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Tumor progression and metastasis are complex processes involving intricate interplay among multiple gene products. Astrocyte Elevated Gene (AEG)-1 was cloned as an HIV-1- and tumor necrosis factor α (TNF-α)-inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. AEG-1 downregulates the expression of the glutamate transporter EAAT2, thus it is implicated in glutamate-induced excitotoxic damage to neurons as evident in HIV-associated neurodegeneration. Interestingly, AEG-1 expression is elevated in subsets of breast cancer, glioblastoma multiforme and melanoma cells and AEG-1 cooperates with Ha-ras to augment the transformed phenotype of normal immortal cells. Moreover, AEG-1 is overexpressed in >95% of human malignant glioma samples when compared with normal human brain. Overexpression of AEG-1 increases and siRNA inhibition of AEG-1 decreases migration and invasion of human glioma cells, respectively. AEG-1 contains a lung-homing domain facilitating breast tumor metastasis to lungs. These findings indicate that AEG-1 might play a pivotal role in the pathogenesis, progression and metastasis of diverse cancers. Our recent observations indicate that AEG-1 exerts its effects by activating the NF-κB pathway and AEG-1 is a downstream target of Ha-ras and plays an important role in Ha-ras-mediated tumorigenesis. These provocative findings are intensifying interest in AEG-1 as a crucial regulator of tumor progression and metastasis and as a potential mediator of neurodegeneration. In this review, we discuss the cloning, structure and function(s) of AEG-1 and provide recent insights into the diverse actions and intriguing properties of this molecule.

Malignant gliomas including glioblastoma multiforme (GBM) and anaplastic astrocytomas are the most common primary brain tumors. Despite multimodal treatment including surgery, chemotherapy and radiation, median survival for patients with GBMs is only 12–15 months. Identifying molecules critical for glioma progression is crucial for devising effective targeted therapy. In the present study, we investigated the potential contribution of Astrocyte Elevated Gene-1 (AEG-1) in gliomagenesis and explored the possibility of AEG-1 as a therapeutic target for malignant glioma. We analyzed the expression levels of AEG-1 in 9 normal brain tissues and 98 brain tumor patient samples by Western blot analysis and immunohistochemistry. AEG-1 expression was significantly elevated in > 90% of diverse human brain tumor samples including GBMs and astrocytic tumors, and also in human glioma cell lines as compared to normal brain tissues and normal astrocytes. Knockdown of AEG-1 by siRNA inhibited cell viability, cloning efficiency, invasive ability of U87 human glioma cells and 9L rat gliosarcoma cells. We also found that matrix metalloproteases (MMP-2 and MMP-9) are involved in AEG-1-mediated invasion of glioma cells. In an orthotopic nude mouse brain tumor model using primary human GBM12 tumor cells, AEG-1 siRNA significantly suppressed glioma cell growth in vivo. Taken together these provocative results indicate that AEG-1 may play a crucial role in the pathogenesis of glioma and that AEG-1 could represent a viable potential target for malignant glioma therapy.

Expression of astrocyte elevated gene-1 (AEG-1) is elevated in multiple human cancers including brain tumors, neuroblastomas, melanomas, breast cancers, non-small cell lung cancers, liver cancers, prostate cancers, and esophageal cancers. This gene plays crucial roles in tumor cell growth, invasion, angiogenesis and progression to metastasis. In addition, over-expression of AEG-1 protects primary and transformed cells from apoptosis-inducing signals by activating PI3K-Akt signaling pathways. These results suggest that AEG-1 is intimately involved in tumorigenesis and may serve as a potential therapeutic target for various human cancers. However, the normal physiological functions of AEG-1 require clarification. We presently analyzed the expression pattern of AEG-1 during mouse development. AEG-1 was expressed in mid-to-hindbrain, fronto-nasal processes, limbs, and pharyngeal arches in the early developmental period from E8.5 to E9.5. In addition, at stages of E12.5-E18.5 AEG-1 was localized in the brain, and olfactory and skeletal systems suggesting a role in neurogenesis, as well as in skin, including hair follicles, and in the liver, which are organ sites in which AEG-1 has been implicated in tumor development and progression. AEG-1 co-localized with Ki-67, indicating a role in cell proliferation, as previously revealed in tumorigenesis. Taken together, these results suggest that AEG-1 may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation, and that temporal regulation of AEG-1 expression may be required during specific stages and in specific tissues during development.

Since its initial identification and cloning in 2002, Astrocyte Elevated Gene-1 (AEG-1), also known as metadherin (MTDH), 3D3 and LYsine-RIch CEACAM1 co-isolated (LYRIC), has emerged as an important oncogene that is overexpressed in all cancers analyzed so far. Examination of a large cohort of patient samples representing diverse cancer indications has revealed progressive increase in AEG-1 expression with stages and grades of the disease and an inverse relationship between AEG-1 expression level and patient prognosis. AEG-1 functions as a bona fide oncogene by promoting transformation. In addition, it plays a significant role in invasion, metastasis, angiogenesis and chemoresistance, all important hallmarks of an aggressive cancer. AEG-1 is also implicated in diverse physiological and pathological processes, such as development, inflammation, neurodegeneration, migraine and Huntington disease. AEG-1 is a highly basic protein with a transmembrane domain and multiple nuclear localization signals and it is present in the cell membrane, cytoplasm, nucleus, nucleolus and endoplasmic reticulum. In each location, AEG-1 interacts with specific proteins thereby modulating diverse intracellular processes the combination of which contributes to its pleiotrophic properties. The present review provides a snapshot of the current literature along with future perspectives on this unique molecule.

Cancer is the result of the progressive acquisition of multiple malignant traits through the accumulation of genetic or epigenetic alterations. Recent studies have established a functional role of MTDH (Metadherin)/AEG-1 (Astrocyte Elevated Gene 1) in several crucial aspects of tumor progression, including transformation, evasion of apoptosis, invasion, metastasis and chemoresistance. Overexpression of MTDH/AEG-1 is frequently observed in melanoma, glioma, neuroblastoma, and carcinomas of breast, prostate, liver and esophagus and is correlated with poor clinical outcomes. MTDH/AEG-1 functions as a downstream mediator of the transforming activity of oncogenic Ha-Ras and c-Myc. Furthermore, MTDH/AEG-1 overexpression activates the PI3K/Akt, NFκB, and Wnt/β-catenin signaling pathways to stimulate proliferation, invasion, cell survival and chemoresistance. The lung-homing domain of MTDH/AEG-1 also mediates the adhesion of tumor cells to the vasculature of distant organs and promotes metastasis. These findings suggest that therapeutic targeting of MTDH/AEG-1 may simultaneously suppress tumor growth, block metastasis and enhance the efficacy of chemotherapeutic treatments.

Glioblastomas continue to carry poor prognoses for patients despite advances in surgical, chemotherapeutic and radiation regimens. One feature of glioblastoma associated with poor prognosis is the degree of hypoxia and expression levels of hypoxia-inducible factor-1α (HIF-1α). HIF-1α expression allows metabolic adaptation to low oxygen availability, partly through upregulation of VEGF and increased tumor angiogenesis. Here, we demonstrate an induced level of astrocyte-elevated gene-1 (AEG-1) by hypoxia in glioblastoma cells. AEG-1 has the capacity to promote anchorage-independent growth and cooperates with Ha-ras in malignant transformation. In addition, AEG-1 was recently demonstrated to serve as an oncogene and can induce angiogenesis in glioblastoma. Results from in vitro studies show that hypoxic induction of AEG-1 is dependent on HIF-1α stabilization during hypoxia and that PI3K inhibition abrogates AEG-1 induction during hypoxia through loss of HIF-1α stability. Furthermore, we show that AEG-1 is induced by glucose deprivation and that prevention of intracellular reactive oxygen species (ROS) production prevents this induction. Additionally, AEG-1 knockdown results in increased ROS production and increased glucose deprivation-induced cytotoxicity. On the other hand, AEG-1 overexpression prevents ROS production and decreases glucose deprivation-induced cytotoxicity, indicating that AEG-1 induction is necessary for cells to survive this type of cell stress. These observations link AEG-1 overexpression in glioblastoma with hypoxia and glucose deprivation and targeting these physiological pathways may lead to therapeutic advances in the treatment of glioblastoma in the future.

Background

Astrocyte elevated gene-1 (AEG-1) was originally characterized as a HIV-1-inducible gene in primary human fetal astrocyte. Recent studies highlight a potential role of AEG-1 in promoting tumor progression and metastasis. The aim of this study was to investigate if AEG-1 serves as a potential therapeutic target of human neuroblastoma.

Methods

We employed RNA interference to reduce AEG-1 expression in human neuroblastoma cell lines and analyzed their phenotypic changes.

Results

We found that the knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibited cell proliferation and apoptosis. The specific downregulation induced cell arrest in the G0/G1 phase of cell cycle. In the present study, we also observed a significant enhancement of chemo-sensitivity to cisplatin and doxorubicin by knockdown of AEG-1.

Conclusion

Our study suggests that overexpressed AEG-1 enhance the tumorogenic properties of neuroblastoma cells. The inhibition of AEG-1 expression could be a new adjuvant therapy for neuroblastoma.

Background

Astrocyte elevated gene 1 (AEG-1), an important oncogene, has been shown to be overexpressed in several types of cancers. In colorectal cancer (CRC), the protein level of AEG-1 is up-regulated in tumour tissue compared to normal mucosa, showing prognostic significance. Since little is known about the transcriptional level of AEG-1 expression and its biological pathway in CRC the aim of the present study was to examine the relationship of AEG-1 mRNA expression, the protein level and clinicopathological variables as well as its biology pathway in CRC.

Material and methods

The mRNA expression of AEG-1 was analysed by qPCR in fresh frozen patient samples including 156 primary tumours, along with the corresponding normal mucosa, and in five colon cancer cell lines, SW480, SW620, KM12C, KM12SM and KM12L4a. AEG-1 protein expression was investigated by immunohistochemistry in paraffin-embedded materials from 74 distant normal mucosa, 107 adjacent mucosa, 158 primary tumour, 35 lymph node metastasis and 9 liver metastasis samples. In addition, the AEG-1 protein expression was elucidated in the cell lines by Western blot.

Results

The lymph node metastatic cell line SW620 had a significantly higher AEG-1 mRNA (0.27 ± 0.02) expression compared to the primary tumour cell line SW480 (0.17 ± 0.04, p = 0.026). AEG-1 expression at the mRNA level and/or the protein level was significantly up-regulated gradually from normal mucosa to primary CRC, and then to lymph node metastasis and finally to liver metastasis (p < 0.05). There were significant associations of AEG-1 mRNA expression with tumour location (p = 0.047), as well as mRNA and protein expression with the tumour stage (p < 0.03). Furthermore AEG-1 protein expression was positively related to biological variables including NF-κB, p73, Rad50 and apoptosis (p < 0.05).

Conclusion

AEG-1 is up-regulated, at the mRNA and the protein level, during CRC development and aggressiveness, and is related to tumour location and stage. It may play its role in CRC through the NF-κB signaling pathway.

Background

Reactive astrogliosis is a ubiquitous but poorly understood hallmark of central nervous system pathologies such as trauma and neurodegenerative diseases. In vitro and in vivo studies have identified proinflammatory cytokines and chemokines as mediators of astrogliosis during injury and disease; however, the molecular mechanism remains unclear. In this study, we identify astrocyte elevated gene-1 (AEG-1), a human immunodeficiency virus 1 or tumor necrosis factor α-inducible oncogene, as a novel modulator of reactive astrogliosis. AEG-1 has engendered tremendous interest in the field of cancer research as a therapeutic target for aggressive tumors. However, little is known of its role in astrocytes and astrocyte-mediated diseases. Based on its oncogenic role in several cancers, here we investigate the AEG-1-mediated regulation of astrocyte migration and proliferation during reactive astrogliosis.

Methods

An in vivo brain injury mouse model was utilized to show AEG-1 induction following reactive astrogliosis. In vitro wound healing and cell migration assays following AEG-1 knockdown were performed to analyze the role of AEG-1 in astrocyte migration. AEG-1-mediated regulation of astrocyte proliferation was assayed by quantifying the levels of cell proliferation markers, Ki67 and proliferation cell nuclear antigen, using immunocytochemistry. Confocal microscopy was used to evaluate nucleolar localization of AEG-1 in cultured astrocytes following injury.

Results

The in vivo mouse model for brain injury showed reactive astrocytes with increased glial fibrillary acidic protein and AEG-1 colocalization at the wound site. AEG-1 knockdown in cultured human astrocytes significantly reduced astrocyte migration into the wound site and cell proliferation. Confocal analysis showed colocalization of AEG-1 to the nucleolus of injured cultured human astrocytes.

Conclusions

The present findings report for the first time the novel role of AEG-1 in mediating reactive astrogliosis and in regulating astrocyte responses to injury. We also report the nucleolar localization of AEG-1 in human astrocytes in response to injury. Future studies may be directed towards elucidating the molecular mechanism of AEG-1 action in astrocytes during reactive astrogliosis.

Astrocyte elevated gene-1 (AEG-1), also known as MTDH and Lyric, is a novel gene that was first cloned by subtraction hybridization in 2002 and has recently been shown to play a role as a crucial oncogene that acts as a promoter of tumor malignancy. Overexpression and inhibition studies both in in vitro and in vivo models have partly shown the oncogenic roles of AEG-1 in a number of crucial aspects of tumor development and progression, including transformation, evasion of apoptosis, proliferation, cell survival, migration, invasion, metastasis, angiogenesis and chemoresistance through the activation of numerous signaling pathways, such as the nuclear factor κB, PI3K/AKT, Wnt/β-catenin and mitogen-activated protein kinase signaling pathways. However the potential roles of AEG-1, particularly in specific organs or tissues, such as breast tissue, require further clarification. Studies have found that in normal human breast tissue, AEG-1 is always expressed at low levels or is absent, while it is widely overexpressed in many breast cancer cell lines and breast tumors. The present review evaluates the current literature with regards to AEG-1 relative to breast cancer development and progression and highlights new perspectives relative to this molecule, indicating its potential to become a new target for the clinical treatment of breast cancer.

Glioblastoma multiforme is the most malignant type of primary brain tumor with a poor prognosis. These tumors consist of a heterogeneous population of malignant cells, including well-differentiated tumor cells and less differentiated cells with stem cell properties. These cancer stem cells, known as brain tumor initiating cells, likely contribute to glioma recurrence, as they are highly invasive, mobile, resistant to radiation and chemotherapy, and have the capacity to self-renew. Glioblastoma tumor cells release excitotoxic levels of glutamate, which may be a key process in the death of peritumoral neurons, formation of necrosis, local inflammation, and glioma-related seizures. Moreover, elevated glutamate levels in the tumor may act in paracrine and autocrine manner to activate glutamate receptors on glioblastoma tumor cells, resulting in proliferation and invasion. Using a previously described culturing condition that selectively promotes the growth of brain tumor initiating cells, which express the stem cell markers nestin and SOX-2, we characterize the expression of α-amino-3-hydroxy-5-methyl-4-isozolepropionic acid (AMPA)-type glutamate receptor subunits in brain tumor initiating cells derived from glioblastomas. Here we show for the first time that glioblastoma brain tumor initiating cells express high concentrations of functional calcium-permeable AMPA receptors, compared to the differentiated tumor cultures consisting of non-stem cells. Up-regulated calcium-permeable AMPA receptor expression was confirmed by immunoblotting, immunocytochemistry, and intracellular calcium imaging in response to specific agonists. Our findings raise the possibility that glutamate secretion in the GBM tumor microenvironment may stimulate brain tumor derived cancer stem cells.

Background

Astrocyte elevated gene-1 (AEG-1) is associated with tumorigenesis and progression in diverse human cancers. The present study was aimed to investigate the clinical and prognostic significance of AEG-1 in salivary gland carcinomas (SGC).

Methods

Real-time PCR and western blot analyses were employed to examine AEG-1 expression in two normal salivary gland tissues, eight SGC tissues of various clinical stages, and five pairs of primary SGC and adjacent salivary gland tissues from the same patient. Immunohistochemistry (IHC) was performed to examine AEG-1 protein expression in paraffin-embedded tissues from 141 SGC patients. Statistical analyses was applies to evaluate the diagnostic value and associations of AEG-1 expression with clinical parameters.

Results

AEG-1 expression was evidently up-regulated in SGC tissues compared with that in the normal salivary gland tissues and in matched adjacent salivary gland tissues. AEG-1 protein level was positively correlated with clinical stage (P < 0.001), T classification (P = 0.008), N classification (P = 0.008) and M classifications (P = 0.006). Patients with higher AEG-1 expression had shorter overall survival time, whereas those with lower tumor AEG-1 expression had longer survival time.

Conclusions

Our results suggest that AEG-1 expression is associated with SGC progression and may represent a novel and valuable predictor for prognostic evaluation of SGC patients.

Since its original cloning by subtraction hybridization in 2002, it is now evident that Astrocyte elevated gene-1 (AEG-1) is a key contributor to the carcinogenic process in diverse organs. AEG-1 protein expression is elevated in advanced stages of many cancers, which correlates with poor survival. In specific cancers, such as breast and liver cancer, the AEG-1 gene itself is amplified further supporting a seminal role in tumorigenesis. Overexpression and inhibition studies both in in vitro and in in vivo models reveal the importance of AEG-1 in regulating multiple physiologically and pathologically relevant processes including proliferation, invasion, metastasis and gene expression. AEG-1 is a single-pass transmembrane protein with multiple nuclear localization signals and no known domains or motifs. Although pertinent roles of AEG-1 in the carcinogenic process are established, its potential function (promotion of metastasis only versus functioning as a bona fide oncogene) as well as localization (cell surface versus nucleus) remain areas requiring further clarification. The present review critically evaluates what is currently known about AEG-1 and provides new perspectives relative to this intriguing molecule that may provide a rational target for intervening in the cancer phenotype.

Our recent findings demonstrate that Astrocyte Elevated Gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) samples and AEG-1 plays a central role in regulating development and progression of HCC. In the present manuscript, we elucidate a molecular mechanism of AEG-1-induced chemoresistance, an important characteristic of aggressive cancers. AEG-1 increases the expression of multidrug resistance gene 1 (MDR1) protein resulting in increased efflux and decreased accumulation of doxorubicin (DOX) promoting DOX-resistance. Suppression of MDR1, by siRNA or by chemical reagents, or inhibition of AEG-1 or a combination of both genes significantly increases in vitro sensitivity to DOX. In nude mice xenograft studies, a lentivirus expressing AEG-1 shRNA, in combination with DOX, profoundly inhibited growth of aggressive human HCC cells compared to either agent alone. We document that although AEG-1 does not affect MDR1 gene transcription, it facilitates association of MDR1 mRNA to polysomes resulting in increased translation and AEG-1 also inhibits ubiquitination and subsequent proteasome-mediated degradation of MDR1 protein. This study is the first documentation of a unique aspect of AEG-1 function, i.e., translational and post-translational regulation of proteins. Inhibition of AEG-1 might provide a means of more effectively using chemotherapy to treat HCC, which displays inherent chemoresistance with aggressive pathology.

The malignant growth of glial support cells causes gliomas, highly invasive, primary brain tumors that are largely resistant to therapy. Individual tumor cells spread by active cell migration, invading diffusely into the normal brain. This process is facilitated by Cl- channels that endow glioma cells with an enhanced ability to quickly adjust their shape and cell volume to fit the narrow and tortuous extracellular brain spaces. Once satellite tumors enlarge, their growth is limited by the spatial constraints imposed by the bony cavity of the skull and spinal column. Glioma cells circumvent this limitation by active destruction of peritumoral neural tissue through the release of glutamate, inducing peritumoral seizures and ultimately excitotoxic neuronal cell death. Hence, primary brain tumors support their unusual biology by taking advantage of ion channels and transporters that are designed to support ion homeostatic functions in normal brain.

Since its discovery, nearly one decade of research on astrocyte elevated gene 1 (AEG-1) has witnessed expanding knowledge of this molecule, ranging from its role in cancer biology to molecular mechanisms underlying the biological functions. As a multifunctional oncoprotein, AEG-1 has been shown to overexpress in multiple types of human cancer, and the elevation of AEG-1 in tumor cells leads to enhanced phenotypes characteristic of malignant aggressiveness, including increased abilities to proliferate robustly, to invade surrounding tissues, to migrate, to induce neovascularization, and to enhance chemoresistance. The multifunctional role of AEG-1 in tumor development and progression has been found to be associated with several signaling cascades, namely, 1) activation of NF-kappa B, partially through direct interaction with p65; 2) PI3K/AKT signaling triggered by AEG-1 indirectly; 3) enhancement of the transcriptional activity of beta-catenin by indirect activation of MAPK and induction of LEF1; 4) regulation of mi/siRNA-mediated gene silencing by interacting with SND1; and 5) promotion of protective autophagy; in addition to possibly unknown mechanisms. Elevated AEG-1 expression is seen in nearly all tumor types, and in most cases AEG-1 positively correlates with tumor progression and poorer patient survival. Taken together, AEG-1 might represent a potential prognostic biomarker and therapeutic target.

Purpose

Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment thus requiring innovative and effective targeted therapies. The oncogene Astrocyte elevated gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates insulin-like growth factor binding protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function.

Experimental Design

IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarray and real-time PCR and ELISA in human HCC cell lines. Dual Fluorescence in situ hybridization was performed to detect loss of heterozygosity at IGFBP7 locus. Stable IGFBP7-overexpressing clones were established in the background of AEG-1-overexpressing human HCC cells and were analyzed for in vitro proliferation and senescence and in vivo tumorigenesis and angiogenesis.

Results

IGFBP7 expression is significantly downregulated in human HCC samples and cell lines compared to normal liver and hepatocytes, respectively, and inversely correlates with the stages and grades of HCC. Genomic deletion of IGFBP7 was identified in 26% of HCC patients. Forced overexpression of IGFBP7 in AEG-1 overexpressing HCC cells inhibited in vitro growth and induced senescence, and profoundly suppressed in vivo growth in nude mice that might be an end result of inhibition of angiogenesis by IGFBP7.

Conclusion

The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Accordingly, targeted overexpression of IGFBP7 might be a potential novel therapy for HCC.

Hepatocellular carcinoma (HCC) is a highly aggressive vascular cancer characterized by diverse etiology, activation of multiple signal transduction pathways, and various gene mutations. Here, we have determined a specific role for astrocyte elevated gene-1 (AEG1) in HCC pathogenesis. Expression of AEG1 was extremely low in human hepatocytes, but its levels were significantly increased in human HCC. Stable overexpression of AEG1 converted nontumorigenic human HCC cells into highly aggressive vascular tumors, and inhibition of AEG1 abrogated tumorigenesis by aggressive HCC cells in a xenograft model of nude mice. In human HCC, AEG1 overexpression was associated with elevated copy numbers. Microarray analysis revealed that AEG1 modulated the expression of genes associated with invasion, metastasis, chemoresistance, angiogenesis, and senescence. AEG1 also was found to activate Wnt/β-catenin signaling via ERK42/44 activation and upregulated lymphoid-enhancing factor 1/T cell factor 1 (LEF1/TCF1), the ultimate executor of the Wnt pathway, important for HCC progression. Inhibition studies further demonstrated that activation of Wnt signaling played a key role in mediating AEG1 function. AEG1 also activated the NF-κB pathway, which may play a role in the chronic inflammatory changes preceding HCC development. These data indicate that AEG1 plays a central role in regulating diverse aspects of HCC pathogenesis. Targeted inhibition of AEG1 might lead to the shutdown of key elemental characteristics of HCC and could lead to an effective therapeutic strategy for HCC.

Gliomas are uniformly fatal forms of primary brain neoplasms that vary from low-grade to high-grade (glioblastoma). While low-grade gliomas are weakly angiogenic, glioblastomas are among the most angiogenic of tumors. Thus, interactions between glioma cells and their tissue microenvironment may play an important role in aggressive tumor formation and progression. To quantitatively explore how tumor cells interact with their tissue microenvironment, we incorporated the interactions of normoxic glioma cells, hypoxic glioma cells, vascular endothelial cells, diffusible angiogenic factors, and necrosis formation into a first-generation, biologically-based mathematical model for glioma growth and invasion. Model simulations quantitatively described the spectrum of in vivo dynamics of gliomas visualized with medical imaging. Further, we investigated how proliferation and dispersal of glioma cells combine to induce increasing degrees of cellularity, mitoses, hypoxia-induced neo-angiogenesis and necrosis, features that characterize increasing degrees of “malignancy”, and we found that changes in the net rates of proliferation ({lower case rho}) and invasion (D) are not always necessary for malignant progression. Thus, although other factors, including the accumulation of genetic mutations, can change cellular phenotype (e.g. proliferation and invasion rates), this study suggests that these are not required for malignant progression. Simulated results are placed in the context of the current clinical World Health Organization grading scheme for studying specific patient examples. This study suggests that through the application of the proposed model for tumor-microenvironment interactions, predictable patterns of dynamic changes in glioma histology distinct from changes in cellular phenotype (e.g. proliferation and invasion rates) may be identified, thus providing a powerful clinical tool.

Several important and previously unrecognized roles for the neurotransmitter glutamate in the biology of primary brain tumors have recently been elucidated. Glutamate is produced and released from glioma cells via the system xc- cystine glutamate transporter as a byproduct of glutathione synthesis. Glutamate appears to play a central role in the malignant phenotype of glioma via multiple mechanisms. By binding to peritumoral neuronal glutamate receptors, glutamate is responsible for seizure induction and similarly causes excitotoxicity which aids the expansion of tumor cells into the space vacated by destroyed tissue. Glutamate also activates ionotropic and metabotropic glutamate receptors on glioma cells in a paracrine and autocrine manner. AMPA glutamate receptors lack the GluR2 subunit rendering them Ca2+ permeable and capable of activating the AKT and MAPK pathways. Furthermore, these receptors are critical in aiding the invasion of glioma cells into normal brain. AMPA-Rs accumulate at focal adhesion sites where they may indirectly mediate interactions between the ECM and integrins. Glutamate receptor stimulation results in activation of focal adhesion kinase (FAK) which is critical to the regulation of growth factor and integrin-stimulated cell motility and invasion. The multitude of effects of glutamate on glioma biology supports the rationale for pharmacological targeting of glutamate receptors and transporters. Several ongoing and recently completed clinical trials are exploring the therapeutic potential of interrupting glutamate-mediated brain tumor growth.

LYRIC/AEG-1 is a unique protein that has been shown to promote tumor cell migration and invasion through activation of the transcription factor NF-κB. We performed yeast two-hybrid screening to detect LYRIC/AEG-1 associated proteins, and identified BCCIP, a CDKN1A and BRCA2-associated protein involved in cell cycle regulation and DNA repair. Here we demonstrate association between LYRIC/AEG-1 and BCCIP in mammalian cells, and define the region of interaction. Co-expression of the two proteins resulted in decreased levels of BCCIPα, an effect partially abrogated by proteasome inhibition. A truncated LYRIC/AEG-1 construct lacking the interaction region did not alter BCCIPα protein levels. Coincidentally, it was observed that overexpression of BCCIPα in DU145 prostate tumor cells induced an apparent neuroendocrine differentiation. In summary, our data suggest LYRIC/AEG-1 is a negative regulator of BCCIPα, promoting proteasomal degradation either through direct interaction, or potentially through an indirect mechanism involving downstream effects of the NF-κB signaling pathway.

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3′-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3′-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.

High-grade gliomas release excitotoxic concentrations of glutamate, which has been shown to enhance tumor proliferation and migration. α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptors are abundantly expressed at the invading edge of glioblastoma specimens, suggesting they may play an important biologic role in tumor invasion. In this study, we examined potential mechanisms by which AMPA receptor (AMPAR) expression and stimulation promote glioma cell migration and invasion. Overexpression of GluR1, the most abundant AMPAR subunit in gliomas, positively correlated with glioma cell adhesion to type I and type IV collagen, which was decreased in cells with knockdown of GluR1 and with blocking antibodies to β1 integrin. Furthermore, stimulation of the AMPAR led to detachment of cells from the extracellular matrix (ECM). Immunoprecipitation studies showed that GluR1 associated with the actin cytoskeleton-linked protein band 4.1B (brain type), which may serve as a link between GluR1 and integrins. Overexpression of GluR1 correlated with increased cell-surface expression of β1 integrin, increased phosphorylation of focal adhesion kinase (FAK-Y397), and enhanced numbers of focal adhesion (FA) complexes. Cells overexpressing GluR1 had increased colocalization of actin and paxillin at FAs and, in several glioma cell lines, significantly increased invasion in an in vitro Matrigel transwell assay. Likewise, in an intracranial xenograft model, overexpression of GluR1 led to perivascular and subependymal glioma cell invasion similar to patterns of tumor dissemination described in human glioblastoma. Together, these results suggest that AMPARs may link signals from the ECM to sites of FA, where signal integration promotes tumor invasion.

GATA4 loss as a result of promoter hypermethylation or somatic mutation promotes growth and chemotherapy resistance of human astrocytomas.

Glioblastoma Multiforme (GBM), the most common and lethal primary human brain tumor, exhibits multiple molecular aberrations. We report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumor suppressor gene (TSG). Although GATA4 was expressed in normal brain, loss of GATA4 was observed in 94/163 GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor-like phenotype, formation of neurospheres, and the ability to differentiate into astrocytes, neurons, and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures, and brain tumor–initiating cells suppressed tumor growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21CIP1, independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT (O-6-methylguanine-DNA-methyltransferase), the DNA repair enzyme which is often implicated in temozolomide resistance. Instead, GATA4 reduced expression of APNG (alkylpurine-DNA-N-glycosylase), a DNA repair enzyme which is poorly characterized in GBM-mediated temozolomide resistance. Identification and validation of GATA4 as a TSG and its downstream targets in GBM may yield promising novel therapeutic strategies.

Malignant gliomas, including glioblastoma multiforme, constitute the most common and aggressive primary brain tumors in adults. The transcription factor signal transducer and activator of transcription 3 (STAT3) plays an essential role in glioblastoma pathogenesis downstream of the major oncogenic protein epidermal growth factor receptor variant III (EGFRvIII). However, the critical gene targets of STAT3 that mediate EGFRvIII-induced glial transformation have remained unknown. Here, we identify inducible nitric oxide synthase (iNOS) as a novel target gene of STAT3 in EGFRvIII-expressing mouse astrocytes. Endogenous STAT3 occupies the endogenous iNOS promoter and stimulates iNOS transcription in EGFRvIII-expressing astrocytes. STAT3 does not appear to control iNOS transcription in astrocytes deficient in the major glioblastoma tumor suppressor protein PTEN, suggesting that STAT3 regulates iNOS transcription specifically in EGFRvIII-expressing astrocytes. Importantly, inhibition of iNOS by distinct approaches including knockdown by RNA interference reduces cell population growth and invasiveness of EGFRvIII-expressing astrocytes. In addition, upon iNOS knockdown or administration of a small molecule inhibitor of iNOS, EGFRvIII-expressing astrocytes form smaller tumors in vivo. These findings suggest that inhibition of iNOS may have potential therapeutic value for EGFRvIII-activated brain tumors.