Identification of miRNA-Mediated Core Gene Module for Glioma Patient Prediction by Integrating High-Throughput miRNA,mRNA Expression and Pathway Structure

The prognosis of glioma patients is usually poor, especially in patients with glioblastoma (World Health Organization (WHO) grade IV). The regulatory functions of microRNA (miRNA) on genes have important implications in glioma cell survival. However, there are not many studies that have investigated glioma survival by integrating miRNAs and genes while also considering pathway structure. In this study, we performed sample-matched miRNA and mRNA expression profilings to systematically analyze glioma patient survival. During this analytical process, we developed pathway-based random walk to identify a glioma core miRNA-gene module, simultaneously considering pathway structure information and multi-level involvement of miRNAs and genes. The core miRNA-gene module we identified was comprised of four apparent sub-modules; all four sub-modules displayed a significant correlation with patient survival in the testing set (P-values≤0.001). Notably, one sub-module that consisted of 6 miRNAs and 26 genes also correlated with survival time in the high-grade subgroup (WHO grade III and IV), P-value = 0.0062. Furthermore, the 26-gene expression signature from this sub-module had robust predictive power in four independent, publicly available glioma datasets. Our findings suggested that the expression signatures, which were identified by integration of miRNA and gene level, were closely associated with overall survival among the glioma patients with various grades.     

PAICS is related to glioma grade and can promote glioma growth and migration

Glioma is a common malignant tumour of the brain. In this study, we aimed to investigate diagnostic biomarkers and its role in glioma. Weighted gene co-expression network analysis (WGCNA) and Cytoscape software were used to screen the marker genes in glioma. RT-qPCR and Western blotting methods were performed to determine the expression of PAICS, ERCC1 and XPA genes in glioma tissues. Expression level of PAICS in different grades of glioma was examined by immunohistochemistry. CCK8 and Colony formation assays were used to detect cell proliferation. Cell adhesion assay was used to detect adhesion ability. Wound healing and transwell tests were used to detect cell migration ability. Flow cytometry was used to detect cell cycle and apoptosis. According to the predicted co-expression network, we identified the hub gene PAICS. Furthermore, we observed that PAICS expression level was up-regulated in glioma tissues compared with normal tissues, and the expression level was correlated with the grade of glioma. Moreover, we found PAICS can promote glioma cells proliferation and migration in vitro. Flow cytometry results showed that si-PAICS cells were stalled at the G1 phase compared with the si-NC cells and knocking down PAICS expression can increase apoptotic rate. PAICS can regulate the mRNA and protein levels of nucleotide excision repair pathway core genes ERCC1 and XPA. l -aspartic acid can affect the expression of PAICS and then inhibit glioma cell proliferation. Our results indicated that PAICS can promote glioma proliferation and migration. PAICS may act as a potential diagnostic marker and a therapeutic target for glioma.     

Identifying core gene modules in glioblastoma based on multilayer factor-mediated dysfunctional regulatory networks through integrating multi-dimensional genomic data

The driver genetic aberrations collectively regulate core cellular processes underlying cancer development. However, identifying the modules of driver genetic alterations and characterizing their functional mechanisms are still major challenges for cancer studies. Here, we developed an integrative multi-omics method CMDD to identify the driver modules and their affecting dysregulated genes through characterizing genetic alteration-induced dysregulated networks. Applied to glioblastoma (GBM), the CMDD identified a core gene module of 17 genes, including seven known GBM drivers, and their dysregulated genes. The module showed significant association with shorter survival of GBM. When classifying driver genes in the module into two gene sets according to their genetic alteration patterns, we found that one gene set directly participated in the glioma pathway, while the other indirectly regulated the glioma pathway, mostly, via their dysregulated genes. Both of the two gene sets were significant contributors to survival and helpful for classifying GBM subtypes, suggesting their critical roles in GBM pathogenesis. Also, by applying the CMDD to other six cancers, we identified some novel core modules associated with overall survival of patients. Together, these results demonstrate integrative multi-omics data can identify driver modules and uncover their dysregulated genes, which is useful for interpreting cancer genome.     

Proliferation: the Most Prominent Predictor of Clinical Outcome in Breast Cancer

We recently identified a gene expression cassette of 97 unique genes that were consistently differentially expressed between low and high grade breast carcinomas. The majority of these genes were overexpressed in high grade tumors and, as expected, they were associated with cell cycle progression and proliferation. Interestingly, by applying this gene expression cassette to several datasets, we demonstrated that intermediate grade tumors were composed of a mixture of well- and poorly- differentiated tumors with statistically distinct clinical outcome similar to those of low and high grade carcinomas. Furthermore, these proliferation-related genes appear to be a common denominator of several existing prognostic gene expression signatures. This recapitulates their prognostic power far beyond the estrogen receptor (ER) status and highlights the importance of proliferation genes in breast cancer biology. Importantly, their weight seems to be far more important in ER-positive than in ER-negative disease.     

Identification of Molecular Pathways Facilitating Glioma Cell Invasion In Situ

Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion.In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.     

Glutamate as chemotactic fuel for diffuse glioma cells: Are they glutamate suckers?

Diffuse gliomas comprise a group of primary brain tumors that originate from glial (precursor) cells and present as a variety of malignancy grades which have in common that they grow by diffuse infiltration. This phenotype complicates treatment enormously as it precludes curative surgery and radiotherapy. Furthermore, diffusely infiltrating glioma cells often hide behind a functional blood–brain barrier, hampering delivery of systemically administered therapeutic and diagnostic compounds to the tumor cells. The present review addresses the biological mechanisms that underlie the diffuse infiltrative phenotype, knowledge of which may improve treatment strategies for this disastrous tumor type. The invasive phenotype is specific for glioma: most other brain tumor types, both primary and metastatic, grow as delineated lesions. Differences between the genetic make-up of glioma and that of other tumor types may therefore help to unravel molecular pathways, involved in diffuse infiltrative growth. One such difference concerns mutations in the NADP⁺-dependent isocitrate dehydrogenase (IDH1 and IDH2) genes, which occur in > 80% of cases of low grade glioma and secondary glioblastoma. In this review we present a novel hypothesis which links IDH1 and IDH2 mutations to glutamate metabolism, possibly explaining the specific biological behavior of diffuse glioma.     

Decreased 5-hydroxymethylcytosine is associated with neural progenitor phenotype in normal brain and shorter survival in malignant glioma

Epigenetic modification of DNA by cytosine methylation to produce 5-methylcytosine (5mC) has become well-recognized as an important epigenetic process in human health and disease. Recently, further modification of 5mC by the ten eleven translocated (TET) family of enzymes to produce 5-hydroxymethylcytosine (5hmC) has been described. In the present study, we used immunohistochemistry to evaluate the distribution of 5hmC in human brain during different periods of development and in a large series of gliomas (n=225). We found that during development, 5hmC levels are high in more differentiated compartments like the fetal cortex, but low in the periventricular progenitor cell regions. In adults, we found 5hmC levels to be highest in the cortex, but present in all intrinsic cell types in the brain including stromal elements. In brain tumors, 5hmC levels were high in low grade tumors and reduced in malignant glioma, but did not exhibit any correlation with IDH1 mutation status. Additionally, we identified a significant relationship between low levels of 5hmC and reduced survival in malignant glioma. This observation was further supported by in silico analysis showing differential expression of genes involved in 5hmC homeostasis in aggressive subsets of glioblastoma. Finally, we show that several genes involved in regulating the levels of 5hmC are also prognostic in malignant glioma. These findings suggest that 5hmC regulation in malignant glioma may represent an important determinant of tumor differentiation and aggressive behavior, as well as a potential therapeutic target.     

Glioma IL13Rα2 Is Associated with Mesenchymal Signature Gene Expression and Poor Patient Prognosis

A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.     

Syndecan-1 expression in human glioma is correlated with advanced tumor progression and poor prognosis

Syndecan-1 has been implicated in tumorigenesis and progression of various human malignancies. Recent studies have demonstrated that syndecan-1 may have a different function and biological activity depending on the specific tumor type. Therefore, the aim of this study was to investigate the clinical significance of syndecan-1 in human gliomas. One hundred and sixteen glioma patients (26 World Health Organization (WHO) grade I, 30 WHO grade II, 30 WHO grade III, and 30 WHO grade IV) and 15 normal brain specimens acquired from 15 patients undergoing surgery for epilepsy as control were collected. Immunohistochemistry assay, quantitative real-time PCR and Western blot analysis were carried out to detect the expression of syndecan-1 at gene and protein levels in glioma samples with different WHO grades. Syndecan-1 gene and protein levels were both higher in glioma tissues compared to controls (both P < 0.001). In addition, its expression levels increased with ascending tumor WHO grades according to the results of immunohistochemistry assay, quantitative real-time PCR and Western blot analysis. Moreover, the survival rate of syndecan-1-positive patients was significantly lower than that of syndecan-1-negative patients (P = 0.006). We further confirmed that the increased expression of syndecan-1 was an independent prognostic indicator in glioma by multivariate analysis (P = 0.01). Our data suggest for the first time that the increased expression of syndecan-1 at gene and protein levels is correlated with advanced tumor progression and poor outcome in patients with glioma. Syndecan-1 might serve as a potential prognosis predictor of this dismal tumor.     

Molecular and cellular biology of neuroendocrine lung tumors: Evidence for separate biological entities

Pulmonary neuroendocrine tumors (NETs) are traditionally described as comprising a spectrum of neoplasms, ranging from low grade typical carcinoids (TCs) via the intermediate grade atypical carcinoids (ACs) to the highly malignant small cell lung cancers (SCLCs) and large cell neuroendocrine carcinomas (LCNECs). Recent data, however, suggests that two categories can be distinguished on basis of molecular and clinical data, i.e. the high grade neuroendocrine (NE) carcinomas and the carcinoid tumors. Bronchial carcinoids and SCLCs may originate from the same pulmonary NE precursor cells, but a precursor lesion has only been observed in association with carcinoids, termed diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. The occurrence of mixed tumors exclusively comprising high grade NE carcinomas also supports a different carcinogenesis for these two groups. Histopathologically, high grade NE lung tumors are characterized by high mitotic and proliferative indices, while carcinoids are defined by maximally 10 mitoses per 2mm(2) (10 high-power fields) and rarely have Ki67-proliferative indices over 10%. High grade NE carcinomas are chemosensitive tumors, although they usually relapse. Surgery is often not an option due to extensive disease at presentation and early metastasis, especially in SCLC. Conversely, carcinoids are often insensitive to chemo- and radiation therapy, but cure can usually be achieved by surgery. A meta-analysis of comparative genomic hybridization studies performed for this review, as well as gene expression profiling data indicates separate clustering of carcinoids and carcinomas. Chromosomal aberrations are much more frequent in carcinomas, except for deletion of 11q, which is involved in the whole spectrum of NE lung tumors. Deletions of chromosome 3p are rare in carcinoids but are a hallmark of the high grade pulmonary NE carcinomas. On the contrary, mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are restricted to carcinoid tumors. Many of the differences between carcinoids and high grade lung NETs can be ascribed to tobacco consumption, which is strongly linked to the occurrence of high grade NE carcinomas. Smoking causes p53 mutations, very frequently present in SCLCs and LCNECs, but rarely in carcinoids. It further results in other early genetic events in SCLCs and LCNECs, such as 3p and 17p deletions. Smoking induces downregulation of E-cadherin and associated epithelial to mesenchymal transition. Also, high grade lung NETs display higher frequencies of aberrations of the Rb pathway, and of the intrinsic and extrinsic apoptotic routes. Carcinoid biology on the other hand is not depending on cigarette smoke intake but rather characterized by aberrations of other specific genetic events, probably including Menin or its targets and interaction partners. This results in a gradual evolution, most likely from proliferating pulmonary NE cells via hyperplasia and tumorlets towards classical carcinoid tumors. We conclude that carcinoids and high grade NE lung carcinomas are separate biological entities and do not comprise one spectrum of pulmonary NETs. This implies the need to reconsider both diagnostic as well as therapeutic approaches for these different groups of malignancies.     

Extracellular Vesicles in Glioma: From Diagnosis to Therapy

Increasing evidence indicates that extracellular vesicles (EVs) secreted from tumor cells play a key role in the overall progression of the disease state. EVs such as exosomes are secreted by a wide variety of cells and transport a varied population of proteins, lipids, DNA, and RNA species within the body. Gliomas constitute a significant proportion of all primary brain tumors and majority of brain malignancies. Glioblastoma multiforme (GBM) represents grade IV glioma and is associated with very poor prognosis despite the cumulative advances in diagnostic procedures and treatment strategies. Here, the authors describe the progress in understanding the role of EVs, especially exosomes, in overall glioma progression, and how new research is unraveling the utilities of exosomes in glioma diagnostics and development of next‐generation therapeutic systems. Finally, based on an understanding of the latest scientific literature, a model for the possible working of therapeutic exosomes in glioma treatment is proposed.     

Chromosome-specific spatial periodicities in gene expression revealed by spectral analysis

Recent years have seen an unprecedented surge of research activity in studies of gene expression. This extensive work, however, has been almost uniformly focused on genome-wide gene expression and has largely ignored the fundamental fact that every gene has a specific chromosome location. We propose a novel method of spectral analysis for detecting hidden periodicities in gene expression signals ordered along the length of each chromosome. Using this method, we have discovered that each chromosome in rodents and humans has a unique periodic pattern of gene expression. The uncovered spatial periodicities in gene expression are tissue-specific in the sense that the largest differences in humans were observed between two normal tissues (brain and mammary gland) as well as between their tumor counterparts (glioma and breast cancer). The smallest differences resulted from the comparison of tumors (glioma and breast cancer) with their normal counterparts. All such effects do not extend to all chromosomes but are limited to only some of them. The estimated periods and amplitudes are identical for the genes located on the positive and negative DNA strands. While precise molecular mechanisms of chromosome-specific periodicities in gene expression have yet to be unraveled, their universal presence in different tissues adds another dimension to the current understanding of the genome organization.     

Grade-dependent Proteomics Characterization of Kidney Cancer

Kidney cancer is frequently metastatic on presentation at which point the disease is associated with a 95% mortality. Assessment of tumor grade on pathological examination is the most powerful means for prognostication as well as for stratification of patients into those who might respond to conventional or targeted therapy. Although there exist several grading systems in common use, all suffer from significant disparity among observers. In an attempt to objectify this process as well as to acquire grade-specific mechanistic information, we performed LC-MS/MS-based proteomics analysis on 50 clear cell kidney cancers equally distributed among normal tissues and Fuhrman grades 1–4. Initial experiments confirmed the utility of using archived formalin-fixed paraffin-embedded samples for LC-MS/MS-based proteomics analysis, and the LC-MS/MS findings were validated by extensive immunoblotting. We now show that changes among many biochemical processes and pathways are strongly grade-dependent with the glycolytic and amino acid synthetic pathways highly represented. In addition, proteins relating to acute phase and xenobiotic metabolism signaling are highly represented. Self-organized mapping of proteins with similar patterns of expression led to the creation of a heat map that will be useful in grade characterization as well as in future research relating to oncogenic mechanisms and targeted therapies for kidney cancer.Kidney cancer (or renal cell carcinoma (RCC)¹) is the seventh most common malignancy, the 10th most common cause of cancer death in men, and the ninth most common cancer in women. In 2009, an estimated 13,000 deaths (8,100 men and 4,900 women) will occur in the United States. The disease is frequently asymptomatic; a third of cases are diagnosed when the disease is already metastatic at which time it has 95% mortality (1).Assessment of tumor grade is the most powerful available means to date of determining tumor prognosis; thus objective criteria for assessing grade are essential such that prognostication is unambiguous. In addition, grade criteria are useful in stratifying patients into those most likely to respond to conventional as well as new targeted therapies. There exist several systems for assigning tumor grade in RCC, although most pathologists utilize the Fuhrman grading system. As is evidenced by the abundance of extant grading systems (2), there appears to be a general lack of consensus and thus considerable variability in assigning tumor grades. Objective criteria for grade assignment utilizing specific protein markers will be useful in objectifying this process and thereby allowing for more accurate prognostication. Furthermore assessment of the biological basis of the differences among grades, as evidenced by diverse biochemical pathways altered in a grade-specific fashion, will lead to the development of novel diagnostic assays as well as therapeutic interventions.Once objective grading criteria are put forth, molecular mechanisms by which tumors transition among grades can be identified and further investigated. Using this information, it might be possible to recapitulate the grade transition in vitro to discover novel mechanisms of oncogenesis or at least of transition from a relatively benign to a highly malignant phenotype. Moreover utilizing a systems biology approach to glean grade-specific network and pathway data has the capability to further the understanding of RCC oncogenesis. This approach can be used to identify novel mechanisms of tumor progression within grades and thereby can yield druggable targets.We now show that validated grade-specific, highly sensitive proteomics analysis of RCC resulted in the identification of proteins that vary in expression in a grade-specific fashion. From these data, we identified pathways and networks that are relevant, and likely critical, to grade transitions, and we discovered markers that, either separately or in combination, are able to assist in differentiation among grades. Furthermore our analysis yielded pathways altered in RCC that can ultimately be used both to stratify patients to grade-specific treatments and to identify new therapeutic targets.