FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

Atopic conditions and brain tumor risk in children and adolescents--an international case-control study (CEFALO)

BACKGROUND A number of epidemiological studies indicate an inverse association between atopy and brain tumors in adults, particularly gliomas. We investigated the association between atopic disorders and intracranial brain tumors in children and adolescents, using international collaborative CEFALO data. PATIENTS AND METHODS CEFALO is a population-based case-control study conducted in Denmark, Norway, Sweden, and Switzerland, including all children and adolescents in the age range 7-19 years diagnosed with a primary brain tumor between 2004 and 2008. Two controls per case were randomly selected from population registers matched on age, sex, and geographic region. Information about atopic conditions and potential confounders was collected through personal interviews. RESULTS In total, 352 cases (83%) and 646 controls (71%) participated in the study. For all brain tumors combined, there was no association between ever having had an atopic disorder and brain tumor risk [odds ratio 1.03; 95% confidence interval (CI) 0.70-1.34]. The OR was 0.76 (95% CI 0.53-1.11) for a current atopic condition (in the year before diagnosis) and 1.22 (95% CI 0.86-1.74) for an atopic condition in the past. Similar results were observed for glioma. CONCLUSIONS There was no association between atopic conditions and risk of all brain tumors combined or of glioma in particular. Stratification on current or past atopic conditions suggested the possibility of reverse causality, but may also the result of random variation because of small numbers in subgroups. In addition, an ongoing tumor treatment may affect the manifestation of atopic conditions, which could possibly affect recall when reporting about a history of atopic diseases. Only a few studies on atopic conditions and pediatric brain tumors are currently available, and the evidence is conflicting.

Mechanisms of Adenovirus-Mediated Autophagy

A patient diagnosed with a glioma, generally, has an average of 14 months year to live after implementation of conventional therapies such as surgery, chemotherapy, and radiation. Glioblastomas are highly lethal because of their aggressive nature and resistance to conventional therapies and apoptosis. Thus other avenues of cell death urgently need to be explored. Autophagy, which is also known as programmed cell death type II, has recently been identified as an alternative mechanism to kill apoptosis- resistant cancer cells. Traditionally, researchers have studied how cells undergo autophagy during viral infection as an immune response mechanism, but recently researchers have discovered how viruses have evolved to manipulate autophagy for their benefit. Extensive studies of viral-induced autophagy provide a rationale to investigate other viruses, such as the adenovirus, which may be developed as part of a therapy against cancers resistant to apoptosis. Despite the present and relatively poor understanding of the mechanisms behind adenoviral-induced autophagy, adenovirus is a promising candidate, because of its ability to efficiently eradicate tumors. A better understanding of how the adenovirus induces autophagy will allow for the development of viruses with increased oncolytic potency. We hypothesized that adenovirus induces autophagy in order to aid in lysis. We found that replication, not infection, was required for adenovirus-mediated autophagy. Loss of function analysis of early genes revealed that, of the early genes tested, no single gene was sufficient to induce autophagy alone. Examination of cellular pathways for their role in autophagy during adenovirus infection revealed a function for the eIF2α pathway and more specifically the GCN2 kinase. Cells lacking GCN2 are more resistant to adenovirus-mediated autophagy in vitro; in vivo we also found these cells fail to undergo autophagy, but display more cell death. We believe that autophagy is a protective mechanism the cell employs during adenoviral infection, and in the in vivo environment, cells cannot recover from virus infection and are more susceptible to death. Congruently, infected cells deficient for autophagy through deletion of ATG5 are not able undergo productive cell lysis, providing evidence that the destruction of the cytoplasm and cell membrane through autophagy is crucial to the viral life cycle. This project is the first to describe a gene, other than a named autophagy gene, to be required for adenovirus- mediated autophagy. It is also the first to examine autophagic cell death as a means to aid in viral-induced cell lysis.

Mechanisms of Adenovirus-Mediated Autophagy

A patient diagnosed with a glioma, generally, has an average of 14 months year to live after implementation of conventional therapies such as surgery, chemotherapy, and radiation. Glioblastomas are highly lethal because of their aggressive nature and resistance to conventional therapies and apoptosis. Thus other avenues of cell death urgently need to be explored. Autophagy, which is also known as programmed cell death type II, has recently been identified as an alternative mechanism to kill apoptosis- resistant cancer cells. Traditionally, researchers have studied how cells undergo autophagy during viral infection as an immune response mechanism, but recently researchers have discovered how viruses have evolved to manipulate autophagy for their benefit. Extensive studies of viral-induced autophagy provide a rationale to investigate other viruses, such as the adenovirus, which may be developed as part of a therapy against cancers resistant to apoptosis. Despite the present and relatively poor understanding of the mechanisms behind adenoviral-induced autophagy, adenovirus is a promising candidate, because of its ability to efficiently eradicate tumors. A better understanding of how the adenovirus induces autophagy will allow for the development of viruses with increased oncolytic potency. We hypothesized that adenovirus induces autophagy in order to aid in lysis. We found that replication, not infection, was required for adenovirus-mediated autophagy. Loss of function analysis of early genes revealed that, of the early genes tested, no single gene was sufficient to induce autophagy alone. Examination of cellular pathways for their role in autophagy during adenovirus infection revealed a function for the eIF2α pathway and more specifically the GCN2 kinase. Cells lacking GCN2 are more resistant to adenovirus-mediated autophagy in vitro; in vivo we also found these cells fail to undergo autophagy, but display more cell death. We believe that autophagy is a protective mechanism the cell employs during adenoviral infection, and in the in vivo environment, cells cannot recover from virus infection and are more susceptible to death. Congruently, infected cells deficient for autophagy through deletion of ATG5 are not able undergo productive cell lysis, providing evidence that the destruction of the cytoplasm and cell membrane through autophagy is crucial to the viral life cycle. This project is the first to describe a gene, other than a named autophagy gene, to be required for adenovirus- mediated autophagy. It is also the first to examine autophagic cell death as a means to aid in viral-induced cell lysis.

Development of a Bayesian Joint Logistic Model to Better Study the Association between Haplotypes and Disease

In 2011, there will be an estimated 1,596,670 new cancer cases and 571,950 cancer-related deaths in the US. With the ever-increasing applications of cancer genetics in epidemiology, there is great potential to identify genetic risk factors that would help identify individuals with increased genetic susceptibility to cancer, which could be used to develop interventions or targeted therapies that could hopefully reduce cancer risk and mortality. In this dissertation, I propose to develop a new statistical method to evaluate the role of haplotypes in cancer susceptibility and development. This model will be flexible enough to handle not only haplotypes of any size, but also a variety of covariates. I will then apply this method to three cancer-related data sets (Hodgkin Disease, Glioma, and Lung Cancer). I hypothesize that there is substantial improvement in the estimation of association between haplotypes and disease, with the use of a Bayesian mathematical method to infer haplotypes that uses prior information from known genetics sources. Analysis based on haplotypes using information from publically available genetic sources generally show increased odds ratios and smaller p-values in both the Hodgkin, Glioma, and Lung data sets. For instance, the Bayesian Joint Logistic Model (BJLM) inferred haplotype TC had a substantially higher estimated effect size (OR=12.16, 95% CI = 2.47-90.1 vs. 9.24, 95% CI = 1.81-47.2) and more significant p-value (0.00044 vs. 0.008) for Hodgkin Disease compared to a traditional logistic regression approach. Also, the effect sizes of haplotypes modeled with recessive genetic effects were higher (and had more significant p-values) when analyzed with the BJLM. Full genetic models with haplotype information developed with the BJLM resulted in significantly higher discriminatory power and a significantly higher Net Reclassification Index compared to those developed with haplo.stats for lung cancer. Future analysis for this work could be to incorporate the 1000 Genomes project, which offers a larger selection of SNPs can be incorporated into the information from known genetic sources as well. Other future analysis include testing non-binary outcomes, like the levels of biomarkers that are present in lung cancer (NNK), and extending this analysis to full GWAS studies.

APPLICATIONS OF EPHB4 RECEPTOR SPECIFIC PEPTIDES IN TARGETED CANCER IMAGING AND THERAPY

EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are found over-expressed in a variety of tumors cells including glioma cells as well as angiogenic blood vessels. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed against EphB4, and improve patient outcomes. Targeted delivery of EphB4 receptor specific peptide conjugated hollow gold nanoshells (HAuNS) into tumors has great potential in cancer imaging and photothermal therapy. In this study, we developed an EphB4 specific peptide named TNYL-RAW and labeled with radioisotope 64Cu and Cy5.5 dye. We also conjugate this specific peptide with hollow gold nanoshells (HAuNS) to evaluate targeted photothermal therapy of cancers. In vitro, 64Cu-DOTA-TNYL- RAW specifically bind to CT26 and PC-3M cells but not to A549 cells. In vivo, Small-animal PET/CT clearly showed the significant uptake of 64Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, µPET/CT and near-infrared optical imaging clearly showed the uptake of the dual labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of nude mice. In U251 tumors, Cy5.5-labeled peptide can bind to EphB4-expressing tumor blood vessels and tumors cells. But in U87 models, dual labeled peptide only could bind to tumor associated blood vessels. Also, Irradiation of PC-3M and CT-26 cell treated with TNYL-PEG-HAuNS nanopatilces with near-infrared (NIR) laser resulted in selective destruction of these cells in vitro. EphB4 targeted TNYL-PEG-HAuNS showed more photothermal killing effect on CT26 tumor model than PEG-HAuNS did. In summary, tumors with overexpression of EphB4 receptors can be noninvasively visualized by micro PET/CT with 64Cu labeled or dual labeled TNYL-RAW peptide. Targeted delivery of TNYL-RAW conjugated HAuNS into tumors can greatly improve the treatment effect of photothermal therapy. The information acquired with this study should be advantageous in improving diagnostics and future applications in photothermal ablation therapy in clinical.

THE ROLE OF RECEPTOR TYROSINE KINASE AXL IN PANCREATIC DUCTAL ADENOCARCINOMA AND ITS REGULATION BY HEMATOPOIETIC PROGENITOR KINASE 1

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies with less than 5% of five year survival rate. New molecular markers and new therapeutic targets are urgently needed for patients with PDA. Oncogenic receptor tyrosine kinase Axl has been reported to be overexpressed in many types of human malignancies, including diffuse glioma, melanoma, osteosarcoma, and carcinomas of lung, colon, prostate, breast, ovary, esophagus, stomach, and kidney. However, the expression and functions of Axl in PDA are unclear. We hypothesized that Axl contributes to the development and progression of PDA. We examined Axl expression in 54 human PDA samples and their paired benign pancreatic tissue by immunohistochemistry, we found that Axl was overexpressed in 70% of stage II PDAs, but only 22% of benign ducts (P=0.0001). Axl overexpression was associated with higher frequencies of distant metastasis and was an independent prognostic factor for both poor overall and recurrence-free survivals in patients with stage II PDA (p = 0.03 and 0.04). Axl silencing by shRNA in pancreatic cancer cell lines, panc-28 and Panc-1, decreased tumor cell migration and invasion and sensitized PDA cells to apoptosis stimuli such as γ-irradiation and serum starvation. In addition, we found that Axl-mediated Akt and NF-κB activation and up regulation of MMP2 were involved in the invasion, migration and survival of PDA cells. Thus, we demonstrate that Axl plays an important role in the development and progression of PDA. Targeting Axl signaling pathway may represent a new approach for the treatment of PDA. To understand the molecular mechanisms of Axl overexpression in PDA, we found that Axl expression was down-regulated by hematopoietic progenitor kinase 1 (HPK1), a newly identified tumor suppressor in PDA. HPK1 is lost in over 95% of PDAs. Restoration of HPK1 in PDA cells down-regulated Axl expression. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1, and monensin, suggesting that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway. HPK1 interacted with and phosphorylated dynamin, a critical component of endocytosis pathway. Overexpression of dominant negative form of dynamin blocked the HPK1-mediated Axl degradation. Therefore we concluded that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway and loss of HPK1 expression may contribute to Axl overexpression in PDAs.

Biologic and molecular analysis of tumor suppressor loci in human glioblastoma multiforme

Molecular and cytogenetic analyses of human glioblastomas have revealed frequent genetic alterations, including major deletions in chromosomes 9, 10, and 17, suggesting the presence of glioma-associated tumor suppressor genes on these chromosomes. To examine this hypothesis, copies of chromosomes 2, 4, and 10 derived from a human fibroblast cell line were independently introduced into a human glioma cell line, U251, by microcell-mediated chromosomal transfer. Successful transfer of chromosomes in each case was confirmed by resistance to the drug G418, indicating the presence of the neomycin-resistance gene previously integrated into each transferred chromosome. The presence of novel chromosomes and or chromosomal fragments was also demonstrated by molecular and karyotypic analyses. The hybrid clones containing either a novel chromosome 4 or chromosome 10 displayed suppression of the tumorigenic phenotype in vivo and suppression of the transformed phenotype in vitro, while cells containing a transferred chromosome 2 failed to alter their tumorigenic phenotype. The hybrid cells containing chromosome 4 or 10 exhibited a significant decrease in their saturation density, altered cellular morphology at high cell density, but only a slight decrease in their exponential growth rate. A dramatic decrease was observed in growth of cells with chromosome 4 or 10 in soft agarose, with the number and size of the colonies being greatly reduced, compared to the parental or chromosome 2 containing cells. The introduction of chromosome 4 or 10 also completely suppressed tumor formation in nude mice. These studies indicate that chromosome 10, as hypothesized, and chromosome 4, a novel finding for gliomas, harbor tumor suppressor loci that may be directly involved in the initiation or progression of normal glial precursors to human glioblastoma multiforme. ^

Analysis of differential gene expression in nontumorigenic glioblastoma multiforme

Loss of chromosome 10 represents the most common cytogenetic abnormality in high grade gliomas (glioblastoma multiforme). To identify genes involved in the malignant progression of human gliomas, a subtractive hybridization was performed between a tumorigenic glioblastoma cell line (LG11) and a nontumorgenic hybrid cell (LG11.3) containing an introduced chromosome 10. LG11 mRNA was subtracted from LG11.3 cDNA to produce cDNA probes enriched for sequences whose expression differs quantitatively from the parental tumorigenic cells. Both known and novel sequences were identified as a result of the subtraction. Northern blot analysis was then used to confirm differential expression of several subtracted clones. One novel clone, clone 17, identified a 2.6 kb message that showed a consistent two to four fold increase in expression in the LG11.3 nontumorigenic cells. Clone 17 (340 bp) was used successfully to screen for a near full-length version, RIG (regulated in glioma), which was 2,569 bp in size. The RIG cDNA sequence showed homology to clone 17 and to an anonymous EST (IB666), but to no previously identified genes. This screening effort also identified several independent clones representing novel sequences, most of which failed to show increased expression in the nontumorigenic GBM cells. Tissue distribution studies of RIG indicated highest levels of expression in human brain with appreciably lower levels in heart and lung. In vitro transcription and translation experiments demonstrated the ability of RIG to direct the synthesis of a 13 kD protein product. However, open reading frame analysis revealed no identify with previously described motifs or any known proteins. Using a combination of somatic cell hybrid panels and in situ hybridization, the RIG gene was mapped to chromosome 11p14-11p15. Further study of RIG and related gene products may provide insight into the negative regulation of glial oncogenesis. ^

Relationships between the alterations of tumor suppressor genes, risk factors and clinical outcomes of gliomas

The relationship between MMAC/PTEN, DMBT1 and the progression and prognosis of glioma, and the association between the alterations of MMAC/PTEN, p53, p16, and Rb and some cancer risk factors, such as smoking, exposure to radiation, family cancer history, and previous cancer history, were assessed in 4 studies. ^ By allelic deletion analysis, MMAC/PTEN locus was shown to be frequently lost in glioblastomas multiforme (GM) but maintained in most lower-grade astrocytic tumors. DMBT1 locus, however, was frequently lost in all grades of gliomas examined. The potential biological significance of these two regions was frontier assessed by examining microcell-hybrids that contained various fragments of 10q. Somatic cell hybrid clones that retained the MMAC/PTEN locus have less transformed phenotypes, exhibiting an inability to grow in soft agarose. On the other hand, the presence or absence of DAMT1 did not correlate with any in vitro phenotype assessed in our model system. Further, Cox proportional hazards regression analysis, adjusted for age at surgery and histologic grades (GM, and non-GM), showed that without LOH at the MMAC/PTEN locus had a significantly better prognosis than did patients with LOH at MMAC/ PTEN (hazard ratio = 0.5; 95% Cl = 0.28–0.89; P = 0.018). Furthermore, status of LOH at MMAC/PTEN was found to be significantly associated with age, while that for DMBT1 was not. These results suggest that the DMBT1 may be involved early in the oncogenesis of gliomas, while alterations in the MMAC /PTEN may be a late event in the oncogenesis related with progression of gliomas and provide a significant prognostic marker for patient survival. ^ The associations between 4 cancer risk factors and 4 tumor suppressor genes were assessed. The expression of p16 was observed to be associated with current smoking (adjusted OR = 1.9, 95% CI = 1.02–3.6) but not the former smoking (adjusted OR = 1.1, 95% Cl = 0.5–3.5). The expression of p53 was found to be associated with the family cancer history (OR = 3.5, 95% Cl = 1.07–11 for patients with first-degree family history of cancer). MMAC/ PTEN was associated with the histologic grade (OR = 2.8, 95% CI = 1.2–6.6) and age (P = 0.035). Also, the OR for LOH around MMAC/PTEN in patients with a family history of cancer was elevated (OR = 1.9, 95% CI = 0.8–4.6 for patients with first-degree family history of cancer). The associations between exposure and the alterations of tumor suppressor genes, between smoking and p16, between family history of cancer and p53 and MMAC/PTEN, provide suggestive evidences that those exposures are related to the development of gliomas. ^

Use of Genetically Modified Glial Cells Overexpressing Laminin α1-Chain Peptides in Neurite Outgrowth Studies

1. C6 glioma cells were transfected with two constructs carrying C-terminal laminin alpha1-chain sequences of 117 and 114 bp length, respectively. These sequences are specifically known to code for peptides which have neurite-promoting activity. 2. The stable expression and secretion of the two peptides was detected by Northern and Western blot analysis. 3. Primary neuronal cultures derived from embryonic mouse forebrain were cocultured with these transfected cells and exhibited a substantial increase in neurite outgrowth and in survival time. Conditioned media from the transfected cells generated similar effects. 4. Organotypic cultures from embryonic mouse brain were used as a second system as being closer to the in vivo situation. Again, coculture of brain slices with transfected cells or treatment with laminin peptide-containing media increased neuronal outgrowth.

Chameleons everywhere

We report the case of an HIV-infected man returning from Thailand with secondary syphilis with general symptoms, hepatitis and a pulmonary mass lesion. A cerebrospinal fluid examination showed no signs of neurosyphilis. Two months after successful treatment with benzathine penicillin he presented with a mass lesion in the brain suspected to be a glioma or glioblastoma, which turned out to be a syphilitic gumma. Syphilis remains a great imitator in clinical medicine. Syphilitic brain gummata can develop within a few months.

Patient-Specific Semi-Supervised Learning for Postoperative Brain Tumor Segmentation

In contrast to preoperative brain tumor segmentation, the problem of postoperative brain tumor segmentation has been rarely approached so far. We present a fully-automatic segmentation method using multimodal magnetic resonance image data and patient-specific semi-supervised learning. The idea behind our semi-supervised approach is to effectively fuse information from both pre- and postoperative image data of the same patient to improve segmentation of the postoperative image. We pose image segmentation as a classification problem and solve it by adopting a semi-supervised decision forest. The method is evaluated on a cohort of 10 high-grade glioma patients, with segmentation performance and computation time comparable or superior to a state-of-the-art brain tumor segmentation method. Moreover, our results confirm that the inclusion of preoperative MR images lead to a better performance regarding postoperative brain tumor segmentation.

Delay effects in the response of low-grade gliomas to radiotherapy: a mathematical model and its therapeutical implications

Low-grade gliomas (LGGs) are a group of primary brain tumours usually encountered in young patient populations. These tumours represent a difficult challenge because many patients survive a decade or more and may be at a higher risk for treatment-related complications. Specifically, radiation therapy is known to have a relevant effect on survival but in many cases it can be deferred to avoid side effects while maintaining its beneficial effect. However, a subset of LGGs manifests more aggressive clinical behaviour and requires earlier intervention. Moreover, the effectiveness of radiotherapy depends on the tumour characteristics. Recently Pallud et al. (2012. Neuro-Oncology, 14: , 1-10) studied patients with LGGs treated with radiation therapy as a first-line therapy and obtained the counterintuitive result that tumours with a fast response to the therapy had a worse prognosis than those responding late. In this paper, we construct a mathematical model describing the basic facts of glioma progression and response to radiotherapy. The model provides also an explanation to the observations of Pallud et al. Using the model, we propose radiation fractionation schemes that might be therapeutically useful by helping to evaluate tumour malignancy while at the same time reducing the toxicity associated to the treatment.

The Multimodal Brain TumorImage Segmentation Benchmark (BRATS)

In this paper we report the set-up and results of the Multimodal Brain Tumor Image Segmentation Benchmark (BRATS) organized in conjunction with the MICCAI 2012 and 2013 conferences. Twenty state-of-the-art tumor segmentation algorithms were applied to a set of 65 multi-contrast MR scans of low- and high-grade glioma patients - manually annotated by up to four raters - and to 65 comparable scans generated using tumor image simulation software. Quantitative evaluations revealed considerable disagreement between the human raters in segmenting various tumor sub-regions (Dice scores in the range 74-85%), illustrating the difficulty of this task. We found that different algorithms worked best for different sub-regions (reaching performance comparable to human inter-rater variability), but that no single algorithm ranked in the top for all subregions simultaneously. Fusing several good algorithms using a hierarchical majority vote yielded segmentations that consistently ranked above all individual algorithms, indicating remaining opportunities for further methodological improvements. The BRATS image data and manual annotations continue to be publicly available through an online evaluation system as an ongoing benchmarking resource.