Ty W. Abel

Ligand-dependent activation of the hedgehog pathway in glioma progenitor cells.

The hedgehog (Hh) signaling pathway regulates progenitor cells during embryogenesis and tumorigenesis in multiple organ systems. We have investigated the activity of this pathway in adult gliomas, and demonstrate that the Hh pathway is operational and activated within grade II and III gliomas, but not grade IV de novo glioblastoma multiforme. Furthermore, our studies reveal that pathway activity and responsiveness is confined to progenitor cells within these tumors. Additionally, we demonstrate that Hh signaling in glioma progenitor cells is ligand-dependent and provide evidence documenting the in vivo source of Sonic hedgehog protein. These findings suggest a regulatory role for the Hh pathway in progenitor cells within grade II and III gliomas, and the potential clinical utility of monitoring and targeting this pathway in these primary brain tumors.

Transforming Growth Factor–β Regulates Mammary Carcinoma Cell Survival and Interaction with the Adjacent Microenvironment

Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.

Junctional Adhesion Molecule-A Is Required for Hematogenous Dissemination of Reovirus

Diverse families of viruses bind immunoglobulin superfamily (IgSF) proteins located in tight junctions (TJs) and adherens junctions of epithelium and endothelium. However, little is known about the roles of these receptors in the pathogenesis of viral disease. Junctional adhesion molecule-A (JAM-A) is an IgSF protein that localizes to TJs and serves as a receptor for mammalian reovirus. We inoculated wild-type (WT) and isogenic JAM-A(-/-) mice perorally with reovirus and found that JAM-A is dispensable for viral replication in the intestine but required for systemic dissemination. Reovirus replication in the brain and tropism for discrete neural regions are equivalent in WT and JAM-A(-/-) mice following intracranial inoculation, suggesting a function for JAM-A in reovirus spread to extraintestinal sites. JAM-A promotes reovirus infection of endothelial cells, providing a conduit for the virus into the bloodstream. These findings indicate that a broadly expressed IgSF viral receptor specifically mediates hematogenous dissemination in the host.

Reovirus Apoptosis and Virulence Are Regulated by Host Cell Membrane Penetration Efficiency

ABSTRACT Apoptosis plays an important role in the pathogenesis of reovirus encephalitis and myocarditis in infected animals. Differences in apoptosis efficiency displayed by reovirus strains are linked to the viral μ1-encoding M2 gene segment. Studies using pharmacologic inhibitors of reovirus replication demonstrate that apoptosis induction by reovirus requires viral disassembly in cellular endosomes but not RNA synthesis. Since the μ1 protein functions to pierce endosomal membranes during this temporal window, these findings point to an important role for μ1 in activating signaling pathways that lead to apoptosis. To understand mechanisms used by μ1 to induce apoptosis, a panel of μ1 mutant viruses generated by reverse genetics was analyzed for the capacities to penetrate host cell membranes, activate proapoptotic signaling pathways, evoke cell death, and produce encephalitis in newborn mice. We found that single amino acid changes within the δ region of μ1 reduce the efficiency of membrane penetration. These mutations also diminish the capacities of reovirus to activate proapoptotic transcription factors NF-κB and IRF-3 and elicit apoptosis. Additionally, we observed that following intracranial inoculation, an apoptosis-deficient μ1 mutant is less virulent in newborn mice in comparison to the wild-type virus. These results indicate a critical function for the membrane penetration activity of μ1 in evoking prodeath signaling pathways that regulate reovirus pathogenesis.

Independent Regulation of Reovirus Membrane Penetration and Apoptosis by the μ1 φ Domain

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein μ1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of μ1 indicates that the μ1 ϕ domain is sufficient to elicit a cell death response. To evaluate the contribution of the μ1 ϕ domain to the induction of apoptosis following reovirus infection, ϕ mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in ϕ diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in ϕ affect the apoptotic potential of reovirus, suggesting that ϕ initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective ϕ mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the ϕ domain of μ1 plays an important regulatory role in reovirus-induced apoptosis and disease.

Neurodegeneration in Mice Resulting From Loss of Functional Selenoprotein P or Its Receptor Apolipoprotein E Receptor 2

Selenoprotein P (Sepp1) is involved in selenium homeostasis. Mice with a deletion of Sepp1, replacement of it by the shortened form Sepp1&Dgr;240-361, or deletion of its receptor apolipoprotein E receptor 2 develop severe neurologic dysfunction when fed low-selenium diet. Because the brainstems of Sepp1−/− mice had been observed to contain degenerated axons, a study of these 3 strains was made under selenium-deficient and high-selenium (control) conditions. Selenium-deficient wild-type mice were additional controls. Serial sections of the brain were evaluated with amino cupric silver degeneration and anti-glial fibrillary acidic protein stains. All 3 strains with altered Sepp1 metabolism developed severe axonal injury when fed selenium deficient diet. This injury was mitigated by high-selenium diet and was absent from selenium-deficient wild-type mice. Injury was most severe in Sepp1−/− mice, with staining in at least 6 brain regions. Injury in Sepp1&Dgr;240-361 and apolipoprotein E receptor 2−/− mice was less severe and occurred only in areas injured in Sepp1−/− mice, suggesting a common selenium-related etiology. Affected brain regions were primarily associated with auditory and motor functions, consistent with the clinical signs. Those areas have high metabolic rates. We conclude that interference with Sepp1 function damages auditory and motor areas, at least in part by restricting selenium supply to the brain regions.

Diagnostic utility of SALL4 in primary germ cell tumors of the central nervous system: A study of 77 cases

Primary germ cell tumors of the central nervous system (CNS) sometimes pose diagnostic difficulty. In this study we analyzed the diagnostic utility of a novel marker, SALL4, in 77 such tumors (59 pure and 18 mixed) consisting of the following tumors/tumor components: 49 germinomas, 7 embryonal carcinomas, 27 yolk sac tumors, 3 choriocarcinomas, and 14 teratomas. We also stained SALL4 in 99 primary non-germ cell tumors to test SALL4 specificity. We compared SALL4 with OCT4 in all germ cell tumors and compared SALL4 with α-fetoprotein and glypican-3 in all yolk sac tumors. The staining was semiquantitatively scored as 0 (no staining), 1+ (<=30%), 2+(31–60%), 3+ (61–90%), and 4+ (>90%). Strong SALL4 staining was observed in all 49 germinomas (4+ in 48, 3+ in 1), 7 embryonal carcinomas (all 4+), and 27 yolk sac tumors (1+ in 1, 2+ in 2, 3+ in 7, 4+ in 17). SALL4 staining, 1+ weak to focally strong, was observed in 2 of 3 choriocarcinomas (in mononucleated trophoblasts) and in 9 of 14 teratomas (in primitive neuroepithelium and teratomatous glands). All germinomas and embryonal carcinomas showed strong OCT4 staining (4+ in all except 1 germinoma with 3+), whereas other germ cell tumors were negative. Out of 27 yolk sac tumors, 26 showed positive α-fetoprotein staining (1+ in 9, 2+ in 7, 3+ in 5, and 4+ in 5). All yolk sac tumors showed positive glypican-3 staining (1+ in 6, 2+ in 6, 3+ in 7, and 4+ in 8). The mean percentage of yolk sac tumor cells stained was 84% with SALL4, 45% with α-fetoprotein, and 63% with glypican-3 (P<0.01). No non-germ cell tumors showed SALL4 staining. Our results indicate that SALL4 is a novel sensitive diagnostic marker for primary germ cell tumors of the CNS with high specificity. SALL4 is a more sensitive marker than α-fetoprotein and glypican-3 for yolk sac tumors.

CXCR4 Expression is Elevated in Glioblastoma Multiforme and Correlates with an Increase in Intensity and Extent of Peritumoral T2-weighted Magnetic Resonance Imaging Signal Abnormalities

OBJECTIVEWith the objective of investigating the utility of CXCR4, a chemokine receptor known to mediate glioma cell invasiveness, as a molecular marker for peritumoral disease extent in high-grade gliomas, we sought to characterize the expression profile of CXCR4 in a large panel of tumor samples and determine whether CXCR4 expression levels within glioblastoma multiforme might correlate with radiological evidence of a more extensive disease process. METHODSFreshly resected tumor tissue samples were processed for immunohistochemical and quantitative polymerase chain reaction analyses to identify and quantify expression levels of CXCR4 and its corresponding ligand CXCL12. T1 postcontrast and T2-weighted magnetic resonance imaging brain scans were used to generate voxel signal intensity histograms that were quantitatively analyzed to determine the extent and intensity of peritumoral signal abnormality as a marker of disseminated disease in the brain. RESULTSCXCR4 expression was markedly elevated in Grade III and IV tumors compared with Grade II gliomas. Significantly, when patients with glioblastoma multiforme were segregated into two groups based on CXCR4 expression level, we observed a statistically significant increase in the intensity and extent of peritumoral magnetic resonance imaging signal abnormalities associated with CXCR4 high-expressing gliomas. CONCLUSIONOur data confirm that high-grade gliomas robustly express CXCR4 and demonstrate a correlative relationship between expression levels of the CXCR4 receptor and the magnetic resonance imaging-based finding of a diffuse and more extensive disease process in the brain. CXCR4 expression status may, therefore, prove useful as a marker of disseminated disease in patients with glioblastoma multiforme.

Targeted inhibition of the Hedgehog pathway in established malignant glioma xenografts enhances survival

Hedgehog pathway activity has been demonstrated in malignant glioma. However, its role in tumor growth has not been determined. Here we demonstrate that pharmacological inhibition of the Hedgehog pathway in established orthotopic malignant glioma xenografts confers a survival advantage. Pathway inhibition is measured in transplanted human tumor cells and not in host mouse brain. Correspondingly, survival benefit is observed only in tumors with an operational Hedgehog pathway. These data indicate that Hedgehog signaling regulates the growth of select malignant gliomas. We also demonstrate that Hedgehog pathway component and gene target expression segregate to CD133+ tumor initiating cells. Treated mice eventually succumb to disease, thus, targeting the Hedgehog pathway in CD133+ cells produces significant, but incomplete tumor regression. Therefore, our studies suggest that more complete tumor regression may require the inclusion of other therapeutic targets, including CD133− cells.

Proopiomelanocortin gene expression is decreased in the infundibular nucleus of postmenopausal women.

Previous studies have shown that estrogen withdrawal decreases the secretion of beta-endorphin from the monkey hypothalamus. In addition, there are consistent age-associated changes in beta-endorphin neurons in the rodent. Based on these findings, we hypothesized that the activity of hypothalamic beta-endorphin neurons would be decreased in the hypothalamus of postmenopausal women. In the present study, we examined the expression of proopiomelanocortin (POMC) mRNA, the precursor mRNA for beta-endorphin, in the medial basal hypothalamus of premenopausal and postmenopausal women. Every 20th sagittal section through the hypothalamus was hybridized with a synthetic [35S]labeled, 48-base oligonucleotide probe complementary to POMC mRNA. Labeled neurons were counted and their somatic profile areas were measured with an image-combining computer microscope system. The number of POMC mRNA-containing neurons/section in the infundibular nucleus was reduced by 65% in postmenopausal women. In contrast, there was no significant difference in the number of neurons expressing POMC gene transcripts in the retrochiasmatic region. The POMC neurons in the retrochiasmatic area were also distinct morphologically from those in the infundibular nucleus. The differences between the infundibular and retrochiasmatic regions suggest that functional subgroups of POMC neurons exist in the human hypothalamus. Our findings provide evidence that the activity of hypothalamic POMC neurons is decreased in the infundibular nucleus of postmenopausal women. Both aging and gonadal steroid withdrawal may contribute to the decline in POMC gene expression in postmenopausal women.