Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.     

A systemic insight into astrocytoma biology across different grades

Astrocytomas are the most common type of brain tumors, which originate from glial cells, and are classified into specific grades based on their histopathological behavior. To develop precise therapeutic strategies for the disease, it is important to identify the molecular signatures specific to each grade as well as the key factors responsible for the transition from one grade to the next. In this study, we have taken a systems approach to investigate the gene expression profiles of each grades of the disease by mapping shortest paths of gene interaction in each grade and also between one grade and the next. Module core genes govern the topology of these networks and serve as important functional players in each grade as well as help in grade transition events. Shortlisted among these module core genes are well-characterized players of glioma as well as novel molecules (32 grade-specific genes and 15 grade transition–specific genes), which influence important prooncogenic functions but have not been linked to glioma biology yet. These module core genes provide interesting insight into the biology of astrocytic tumors and are potential therapeutic targets for astrocytoma.     

Quantitation of angiogenesis and its correlation with vascular endothelial growth factor expression in astrocytic tumors

OBJECTIVE: To quantitate tumor angiogenesis by establishing intratumoral microvessel density (IMD), to study vascular endothelial growth factor (VEGF) expression in different grades of astrocytomas and to correlate VEGF expression with tumor angiogenesis. STUDY DESIGN: Forty cases of astrocytic neoplasms (10 of each grade) were assessed for tumor angiogenesis and VEGF expression. The panendothelial marker CD31 was used to highlight microvessels. Tumor angiogenesis was quantitated as IMD count per square millimeter in areas of high vascularity, or "hot spots," using an image analyzer. VEGF expression was studied in sections of the tumors. IMD counts per square millimeter and VEGF expression were correlated with histologic grade. The angiogenic potential of tumors as reflected by IMD counts per square millimeter was correlated with the intensity of VEGF expression. RESULTS: Vascular proliferation in high grade gliomas was significantly higher as compared to that in low grade gliomas. IMD count per square millimeter revealed a positive correlation with histologic grade in high grade gliomas. Pilocytic astrocytoma and low grade astrocytoma as a group had comparable IMD counts per square millimeter. VEGF expression paralleled IMD counts in rare high grade gliomas only. CONCLUSION: Malignant progression in astrocytoma is heralded and accompanied by increased angiogenesis. VEGF is an important angiogenic factor in high grade gliomas since its expression parallels the increased IMD counts in these tumors. In contrast, in low grade gliomas, angiogenic factors other than VEGF may contribute to vascular proliferation. The results emphasize the role of antiangiogenic therapy as an optimal tool in therapeutic strategies as they become available.     

Grade-specific diagnostic and prognostic biomarkers in breast cancer

An integrative approach is presented to identify grade-specific biomarkers for breast cancer. Grade-specific molecular interaction networks were constructed with differentially expressed genes (DEGs) of cancer grade 1, 2, and 3. We observed that the molecular network of grade3 is predominantly associated with cancer-specific processes. Among the top ten connected DEGs in the grade3, the increase in the expression of UBE2C and CCNB2 genes was statistically significant across different grades. Along with UBE2C and CCNB2 genes, the CDK1, KIF2C, NDC80, and CCNB2 genes are also profoundly expressed in different grades and reduce the patient's survival. Gene set enrichment analysis of these six genes reconfirms their role in metastatic phenotype. Moreover, the coexpression network shows a strong association of these six genes promotes cancer specific biological processes and possibly drives cancer from lower to a higher grade. Collectively the identified genes can act as potential biomarkers for breast cancer diagnosis and prognosis.     

Proteomic identification of biomarkers in the cerebrospinal fluid (CSF) of astrocytoma patients

The monitoring of changes in the protein composition of the cerebrospinal fluid (CSF) can be used as a sensitive indicator of central nervous system (CNS) pathology, yet its systematic application to analysis of CNS neoplasia has been limited. There is a pressing need for both a better understanding of gliomagenesis and the development of reliable biomarkers of the disease. In this report, we used two proteomic techniques, two-dimensional gel electrophoresis (2-DE), and cleavable Isotope-Coded Affinity Tag (cICAT) to compare CSF proteomes to identify tumor- and grade-specific biomarkers in patients bearing brain tumors of differing histologies and grades. Retrospective analyses were performed on 60 samples derived from astrocytomas WHO grade II, III, and IV, schwannomas, metastastic brain tumors, inflammatory samples, and non-neoplastic controls. We identified 103 potential tumor-specific markers of which 20 were high-grade astrocytoma-specific. These investigations allowed us to identify a spectrum of signature proteins that could be used to distinguish CSF derived from control patients versus those with low- (AII) or high-grade (AIV) astrocytoma. These proteins may represent new diagnostic, prognostic, and disease follow-up markers when used alone or in combination. These candidate biomarkers may also have functional properties that play a critical role in the development and malignant progression of human astrocytomas, thus possibly representing novel therapeutic targets for this highly lethal disease.     

Autocrine regulation of glioblastoma cell-cycle progression,viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay

Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and progression of malignant brain tumors. Given the significance of tumor microenvironment in general, and the established role of paracrine VEGF signaling in glioblastoma (GBM) biology in particular, we explored the potential autocrine control of human astrocytoma behavior by VEGF. Using a range of cell and molecular biology approaches to study a panel of astrocytoma (grade III and IV/GBM)-derived cell lines and a series of clinical specimens from low- and high-grade astrocytomas, we show that co-expression of VEGF and VEGF receptors (VEGFRs) occurs commonly in astrocytoma cells. We found VEGF secretion and VEGF-induced biological effects (modulation of cell cycle progression and enhanced viability of glioblastoma cells) to function in an autocrine manner. Morevover, we demonstrated that the autocrine VEGF signaling is mediated via VEGFR2 (KDR), and involves co-activation of the c-Raf/MAPK, PI3K/Akt and PLC/PKC pathways. Blockade of VEGFR2 by the selective inhibitor (SU1498) abrogated the VEGF-mediated enhancement of astrocytoma cell growth and viability under unperturbed culture conditions. In addition, such interference with VEGF-VEGFR2 signaling potentiated the ionizing radiation-induced tumor cell death. In clinical specimens, both VEGFRs and VEGF were co-expressed in astroglial tumor cells, and higher VEGF expression correlated with tumor progression, thereby supporting the relevance of functional VEGF-VEGFR signaling in vivo. Overall, our results are consistent with a potential autocrine role of the VEGF-VEGFR2 (KDR) interplay as a factor contributing to malignant astrocytoma growth and radioresistance, thereby supporting the candidacy of this signaling cascade as a therapeutic target, possibly in combination with radiotherapy.     

Telomerase Activity in Human Brain Tumors: Astrocytoma and Meningioma

Somatic cells do not have telomerase activity but immortalized cell lines and more than 85 % of the cancer cells show telomerase activation to prevent the telomere from progressive shortening. The activation of this enzyme has been found in a variety of human tumors and tumor-derived cell lines, but only few studies on telomerase activity in human brain tumors have been reported. Here, we evaluated telomerase activity in different grades of human astrocytoma and meningioma brain tumors. In this study, assay for telomerase activity performed on 50 eligible cases consisted of 26 meningioma, 24 astrocytoma according to the standard protocols. In the brain tissues, telomerase activity was positive in 39 (65 %) of 50 patients. One sample t test showed that the telomerase activity in meningioma and astrocytoma tumors was significantly positive entirely (P < 0.001). Also, grade I of meningioma and low grades of astrocytoma (grades I and II) significantly showed telomerase activity. According to our results, we suggest that activation of telomerase is an event that starts mostly at low grades of brain including meningioma and astrocytoma tumors.     

Grade-specific expression profiles of miRNAs/mRNAs and docking study in human grade I-III astrocytomas

Although several miRNAs have been identified to be involved in glioblastoma tumorigenesis, little is known about the global expression profiles of miRNAs and their functional targets in astrocytomas at earlier stages of malignancy. In this study the global expression of miRNAs and mRNAs in normal brain tissue samples and grade I-III astrocytomas were analyzed parallelly using microarrays, and the grade-specific expression profiles of them were obtained by unsupervised hierarchical clustering. It was also confirmed that miR-107, miR-124, miR-138, and miR-149 were downregulated significantly in grade I-IV astrocytomas, and overexpression of miR-124 and miR-149 inhibited glioblastoma cell proliferation and migration. Furthermore, grade-specific changes were discovered in the central biological processes, regulatory networks, and signaling pathways associated with dysregulated genes, and a regulatory network of putative functional miRNA-mRNA pairs was defined. In conclusion, our results may contribute to a better understanding of the molecular mechanisms involved in astrocytoma tumorigenesis and malignant progression.     

Exploration of Involved Key Genes and Signaling Diversity in Brain Tumors

Brain tumors are becoming a major cause of death. The classification of brain tumors has gone through restructuring with regard to some criteria such as the presence or absence of a specific genetic alteration in the 2016 central nervous system World Health Organization update. Two categories of genes with a leading role in tumorigenesis and cancer induction include tumor suppressor genes and oncogenes; tumor suppressor genes are inactivated through a variety of mechanisms that result in their loss of function. As for the oncogenes, overexpression and amplification are the most common mechanisms of alteration. Important cell cycle genes such as p53, ATM, cyclin D2, and Rb have shown altered expression patterns in different brain tumors such as meningioma and astrocytoma. Some genes in signaling pathways have a role in brain tumorigenesis. These pathways include hedgehog, EGFR, Notch, hippo, MAPK, PI3K/Akt, and WNT signaling. It has been shown that telomere length in some brain tumor samples is shortened compared to that in normal cells. As the shortening of telomere length triggers chromosome instability early in brain tumors, it could lead to initiation of cancer. On the other hand, telomerase activity was positive in some brain tumors. It is suggestive that telomere length and telomerase activity are important diagnostic markers in brain tumors. This review focuses on brain tumors with regard to the status of oncogenes, tumor suppressors, cell cycle genes, and genes in signaling pathways as well as the role of telomere length and telomerase in brain tumors.     

Array-Based Approaches for the Identification of Epigenetic Silenced Tumor Suppressor Genes

Carcinogenesis involves the inactivation or inhibition of genes that function as tumor suppressors. Deletions, mutations, or epigenetic silencing of tumor suppressor genes can lead to altered growth, differentiation, and apoptosis. DNA methylation and histone modifications are important epigenetic mechanisms of gene regulation and play essential roles both independently and cooperatively in tumor initiation and progression. Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated discovery of novel tumor suppressor genes. One of the most useful of these approaches is an epigenetic reactivation screening strategy that combines treatment of cancer cells in vitro with DNA methyltransferase and/or histone deacetylase (HDAC) inhibitors, followed by global gene expression analysis using microarrays, to identify upregulated genes. This approach is most effective when complemented by microarray analyses to identify genes repressed in primary tumors. Recently, using cancer cell lines treated with a DNA methylation inhibitor and/or a HDAC inhibitor in conjunction with cDNA microarray analysis, candidate tumor suppressor genes, which are subject to epigenetic silencing, have been identified in endometrial, colorectal, esophageal, and pancreatic cancers. An increasing number of studies have utilized epigenetic reactivation screening to discover novel tumor suppressor genes in cancer. The results of some of the most recent studies are highlighted in this review.     

Gene expression profiling in a renal cell carcinoma cell line: dissecting VHL and hypoxia-dependent pathways

The von Hippel-Lindau tumor suppressor, pVHL, is a key player in one of the best characterized hypoxia signaling pathways, the VHL-hypoxia-inducible factor (VHL-HIF) pathway. To better understand the role of VHL in the hypoxia signaling pathways of tumor cells, we used serial analysis of gene expression (SAGE) to investigate hypoxia-regulated gene expression in renal carcinoma cells (786-0), with and without VHL. The gene expression profiles of the cancer cells were compared to SAGE profiles from normal renal proximal tubule cells grown under both normoxia and hypoxia. The data suggest that the role of VHL as a tumor suppressor may be more complex than previously thought. Further, the data reveal that renal carcinoma cells have evolved an alternative hypoxia signaling pathway(s) compared with normal renal cells. These alternative hypoxia pathways demonstrate VHL-dependent and VHL-independent regulation. The genes involved in such pathways include those with potential importance in the physiological and pathological regulation of tumor growth and angiogenesis. Some of the genes identified as showing overexpression in the cancer cells, particularly those encoding secreted or membrane-bound proteins, could be potential biomarkers for tumors or targets for rational therapeutics that are dependent on VHL status.     

Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility

Originally thought of as a stomach-derived endocrine peptide acting via its receptors in the central nervous system to stimulate food intake and growth hormone expression, ghrelin and its receptor (growth hormone secretagogue receptor (GHS-R)) are widely expressed in a number of organ systems, including cancer cells. However, the direct functional role of ghrelin and its receptor in tumors of central nervous system origin remains to be defined. Here, we demonstrate that the human astrocytoma cell lines U-118, U-87, CCF-STTG1, and SW1088 express 6-, 11-, 15-, and 29-fold higher levels of GHS-R compared with primary normal human astrocytes. The ligation of GHS-R by ghrelin on these cells resulted in an increase in intracellular calcium mobilization, protein kinase C activation, actin polymerization, matrix metalloproteinase-2 activity, and astrocytoma motility. In addition, ghrelin led to actin polymerization and membrane ruffling on cells, with the specific co-localization of the small GTPase Rac1 with GHS-R on the leading edge of the astrocytoma cells and imparting the tumor cells with a motile phenotype. Disruption of the endogenous ghrelin/GHS-R pathway by RNA interference resulted in diminished motility, matrix metalloproteinase activity, and Rac expression, whereas tumor cells stably overexpressing GHS-R exhibited increased cell motility. The relevance of ghrelin and GHS-R expression was verified in clinically relevant tissues from 20 patients with oligodendrogliomas and grade II-IV astrocytomas. Analysis of a central nervous system tumor tissue microarray revealed that strong GHS-R and ghrelin expression was significantly more common in high grade tumors compared with low grade ones. Together, these findings suggest a novel role for the ghrelin/GHS-R axis in astrocytoma cell migration and invasiveness of cancers of central nervous system origin.     

MicroRNA-542-3p Suppresses Tumor Cell Invasion via Targeting AKT Pathway in Human Astrocytoma

The molecular mechanism underlying constitutive activation of AKT signaling, which plays essential roles in astrocytoma progression, is not fully characterized. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of astrocytoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we found that microRNA (miR)-542-3p expression was decreased in glioblastoma cell lines and astrocytoma tissues, and reduced levels of miR-542-3p expression correlated with high histopathological grades and poor prognosis of astrocytoma patients. Exogenous miR-542-3p suppressed glioblastoma cell invasion through not only targeting AKT1 itself but also directly down-regulating its two important upstream regulators, namely, integrin-linked kinase and PIK3R1. Notably, overexpressing miR-542-3p decreased AKT1 phosphorylation and directly and indirectly repressed nuclear translocation and transactivation activity of β-catenin to exert its anti-invasive effect. Furthermore, the miR-542-3p expression level negatively correlated with AKT activity as well as levels of integrin-linked kinase and PIK3R1 in human astrocytoma specimens. These findings suggest that miR-542-3p acts as a negative regulator in astrocytoma progression and that miR-542-3p down-regulation contributes to aberrant activation of AKT signaling, leaving open the possibility that miR-542-3p may be a potential therapeutic target for high grade astrocytoma.