Secretion-mediated STAT3 activation promotes self-renewal of glioma stem-like cells during hypoxia.

High-grade gliomas (HGGs) include the most common and the most aggressive primary brain tumor of adults and children. Despite multimodality treatment, most high-grade gliomas eventually recur and are ultimately incurable. Several studies suggest that the initiation, progression, and recurrence of gliomas are driven, at least partly, by cancer stem-like cells. A defining characteristic of these cancer stem-like cells is their capacity to self-renew. We have identified a hypoxia-induced pathway that utilizes the Hypoxia Inducible Factor 1α (HIF-1α) transcription factor and the JAK1/2-STAT3 (Janus Kinase 1/2 - Signal Transducer and Activator of Transcription 3) axis to enhance the self-renewal of glioma stem-like cells. Hypoxia is a commonly found pathologic feature of HGGs. Under hypoxic conditions, HIF-1α levels are greatly increased in glioma stem-like cells. Increased HIF-1α activates the JAK1/2-STAT3 axis and enhances tumor stem-like cell self-renewal. Our data further demonstrate the importance of Vascular Endothelial Growth Factor (VEGF) secretion for this pathway of hypoxia-mediated self-renewal. Brefeldin A and EHT-1864, agents that significantly inhibit VEGF secretion, decreased stem cell self-renewal, inhibited tumor growth, and increased the survival of mice allografted with S100ß-v-erbB/p53-/- glioma stem-like cells. These agents also inhibit the expression of a hypoxia gene expression signature that is associated with decreased survival of HGG patients. These findings suggest that targeting the secretion of extracellular, autocrine/paracrine mediators of glioma stem-like cell self-renewal could potentially contribute to the treatment of HGGs.

Id4 suppresses MMP2-mediated invasion of glioblastoma-derived cells by direct inactivation of Twist1 function.

Tumor cell invasion is a major contributor to cancer morbidity, and is of particular importance in patients with glioblastoma multiforme (GBM), the highest grade and most aggressive primary brain tumor. Tumor cell invasion and the expression of matrix metalloproteinases (MMPs), which are required for GBM invasion, are enhanced by inhibitor of DNA binding (Id) gene family members, Id1, Id2 and Id3, which can be highly expressed in glioma. Id4 is expressed in GBM at more variable levels than these other family members and we sought to determine its role in invasion. We found, unexpectedly, that invasion was dramatically inhibited in cells expressing Id4 as a result of decreased MMP2, a secreted proteinase key for brain tumor invasion. We demonstrate that Id4 decreased MMP2 expression by a direct inhibitory interaction with Twist1, a basic helix-loop-helix transcription factor known to increase MMP2 expression. Importantly, using data from The Cancer Genome Atlas, we show that Id4 expression correlates with survival of glioblastoma patients and inversely correlates with MMP2 expression. These data suggest that the upregulation of MMP2 resulting from decreased Id4 expression in GBM may contribute to the morbidity and mortality of GBM patients.

Inhibitor of DNA binding-4 promotes angiogenesis and growth of glioblastoma multiforme by elevating matrix GLA levels.

Inhibitor of differentiation-4 is highly expressed in glioblastoma multiforme (GBM). We report a novel pro-angiogenic function for inhibitor of differentiation-4 in the growth of glioblastoma xenografts. Tumor-derived cell cultures expressing elevated levels of ID4 produced enlarged xenografts in immunosuppressed mice that were better vascularized than corresponding control tumors and expressed elevated matrix GLA protein (MGP) that mediated enhanced tumor angiogenesis. Inhibition of MGP resulted in smaller and less vascularized xenografts. Our finding shows a novel function for ID4 in tumor angiogenesis, and identifies ID4 and MGP as possible therapeutic targets for GBM.

EGFR overexpression and radiation response in glioblastoma multiforme.

PURPOSE: Recent studies have suggested relative radioresistance in glioblastoma multiforme (GM) tumors in older patients, consistent with their shorter survival. Two common molecular genetic abnormalities in GM are age related: epidermal growth factor receptor (EGFR) overexpression in older patients and p53 mutations in younger patients. We tested whether these abnormalities correlated with clinical heterogeneity in GM response to radiation treatment. METHODS AND MATERIALS: Radiographically assessed radiation response (5-level scale) was correlated with EGFR immunoreactivity, p53 immunoreactivity, and p53 exon 5-8 mutation status in 170 GM patients treated using 2 prospective clinical protocols. Spearman rank correlation and proportional-odds ordinal regression were used for univariate and multivariate analysis. RESULTS: Positive EGFR immunoreactivity predicted poor radiographically assessed radiation response (p = 0.046). Thirty-three percent of tumors with no EGFR immunoreactivity had good radiation responses (>50% reduction in tumor size by CT or MRI), compared to 18% of tumors with intermediate EGFR staining and 9% of tumors with strong staining. There was no significant relationship between p53 immunoreactivity or mutation status and radiation response. Significant relationships were noted between EGFR score and older age and between p53 score or mutation status and younger age. CONCLUSION: The observed relative radioresistance of some GMs is associated with overexpression of EGFR.

(E)-5-(2-bromovinyl)-2'-deoxyuridine potentiates ganciclovir-mediated cytotoxicity on herpes simplex virus-thymidine kinase--expressing cells.

Tumor cells expressing the thymidine kinase gene of the herpes simplex virus (HSV-tk) are rendered highly susceptible to the cytotoxic effects of different antiherpes drugs. In an attempt to enhance cytotoxicity of this therapeutic approach in glioma and other tumor cell lines transduced with the HSV-tk gene, we evaluated tumor cell killing following co-administration of two different prodrugs metabolized by HSV-tk, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), and ganciclovir (GCV). In 8 of 12 cell lines investigated, addition of BVDU in concentrations showing no cytotoxic effect or only limited cytotoxicity could enhance GCV-mediated cell killing by as much as one order of magnitude. In co-cultures consisting of HSV-tk(+) (9L STK) and HSV-tk(-) (9L wild-type) cells, we also observed potentiation of GCV-mediated cytotoxicity in the presence of BVDU, suggesting strongly enhanced bystander cell killing. BVDU is thought to exert its cytotoxic effect through inhibition of thymidylate synthase activity or by incorporation into replicating DNA. Both effects could be observed in all HSV-tk--expressing cells investigated, including cell lines which did not exhibit cytotoxicity after incubation with BVDU. These findings argue against current concepts of BVDU-mediated cytotoxicity in HSV-tk--expressing cells. Taken together, our data suggest that gene therapy utilizing prodrug activating enzymes may be rendered more effective by simultaneous treatment with two different prodrugs metabolized by the same enzyme.

Protease pretreatment increases the efficacy of adenovirus-mediated gene therapy for the treatment of an experimental glioblastoma model.

Effective virus-mediated gene therapy for cancer will be facilitated by procedures that enhance the low level of gene transfer mediated by replication-deficient, recombinant viral vectors. We found recently that protease pretreatment of solid tumors is a useful strategy for enhancing virus-mediated gene transduction in vivo. In this study, we examined the potential of protease pretreatment to improve the efficacy of a gene therapy strategy for prodrug activation that depends on infection with a recombinant adenovirus encoding herpes simplex virus thymidine kinase (Ad-HSV-tk). Trypsin or a dissolved mixture of collagenase/dispase was inoculated into xenografts derived from the human glioblastoma multiforme-derived cell lines, U87 or U251. Ad-HSV-tk was administered 24 h after protease pretreatment, and animals were then treated for 10 days with ganciclovir (GCV). We found that protease pretreatment increased the efficacy of adenovirus mediated HSV-tk/GCV gene therapy in these experimental tumor models. Mice receiving Ad-HSV-tk/GCV after protease pretreatment demonstrated a significantly greater regression of tumors compared with those treated with Ad-HSV-tk/GCV alone. No adverse effects of protease pretreatment were observed. No signs of metastasis were seen either by histological inspection of lymph nodes or by a PCR-based analysis of selected mouse tissues to detect human tumor cells. Our findings indicate that protease pretreatment may be a useful strategy to enhance the efficacy of virus-mediated cancer gene therapy.

The gene for the axonal cell adhesion molecule TAX-1 is amplified and aberrantly expressed in malignant gliomas.

The human TAX-1 gene encodes a Mr 135,000 glycoprotein that is transiently expressed on the surface of a subset of neurons during development and is involved in neurite outgrowth. The TAX-1 gene has been mapped to a region on chromosome 1 that has been implicated in microcephaly and the Van der Woude syndrome. Using restriction landmark genome scanning to search for amplified genes in gliomas, we found TAX-1 to be amplified in 2 high-grade gliomas among a group of 26 gliomas investigated. Real-time reverse transcription-quantitative PCR analysis detected high levels of TAX-1 mRNA in glial tumors, even in the absence of TAX-1 gene amplification. Immunohistochemical analysis revealed abundant levels of TAX-1 in neoplastic glial cells of glioblastoma multiforme tumors. Because glial tumors are highly invasive and in view of the role of TAX-1 in neurite outgrowth, we investigated the potential role of TAX-1 in glioma cell migration. Using an in vitro assay, we found that the migration of glioma tumor cells is profoundly reduced in the presence of either an anti-TAX-1 antibody or a TAX-1 antisense oligonucleotide. Our findings suggest that TAX-1 plays a role in glial tumorigenesis and may provide a potential target for therapeutic intervention.

Analysis of complex relationships between age, p53, epidermal growth factor receptor, and survival in glioblastoma patients.

Glioblastoma multiforme (GBM) carries a dismal prognosis. However, a range of survival times exists, and parameters that define prognostic groups may help to optimize treatment. To identify such prognostic groups, we analyzed tumor tissue from 110 cases of newly diagnosed GBM from two clinical protocols. Similar to other studies, we found no association of epidermal growth factor receptor (EGFR) overexpression (as assessed by immunohistochemistry), p53 immunopositivity, or p53 mutation with survival in the entire sample. However, EGFR overexpression showed trends toward worse prognosis in patients younger than the median age, but better prognosis in patients older than the median age. This interaction of EGFR with age group was statistically significant and led us to focus our further analyses on the younger patients. In this group, a statistically significant association of EGFR overexpression with worse survival was identified in the p53-negative but not p53-positive tumors. We found a similar result after screening these cases for mutations in p53: EGFR overexpression was negatively associated with survival only in the p53 wild-type cases. To confirm this unexpected result, this finding was reproduced in a validation sample of an additional 42 tumors from younger patients on the same two clinical protocols. This complex relationship between EGFR and p53 in younger patients remained in a multivariate analysis that incorporated additional prognostic variables. The results suggest that analysis of prognostic markers in GBM is complex, and maximal information may require analysis of subgroups based on age and the status of specific markers such as p53. In addition, they suggest a specific group of patients on which to focus promising therapies targeting EGFR.

Pretreatment with protease is a useful experimental strategy for enhancing adenovirus-mediated cancer gene therapy.

A key impediment to the development of effective virus-mediated gene therapy for cancer is the low level of gene transfer that occurs after the administration of recombinant viral vectors. Improving in vivo infection and transduction efficiency is an important goal for gene therapy. The limited distribution of gene delivery is particularly problematic when large vectors such as recombinant adenoviruses and retroviruses are used to mediate transgene delivery to solid tumors. To facilitate the spread of virus, we have investigated the potential of administering proteases prior to the intratumoral inoculation of recombinant replication deficient adenovirus. For these studies, we chose proteases that are active against collagen and the other extracellular matrix proteins found in primary brain tumor tissue, but are not widely expressed in normal brain. Various concentrations of a mixture of collagenase/dispase or trypsin were inoculated into xenografts of human glioblastoma multiforme-derived brain tumor cell lines U87, U251, and SF767. Subsequently, recombinant adenovirus encoding the beta-galactosidase gene was administered and tumor tissue was examined for evidence of virus infection. Both collagenase/dispase and trypsin enhanced virus infection, indicating that protease pretreatment may be a useful strategy for enhancing virus-mediated gene transduction for many in vivo applications.

The basic helix-loop-helix transcription factors myogenin and Id2 mediate specific induction of caveolin-3 gene expression during embryonic development.

Caveolin-3 protein is the only member of the caveolin family that shows a unique muscle-specific expression pattern, and loss of its functional activity causes muscular dystrophy. Caveolin-3 mRNA levels are dramatically increased during the formation of myotubes in the C2C12 cell line. In this study, we characterized the human caveolin-3 5'-flanking region. Promoter analyses demonstrate that the proximal E box element serves as a myogenin binding site and is both necessary and sufficient to control caveolin-3 gene transcription. Transient transfection assays indicated that overexpression of myogenin activates caveolin-3 reporter gene expression, whereas Id2 overexpression inhibited caveolin-3 promoter activation by myogenin. A mutant Id2 protein lacking the HLH domain was not capable of suppressing myogenin-mediated activation. Determination of caveolin-3 transcript distribution patterns in vivo revealed that mRNA was first detectable at day 10 of gestation in the developing somites and heart. Caveolin-3 protein in myoblasts and myotubes was expressed in both the plasma membrane and vesicular structures. During skeletal myogenesis the level of Id2, an inhibitor of differentiation, decreases, allowing the induced basic helix-loop-helix transcription factor myogenin to form transcriptionally active heterodimers that bind to the caveolin-3 promoter and thereby mediate its transcription.

Id genes in nervous system development.

Id genes encode helix-loop-helix proteins that function to mediate processes important for normal development including cellular differentiation, proliferation and apoptosis. Id proteins act as negative regulators of other transcription factors, which are essential for cell determination and differentiation in diverse cell types, and interact with proteins important for cell cycle regulation. Studies of Id gene expression in the nervous system and in neural cells in culture indicate that Id proteins contribute to the regulation of mammalian nervous system development. Also, recognition of a wide variety of proteins with which Id transcription factors are capable of interacting suggests that it will be possible to understand more precisely their specific functions and importantly how these are integrated.

Id-2 regulates critical aspects of human cytotrophoblast differentiation, invasion and migration.

During early human placental development, the conceptus attaches itself to the uterus through cytotrophoblast invasion. Invasive cytotrophoblast cells differentiate from precursor villous cytotrophoblasts, but the essential regulating factors in this process are unknown. Basic helix-loop-helix (bHLH) transcription factor dimers are essential regulators of mouse trophoblast development. We therefore examined the importance of this family of factors in the human placenta. In many cell lineages, bHLH factors are sequestered by members of the Id family, HLH proteins that lack the basic DNA binding domain (Inhibitor of DNA binding proteins (Id-1 to Id-4)). During differentiation of some tissues, Id expression declines, allowing bHLH factors to dimerize, bind DNA and trans-activate lineage-specific genes. To begin to study the role of bHLH transcription factors in human placental development, we first characterized Id expression in cytotrophoblast cells. The cells expressed Id-3 constitutively; Id-2 was downregulated, at the mRNA and protein levels, as the cells differentiated in culture and in situ, respectively. In cases when cytotrophoblast differentiation was compromised (in placentas from women with preeclampsia, or in cells grown under hypoxic conditions in culture), Id-2 expression was maintained. To assess the functional relevance of these correlations, we used an adenovirus vector to maintain Id-2 protein expression in cultured cytotrophoblasts. Compared to control (lacZ-expressing) cells, cytotrophoblasts transduced to constitutively express Id-2 retained characteristics of undifferentiated cells: (alpha)1 integrin expression was low and cyclin B expression was retained. Furthermore, invasion through Matrigel was partially inhibited and migration was strikingly enhanced in Id-2-expressing cells. These results suggest that Id-2 and the bHLH factors that it partners play important roles in human cytotrophoblast development.

Overexpression of E2F1 in glioma-derived cell lines induces a p53-independent apoptosis that is further enhanced by ionizing radiation.

Glioma cell lines show variable responses to radiation in a manner influenced by their p53 status. Irradiation of glioma cell lines does not generally induce apoptosis. When wild-type p53 is present, these cells undergo a G1 arrest that is closely associated with increased radiosensitivity as measured by clonogenic survival. Previously, others have shown that dysregulated overexpression of E2F1 induces apoptosis in cell lines with either functional or inactivated p53. We found that regardless of p53 status, apoptosis induced by overexpression of E2F1 in glioma cell lines was further enhanced by treatment with ionizing radiation. BAX induction did not follow E2F1 overexpression or irradiation in the glioma cell lines tested. Thus, the apoptotic response of glioma-derived cells to irradiation can be enhanced by E2F1 by a mechanism that does not involve the induction of BAX.

Id-1 and Id-2 are overexpressed in pancreatic cancer and in dysplastic lesions in chronic pancreatitis.

Id proteins antagonize basic helix-loop-helix proteins, inhibit differentiation, and enhance cell proliferation. In this study we compared the expression of Id-1, Id-2, and Id-3 in the normal pancreas, in pancreatic cancer, and in chronic pancreatitis (CP). Northern blot analysis demonstrated that all three Id mRNA species were expressed at high levels in pancreatic cancer samples by comparison with normal or CP samples. Pancreatic cancer cell lines frequently coexpressed all three Ids, exhibiting a good correlation between Id mRNA and protein levels, as determined by immunoblotting with highly specific anti-Id antibodies. Immunohistochemistry using these antibodies demonstrated the presence of faint Id-1 and Id-2 immunostaining in pancreatic ductal cells in the normal pancreas, whereas Id-3 immunoreactivity ranged from weak to strong. In the cancer tissues, many of the cancer cells exhibited abundant Id-1, Id-2, and Id-3 immunoreactivity. Scoring on the basis of percentage of positive cells and intensity of immunostaining indicated that Id-1 and Id-2 were increased significantly in the cancer cells by comparison with the respective controls. Mild to moderate Id immunoreactivity was also seen in the ductal cells in the CP-like areas adjacent to these cells and in the ductal cells of small and interlobular ducts in CP. In contrast, in dysplastic and atypical papillary ducts in CP, Id-1 and Id-2 immunoreactivity was as significantly elevated as in the cancer cells. These findings suggest that increased Id expression may be associated with enhanced proliferative potential of pancreatic cancer cells and of proliferating or dysplastic ductal cells in CP.

A repertoire of differentially expressed transcription factors that offers insight into mechanisms of human cytotrophoblast differentiation.

During human placental development, specialized cells allocated to the extraembryonic lineage (cytotrophoblasts) invade the uterus, anchoring the conceptus to the decidua and tapping a supply of maternal blood. This unusual behavior requires cytotrophoblasts to assume highly specialized characteristics; some are commonly associated with tumor cells, while others are typical of endothelia. Here we investigated the transcriptional mechanisms that control cytotrophoblast differentiation/invasion. Specifically, we examined the cells' expression of a number of transcription factors, at the RNA level, as they differentiated along the invasive pathway in vitro. Since basic helix-loop-helix (bHLH) proteins play important roles in murine trophoblast differentiation, we first examined their expression by cytotrophoblasts. As in murine placental development, expression of the human homologue of Mash-2 was confined to progenitor cells. But expression of Hand-1, which promotes differentiation of murine trophoblast giant cells, was not detected. We also found that cytotrophoblasts upregulated the expression of bHLH/PAS factors that function in adaptive responses to hypoxia, including hEPAS-1, which is expressed primarily in endothelial cells. Quite unexpectedly, we discovered that cytotrophoblasts express high levels of mRNA encoding the human homologue of the Drosophila neuronal fate gene, glial cells missing-1 (gcm-1). We also found evidence of crosstalk between the bHLH and GCM-1 regulatory networks. Together, these results offer insights into the transcriptional mechanisms that govern cytotrophoblast differentiation/invasion. Interestingly, these mechanisms suggest analogies with those that govern differentiation of murine stem cells allocated to both the intra- and extraembryonic lineages.

Immortalization of primary human keratinocytes by the helix-loop-helix protein, Id-1.

Basic helix-loop-helix (bHLH) DNA-binding proteins have been demonstrated to regulate tissue-specific transcription within multiple cell lineages. The Id family of helix-loop-helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins. Overexpression of Id proteins within a variety of cell types has been shown to inhibit their ability to differentiate under appropriate conditions. We demonstrate that ectopic expression of Id-1 leads to activation of telomerase activity and immortalization of primary human keratinocytes. These immortalized cells have a decreased capacity to differentiate as well as activate phosphorylation of the retinoblastoma protein. Additionally, these cells acquire an impaired p53-mediated DNA-damage response as a late event in immortalization. We conclude that bHLH proteins play a pivotal role in regulating normal keratinocyte growth and differentiation, which can be disrupted by the immortalizing functions of Id-1 through activation of telomerase activity and inactivation of the retinoblastoma protein.

Id gene expression as a key mediator of tumor cell biology.

Id genes encode members of the helix-loop-helix (HLH) family of transcription factors that inhibit transcription by forming inactive heterodimers with basic HLH (bHLH) proteins. There are four members of the Id gene family recognized in mammals, and the proteins they encode share homology primarily in their HLH domain. bHLH proteins typically form heterodimers with other bHLH proteins, and their basic domain binds to a DNA sequence element, the E-box, activating transcription. Products of Id genes lack the basic DNA binding domain of the bHLH transcription factors, and when they heterodimerize with bHLH proteins, the complexes are inactive. Generally, high levels of Id mRNA are detected in proliferative undifferentiated, embryonal cells and lower levels are detected in well-differentiated, mature, adult tissues. In vitro, these genes are generally expressed at lower levels in cells after the induction of differentiation. Recently, high levels of expression of Id genes have been identified in cell lines derived from a wide variety of different tumors and in tumor tissues as well. These findings suggest that not only the inappropriate proliferation of tumors but also the anaplastic characteristics that contribute to their malignant behavior may be regulated by Id gene expression.

Molecular genetics of brain tumors.

As many as 40000 patients are newly diagnosed each year as having brain tumors. About half of these are metastatic foci of tumors originating outside the central nervous system, while the other half are primary tumors of central nervous system tissues. These are a diverse group of neoplasms. Currently, primary brain tumors are classified in a manner that reflects their histological appearance and location. The identification of cancer as a disorder of genes, however, has opened the possibility of classifying tumors according to the genetic alterations that underlie their pathogenesis and that regulate their malignant behavior. Two major classes of genes critical for the development of all types of cancer, including brain tumors, are now recognized: tumor suppressor genes, which encode genes that function to inhibit cell proliferation and tumor development, and oncogenes, which encode proteins that stimulate proliferation and mediate biological activities important for invasion, neoangiogenesis, immune escape, and other characteristics of malignancy. While in most cases the specific pathways regulating tumor characteristics such as tumor neoangiogenesis and tissue invasion remain to be defined, recognition of the genetic changes characteristic of individual tumor types should provide opportunities to develop more effective, less toxic therapies.

Id4 expression induces apoptosis in astrocytic cultures and is down-regulated by activation of the cAMP-dependent signal transduction pathway.

The Id family of helix-loop-helix transcription factors has been implicated in the regulation of cellular differentiation in several different lineages. We have explored the potential regulatory role of the cyclic AMP-dependent signaling pathway on Id gene expression in astroglial primary cultures. We found that primary cultures of mouse forebrain astrocytes constitutively expressed the four known members of the Id gene family, Id1, Id2, Id3, and Id4. During culture in presence of serum for 4 weeks, the expression of Id4 was up-regulated. In these same cultures, treatment with dibutyryl-cyclic AMP, a cyclic AMP analogue known to promote astrocyte differentiation, dramatically and selectively decreased Id4 gene expression. This effect was detectable after short-term treatment and was maintained during long-term treatment. Forskolin and pentoxifylline, two other agents known to elevate intracellular cyclic AMP through different mechanisms, also potently decreased Id4 gene expression. Furthermore, overexpression of Id4 in an astrocyte-derived cell line induced cells to round up and die by apoptosis. These results indicate that the cyclic AMP pathway acts as an inhibitor of Id4 gene expression in astrocytes, identify a new function for Id4, and suggest that Id4 is strategically positioned in the chain of molecular events regulating astrocyte differentiation and apoptosis.