Balveen Kaur

Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis

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 alpha and beta subunits. The alpha 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 alpha and beta, 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.

Pseudopalisades in Glioblastoma Are Hypoxic, Express Extracellular Matrix Proteases, and Are Formed by an Actively Migrating Cell Population

Necrosis and vascular proliferation are the pathologic features that distinguish the most malignant infiltrative astrocytoma, glioblastoma (GBM), from those of lower grades. In GBM, hypercellular zones called pseudopalisades typically surround necrotic foci. Although these cells are known to secrete high levels of proangiogenic factors that promote tumor growth, their origins are ill defined. We propose that pseudopalisades represent differing stages and histologic samplings of astrocytoma cells migrating away from a hypoxic/anoxic focus, often triggered by a central vaso-occlusive event. This proposition is based on our findings that pseudopalisading cells are 5–50% less proliferative and 6–20 times more apoptotic than adjacent astrocytoma, indicating that cell accumulation does not result from increased proliferation or resistance to apoptosis. Coexisting inflammatory cells account for <2% of pseudopalisading cells and cannot account for hypercellularity. Pseudopalisading cells show nuclear expression of hypoxia-inducible factor 1α, consistent with their hypoxic nature, and hypoxia induces a 20–60% increase in glioma cell migration in vitro. Hypoxic cells in vitro and pseudopalisades in GBM specimens show enhanced gelatinase activity, typical of an invasive phenotype. These results suggest that pseudopalisading cells are migrating at the periphery of a hypoxic center. To uncover a potential source of hypoxia and sequence of structural events leading to pseudopalisade formation, we performed a morphometric analysis of 234 pseudopalisades from 85 pretreatment GBMs. We found distorted, degenerating, or thrombosed blood vessels within the center of more than half the pseudopalisades, suggesting that at least a subset of pseudopalisades are two-dimensional histologic representations of tumor cells migrating away from a vaso-occlusive event.

Cyclophosphamide enhances glioma virotherapy by inhibiting innate immune responses.

Clinical trials are testing oncolytic viruses (OVs) as therapies for cancer. We have shown that animals that have brain tumors and are treated with a herpes simplex virus (HSV)-derived OV live significantly longer when cyclophosphamide (CPA) is preadministered. Here, we explore the mechanisms behind this finding. In a syngeneic rat glioma model, intratumoral HSV administration is associated with rapid increase of natural killer cells, microglia/macrophages (CD68+ and CD163+), and IFN-γ. Pretreatment with CPA enhances HSV replication and oncolysis and reduces an HSV-mediated increase in CD68+ and CD163+ cells and intratumoral IFN-γ. Molecular imaging shows CPA pretreatment to inhibit HSV-induced infiltration of tumor-associated phagocytic cells. Our results reveal molecular and cellular mechanisms that inhibit intratumoral spread of HSV and suggest a therapeutic path for improving the efficacy of virotherapy as a treatment for cancer.

Genetic and hypoxic regulation of angiogenesis in gliomas

Infiltrative astrocytic neoplasms are by far the most common malignant brain tumors in adults. Clinically, they are highly problematic due to their widely invasive nature which makes a complete resection almost impossible. Biologic progression of these tumors is inevitable and adjuvant therapies are only moderately effective in prolonging survival. Glioblastoma multiforme (GBM WHO grade IV), the most malignant form of infiltrating astrocytoma, can evolve from a lower grade precursor tumor (secondary GBM) or can present as high grade lesion from the outset, so-called de novoGBM. Molecular genetic investigations suggest that GBMs are comprised of multiple molecular genetic subsets. Notwithstanding the diversity of genetic alterations leading to the GBM phenotype, the vascular changes that evolve in this disease, presumably favoring further growth, are remarkably similar. Underlying genetic alterations in GBM may tilt the balance in favor of an angiogenic phenotype by upregulation of pro-angiogenic factors and down-regulation of angiogenesis inhibitors. Increased vascularity and endothelial cell proliferation in GBMs are also driven by hypoxia-induced expression of pro-angiogenic cytokines, such vascular endothelial growth factor (VEGF). Understanding the contribution of genetic alterations and hypoxia in angiogenic dysregulation in astrocytic neoplasms will lead to the development of better anti-angiogenic therapies for this disease. This review will summarize the properties of angiogenic dysregulation that lead to the highly vascularized nature of these tumors.

Vasculostatin, a proteolytic fragment of brain Angiogenesis inhibitor 1, is an antiangiogenic and antitumorigenic factor

Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with unknown function expressed primarily in normal but not tumoral brain. The finding of thrombospondin type 1 repeats in its extracellular domain suggested an antiangiogenic function, but the mechanisms by which a transmembrane receptor could inhibit angiogenesis remained unexplained. Here we demonstrate that BAI1 is proteolytically cleaved at a conserved G-protein-coupled receptor proteolytic cleavage site (GPS), releasing its 120 kDa extracellular domain. We named this secreted fragment Vasculostatin as it inhibited migration of endothelial cells in vitro and dramatically reduced in vivo angiogenesis. Both constitutive and doxycycline-induced expression of Vasculostatin elicited dose-dependent suppression of tumor growth and vascular density in mice, implicating Vasculostatin in the regulation of vascular homeostasis and tumor prevention. Generation of a soluble antiangiogenic factor by cleavage of a pre-existing transmembrane protein represents a novel mechanism for regulating vascular homeostasis and preventing tumorigenesis. Modulation of this cleavage or delivery of Vasculostatin may constitute novel treatment modalities for cancer and other diseases of aberrant angiogenesis, especially in the brain.

MicroRNAs/TP53 feedback circuitry in glioblastoma multiforme

MicroRNAs (miRNAs) are increasingly implicated in regulating cancer initiation and progression. In this study, two miRNAs, miR-25 and -32, are identified as p53-repressed miRNAs by p53-dependent negative regulation of their transcriptional regulators, E2F1 and MYC. However, miR-25 and -32 result in p53 accumulation by directly targeting Mdm2 and TSC1, which are negative regulators of p53 and the mTOR (mammalian target of rapamycin) pathway, respectively, leading to inhibition of cellular proliferation through cell cycle arrest. Thus, there is a recurrent autoregulatory circuit involving expression of p53, E2F1, and MYC to regulate the expression of miR-25 and -32, which are miRNAs that, in turn, control p53 accumulation. Significantly, overexpression of transfected miR-25 and -32 in glioblastoma multiforme cells inhibited growth of the glioblastoma multiforme cells in mouse brain in vivo. The results define miR-25 and -32 as positive regulators of p53, underscoring their role in tumorigenesis in glioblastoma.

NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors

The role of the immune response to oncolytic Herpes simplex viral (oHSV) therapy for glioblastoma is controversial because it might enhance or inhibit efficacy. We found that within hours of oHSV infection of glioblastomas in mice, activated natural killer (NK) cells are recruited to the site of infection. This response substantially diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on the NK cell natural cytotoxicity receptors (NCRs) NKp30 and NKp46, whose ligands are upregulated in oHSV-infected glioblastoma cells. We found that HSV titers and oHSV efficacy are increased in Ncr1−/− mice and a Ncr1−/− NK cell adoptive transfer model of glioma, respectively. These results demonstrate that glioblastoma virotherapy is limited partially by an antiviral NK cell response involving specific NCRs, uncovering new potential targets to enhance cancer virotherapy.

Genetic and biologic progression in astrocytomas and their relation to angiogenic dysregulation

Infiltrative astrocytic neoplasms are the most common malignancies of the central nervous system. They remain clinically problematic because of their involvement of brain structures critical to proper cognitive, behavioral, and motor function; their widely invasive properties, which make them difficult to resect totally; and their nearly inevitable biologic progression in spite of adjuvant therapy. Glioblastoma multiforme (GBM, World Health Organization grade IV), the most malignant form of infiltrating astrocytoma, can present as a high-grade lesion from the outset (so-called de novo GBM) or can evolve from a lower grade precursor lesion (secondary GBM). Molecular genetic investigations suggest that GBM is best regarded as a clinicopathologic entity composed of multiple molecular genetic subsets. Molecular alterations associated with progression to GBM and that define genetic subsets include epidermal growth factor receptor amplifications, p53 mutations, retinoblastoma pathway alterations [most commonly, p16(CDKN2A) losses], and chromosome 10 alterations, including PTEN mutations. Despite the wide range of genetic events that ultimately lead to GBM, the vascular changes that evolve are remarkably similar. Microvascular hyperplasia is spatially and temporally associated with pseudopalisading necrosis in GBM and is believed to be driven by hypoxia-induced expression of proangiogenic cytokines such vascular endothelial growth factor. In addition, genetic alterations in GBM are thought to contribute directly or indirectly to angiogenic dysregulation. Both p53 mutations and genetic losses on chromosome 10 may tip the balance toward an angiogenic phenotype through upregulation of proangiogenic factors and/or downregulation of angiogenesis inhibitors. Understanding genetic events and their relation to angiogenic regulation in astrocytic neoplasms may eventually lead to therapies that are specifically directed at molecularly defined subsets of these diseases.

Phase IB Study of Gene-Mediated Cytotoxic Immunotherapy Adjuvant to Up-Front Surgery and Intensive Timing Radiation for Malignant Glioma

PURPOSE Despite aggressive therapies, median survival for malignant gliomas is less than 15 months. Patients with unmethylated O(6)-methylguanine-DNA methyltransferase (MGMT) fare worse, presumably because of temozolomide resistance. AdV-tk, an adenoviral vector containing the herpes simplex virus thymidine kinase gene, plus prodrug synergizes with surgery and chemoradiotherapy, kills tumor cells, has not shown MGMT dependency, and elicits an antitumor vaccine effect. PATIENTS AND METHODS Patients with newly diagnosed malignant glioma received AdV-tk at 3 × 10(10), 1 × 10(11), or 3 × 10(11) vector particles (vp) via tumor bed injection at time of surgery followed by 14 days of valacyclovir. Radiation was initiated within 9 days after AdV-tk injection to overlap with AdV-tk activity. Temozolomide was administered after completing valacyclovir treatment. RESULTS Accrual began December 2005 and was completed in 13 months. Thirteen patients were enrolled and 12 completed therapy, three at dose levels 1 and 2 and six at dose level 3. There were no dose-limiting or significant added toxicities. One patient withdrew before completing prodrug because of an unrelated surgical complication. Survival at 2 years was 33% and at 3 years was 25%. Patient-reported quality of life assessed with the Functional Assessment of Cancer Therapy-Brain (FACT-Br) was stable or improved after treatment. A significant CD3(+) T-cell infiltrate was found in four of four tumors analyzed after treatment. Three patients with MGMT unmethylated glioblastoma multiforme survived 6.5, 8.7, and 46.4 months. CONCLUSION AdV-tk plus valacyclovir can be safely delivered with surgery and accelerated radiation in newly diagnosed malignant gliomas. Temozolomide did not prevent immune responses. Although not powered for efficacy, the survival and MGMT independence trends are encouraging. A phase II trial is ongoing.

Extracellular Vesicles and Their Convergence with Viral Pathways

Extracellular vesicles (microvesicles), such as exosomes and shed microvesicles, contain a variety of molecules including proteins, lipids, and nucleic acids. Microvesicles appear mostly to originate from multivesicular bodies or to bud from the plasma membrane. Here, we review the convergence of microvesicle biogenesis and aspects of viral assembly and release pathways. Herpesviruses and retroviruses, amongst others, recruit several elements from the microvesicle biogenesis pathways for functional virus release. In addition, noninfectious pleiotropic virus-like vesicles can be released, containing viral and cellular components. We highlight the heterogeneity of microvesicle function during viral infection, addressing microvesicles that can either block or enhance infection, or cause immune dysregulation through bystander action in the immune system. Finally, endogenous retrovirus and retrotransposon elements deposited in our genomes millions of years ago can be released from cells within microvesicles, suggestive of a viral origin of the microvesicle system or perhaps of an evolutionary conserved system of virus-vesicle codependence. More research is needed to further elucidate the complex function of the various microvesicles produced during viral infection, possibly revealing new therapeutic intervention strategies.