[show abstract][hide abstract] ABSTRACT: Despite progress in the determination of miR interactions, their regulatory role in cancer is only beginning to be unraveled. Utilizing gene expression data from 27 glioblastoma samples we found that the mere knowledge of physical interactions between specific mRNAs and miRs can be used to determine associated regulatory interactions, allowing us to identify 626 associated interactions, involving 128 miRs that putatively modulate the expression of 246 mRNAs. Experimentally determining the expression of miRs, we found an over-representation of over(under)-expressed miRs with various predicted mRNA target sequences. Such significantly associated miRs that putatively bind over-expressed genes strongly tend to have binding sites nearby the 3'UTR of the corresponding mRNAs, suggesting that the presence of the miRs near the translation stop site may be a factor in their regulatory ability. Our analysis predicted a significant association between miR-128 and the protein kinase WEE1, which we subsequently validated experimentally by showing that the over-expression of the naturally under-expressed miR-128 in glioma cells resulted in the inhibition of WEE1 in glioblastoma cells.
PLoS ONE 01/2011; 6(2):e14681. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: ST7 has been proposed to be a tumor suppressor gene in the chromosome region 7q31.1-q31.2. In order to gain some insight into its role in cancer, the localization and verification of the ST7 expression levels were determined.
Various types of ST7 expression vectors tagged with the sequences of GFP, YFP or V5 were created using a gateway cloning system and full-length ST7 cDNA isolated from a human adult brain cDNA library. Cell cycle synchronization was also performed to analyze the expression of endogenous ST7 and its potentially related genes at each stage of the cell cycle.
Cytosolic ST7 expression in HCT-116, MCF-7 and PC-3 cancer cell lines was detected via the fluorescence signal of the fusion proteins. ST7 translocation from the cytoplasm to the nucleus has not been observed in any of the conditions assayed. A cell cycle synchronization study demonstrated that both ST7 and SERPINE1 were overexpressed when cells were arrested. Expression of these genes was found to be diminished when the cells re-entered cell division status. In addition, we also found that Survivin, MMP-13 and Cyclin D1 were differentially expressed during the cell cycle.
Our findings suggest that ST7 mediates tumor suppression through the regulation of the genes involved in maintaining the cellular structure of the cell and involved in oncogenic pathways.
Journal of Cancer Research and Clinical Oncology 03/2010; 137(1):89-97. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gliomas are the most common type of primary brain tumors in adults and a significant cause of cancer-related mortality. Defining glioma subtypes based on objective genetic and molecular signatures may allow for a more rational, patient-specific approach to therapy in the future. Classifications based on gene expression data have been attempted in the past with varying success and with only some concordance between studies, possibly due to inherent bias that can be introduced through the use of analytic methodologies that make a priori selection of genes before classification. To overcome this potential source of bias, we have applied two unsupervised machine learning methods to genome-wide gene expression profiles of 159 gliomas, thereby establishing a robust glioma classification model relying only on the molecular data. The model predicts for two major groups of gliomas (oligodendroglioma-rich and glioblastoma-rich groups) separable into six hierarchically nested subtypes. We then identified six sets of classifiers that can be used to assign any given glioma to the corresponding subtype and validated these classifiers using both internal (189 additional independent samples) and two external data sets (341 patients). Application of the classification system to the external glioma data sets allowed us to identify previously unrecognized prognostic groups within previously published data and within The Cancer Genome Atlas glioblastoma samples and the different biological pathways associated with the different glioma subtypes offering a potential clue to the pathogenesis and possibly therapeutic targets for tumors within each subtype.
Cancer Research 03/2009; 69(5):2091-9. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Primary brain tumors are a major cause of cancer mortality in the United States. Therapy for gliomas, the most common type of primary brain tumors, remains suboptimal. The development of improved therapeutics will require greater knowledge of the biology of gliomas at both the genomic and transcriptional levels. We have previously reported whole genome profiling of chromosome copy number alterations (CNA) in gliomas, and now present our findings on how those changes may affect transcription of genes that may be involved in tumor induction and progression. By calculating correlation values of mRNA expression versus DNA copy number average in a moving window around a given RNA probe set, biologically relevant information can be gained that is obscured by the analysis of a single data type. Correlation coefficients ranged from -0.6 to 0.7, highly significant when compared with previous studies. Most correlated genes are located on chromosomes 1, 7, 9, 10, 13, 14, 19, 20, and 22, chromosomes known to have genomic alterations in gliomas. Additionally, we were able to identify CNAs whose gene expression correlation suggests possible epigenetic regulation. This analysis revealed a number of interesting candidates such as CXCL12, PTER, and LRRN6C, among others. The results have been verified using real-time PCR and methylation sequencing assays. These data will further help differentiate genes involved in the induction and/or maintenance of the tumorigenic process from those that are mere passenger mutations, thereby enriching for a population of potentially new therapeutic molecular targets.
Cancer Research 03/2009; 69(4):1596-603. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, is involved in diverse cellular processes ranging from nutrient and energy homeostasis to proliferation and apoptosis. Its role in glioblastoma multiforme has yet to be elucidated. We identified GSK3 as a regulator of glioblastoma multiforme cell survival using microarray analysis and small-molecule and genetic inhibitors of GSK3 activity. Various molecular and genetic approaches were then used to dissect out the molecular mechanisms responsible for GSK3 inhibition-induced cytotoxicity. We show that multiple small molecular inhibitors of GSK3 activity and genetic down-regulation of GSK3alpha/beta significantly inhibit glioma cell survival and clonogenicity. The potency of the cytotoxic effects is directly correlated with decreased enzyme activity-activating phosphorylation of GSK3alpha/beta Y276/Y216 and with increased enzyme activity inhibitory phosphorylation of GSK3alpha S21. Inhibition of GSK3 activity results in c-MYC activation, leading to the induction of Bax, Bim, DR4/DR5, and tumor necrosis factor-related apoptosis-inducing ligand expression and subsequent cytotoxicity. Additionally, down-regulation of GSK3 activity results in alteration of intracellular glucose metabolism resulting in dissociation of hexokinase II from the outer mitochondrial membrane with subsequent mitochondrial destabilization. Finally, inhibition of GSK3 activity causes a dramatic decrease in intracellular nuclear factor-kappaB activity. Inhibition of GSK3 activity results in c-MYC-dependent glioma cell death through multiple mechanisms, all of which converge on the apoptotic pathways. GSK3 may therefore be an important therapeutic target for gliomas. Future studies will further define the optimal combinations of GSK3 inhibitors and cytotoxic agents for use in gliomas and other cancers.
Cancer Research 09/2008; 68(16):6643-51. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: FK506 binding protein 5 (FKBP5) belongs to a family of immunophilins named for their ability to bind immunosuppressive drugs, also known as peptidyl-prolyl cis-trans isomerases, and also with chaperones to help protein folding. Using glioma cDNA microarray analysis, we found that FKBP5 was overexpressed in glioma tumors. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The roles of FKBP5 in glioma cells were then examined. We found that cell growth was suppressed after FKBP5 expression was inhibited by short interfering RNA transfection and enhanced by FKBP5 overexpression. Electrophoretic mobility shift assay showed that nuclear factor-kappa B (NF-kappaB) and DNA binding was enhanced by FKBP5 overexpression. The expression level of I-kappa B alpha and phosphorylated NF-kappaB was regulated by the expression of FKBP5. These data suggest that FKBP5 is involved in NF-kappaB pathway activation in glioma cells. In addition, FKBP5 overexpression in rapamycin-sensitive U87 cells blocked the cells' response to rapamycin treatment, whereas rapamycin-resistant glioma cells, both PTEN-positive and -negative, were synergistically sensitive to rapamycin after FKBP5 was knocked down, suggesting that the FKBP5 regulates glioma cell response to rapamycin treatment. In conclusion, our study demonstrates that FKBP5 plays an important role in glioma growth and chemoresistance through regulating signal transduction of the NF-kappaB pathway.
Neoplasia (New York, N.Y.) 04/2008; 10(3):235-43. · 5.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: Despite similarities between tumor-initiating cells with stem-like properties (TICs) and normal neural stem cells, we hypothesized that there may be differences in their differentiation potentials. We now demonstrate that both bone morphogenetic protein (BMP)-mediated and ciliary neurotrophic factor (CNTF)-mediated Jak/STAT-dependent astroglial differentiation is impaired due to EZH2-dependent epigenetic silencing of BMP receptor 1B (BMPR1B) in a subset of glioblastoma TICs. Forced expression of BMPR1B either by transgene expression or demethylation of the promoter restores their differentiation capabilities and induces loss of their tumorigenicity. We propose that deregulation of the BMP developmental pathway in a subset of glioblastoma TICs contributes to their tumorigenicity both by desensitizing TICs to normal differentiation cues and by converting otherwise cytostatic signals to proproliferative signals.
Cancer Cell 02/2008; 13(1):69-80. · 24.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genetic aberrations, such as gene amplification, deletions, and loss of heterozygosity, are hallmarks of cancer and are thought to be major contributors to the neoplastic process. Established cancer cell lines have been the primary in vitro and in vivo models for cancer for more than 2 decades; however, few such cell lines have been extensively characterized at the genomic level. Here, we present a high-resolution genome-wide chromosomal alteration and gene expression analyses of five of the most commonly used glioma cell lines and compare the findings with those observed in 83 primary human gliomas. Although genomic alterations known to occur in primary tumors were identified in the cell lines, we also observed several novel recurrent aberrations in the glioma cell lines that are not frequently represented in primary tumors. Additionally, a global gene expression cluster distinct from primary tumors was identified in the glioma cell lines. Our results indicate that established cell lines are generally a poor representation of primary tumor biology, presenting a host of genomic and gene expression changes not observed in primary tissues, although some discrete features of glioma biology were conserved in the established cell lines. Refined maps of genetic alterations and transcriptional divergence from the original tumor type, such as the one presented here, may help serve as a guideline for a more biologically rational and clinically relevant selection of the most appropriate glioma model for a given experiment.
Molecular Cancer Research 02/2008; 6(1):21-30. · 4.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Malignant gliomas are generally resistant to all conventional therapies. Notable exceptions are anaplastic oligodendrogliomas with loss of heterozygosity on chromosome 1p (1p+/-). Patients with 1p+/- anaplastic oligodendroglioma frequently respond to procarbazine, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and vincristine. Because the underlying biologic basis for this clinical finding is unclear, we evaluated differentially expressed 1p-encoded proteins in 1p+/- and 1p+/+ malignant glioma cell lines and then examined whether their expression was associated with outcome of patients with anaplastic oligodendroglioma.
We used a comparative proteomic screen of A172 (1p+/-) and U251 (1p+/+) malignant glioma cell lines to identify differentially expressed 1p-encoded proteins, including stathmin, a microtubule-associated protein. 1p+/- and 1p+/+ anaplastic oligodendroglioma specimens from 24 patients were assessed for stathmin expression by immunohistochemistry. The relationship between stathmin expression and clinical outcome was assessed with Kaplan-Meier analyses. RNA inhibition and cDNA transfection experiments tested effects of stathmin under- and overexpression, respectively, on the in vitro and in vivo resistance of malignant glioma cells to treatment with nitrosourea. For in vivo resistance studies, 36 mice with intracranial and 16 mice with subcutaneous xenograft tumor implants were used (one tumor per mouse). Flow cytometry was used for cell cycle analysis. Immunoblotting was used to assess protein expression. All statistical tests were two-sided.
Decreased stathmin expression in tumors was statistically significantly associated with loss of heterozygosity in 1p (P<.001) and increased recurrence-free survival (P<.001). The median recurrence-free survival times for patients with tumors expressing low, intermediate, or high stathmin levels were 45 months (95% confidence interval [CI] = 0 to 90 months), 17 months (95% CI = 10.6 to 23.4 months), and 6 months (95% CI = 1.7 to 10.3 months), respectively. Expression of stathmin was inversely associated with overall survival of nitrosourea-treated mice carrying xenograft tumors. Median survival of mice with stathmin+/- tumors was 95 days (95% CI = 68.7 to 121.3 days) and that of mice with stathmin+/+ tumors was 64 days (95% CI = 58.2 to 69.8 days) (difference = 31 days, 95% CI = 4.1 to 57.9 days; P<.001, log-rank test). Nitrosoureas induced mitotic arrest in malignant glioma cells, and this effect was greater in cells with decreased stathmin expression.
Loss of heterozygosity for the stathmin gene may be associated with improved outcomes of patients with 1p+/- anaplastic oligodendroglioma tumors.
[show abstract][hide abstract] ABSTRACT: Primary brain tumors are the fourth leading cause of cancer mortality in adults under the age of 54 years and the leading cause of cancer mortality in children in the United States. Therapy for the most common type of primary brain tumors, gliomas, remains suboptimal. The development of new and more effective treatments will likely require a better understanding of the biology of these tumors. Here, we show that use of the high-density 100K single-nucleotide polymorphism arrays in a large number of primary tumor samples allows for a much higher resolution survey of the glioma genome than has been previously reported in any tumor type. We not only confirmed alterations in genomic areas previously reported to be affected in gliomas, but we also refined the location of those sites and uncovered multiple, previously unknown regions that are affected by copy number alterations (amplifications, homozygous and heterozygous deletions) as well as allelic imbalances (loss of heterozygosity/gene conversions). The wealth of genomic data produced may allow for the development of a more rational molecular classification of gliomas and serve as an important starting point in the search for new molecular therapeutic targets.
Cancer Research 11/2006; 66(19):9428-36. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, is involved in diverse cellular processes ranging from nutrient and energy homeostasis to proliferation and apoptosis. Its role in glioblastoma multiforme has yet to be elucidated. We identified GSK3 as a regulator of glioblastoma multiforme cell survival using microarray analysis and small-molecule and genetic inhibitors of GSK3 activity. Various molecular and genetic approaches were then used to dissect out the molecular mechanisms responsible for GSK3 inhibition- induced cytotoxicity. We show that multiple small molecular inhibitors of GSK3 activity and genetic down-regulation of GSK3A/B significantly inhibit glioma cell survival and clonogenicity. The potency of the cytotoxic effects is directly correlated with decreased enzyme activity-activating phos- phorylation of GSK3A/B Y276/Y216 and with increased enzyme activity inhibitory phosphorylation of GSK3A S21. Inhibition of GSK3 activity results in c-MYC activation, leading to the induction of Bax, Bim, DR4/DR5, and tumor necrosis factor-related apoptosis-inducing ligand expression and sub- sequent cytotoxicity. Additionally, down-regulation of GSK3 activity results in alteration of intracellular glucose metabo- lism resulting in dissociation of hexokinase II from the outer mitochondrial membrane with subsequent mitochondrial destabilization. Finally, inhibition of GSK3 activity causes a dramatic decrease in intracellular nuclear factor-KB activity. Inhibition of GSK3 activity results in c-MYC-dependent glioma cell death through multiple mechanisms, all of which converge on the apoptotic pathways. GSK3 may therefore be an important therapeutic target for gliomas. Future studies will further define the optimal combinations of GSK3 inhibitors and cytotoxic agents for use in gliomas and other cancers. (Cancer Res 2008;68(16):6643-51)