Glioblastoma remains one of the deadliest forms of cancer. Infiltrating cancer cells in the surrounding brain prevent complete resection and tumor cell resistance to chemoradiation results in the poor prognosis of the glioblastoma patient. Much research has been devoted over the years to the pathogenesis and treatment of glioblastoma. The tumor stem cell hypothesis, which was initially described in hematopoietic cell malignancies, may explain the resistance of these tumors to conventional therapies. In this model, a certain subset of tumor cells, with characteristics similar to normal neural stem cells, is capable of producing the variety of cell types, which constitute the bulk of a tumor. As these tumor cells have properties distinct from those constituting the bulk of the tumor, a different approach may be required to eradicate these residual infiltrating cells from the brain. Here we outline the history behind the theory of glioblastoma cancer stem-like cells, as they are now referred to. We will also discuss the implications of their existence on commonly held beliefs about glioblastoma pathogenesis and how they might influence future treatment strategies.

Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels, but interfere with the HIF-1α/HIF-1β/p300/CBP complex formation by interacting with p300 and CBP.

Solid tumors generally grow under hypoxic conditions, a pathophysiological change, which activates the expression of genes responsible for malignant, aggressive, and treatment-refractory properties. Hypoxia inducible factor (HIF) is the chief transcription factor regulating hypoxia-driven gene expression. Therefore, the HIF pathway has become a critical target for cancer therapeutics development. We screened a privileged library of about 10,000 natural-product-like compounds using a cell-based assay for HIF-dependent transcriptional activity and identified several arylsulfonamide HIF pathway inhibitors. Among these compounds, the most potent ones showed an IC50 of ~0.5 μM in the hypoxia-responsive element (HRE)-luciferase reporter system. Further studies are needed to fully elucidate the mechanism of action of this class of compounds and their structure-activity relationship.

Gliomas are a group of aggressive brain tumors that diffusely infiltrate adjacent brain tissues, rendering them largely incurable, even with multiple treatment modalities and agents. Mostly asymptomatic at early stages, they present in several subtypes with astrocytic or oligodendrocytic features and invariably progress to malignant forms. Gliomas are difficult to classify precisely because of interobserver variability during histopathologic grading. Identifying biological signatures of each glioma subtype through protein biomarker profiling of tumor or tumor-proximal fluids is therefore of high priority. Such profiling not only may provide clues regarding tumor classification but may identify clinical biomarkers and pathologic targets for the development of personalized treatments. In the past decade, differential proteomic profiling techniques have utilized tumor, cerebrospinal fluid, and plasma from glioma patients to identify the first candidate diagnostic, prognostic, predictive, and therapeutic response markers, highlighting the potential for glioma biomarker discovery. The number of markers identified, however, has been limited, their reproducibility between studies is unclear, and none have been validated for clinical use. Recent technological advancements in methodologies for high-throughput profiling, which provide easy access, rapid screening, low sample consumption, and accurate protein identification, are anticipated to accelerate brain tumor biomarker discovery. Reliable tools for biomarker verification forecast translation of the biomarkers into clinical diagnostics in the foreseeable future. Herein we update the reader on the recent trends and directions in glioma proteomics, including key findings and established and emerging technologies for analysis, together with challenges we are still facing in identifying and verifying potential glioma biomarkers.

Brain Angiogenesis Inhibitor 1 (BAI1) is a putative G protein-coupled receptor with potent anti-angiogenic and anti-tumorigenic properties that is mutated in certain cancers. BAI1 is expressed in normal human brain, but it is frequently silenced in glioblastoma multiforme (GBM). In this study we show this silencing event is regulated by overexpression of methyl-CpG-binding domain protein 2 (MBD2), a key mediator of epigenetic gene regulation, which binds to the hypermethylated BAI1 gene promoter. In glioma cells, treatment with the DNA demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dC) was sufficient to reactivate BAI1 expression. Chromatin immunoprecipitation (ChIP) showed that MBD2 was enriched at the promoter of silenced BAI1 in glioma cells and that MBD2 binding was released by 5-Aza-dC treatment. RNAi-mediated knockdown of MBD2 expression led to reactivation of BAI1 gene expression and restoration of BAI1 functional activity, as indicated by increased anti-angiogenic activity in vitro and in vivo. Taken together, our results suggest that MBD2 overexpression during gliomagenesis may drive tumor growth by suppressing the anti-angiogenic activity of a key tumor suppressor. These findings have therapeutic implications since inhibiting MBD2 could offer a strategy to reactivate BAI1 and suppress glioma pathobiology.

While G-protein-coupled receptors (GPCRs) have received considerable attention for their biological activity in a diversity of physiological functions and have become targets for therapeutic intervention in many diseases, the function of the cell adhesion subfamily of GPCRs remains poorly understood. Within this group, the family of brain angiogenesis inhibitor molecules (BAI1-3) has become increasingly appreciated for their diverse roles in biology and disease. In particular, recent findings suggest emerging roles for BAI1 in the regulation of phenomena including phagocytosis, synaptogenesis, and the inhibition of tumor growth and angiogenesis via the processing of its extracellular domain into secreted vasculostatins. Here we summarize the known biological features of the BAI proteins, including their structure, proteolysis events, and interacting partners, and their recently identified ability to regulate certain signaling pathways. Finally, we discuss the potential of the BAIs as therapeutics or targets for diseases as varied as cancer, stroke, and schizophrenia.

Malignant gliomas are the most common and the most lethal primary brain tumors in adults. Among malignant gliomas, 60%–80% show loss of P14ARF tumor suppressor activity due to somatic alterations of the INK4A/ARF genetic locus. The tumor suppressor activity of P14ARF is in part a result of its ability to prevent the degradation of P53 by binding to and sequestering HDM2. However, the subsequent finding of P14ARF loss in conjunction with TP53 gene loss in some tumors suggests the protein may have other P53-independent tumor suppressor functions. Here, we report what we believe to be a novel tumor suppressor function for P14ARF as an inhibitor of tumor-induced angiogenesis. We found that P14ARF mediates antiangiogenic effects by upregulating expression of tissue inhibitor of metalloproteinase–3 (TIMP3) in a P53-independent fashion. Mechanistically, this regulation occurred at the gene transcription level and was controlled by HDM2-SP1 interplay, where P14ARF relieved a dominant negative interaction of HDM2 with SP1. P14ARF-induced expression of TIMP3 inhibited endothelial cell migration and vessel formation in response to angiogenic stimuli produced by cancer cells. The discovery of this angiogenesis regulatory pathway may provide new insights into P53-independent P14ARF tumor-suppressive mechanisms that have implications for the development of novel therapies directed at tumors and other diseases characterized by vascular pathology.

SUMMARY

Two abundant classes of mobile elements, namely Alu and L1 elements, continue to generate new retrotransposon insertions in human genomes. Estimates suggest that these elements have generated millions of new germline insertions in individual human genomes worldwide. Unfortunately, current technologies are not capable of detecting most of these young insertions, and the true extent of germline mutagenesis by endogenous human retrotransposons has been difficult to examine. Here, we describe new technologies for detecting these young retrotransposon insertions and demonstrate that such insertions indeed are abundant in human populations. We also found that new somatic L1 insertions occur at high frequencies in human lung cancer genomes. Genome-wide analysis suggests that altered DNA methylation may be responsible for the high levels of L1 mobilization observed in these tumors. Our data indicate that transposon-mediated mutagenesis is extensive in human genomes, and is likely to have a major impact on human biology and diseases.

Purpose

Hypoxia inducible factor-1 (HIF-1) is the central mediator of the cellular response to low oxygen and functions as a transcription factor for a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1 is over-expressed in cancer and has become an important therapeutic target in solid tumors. In this study, a novel HIF-1α inhibitor was identified and its molecular mechanism was investigated.

Experimental Design

Using a HIF-responsive reporter cell-based assay, a 10,000-membered natural product-like chemical compound library was screened to identify novel HIF-1 inhibitors. This led us to discover KC7F2, a lead compound with a central structure of cystamine. The effects of KC7F2 on HIF-1 transcription, translation and protein degradation processes were analyzed.

Results

KC7F2 markedly inhibited HIF-mediated transcription in cells derived from different tumor types, including glioma, breast and prostate cancers and exhibited enhanced cytotoxicity under hypoxia. KC7F2 prevented the activation of HIF-target genes such as Carbonic Anhydrase IX, Matrix Metalloproteinase 2 (MMP2), Endothelin 1 and Enolase 1. Investigation of the mechanism of action of KC7F2 showed that it worked through the down-regulation of HIF-1α protein synthesis, an effect accompanied by the suppression of the phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and p70 S6 kinase (S6K), key regulators of HIF-1α protein synthesis.

Conclusion

These results show that KC7F2 is a potent HIF-1 pathway inhibitor and that its potential as a cancer therapy agent warrants further study.

Malignant gliomas are the most common and deadly brain tumors. Nevertheless, survival for patients with glioblastoma, the most aggressive glioma, although individually variable, has improved from an average of 10 months to 14 months after diagnosis in the last 5 years due to improvements in the standard of care. Radiotherapy has been of key importance to the treatment of these lesions for decades, and the ability to focus the beam and tailor it to the irregular contours of brain tumors and minimize the dose to nearby critical structures with intensity-modulated or image-guided techniques has improved greatly. Temozolomide, an alkylating agent with simple oral administration and a favorable toxicity profile, is used in conjunction with and after radiotherapy. Newer surgical techniques, such as fluorescence-guided resection and neuroendoscopic approaches, have become important in the management of malignant gliomas. Furthermore, new discoveries are being made in basic and translational research, which are likely to improve this situation further in the next 10 years. These include agents that block 1 or more of the disordered tumor proliferation signaling pathways, and that overcome resistance to already existing treatments. Targeted therapies such as antiangiogenic therapy with antivascular endothelial growth factor antibodies (bevacizumab) are finding their way into clinical practice. Large-scale research efforts are ongoing to provide a comprehensive understanding of all the genetic alterations and gene expression changes underlying glioma formation. These have already refined the classification of glioblastoma into 4 distinct molecular entities that may lead to different treatment regimens. The role of cancer stem-like cells is another area of active investigation. There is definite hope that by 2020, new cocktails of drugs will be available to target the key molecular pathways involved in gliomas and reduce their mortality and morbidity, a positive development for patients, their families, and medical professionals alike.

Cancer propagating cells (CPCs) within primary central nervous system (CNS) tumors (glioblastoma multiforme (GBM), medulloblastoma (MB) and ependymoma) might be integral to tumor development and perpetuation. These cells, also known as brain cancer propagating cells (BCPCs), have the ability to self-renew and proliferate. BCPCs can initiate new tumors in mice with high efficiency and these exhibit many features that are characteristic of patient's brain tumors. Accumulating evidence suggests that BCPCs might originate from the transformation of neural stem cells (NSCs) and their progenitors. Furthermore, recent studies have shown that NSC surface markers also define BCPCs. Ultimately, treatments that include specific targeting of BCPCs might potentially be more effective at treating the entire tumor mass, translating to improved patient survival and quality of life.

Hypoxia and necrosis are fundamental features of glioblastoma (GBM) and their emergence is critical for the rapid biological progression of this fatal tumor; yet, underlying mechanisms are poorly understood. We have suggested that vaso-occlusion following intravascular thrombosis could initiate or propagate hypoxia and necrosis in GBM. Tissue factor (TF), the main cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Epidermal growth factor receptor (EGFR) amplification and PTEN loss are two common genetic alterations seen in GBM but not in lower-grade astrocytomas that could be responsible for TF up-regulation. The most frequent EGFR mutation in GBM involves deletion of exons 2 to 7, resulting in the expression of a constitutively active receptor, EGFRvIII. Here, we show that overexpression of EGFR or EGFRvIII in human glioma cells causes increased basal TF expression and that stimulation of EGFR by its ligand, EGF, leads to a marked dose-dependent up-regulation of TF. In all cases, increased TF expression led to accelerated plasma coagulation in vitro. EGFR-mediated TF expression depended most strongly on activator protein-1 (AP-1) transcriptional activity and was associated with c-Jun NH2-terminal kinase (JNK) and JunD activation. Restoration of PTEN expression in PTEN-deficient GBM cells diminished EGFR-induced TF expression by inhibiting JunD/AP-1 transcriptional activity. PTEN mediated this effect by antagonizing phosphatidylinositol 3-kinase activity, which in turn attenuated both Akt and JNK activities. These mechanisms are likely at work in vivo, as EGFR expression was highly correlated with TF expression in human high-grade astrocytoma specimens.

p53 protein is a transcription factor involved in multiple tumor-suppressor activities including cell cycle control and apoptosis. TP53 gene is frequently mutated in glioblastoma, suggesting the importance of inactivation of this gene product in gliomagenesis. Restoration of p53 function in glioblastoma cell lines deficient for p53 has shown that p53 induces growth arrest or apoptosis depending on the cell line and vector used to transduce wild-type TP53 alleles. Considering that astrocytes grow and express p53, it is not clear whether these results reflect physiologic responses or the result of p53 overexpression in combination with cellular responses to viral vector infection. Here, we reassessed this issue using a glioblastoma cell line (LN382) that expresses an endogenous temperature-sensitive mutant p53. This cell line expresses TP53 alleles (100% as determined by a p53 transcriptional assay in yeast) mutated at codon 197 GTG (Val) > CTG (Leu). We found that the p53 protein in these cells acted as an inactive mutant at 37°C and as a functional wild-type p53 below 34°C as demonstrated by several lines of evidence, including (i) restoration of transactivating ability in yeast, (ii) induction of p53-modulated genes such as CDKN1p21 and transforming growth factor-α, (iii) disappearance of accumulated p53 protein in the nucleus and (iv) decrease in steady state p53 protein levels. This temperature switch allowed p53 levels, which were close to physiological levels to dramatically reduce LN382 cell proliferation by inducing a G1/S cell cycle block, but not to induce apoptosis. The lack of apoptosis was considered to be a result of the low level p53 expression, because increasing wild-type p53 levels by adenoviral-mediated gene transfer caused apoptosis in these cells. The LN382 cell line will be extremely useful for investigations into the roles of p53 in cellular responses to a variety of stimuli or damages.

Angiogenesis is a critical physiological process that is appropriated during tumorigenesis. Little is known about how this process is specifically regulated in the brain. Brain Angiogenesis Inhibitor-1 (BAI1) is a primarily brain specific seven-transmembrane protein that contains five anti-angiogenic thrombospondin type-1 repeats (TSR). We recently showed that BAI1 is cleaved at a conserved proteolytic cleavage site releasing a soluble, 120 kDa anti-angiogenic factor called Vasculostatin (Vstat120). Vstat120 has been shown to inhibit in vitro angiogenesis and suppress subcutaneous tumor growth. Here, we examine its effect on intracranial growth of malignant gliomas and further study the mechanism of its anti-tumor effects. First, we show that expression of Vstat120 strongly suppresses the intracranial growth of malignant gliomas, even in the presence of the strong pro-angiogenic stimulus mediated by the oncoprotein Epidermal Growth Factor Receptor variant III (EGFRvIII). This tumor suppressive effect is accompanied by a decrease in vascular density in the tumors, suggesting a potent anti-angiogenic effect in the brain. Second, and consistent with this interpretation, we find that treatment with Vstat120 reduces the migration of cultured microvascular endothelial cells in vitro and inhibits corneal angiogenesis in vivo. Third, we demonstrate that these anti-vascular effects are critically dependent on the presence of the cell surface receptor CD36 on endothelial cells in vitro and in vivo, supporting a role of the Vstat120 TSRs in mediating these effects. These results advance the understanding of brain-specific angiogenic regulation, and suggest that Vstat120 has therapeutic potential in the treatment of brain tumors and other intra-cerebral vasculopathies.

A rare subpopulation of cells within malignant gliomas, which shares canonical properties with neural stem cells (NSCs), may be integral to glial tumor development and perpetuation. These cells, also known as tumor initiating cells (TICs), have the ability to self-renew, develop into any cell in the overall tumor population (multipotency), and proliferate. A defining property of TICs is their ability to initiate new tumors in immunocompromised mice with high efficiency. Mounting evidence suggests that TICs originate from the transformation of NSCs and their progenitors. New findings show that TICs may be more resistant to chemotherapy and radiation than the bulk of tumor cells, thereby permitting recurrent tumor formation and accounting for the failure of conventional therapies. The development of new therapeutic strategies selectively targeting TICs while sparing NSCs may provide for more effective treatment of malignant gliomas.

BAP1 (BRCA1-associated protein-1), a deubiquitinating enzyme of unknown cellular function, is mutated in breast and lung cancers. In this study, we have demonstrated for the first time that BAP1 has tumor suppressor activity in vivo by showing that BAP1 can suppress tumorigenicity of lung cancer cells in athymic nude mice. We show that BAP1 fulfills another criterion of a genuine tumor suppressor because cancer-associated mutations in BAP1 result in a protein deficient in deubiquitinating activity. We show for the first time that one of the two predicted nuclear targeting motifs is required for nuclear localization of BAP1 and that a truncation mutant found in a lung cancer cell line results in BAP1 that fails to localize to the nucleus. Furthermore, we demonstrate that deubiquitinating activity and nuclear localization are both required for BAP1-mediated tumor suppression in nude mice. We show that BAP1 exerts its tumor suppressor functions by affecting the cell cycle; speeding the progression through the G1/S checkpoint and inducing cell death via a process that has characteristics of both apoptosis and necrosis. Surprisingly, BAP1-mediated growth suppression is independent of wild-type BRCA1. Since deubiquitinating enzymes are components of the ubiquitin proteasome system, this pathway has emerged as an important target for anti-cancer drugs. The identification of the deubiquitinating enzyme BAP1 as a tumor suppressor may lead to further understanding of how the ubiquitin proteasome system contributes to cancer and aid in the identification of new targets for cancer therapy.

Hypoxia strongly up-regulates tissue factor and promotes plasma clotting by glioblastoma multiforme, but transcriptional mechanisms remain undefined. Here, we investigated the potential roles of early growth response gene-1 (Egr-1), Sp1, nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and hypoxia-inducible factor-1 (HIF-1) in the hypoxic regulation of tissue factor by glioblastoma multiforme cells in vitro. Hypoxia (1% O2) strongly induced Egr-1 mRNA within 1 hour and led to nuclear localization of Egr-1 protein. Using luciferase reporter plasmids in glioma cells, we found that hypoxia dramatically increased luciferase activity in cells with constructs containing Egr-1-binding sites but not in cells with constructs containing AP-1- or NF-κB-binding sites. Electrophoretic mobility shift assays revealed hypoxia-induced Egr-1, but not Sp1, binding to oligonucleotides containing the Egr-1/Sp1 motif of tissue factor gene promoter. Using an expression vector containing the minimal tissue factor promoter (−111 to +14 bp) and small interfering RNA (siRNA) directed at Egr-1 and Sp1 mRNAs, we found that Egr-1 was required for maximal hypoxic induction of promoter activity. Forced overexpression of Egr-1 but not Sp1 by cDNA transfection caused up-regulation of tissue factor in glioma cells under normoxia (21% O2), whereas siRNA directed at Egr-1 strongly attenuated hypoxia-induced tissue factor expression. To examine the effects of HIF-1α on tissue factor expression, we used glioma cells stably transfected with a HIF-1α siRNA expression vector and found that HIF-1α mRNA silencing did not affect tissue factor expression under hypoxia. We conclude that hypoxic up-regulation of tissue factor in glioblastoma multiforme cells depends largely on Egr-1 and is independent of HIF-1.

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

There is a need for novel therapies targeting hypoxic cells in tumors. These cells are associated with tumor resistance to therapy and express hypoxia inducible factor-1 (HIF-1), a transcription factor that mediates metabolic adaptation to hypoxia and activates tumor angiogenesis. We previously developed an oncolytic adenovirus (HYPR-Ad) for the specific killing of hypoxic/HIF-active tumor cells, which we now armed with an interleukin-4 gene (HYPR-Ad-IL4). We designed HYPR-Ad-IL4 by cloning the Ad E1A viral replication and IL-4 genes under the regulation of a bidirectional hypoxia/HIF-responsive promoter. The IL-4 cytokine was chosen for its ability to induce a strong host antitumor immune response and its potential antiangiogenic activity. HYPR-Ad-IL4 induced hypoxia-dependent IL-4 expression, viral replication, and conditional cytolysis of hypoxic, but not normoxic cells. The treatment of established human tumor xenografts with HYPR-Ad-IL4 resulted in rapid and maintained tumor regression with the same potency as that of wild-type dl309-Ad. HYPR-Ad-IL4–treated tumors displayed extensive necrosis, fibrosis, and widespread viral replication. Additionally, these tumors contained a distinctive leukocyte infiltrate and prominent hypoxia. The use of an oncolytic Ad that locally delivers IL-4 to tumors is novel, and we expect that HYPR-Ad-IL4 will have broad therapeutic use for all solid tumors that have hypoxia or active HIF, regardless of tissue origin or genetic alterations.

Interleukin-8 (IL-8, or CXCL8), which is a chemokine with a defining CXC amino acid motif that was initially characterized for its leukocyte chemotactic activity, is now known to possess tumorigenic and proangiogenic properties as well. In human gliomas, IL-8 is expressed and secreted at high levels both in vitro and in vivo, and recent experiments suggest it is critical to glial tumor neovascularity and progression. Levels of IL-8 correlate with histologic grade in glial neoplasms, and the most malignant form, glioblastoma, shows the highest expression in pseudopalisading cells around necrosis, suggesting that hypoxia/anoxia may stimulate expression. In addition to hypoxia/anoxia stimulation, increased IL-8 in gliomas occurs in response to Fas ligation, death receptor activation, cytosolic Ca2+, TNF-α, IL-1, and other cytokines and various cellular stresses. The IL-8 promoter contains binding sites for the transcription factors NF-κB, AP-1, and C-EBP/NF-IL-6, among others. AP-1 has been shown to mediate IL-8 upregulation by anoxia in gliomas. The potential tumor suppressor ING4 was recently shown to be a critical regulator of NF-κB-mediated IL-8 transcription and subsequent angiogenesis in gliomas. The IL-8 receptors that could contribute to IL-8-mediated tumorigenic and angiogenic responses include CXCR1 and CXCR2, both of which are G-protein coupled, and the Duffy antigen receptor for cytokines, which has no defined intracellular signaling capabilities. The proangiogenic activity of IL-8 occurs predominantly following binding to CXCR2, but CXCR1 appears to contribute as well through independent, small-GTPase activity. A precise definition of the mechanisms by which IL-8 exerts its proangiogenic functions requires further study for the development of effective IL-8-targeted therapies.

Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of α and β subunits. The α subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors α and β, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.

Background

The ADAMTS (A Disintegrin-like and Metalloprotease with Thrombospondin motifs) proteins are a family of metalloproteases with sequence similarity to the ADAM proteases, that contain the thrombospondin type 1 sequence repeat motifs (TSRs) common to extracellular matrix proteins. ADAMTS proteins have recently gained attention with the discovery of their role in a variety of diseases, including tissue and blood disorders, cancer, osteoarthritis, Alzheimer's and the genetic syndromes Weill-Marchesani syndrome (ADAMTS10), thrombotic thrombocytopenic purpura (ADAMTS13), and Ehlers-Danlos syndrome type VIIC (ADAMTS2) in humans and belted white-spotting mutation in mice (ADAMTS20).

Results

Phylogenetic analysis and comparison of the exon/intron organization of vertebrate (Homo, Mus, Fugu), chordate (Ciona) and invertebrate (Drosophila and Caenorhabditis) ADAMTS homologs has elucidated the evolutionary relationships of this important gene family, which comprises 19 members in humans.

Conclusions

The evolutionary history of ADAMTS genes in vertebrate genomes has been marked by rampant gene duplication, including a retrotransposition that gave rise to a distinct ADAMTS subfamily (ADAMTS1, -4, -5, -8, -15) that may have distinct aggrecanase and angiogenesis functions.

Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, results from a disruption of the balance between stimulatory and inhibitory factors. Here, we show that anoxia reduces expression of thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, in glioblastoma cells. This suggests that reduced oxygen tension can promote angiogenesis not only by stimulating the production of inducers, such as vascular endothelial growth factor, but also by reducing the production of inhibitors. This downregulation may significantly contribute to glioblastoma development, since we show that an increase in TSP-1 expression is sufficient to strongly suppress glioblastoma cell tumorigenicity in vivo.

The purpose of the present study was to determine the in vitro and in vivo anti-cancer activity and pharmacological properties of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1. In the present study, we investigated the in vitro activity of KCN1 on cell proliferation and cell cycle distribution of pancreatic cancer cells, using the MTT and BrdUrd assays, and flow cytometry. The in vivo anti-cancer effects of KCN1 were evaluated in two distinct xenograft models of pancreatic cancer. We also developed an HPLC method for the quantitation of the compound, and examined its stability in mouse plasma, plasma protein binding, and degradation by mouse S9 microsomal enzymes. Furthermore, we examined the pharmacokinetics of KCN1 following intravenous or intraperitoneal injection in mice. Results showed that, in a dose-dependent manner, KCN1 inhibited cell growth and induced cell cycle arrest in human pancreatic cancer cells in vitro, and showed in vivo anticancer efficacy in mice bearing Panc-1 or Mia Paca-2 tumor xenografts. The HPLC method provided linear detection of KCN1 in all of the matrices in the range from 0.1 to 100 µM, and had a lower limit of detection of 0.085 µM in mouse plasma. KCN1 was very stable in mouse plasma, extensively plasma bound, and metabolized by S9 microsomal enzymes. The pharmacokinetic studies indicated that KCN1 could be detected in all of the tissues examined, most for at least 24 h. In conclusion, our preclinical data indicate that KCN1 is a potential therapeutic agent for pancreatic cancer, providing a basis for its future development.