invariable co-amplification of the adjacent CDK4 gene. All tumours with PIKE amplification as well as the vast majority of glioblastomas without amplification demonstrated increased expression of PIKE-A but not PIKE-S/L transcripts as compared with non-neoplastic brain tissue. Taken together, our data support an important role of PIK3CA and PIKE gene aberrations in the molecular pathogenesis of primary glioblastomas.
Article
Astrocytic gliomas are the most common primary brain tumours. Here we summarize the characteristic neuropathological features of the different types of astrocytic neoplasms according to the World Health Organization classification of tumours of the nervous system. In addition, we report on the present state of the art concerning the molecular genetics of these tumours. Over the past 20 years a number of recurrent chromosomal,genetic and epigenetic alterations have been found to be associated with the different histological types and malignancy grades of astrocytic tumours. However, we are still far from understanding the complex mechanisms that underly tumour initiation and progression in the individual case. Furthermore, the clinical significance of molecular parameters for the diagnostic and prognostic assessment of astrocytic gliomas is still limited. Therefore further investigation of the molecular mechanisms underlying oncogenesis and progression of these most common brain tumours is necessary to improve their diagnostic assessment and to devise novel, individually tailored treatment strategies.
Article
Conventional surgery, radio- and chemotherapy have failed to significantly improve the prognosis of patients with malignant astrocytomas--hence the need for understanding their molecular biology. Harvesting this understanding to yield novel biological targeted therapies has approached the clinical doorstep. Therapeutic efficacy will likely require combinatorial therapy involving biologicals and conventional therapies, with small incremental efficacy in selected sub-groups. This review highlights some of the findings over the past year (June 2003-2004) that have contributed to this slow but essential journey towards our understanding of the biology of astrocytomas. The accumulation of loss and/or gain of function molecular alterations underlying astrocytoma formation, progression and key growth parameters including proliferation, angiogenesis, apoptosis, invasion and resistance are emerging. These alterations involve those regulating the growth factor/receptor and downstream signaling networks, cell cycle, immune modulators and other key biological processes. The advances are facilitated by interactions amongst clinician and basic scientists, in both academia and industry. They have incorporated high-throughput bioinformatics analysis of genomic and expression array data, the emerging field of proteomics and development of various genetically engineered models of astrocytomas. Astrocytomas, like other cancers, are a result of several molecular alterations, some of which strongly correlate to their pathological grade. However, molecular heterogeneity exists between astrocytomas of similar grades and likely between varying micro-environmental regions of a single tumor. Characterization of the molecular signature of an astrocytoma and linking with the appropriate 'tailored' therapie(s) is the hope of the future.
Article
Enolase‐phosphatase 1 (ENOPH1), a newly identified enzyme involved in l‐methionine biosynthesis, is associated with anxiety and depression. In this study, ENOPH1 was found to play a crucial role in promoting the proliferation and migration of glioma cells. Among high‐grade glioma patients, the overall survival of the group showing high ENOPH1 expression was shorter than that of the group showing low ENOPH1 expression. ENOPH1 knockdown inhibited glioma cell proliferation and migration. In parallel, ENOPH1 knockdown suppressed tumor growth capacity and prolonged survival in an orthotopic glioma model. Mechanistically, we found that ENOPH1 activates the PI3K/AKT/mTOR signaling pathway by regulating THEM4. In conclusion, ENOPH1 is an important mediator that promotes glioma cell proliferation and migration. ENOPH1 knockdown inhibited glioma cell proliferation and migration. ENOPH1 activates the PI3K/AKT/mTOR signaling pathway.
Article
Epigenetic regulation of gene expression by DNA methylation and histone modification is frequently altered in human cancers including gliomas, the most common primary brain tumors. In diffuse astrocytic and oligodendroglial gliomas, epigenetic changes often present as aberrant hypermethylation of 5'-cytosine-guanine (CpG)-rich regulatory sequences in a large variety of genes, a phenomenon referred to as glioma CpG island methylator phenotype (G-CIMP). G-CIMP is particularly common but not restricted to gliomas with isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) mutation. Recent studies provided a mechanistic link between these genetic mutations and the associated widespread epigenetic modifications. Specifically, 2-hydroxyglutarate, the oncometabolite produced by mutant IDH1 and IDH2 proteins, has been shown to function as a competitive inhibitor of various α-ketoglutarate (α-KG)-dependent dioxygenases, including histone demethylases and members of the ten-eleven-translocation (TET) family of 5-methylcytosine (5mC) hydroxylases. In this review article, we briefly address (i) the basic principles of epigenetic control of gene expression; (ii) the most important methods to analyze focal and global epigenetic alterations in cells and tissues; and (iii) the involvement of epigenetic alterations in the molecular pathogenesis of gliomas. Moreover, we discuss the promising roles of epigenetic alterations as molecular diagnostic markers and novel therapeutic targets, and highlight future perspectives toward unraveling the "glioma epigenome."
Article
Protein kinase B (PKB/Akt) is a serine/threonine protein kinase that created serious interest when it was revealed as a mediator of the PI3K pathway. It comprises three isoforms that play both unique and redundant roles. Upon binding to phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) generated by PI3K, PKB is phosphorylated by PDK1 at T308. To achieve full kinase activity, PKB needs to be phosphorylated at a second key residue, S473, by members of the PI3K-related kinase family mTORC2 or DNA-PK, depending on the stimulus and the context. Besides, a number of phosphatases and interacting partners have been shown to further modulate its subcellular localization, phosphorylation, and kinase activity. This review aims at illustrating the remarkable complexity in the regulation of PKB signaling downstream of PI3K. Such regulation could be attributed to the specific roles of the PKB isoforms, their expression pattern, subcellular localization, targets, phosphorylation by upstream kinases in a stimulus- and context-dependent manner and by phosphatases, and interaction with binding partners. This allows this key kinase to fulfill physiological functions in numerous processes, including embryonic development, thymocyte development, adipocyte differentiation, glucose homeostasis, and to avoid pathological loss of control such as tumor formation.
Article
This chapter provides an outline of the surgical pathology and the recognized genetic and molecular changes of common tumors of the nervous system in children and adults. The histological basis for classification and malignancy grading of the tumors and some common diagnostic problems are also addressed. Many brain tumors are morphologically heterogeneous, and classification of brain tumors is dependent on the recognition of areas with the characteristic histology for a particular tumor type. However, molecular data is more and more often being requested in addition to the histopathological findings. In many other areas of oncology, such as lymphoma, breast cancer, and sarcoma diagnosis, molecular data are the norm. The increasing number of evidence indicate that mutation at codon 132 of IDH1 (or rarely mutation of codon 172 of IDH2) combined with either TP53 mutation or total 1p/19q loss is a frequent and early change in the majority of oligodendroglial tumors, diffuse astrocytomas, anaplastic astrocytomas, and secondary glioblastomas, but not in primary glioblastomas. As new forms of molecular targeted therapy are introduced and found to be effective, the analysis of tumor specimens will have to be extended to provide clinically relevant data; medical professionals must be able to both provide this information and interpret the findings.
Chapter
Aberrations in the epigenetic machinery of the genome result in inactivation of critical genes and are important mechanisms in the evolution of malignancies that not only contributes to tumorigenesis but may also precede genetic changes. Several such epigenetic mechanisms have been observed in gliomas including DNA hypermethylation at the promoter or the coding regions of genes, histone modifications including methylation and acetylation, nucleosomal rearrangement, and dysregulation of noncoding RNA expression; these changes have been shown to play a critical role in the biology of gliomas and to contribute to the clinical outcome. This review examines the general role of epigenetic changes in the malignant process and focuses on the known changes in gene expression in gliomas due to epigenetic modifications, both in the context of gliomagenesis and in the development of new therapeutic strategies against these malignancies. © Springer Science+Business Media, LLC 2010. All rights reserved.
Article
Classical molecular genetics as a descriptive science has afforded insight into the events that underlie tumorigenesis of intracranial neoplasms. From comparative genomic hybridization (CGH) studies, a coherent model for the role of different genes and chromosomal regions in gliomagenesis has been developed which informs changes based on the age of tumor onset, the pattern of progression of the disease, and the histological subtype at diagnosis. The histological and molecular genetic descriptors of human brain tumors are maturing into routine applications in diagnostic and prognostic practices. The tools for such descriptions continue to expand in their pace, precision, and practice, warranting a review of the current understanding of the genetic and genomic basis for glioma behavior. Due to the distorted genetics of astrocytic tumors, it remains of interest to gain an appreciation for the underlying mechanisms of the genetic instability. Chromosomal instability in glioblastoma multiforme (GBM) is found in a large percentage of specimens (up to 70 per cent), resulting in a tumor process with a rich repertoire of permutations for both microenvironmental adaptation and survival under selection pressure such as chemotherapy.
Article
Objective: Protein kinase B (PKB/Akt), which is phosphorylated and activated by upstream activators, exerts critical neuroprotective effects by phosphorylating downstream targets after traumatic brain injury (TBI). Studies on the regulation of Akt will be crucial for our understanding of neuronal survival. The goal of this study is to investigate the effects of carboxyl-terminal modulator protein (CTMP) on phosphorylation of Akt and neurological function in a mouse model of TBI. Methods: Traumatic brain injury in mice was performed by a controlled cortical impact device. The expression of Akt, phospho-Akt, and CTMP was examined in the injured cortices by immunohistochemistry and Western blot analysis. To determine the effects of CTMP, small interfering RNAs (siRNAs) directed against CTMP were injected in mice with TBI, and the expression of phosphorylated Akt and neurological function were evaluated. Results: Phospho-Akt significantly increased at 4 hours post-TBI in the nucleus (P < 0.01) and remained at high levels until 72 hours after TBI, as shown by Western blot analysis. In the cytosol, the expression of phospho-Akt reached its peak at 4 hours post-TBI, but decreased markedly at 24 hours and maintained below pre-TBI levels until 72 hours post-TBI. Interestingly, the expression of CTMP significantly increased 4 hours after TBI (P < 0.01) and sustained those levels until 72 hours without dramatic changes. Treatment with CTMP siRNA effectively augmented the phosphorylation of Akt and significantly improved the neurological functional recovery up to 28 days post-TBI. Conclusion: We conclude that Akt is phosphorylated and translocated to nucleus after TBI to exert neuroprotective effects. However, CTMP is simultaneously triggered to inhibit the phosphorylation of Akt. Inhibition of CTMP by siRNA improves the recovery of neurological functions after TBI.
Article
Brain tumors encompass a heterogeneous group of malignant tumors with variable histopathology, aggressiveness, clinical outcome and prognosis. Current gene expression profiling studies indicate interplay of genetic and epigenetic alterations in their pathobiology. A central molecular event underlying epigenetics is the alteration of chromatin structure by post-translational modifications of DNA and histones as well as nucleosome repositioning. Dynamic remodeling of the fundamental nucleosomal structure of chromatin or covalent histone marks located in core histones regulate main cellular processes including DNA methylation, replication, DNA-damage repair as well as gene expression. Deregulation of these processes has been linked to tumor suppressor gene silencing, cancer initiation and progression. The reversible nature of deregulated chromatin structure by DNA methylation and histone deacetylation inhibitors, leading to re-expression of tumor suppressor genes, makes chromatin-remodeling pathways as promising therapeutic targets. In fact, a considerable number of these inhibitors are being tested today either alone or in combination with other agents or conventional treatments in the management of brain tumors with considerable success. In this review, we focus on the mechanisms underpinning deregulated chromatin remodeling in brain tumors, discuss their potential clinical implications and highlight the advances toward new therapeutic strategies.
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The prominence of the PI3K-Akt signaling pathway in several tumors indicates a relationship with tumor grade and proliferation. Critical cellular processes are driven through this pathway. More detailed knowledge of the pathogenesis of tumors would enable us to design targeted drugs to block both membrane tyrosine kinase receptors and the intracellular kinases involved in the transmission of the signal. The newly approved molecular inhibitors sunitinib (an inhibitor of vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and other tyrosine kinase receptors), sorafenib (a serine-threonine kinase inhibitor that acts against B-Raf) and temsirolimus (an mTOR inhibitor) shown clinical activity in advanced kidney cancer. Chronic myeloid leukemia has changed its natural history thanks to imatinib and dasatinib, both of which inhibit the intracellular bcr/abl protein derived from the alteration in the Philadelphia chromosome. Intracellular pathways are still important in cancer development and their blockade directly affects outcome. Cross-talk has been observed but is not well understood. Vertical and horizontal pathway blockade are promising anticancer strategies. Indeed, preclinical and early clinical data suggest that combining superficial and intracellular blocking agents can synergize and leverage single-agent activity. The implication of the Akt signaling pathway in cancer is well established and has led to the development of new anticancer agents that block its activation.
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Human THEM4 (hTHEM4) is comprised of a catalytically active hotdog-fold acyl-CoA thioesterase domain and an N-terminal domain of unknown fold and function. hTHEM4 has been linked to Akt1 regulation and cell apoptosis. Herein, we report the X-ray structure of hHTEM4 bound with undecan-2-one-CoA. Structure guided mutagenesis was carried out to confirm the catalytic residues. The N-terminal domain is shown to be partially comprised of irregular and flexible secondary structure, reminiscent of a protein-binding domain. We demonstrate direct hTHEM4-Akt1 binding by immunoprecipitation and by inhibition of Akt1 kinase activity, thus providing independent evidence that hTHEM4 is an Akt1 negative regulator.
Chapter
Phosphoinositide 3-kinase (PI3K) was discovered over 20 years ago as an enzyme that was active in growth factor-stimulated and oncogene-transformed cells. Ten years later, the PTEN gene was isolated by its deletion in a large proportion of human cancers, including glioblastoma. These two areas of research converged when it was shown that PTEN dephosphorylated the lipid product of PI3K activity, phosphatidylinositol (3,4,5) trisphosphate, or PIP3. Furthermore, it has since become clear that PTEN has tumor-suppressive activities that are independent of its lipid phosphatase activity. This chapter reviews the importance of PI3K activity in the development of glioblastoma, by describing the different genetic and epigenetic alterations that occur to deregulate the activity of this pathway. It will also describe the regulation of PTEN expression and activity, by transcriptional and posttranslational processes. Recent results implicating PI3K activity and PTEN in the cells that are thought to initiate early brain tumor development (brain tumor stem cells) are also reviewed. Finally, the possibilities of using this pathway both as a direct therapeutic target and as a way of predicting response to recently developed drugs are discussed.
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Identification of methylation-silenced genes in colorectal cancer (CRC) is of great importance. We employed oligonucleotide microarrays to identify differences in global gene expression of five CRC cell lines (HCT116, RKO, Colo320, SW480 and HT29) that were analyzed before and after treatment with 5-aza-2'-deoxycitidine. Selected candidates were subjected to methylation-specific PCR and real-time quantitative reverse transcription-PCR using 15 CRC cell lines and 23 paired tumor and normal samples from CRC patients. After 5-aza-2'-deoxycitidine treatment, 139 genes were re-expressed in all 5 CRC cell lines collectively with a fold change of more than 1.5 in at least one cell line. These genes include known methylated and silenced genes in CRC. After applying study selection criteria we identified 20 candidates. The GADD45B and THSD1 genes were selected for further analysis. Among 15 colon cancer cell lines, methylation was only identified in THSD1 (27%). THSD1 methylation was subsequently investigated in 23 colorectal tumors and methylation was detected in 9% of the analyzed samples; the observed promoter hypermethylation was cancer-specific. THSD1 mRNA down-regulation was observed in tumor tissues. This genome-wide screening led to the identification of genes putatively affected by methylation in CRC. The THSD1 gene may play a role in the tumorigenesis of CRC.
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Treated glioblastoma patients survive from 6 to 14 months. In the first part of this review, we describe glioma origins, cancer stem cells and the genomic alterations that generate dysregulated cell division, with enhanced proliferation and diverse response to radiation and chemotherapy. We review the pathways that mediate tumour cell proliferation, neo-angiogenesis, tumor cell invasion, as well as necrotic and apoptotic cell death. Then, we examine the ability of gliomas to evade and suppress the host immune system, exhibited at the levels of antigen recognition and immune activation, limiting the effective signaling between glioma and host immune cells. The second part of the review presents current therapies and their drawbacks. This is followed by a summary of the work of our laboratory during the past 20 years, on oligosaccharide and glycosphingolipid inhibitors of astroblast and astrocytoma division. Neurostatins, the O-acetylated forms of gangliosides GD1b and GT1b naturally present in mammalian brain, are cytostatic for normal astroblasts, but cytotoxic for rat C6 glioma cells and human astrocytoma grades III and IV, with ID50 values ranging from 200 to 450 nM. The inhibitors do not affect neurons or fibroblasts up to concentrations of 4 μM or higher. At least four different neurostatin-activated, cell-mediated antitumoral processes, lead to tumor destruction: (i) inhibition of tumor neovascularization; (ii) activation of microglia; (iii) activation of natural killer (NK) cells; (iv) activation of cytotoxic lymphocytes (CTL). The enhanced antigenicity of neurostatin-treated glioma cells, could be related to their increased expression of connexin 43. Because neurostatins and their analogues show specific activity and no toxicity for normal cells, a clinical trial would be the logical next step.
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One major challenge in the development of cancer therapeutics is the selective delivery of the drugs to their cellular targets. In the case of pancreatic cancer, the σ-2 receptor is a unique target that triggers apoptosis upon activation. We have previously developed a series of chemical compounds with high affinity for the σ-2 receptor and showed rapid internalization of the ligands. One particular specific ligand of the σ-2 receptor, SV119, binds to pancreatic cancer cells and induces target cell death in vitro and in vivo. In this study, we characterized the ability of SV119 to selectively deliver other death-inducing cargos to augment the cytotoxic properties of SV119 itself. When conjugated to SV119, small molecules that are known to interfere with intracellular prosurvival pathways retained their ability to induce cell death, the efficiency of which was enhanced by the combinatorial effect of SV119 delivered with its small molecule cargo. Our findings define a simple platform technology to increase the tumor-selective delivery of small molecule therapeutics via σ-2 ligands, permitting chemotherapeutic synergy that can optimize efficacy and patient benefit.
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Reproductive cloning by somatic cell nuclear transfer (SCNT) is a valuable method to propagate service dogs with desirable traits because of higher selection rates in cloned dogs. However, incomplete reprogramming is a major barrier to SCNT, and the assessment of reprogramming is limited to preimplantation embryos and tissues from dead and/or adult tissue. Thus, lifelong monitoring in SCNT dogs can be useful to evaluate the SCNT service dogs for propagation. We applied microarray and qRT-PCR to profile of mRNA and miRNA in whole blood samples collected from four cloned dogs (S), three age-matched control dogs (A), and a donor dog (D). In the analysis of differentially expressed genes in S-A, A-D, and S-D pairs, most genomes were completely reprogrammed and rejuvenated in the cloned offspring. However, several RNAs were differentially expressed. Interestingly, the altered genes are associated with aging and senescence. Furthermore, we identified potential biomarkers such as mirR-223 (NFIB; CLIC4), miRN-494 (ARHGEF12), miR-106b (PPP1R3B; CC2D1A), miR-20a (CC2D1A; PPP1R3B), miR-30e (IGJ; HIRA), and miR-19a (TNRC6A) by miRNA-target mRNA pairing for monitoring rejuvenation, aging/senescence, and reprogramming in cloned dogs. The novel comparative transcriptomic information about SCNT and age-matched dogs can be used to assess the lifelong health of cloned dogs and to facilitate the selection of training animals with minimal invasive procedures.
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Promoter hypermethylation and histone deacetylation are common epigenetic mechanisms implicated in the transcriptional silencing of tumor suppressor genes in human cancer. We treated two immortalized glioma cell lines, T98 and U87, and 10 patient-derived primary glioma cell lines with trichostatin A (TSA), a histone deacetylase inhibitor, or 5-aza-2'-deoxycytidine (5-AzaC), a DNA methyltransferase inhibitor, to comprehensively identify the cohort of genes reactivated through the pharmacologic reversal of these distinct but related epigenetic processes. Whole-genome microarray analysis identified genes induced by TSA (653) or 5-AzaC treatment (170). We selected a subset of reactivated genes that were markedly induced (greater than two-fold) after treatment with either TSA or 5-AzaC in a majority of glioma cell lines but not in cultured normal astrocytes. We then characterized the degree of promoter methylation and transcriptional silencing of selected genes in histologically confirmed human tumor and nontumor brain specimens. We identified two novel brain expressed genes, BEX1 and BEX2, which were silenced in all tumor specimens and exhibited extensive promoter hypermethylation. Viral-mediated reexpression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis and potent tumor suppressor effects in vitro and in a xenograft mouse model. Using an integrated approach, we have established a novel platform for the genome-wide screening of epigenetically silenced genes in malignant glioma. This experimental paradigm provides a powerful new method for the identification of epigenetically silenced genes with potential function as tumor suppressors, biomarkers for disease diagnosis and detection, and therapeutically reversible modulators of critical regulatory pathways important in glioma pathogenesis.
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We investigated 34 oligodendroglial tumors (7 oligodendrogliomas, 11 anaplastic oligodendrogliomas, 8 oligoastrocytomas, and 8 anaplastic oligoastrocytomas) for deletion, mutation, hypermethylation, and expression of the CDKN2A (MTS1, p16(INK4a)), p14(ARF), and CDKN2B (MTS2, p15(INK4b)) tumor suppressor genes at 9p21. One anaplastic oligoastrocytoma carried a homozygous deletion including all 3 genes. None of the tumors demonstrated point mutations in any of the genes. Methylation-specific polymerase chain reaction (MSP) analysis and sequencing of bisulfite-modi fled DNA, however, revealed frequent hypermethylation of the 5'-CpG islands in CDKN2A, p14(ARF), and CDKN2B. Partial or complete methylation of the majority of CpG sites analyzed from each gene was detected in 32% of the tumors at the CDKN2A gene and at a similar Percentage (41%) of the tumors at the p14(ARF) gene and the CDKN2B gene. Most tumors with CDKN2A, p14(ARF), and/or CDKN2B hypermethylation either lacked detectable transcripts from these genes or had lower mRNA levels than those determined for non-neoplastic brain tissue. There was a significant correlation between hypermethylation. of these genes and the presence of allelie losses on chromosomal arms 1p and 19q. In addition, p14(ARF) hypermethylation was predominantly found in tumors without a demonstrated TP53 mutation. Taken together, our results indicate that hypermethylation of CDKN2A, p14(ARF), and CDKN2B is an important epigenetic mechanism by which oligodendroglial tumors may escape from p53- and pRb-dependent growth control.
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Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma (WHO grade III) and/or glioblastoma (WHO grade IV). The molecular basis of astrocytoma progression is still poorly understood, in particular with respect to the progression-associated changes at the mRNA level. Therefore, we compared the transcriptional profile of approximately 6800 genes in primary WHO grade II gliomas and corresponding recurrent high-grade (WHO grade III or IV) gliomas from eight patients using oligonucleotide-based microarray analysis. We identified 66 genes whose mRNA levels differed significantly (P < 0.01, > or =2-fold change) between the primary and recurrent tumors. The microarray data were corroborated by real-time reverse transcription-polymerase chain reaction analysis of 12 selected genes, including 7 genes with increased expression and 5 genes with reduced expression on progression. In addition, the expression of these 12 genes was determined in an independent series of 43 astrocytic gliomas (9 diffuse astrocytomas, 10 anaplastic astrocytomas, 17 primary, and 7 secondary glioblastomas). These analyses confirmed that the transcript levels of nine of the selected genes (COL4A2, FOXM1, MGP, TOP2A, CENPF, IGFBP4, VEGFA, ADD3, and CAMK2G) differed significantly in WHO grade II astrocytomas as compared to anaplastic astrocytomas and/or glioblastomas. Thus, we identified and validated a set of interesting candidate genes whose differential expression likely plays a role in astrocytoma progression.
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Glioblastomas frequently carry mutations in the PTEN tumor suppressor gene on 10q23.3. The tumor suppressor properties of Pten are closely related to its inhibitory effect on the phosphatidyl-inositol-3'-kinase (Pi3k)-dependent activation of protein kinase B (Akt) signalling. Here, we report on the analysis of 17 genes related to the Pi3k/Akt signalling pathway for genetic alteration and aberrant expression in a series of 103 glioblastomas. Mutation, homozygous deletion or loss of expression of PTEN was detected in 32% of the tumors. In contrast, we did not find any aberrations in the inositol polyphosphate phosphatase like-1 gene (INPPL1), whose gene product may also counteract Pi3k-dependent Akt activation. Analysis of genes encoding proteins that may activate the pathway upstream of Pi3k revealed variable fractions of tumors with EGFR amplification (31%), PDGFRA amplification (8%), and IRS2 amplification (2%). The protein tyrosine kinase 2 (PTK2/FAK1) gene was neither amplified nor overexpressed at the mRNA level. Investigation of three genes encoding catalytic subunits of Pi3k (PIK3CA, PIK3CD, and PIK3C2B) revealed amplification of PIK3C2B (1q32) in 6 tumors (6%). Overexpression of PIK3C2B mRNA was detected in 4 of these cases. PIK3CD (1p36.2) and PIK3CA (3q26.3) were not amplified but PIK3CD mRNA was overexpressed in 6 tumors (6%). Amplification and overexpression of AKT1 was detected in a single case of gliosarcoma. The IRS1, PIK3R1, PIK3R2, AKT2, AKT3, FRAP1, and RPS6KB1 genes were neither amplified nor overexpressed in any of the tumors. Taken together, our data indicate that different genes related to the Pi3k/Akt signalling pathway may be aberrant in glioblastomas.
Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice
  • Ec Holland
  • J Celestino
  • C Dai
  • L Schaefer
  • Re Sawaya
  • Gn Fuller
Holland EC, Celestino J, Dai C, Schaefer L, Sawaya RE, Fuller GN. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat Genet 2000;25:55–7.