Astrocyte Differentiation Marker Research Articles

Nuclear Factor I Represses the Notch Effector HEY1 in Glioblastoma

Glioblastomas (GBMs) are highly aggressive brain tumors with a dismal prognosis. Nuclear factor I (NFI) is a family of transcription factors that controls glial cell differentiation in the developing central nervous system. NFIs have previously been shown to regulate the expression of astrocyte markers such as glial fibrillary acidic protein (GFAP) in both normal brain and GBM cells. We used chromatin immunoprecipitation (ChIP)–on-chip to identify additional NFI targets in GBM cells. Analysis of our ChIP data revealed ~400 putative NFI target genes including an effector of the Notch signaling pathway, HEY1, implicated in the maintenance of neural stem cells. All four NFIs (NFIA, NFIB, NFIC, and NFIX) bind to NFI recognition sites located within 1 kb upstream of the HEY1 transcription site. We further showed that NFI negatively regulates HEY1 expression, with knockdown of all four NFIs in GBM cells resulting in increased HEY1 RNA levels. HEY1 knockdown in GBM cells decreased cell proliferation, increased cell migration, and decreased neurosphere formation. Finally, we found a general correlation between elevated levels of HEY1 and expression of the brain neural stem/progenitor cell marker B-FABP in GBM cell lines. Knockdown of HEY1 resulted in an increase in the RNA levels of the GFAP astrocyte differentiation marker. Overall, our data indicate that HEY1 is negatively regulated by NFI family members and is associated with increased proliferation, decreased migration, and increased stem cell properties in GBM cells.

Abstract 5014: Reprogramming of mature astrocytes by abrogation of Rb tumor suppression leads to astrocytoma initiation in GEMMs.

Abstract Astrocytomas are the most common malignant brain tumors which maintain a poor survival rate despite decades of research effort. Understanding the initiation process and identifying astrocytoma cell(s) of origin have been of immense interest. Genetically engineered mouse models (GEMMs) are ideal for studying the initiation process compared to evolved human tumor cells. Our lab has developed an adult inducible GEMM that alters key human glioblastoma (Grade IV) pathways. Inactivation of Rb tumor suppression (TS) by expressing N-terminal 121aa of large T antigen (T121) under GFAP promoter is sufficient to initiate grade II astrocytoma. Addition of mutant KrasG12D leads to grade III and further PTEN deletion to glioblastomas. KrasG12D activation and PTEN deletion alone or in combination failed to initiate astrocytomas, suggesting that Rb TS inactivation is essential for its initiation. Studies from several cancers have identified tumor-initiating cells, a subset of tumor cells with stem cell-like properties responsible for generating the entire tumor mass. Their origin remains a mystery. We hypothesize that Rb TS inactivation reprograms astrocytes into stem/progenitor-like state generating potential cell(s) of origin for astrocytoma. Immunostaining of progenitor and proliferation markers (e.g. nestin and Ki-67) showed their co-expression in the cortex with T121, suggesting a possible reprogramming of mature astrocytes. Moreover, astrocyte differentiation marker S100beta was downregulated in T121 cells. To rule out the possibility of migration resulting in the observed cortical progenitor cells, focal induction of T121 in cortex using lenti-cre injection was performed. We found expression of Ki-67 and nestin and reduced expression of S100beta, suggesting that the reprogramming was independent of the germinal zones in the brain. Furthermore, cortical T121 expressing astrocytes were able to generate neurospheres but astrocytes from wildtype cortex failed to do so. These cells also showed self-renewability and multilineage ability, emphasizing their dedifferentiated status upon Rb-TS inactivation. We were able to achieve in-vitro reprogramming of mature astrocytes upon T121 expression suggesting that the reprogramming does not require brain microenvironment. Sphere assays using cortical cells from mice where individual Rb family member was inactivated suggests that loss of individual Rb family members alone is insufficient in reprogramming the mature astrocytes. With the Rb pathway known to be altered in more than 75% of glioblastomas, our results are a step in the direction of unraveling the process of tumor initiation for this deadly disease. Citation Format: Amit S. Adhikari, Teresa Sullivan, Yurong Song, Xiuyun Lu, Norene O'Sullivan, Terry Van Dyke. Reprogramming of mature astrocytes by abrogation of Rb tumor suppression leads to astrocytoma initiation in GEMMs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5014. doi:10.1158/1538-7445.AM2013-5014

A BMP7 variant inhibits the tumorigenic potential of glioblastoma stem-like cells

Glioblastoma multiforme (GBM) is among the most aggressive tumor types and is essentially an incurable malignancy characterized by resistance to chemo-, radio-, and immunotherapy. GBM is maintained by a hierarchical cell organization that includes stem-like, precursor, and differentiated cells. Recurrence and maintenance of the tumor is attributed to a small population of undifferentiated tumor-initiating cells, defined as glioblastoma stem-like cells (GSLCs). This cellular hierarchy offers a potential treatment to induce differentiation of GSLCs away from tumor initiation to a more benign phenotype or to a cell type more amenable to standard therapies. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth and differentiation. In vitro, a BMP7 variant (BMP7v) decreased primary human GSLC proliferation, endothelial cord formation, and stem cell marker expression while enhancing neuronal and astrocyte differentiation marker expression. In subcutaneous and orthotopic GSLC xenografts, which closely reproduce the human disease, BMP7v decreased tumor growth and stem cell marker expression, while enhancing astrocyte and neuronal differentiation compared with control mice. In addition, BMP7v reduced brain invasion, angiogenesis, and associated mortality in the orthotopic model. Inducing differentiation of GSLCs and inhibiting angiogenesis with BMP7v provides a potentially powerful and novel approach to the treatment of GBM.

Resveratrol Enhances the Antitumor Effects of Temozolomide in Glioblastoma via ROS‐dependent AMPK‐TSC‐mTOR Signaling Pathway

Resveratrol has been regarded as a promising candidate for cancer prevention and treatment. The present study was to investigate the impact of resveratrol on the antitumor effects of temozolomide (TMZ), a standard treatment regiment of glioblastoma (GBM), in vitro and in vivo. We found that the combination of resveratrol and TMZ significantly resulted in G(2)/M cell cycle arrest by flow cytometry, triggered a robust increase in expression of astrocyte differentiation marker glial fibrillary acid protein (GFAP), downregulated the expression of matrix metalloproteinase-9 (MMP-9) by immunohistochemistry and western blot analysis as well as inhibited cell migration by scratch wound assay. Further study revealed that TMZ in combination with resveratrol remarkably increased reactive oxygen species (ROS) production, which serves as an upstream signal for AMP-activated protein kinase (AMPK) activation. Subsequently, activated AMPK inhibited mTOR signaling and downregulated antiapoptosis protein Bcl-2, which was contributed to the additive antiproliferation effects of combination treatment. In an orthotopic xenograft model of GBM, TMZ plus resveratrol treatment significantly reduced the volume of tumor, which was confirmed by decreased expression of Ki-67, a marker of proliferation index. Our findings demonstrate for the first time that resveratrol can enhance TMZ-mediated antitumor effects in GBM in vitro and in vivo, via ROS-dependent AMPK-TSC-mTOR signaling pathway.

ALDH1A1 is a Marker of Astrocytic Differentiation during Brain Development and Correlates with Better Survival in Glioblastoma Patients

Glioblastoma is the most common malignant brain tumor and patients usually succumb to their disease within 2 years. Aldehyde dehydrogenase 1A1 (ALDH1A1) has been suggested as a marker for cancer stem cells that is associated with poor prognosis in human gliomas. However, little is known about the expression and the function of ALDH1A1 in early stages of brain development. We analyzed ALDH1A1 expression in developing and mature central nervous system (CNS) as well as in 93 cases of primary glioblastomas. Surprisingly, ALDH1A1 was absent in the stem cell niches at varying stages of CNS development, but strong ALDH1A1 expression was observed in mature astrocytes coexpressing GFAP and S100. There were 92 out of 93 glioblastomas (99%) that showed ALDH1A1 protein expression in up to 49% of tumor cells. The majority of these cells co-expressed GFAP, but not established stem cell markers such as Nestin, OLIG2 or SOX2. Finally, strong expression of ALDH1A1 correlated with a significantly better survival of the patients and proved to be an independent prognostic marker in our series (P < 0.01). In contrast to other published data, we therefore provide evidence for ALDH1A1 as a marker of astrocytic differentiation during brain development and of better prognosis in patients suffering from primary glioblastoma.

Abstract 3346: Astrocytoma initiation by Rb family inactivation induced dedifferentiation of mature astrocytes.

Abstract Astrocytomas are the most common primary human malignant brain tumors. Despite standard-of-care surgery, radiation, and chemotherapy, no curative treatment for these devastating diseases is currently available. Tumor-initiating cells (TICs), a small population of tumor cells is shown to be responsible for astrocytoma initiation and progression. Though their presence has been convincingly shown, there is considerable debate about their origin. By using genetically engineered mice, we hypothesis that loss of Rb family, induces dedifferentiation of mature astrocytes which are capable of evolving into TICs. Our lab has developed an adult mouse model where inactivation of retinoblastoma (Rb) family proteins by expressing N-terminal 121aa of large T antigen (T121), initiates grade II astrocytoma. pRb inactivation and mutant KrasG12D activation leads to grade III astrocytoma and further PTEN inactivation results in Grade IV glioblastomas. We have utilized the Rb family inactivation model to investigate the dedifferentiation of mature astrocytes. We have performed IHC using progenitor markers, Sox2 and nestin and a proliferation marker Ki67 and observe the presence of these markers coinciding with T121 expression suggesting a possible dedifferentiation of mature astrocytes. Quantitative analysis of the overlap between Sox2 and Ki-67 with T121 indicates that the marker presence is specific to T121 expression. In addition to these markers, we also show that S100α an astrocyte differentiation marker is downregulated in pRb inactivated cells further supporting the hypothesis of possible dedifferentiation of mature astrocytes. Since T121 expression is induced in all GFAP expressing cells, there is a possibility that the GFAP expressing cells in the germinal areas such as SVZ and dentate gyrus could be migrating into the cortex and form the observed progenitor cells. To address this issue we used lenti-Cre to focally induce T121 expression in the cortex. Sox2 expression, along with the reduced expression of differentiation marker S100α in these cells, suggests that the dedifferentiation events are independent of the germinal zones in the adult brain. As a functional assay to identify progenitor/ stem cells we performed neurosphere assay. In our assay conditions, wildtype adult cortex was unable to generate any spheres compared to T121 expressing cells. We found that the longer the post induction time the higher the sphere forming ability of T121 cells. The multilineage ability of these T spheres cells was confirmed by inducing differentiation into different neural lineages. To test the tumor forming ability of the sphere cells, we have performed subcutaneous injections and awaiting the results. Thus we conclude that in our system inactivation of Rb family members induced dedifferentiation in mature astrocytes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3346. doi:1538-7445.AM2012-3346

Biomarkers of glial cell proliferation and differentiation in culture

The two major intermediate filament (IF) proteins of astrocytes are vimentin and GFAP. Early during development, radial glia and immature astrocytes express mainly vimentin. Towards the end of gestation, a switch occurs whereby vimentin is progressively replaced by GFAP in differentiated astroglial cells. The expression of vimentin and GFAP increased markedly after injury to CNS. GFAP has been widely recognized as an astrocyte differentiation marker, constituting the major IF protein of mature astrocyte. In our recent researches we investigated the interactions between growth factors and dexamethasone on cytoskeletal proteins GFAP and vimentin expression under different experimental conditions. In addition, nestin, a currently used marker of neural stem cells, is transiently co-expressed with GFAP during development and is induced in reactive astrocytes following brain injury. The role of S100B in astrocytes, neurons, and microglia is particularly studied in Alzheimer's disease. In conclusion, such glial biomarkers will help us to understand the more general mechanisms involved in CNS development and can open new perspectives for the control of the neurologic diseases.

The immunoglobulin‐like cell adhesion molecule hepaCAM induces differentiation of human glioblastoma U373‐MG cells

Subsequent to our identification of a novel immunoglobulin-like cell adhesion molecule hepaCAM, we showed that hepaCAM is frequently lost in diverse human cancers and is capable of modulating cell motility and growth when re-expressed. Very recently, a molecule identical to hepaCAM (designated as GlialCAM) was found highly expressed in glial cells of the brain. Here, we demonstrate that hepaCAM is capable of inducing differentiation of the human glioblastoma U373-MG cells. Expression of hepaCAM resulted in a significant increase in the astrocyte differentiation marker glial fibrillary acid protein (GFAP), indicating that hepaCAM promotes glioblastoma cells to undergo differentiation. To determine the relationship between hepaCAM expression level and cell differentiation, we established two U373-MG cell lines expressing hepaCAM at different levels. The results revealed that high-level hepaCAM triggered a clear increase in GFAP expression as well as morphological changes characteristic of glioblastoma cell differentiation. Furthermore, high expression of hepaCAM significantly accelerated cell adhesion but inhibited cell proliferation and migration. Concomitantly, deregulation of cell cycle regulatory proteins was detected. Expectedly, the differentiation was noticeably less apparent in cells expressing low-level hepaCAM. Taken together, our findings suggest that hepaCAM induces differentiation of the glioblastoma U373-MG cells. The degree of cell differentiation is dependent on the expression level of hepaCAM.

OmpA Is the Critical Component for Escherichia coli Invasion-Induced Astrocyte Activation

Escherichia coli is the major Gram-negative bacterial pathogen in neonatal meningitis. Outer membrane protein A (OmpA) is a conserved major protein in the E. coli outer membrane and is involved in several host-cell interactions. To characterize the role of OmpA in the invasion of astrocytes by E. coli, we investigated OmpA-positive and OmpA-negative E. coli strains. Outer membrane protein A E44, E105, and E109 strains adhered to and invaded C6 glioma cells 10- to 15-fold more efficiently than OmpA-negative strains. Actin rearrangement, protein tyrosine kinase, and phosphoinositide 3-kinase activation were required for OmpA-mediated invasion by E. coli. In vitro infection of C6 cells and intracerebral injection into mice of the E44 strain induced expression of the astrocyte differentiation marker glial fibrillary acidic protein and the inflammatory mediators cyclooxygenase 2 and nitric oxide synthase 2. After intracerebral infection with E44, all C57BL/6 mice died within 36hours, whereas 80% of mice injected with E44 premixed with recombinant OmpA protein survived. Astrocyte activation and neutrophil infiltration were reduced in brain tissue sections in the mice given OmpA. Taken together, these data suggest that OmpA-mediated invasion plays an important role in the early stage of E.coli-induced brain damage, and that it may have therapeutic use in E. coli meningitis.

The mitogen-activated/extracellular signal-regulated kinase kinase 1/2 inhibitor U0126 induces glial fibrillary acidic protein expression and reduces the proliferation and migration of C6 glioma cells

The extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in diverse cellular functions. ERK and its activating kinase, mitogen-activated/extracellular signal-regulated kinase kinase (MEK), are downstream of cell surface receptors known to be up-regulated in many malignant gliomas. We sought to investigate the role of ERK in glioma cell migration, proliferation and differentiation using the rat-derived C6 glioma cell line and the MEK inhibitor, U0126. Treatment of C6 cells with U0126 caused a significant concentration-dependent reduction in cell proliferation and migration and also induced expression of glial fibrillary acidic protein, a marker of astrocytic differentiation. These results suggest that the ERK pathway regulates glioma cell proliferation, migration and differentiation.

HERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines

Recent studies have led to considerable advancement in our understanding of the molecular mechanisms that underlie the relentless cell growth and invasiveness of human gliomas. Partial understanding of these mechanisms has (1) improved the classification for gliomas, by identifying prognostic subgroups, and (2) pointed to novel potential therapeutic targets. Some classes of ion channels have turned out to be involved in the pathogenesis and malignancy of gliomas. We studied the expression and properties of K+ channels in primary cultures obtained from surgical specimens: human ether a gò-gò related (hERG)1 voltage-dependent K+ channels, which have been found to be overexpressed in various human cancers, and human ether a gò-gò-like 2 channels, that share many of hERG1's biophysical features. The expression pattern of these two channels was compared to that of the classical inward rectifying K+ channels, IRK, that are widely expressed in astrocytic cells and classically considered a marker of astrocytic differentiation. In our study, hERG1 was found to be specifically overexpressed in high-grade astrocytomas, that is, glioblastoma multiforme (GBM). In addition, we present evidence that, in GBM cell lines, hERG1 channel activity actively contributes to malignancy by promoting vascular endothelial growth factor secretion, thus stimulating the neoangiogenesis typical of high-grade gliomas. Our data provide important confirmation for studies proposing the hERG1 channel as a molecular marker of tumour progression and a possible target for novel anticancer therapies.

TGF-α Differentially Regulates GFAP, Vimentin, and Nestin Gene Expression in U-373 MG Glioblastoma Cells: Correlation with Cell Shape and Motility

To begin understanding the regulation and biological significance of changes in the expression of intermediate filament proteins in astrocytic tumors, we have recently shown that TGF-α alters the protein level of glial fibrillary acidic protein (GFAP), nestin, and vimentin in U-373 MG glioblastoma cells. Here, we have determined the molecular mechanisms regulating these changes. In addition, to evaluate the significance of these changes we have examined whether TGF-α affects various cellular properties related to differentiation. Our results show that, in U-373 MG cells treated with TGF-α, GFAP gene transcription, mRNA level, and specific protein synthesis decrease by ∼50%. This suggests that, in U-373 MG cells, TGF-α down-regulates the expression of this marker of astrocytic differentiation at the transcriptional level, resulting in decreased GFAP mRNA level and specific protein synthesis. In contrast, TGF-α does not change vimentin gene transcription, but increases by about 50% the transcription of the gene for nestin, a marker for undifferentiated astrocytic precursors. This differential regulation of GFAP, nestin, and vimentin gene expression indicates that TGF-α induces further dedifferentiation of U-373 MG cells. This notion is also supported by our findings that TGF-α increases the motility of U-373 MG cells and induces a less stellate morphology.

Regulation of gene expression in mature oligodendrocytes by the specialized myelin-like membrane environment: antibody perturbation in culture with the monoclonal antibody R-mAb.

We have previously shown that the growth of oligodendrocyte progenitors in the presence of a monoclonal antibody (R-mAb) reacting with a cell surface component reversibly blocks their further differentiation at a specific, late progenitor stage of the lineage. This block is characterized by a nearly complete elimination of the onset of terminal differentiation at the level of RNA expression. In the present study, mature oligodendrocytes already expressing markers of terminal differentiation were exposed to R-mAb. This resulted in a retraction of cell processes and the formation of round, swollen cells, and a dose-dependent, antibody-specific partial reduction (30-50%) in the steady state levels of markers of terminal differentiation. Upon removing the perturbing antibody, all markers returned to control levels within 2 days. This inhibition was due to modulations of the levels of the specific mRNAs and proteins, not to cell loss. Total protein and levels of a marker of astrocytic differentiation were not affected by the treatment. Monoclonal antibody O1 did not cause the effects observed with R-mAb. We conclude that the response of terminally differentiating oligodendrocytes to the effects of R-mAb is different from that of oligodendrocyte late progenitors. Whereas the latter appears to operate through perturbation of the onset of gene expression (mRNA transcription and/or stability), the partial down-regulation of previously activated myelinogenic gene expression appears to be due to the loss of a normal, myelin-like, membrane environment needed for the stability of myelin mRNA and protein components.

Cerebral glioblastoma with cerebrospinal fluid dissemination

During the last 17 years, complete autopsies were performed on 51 patients who died of cerebral glioblastoma, and 14 were found to have dissemination by cerebrospinal fluid (CSF). In these 14 cases of glioma, the extent of intraparenchymal invasion by the primary tumor and the degree of seeding were studied in connection with histological findings and immunohistochemical staining for glial fibrillary acidic protein (GFAP) as the most reliable marker of astrocytic differentiation. From the findings obtained, the cases were divided into two groups. In one group, consisting of 7 gliomas, autopsy revealed intense seeding, despite only slight invasion by the primary tumor. Among these 7 extensively disseminated gliomas, 4 expressed almost no GFAP, 2 contained only a few GFAP-positive cells, and only 1 displayed an immunohistochemically high degree of astrocytic differentiation. Clinically, 6 of the 7 affected patients developed symptoms attributable to CSF seeding. In the other group consisting of the remaining 7 gliomas, only slight dissemination was seen, despite extensive infiltration of the primary tumor. Each of these 7 gliomas contained many GFAP-positive cells. None of the affected patients developed symptomatic seeding. This study shows the existence of two clinicopathologically distinct groups of disseminated cerebral glioblastomas and suggests that, regardless of morphological features, glioblastomas showing immunohistochemically poor astrocytic differentiation tend to shed tumor cells more vigorously but are less invasive at the primary site than those with many GFAP-positive cells. It is also suggested that, as a consequence, the former glioma type produces symptomatic seeding more frequently than the latter type.