Faculty Bibliography

Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1alpha (Hif1alpha)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis.

Background. Extraosseous Ewing's sarcoma in the spinal epidural space is a rare malignancy, especially in adults. Case Presentation. A 40-year-old male presented with back pain and urinary hesitancy. MRI revealed a thoracic extradural mass with no osseous involvement. He underwent surgery for gross total resection of the mass, which was diagnosed as Ewing's sarcoma. He was subsequently treated with chemoradiotherapy. He remains disease-free 1 year after surgery. Review of the literature indicated only 45 previously reported cases of spinal epidural extraosseous Ewing's sarcoma in adults. Conclusions. Extraosseous Ewing's sarcoma in the spinal epidural space is a rare clinical entity that should be included in the differential for spinal epidural masses. Its treatment is multidisciplinary but frequently requires surgical intervention due to compressive neurologic symptoms. Gross total resection appears to correlate with improved outcomes.

BACKGROUND: Previous reports have proposed an association between traumatic brain injury (TBI) and subsequent glioblastoma (GBM) formation. METHODS: We used literature searches and radiographic evidence from two patients to assess the possibility of a link between TBI and GBM. RESULTS: Epidemiological studies are equivocal on a possible link between brain trauma and increased risk of malignant glioma formation. We present two case reports of patients with GBM arising at the site of prior brain injury. CONCLUSION: The hypothesis that TBI may predispose to gliomagenesis is disputed by several large-scale epidemiological studies, but supported by some. Radiographic evidence from two cases presented here suggest that GBM formed at the site of brain injury. We propose a putative pathogenesis model that connects post-traumatic inflammation, stem and progenitor cell transformation, and gliomagenesis.

Mutations in genes encoding Isocitrate Dehydrogenase (IDH) isoforms are found in80%of low-grade gliomas (LGGs). Sequencing of LGGs has revealed branching cancer genetics; mutant IDH1 astrocytomas contain p53 and ATRX loss of function mutations, while IDH1-mutated oligodendrogliomas have a different set of mutations that includes chr 1p/19q co-deletion. The IDH mutation is a gain-of-function change at its catalytic core that results in the production of (R)-2-hydroxyglutarate, an oncometabolite, which causes characteristic DNA and histone hypermethylation changes that may contribute to tumorigenesis. Mouse models have thus far failed to demonstrate the role of IDH1 mutations in LGG formation. To test the hypothesis that mutant IDH1 is a driver of gliomagenesis, we use human embryonic stem cell (hESC)-derived neural stem cells (NSCs) to overexpress mutant IDH1 protein in combination with p53 and ATRX knockdown. We have generated twelve NSC lines that harbor combinations of mutant IDH1, wt IDH1 or an empty vector, in combination with ATRX and/or p53 knockdown. Our preliminary data indicate that mutantIDH1 does not alter the proliferative capacity of NSCs, as shown by cell cycle analysis and Ki67 staining, but paradoxically increases their apoptotic rate (15.8% vs 5.9% n = 4), a phenotype that is exacerbated by ATRX knockdown, as detected by annexin V and TUNEL staining (17.7% vs 2.6% n = 3). shRNA-mediated knockdown of p53 salvages the pro-apoptotic phenotype of mutant IDH1 and ATRX NSCs. Furthermore, initial observations suggest that mutant IDH1 biases NSCs toward glial fates, as evidenced by upregulation of the CD44 cell surface marker. We are currently testing the effects of IDH1 mutation on i) NSC differentiation to astrocytic and neuronal lineages, ii) NSC metabolism via metabolomics profiling and iii) in vivo tumorigenesis. We propose that mutant IDH1 alters the differentiation program of human NSCs toward glial rather than neuronal fates

BACKGROUND: Long considered to be inert organelles for lipid storage, lipid droplets (LDs) have recently attracted great interest as dynamic structures central to cellular lipid and energy metabolism. hypoxia-inducible gene (HIG2) and perilipin 2 (PLIN2, adipophilin) are LD-associated proteins known to be upregulated by hypoxia (Bensaad, 2014) and/or following von Hippel-Lindau (VHL) gene inactivation (Togashi, 2005; Yao, 2005). We sought to determine whether overexpression of HIG2 and PLIN2 in response to hypoxia or pseudohypoxia may be involved in these histopathologic features of glioma and hemangioblastoma. METHODS: Tumor specimens from 12 patients with glioma grade II-IV (age 3-59 y) and 23 patients with CNS hemangioblastoma (age 15-63 y) were analyzed by immunohistochemistry (IHC) to delineate their expression of HIG2 and PLIN2. To evaluate the role of hypoxia, glioblastoma (GBM) tissues (n = 2) were double-label immunostained for HIF-1alpha and PLIN2 (Zagzag, 2008). Additionally, cultures of tumor spheres isolated from GBM patients (n = 2) (Bayin, 2014) were exposed to hypoxic (1% O2) conditions for 24-72 h, and the cell proteins analyzed by Western blots. RESULTS: HIG2 and PLIN2 were consistently expressed on LDs in hypoxic glioma tumor cells, including pseudopalisading cells in GBMs, but not in adjacent hyperplastic vessels, inflammatory cells or normal brain tissue, independently of tumor grade or the presence of IDH1 (n = 3) and/or TP53 (n = 7) mutations. Likewise, LDs in stromal tumor cells in hemangioblastoma were intensely immunopositive for HIG2 and PLIN2. Double-label IHC showed tight co-expression of HIF-1a and PLIN2 in glioma tumor cells, consistent with the hypoxic regulation of PLIN2. Similarly, expression of HIF-1a and HIG2 proteins was upregulated in GBM tumor spheres under hypoxic conditions. CONCLUSIONS: Our results suggest that HIG2 and PLIN2 are involved in the hypoxic adaptation of lipid metabolism during tumorigenesis, and may serve as specific biomarkers of glioma tumor cells and stromal cells in CNS hemangioblastoma

Intratumoral heterogeneity in glioblastoma (GBM) is exemplified by the diversity of tumor microenvironments, which include normoxic hypervascular areas and necrotic regions, which are considered hypoxic. GBM stem cells (GSCs) play a central role in tumor growth and therapy resistance. How GSCs adapt to diverse GBM microenvironments remains an important and unanswered question. We recently discovered that CD133-expressing GSCs are metabolically adept at expanding in hypoxic conditions and do not require Notch signaling for their self-renewal. Transcriptional analysis indicated that CD133+ GSCs have 17.8+/-8.8-fold enriched expression of GPR133 (n = 3 biospecimens), a member of the adhesion family of Gproteincoupled receptors. Immunostaining with GPR133 antibody revealed that GPR133 expression is restricted to hypoxic regions within GBM tumors (12/12 GBM biospecimens) and not present in normal brain. We observed that GPR133 mRNA expression correlates with hypoxia-induced transcripts, such as CA9 and VEFGA (n = 3 primary cultures). To test the hypothesis that GPR133 expression is regulated by oxygen tension, we subjected GBM cultures to 1% O2 in vitro and found that GPR133 transcript was consistently upregulated (n = 5 cultures). To examine whether GPR133 is important for GSC self-renewal, we tested the effect of shRNA-mediated knockdown on in vitro tumorsphere formation ability. GPR133 knockdown depleted CD133+ GSCs and inhibited tumorsphere formation under both normoxic and hypoxic conditions (p< 0.05). GPR133 knockdown also reduced in vivo tumorigenicity and increased survival of implanted mice (n = 3). Using colorimetric assays, we found that CD133+ GSCs have 26.2+/-12.53% higher cAMP levels compared to CD133- GBM cells (n = 3) and that GPR133 knockdown downregulated cAMP levels to 47.25+/-27.27% of scramble control (n = 3), suggesting that GPR133 signals through activation of adenylate cyclase and cAMP elevation

A major impeding factor in designing effective therapies against glioblastoma (GBM) is its extensive molecular heterogeneity and the diversity of microenvironmental conditions within any given tumor. To test whether heterogeneity with the GBM stem cell (GSC) population is required to ensure tumor growth in such diverse microenvironments, we used human GBM biospecimens to examine the identity of cells marked by two established GSC markers: CD133 and activation of the Notch pathway. Using primary GBM cultures engineered to express GFP upon activation of Notch signaling, we observed only partial overlap between cells expressing cell surface CD133 and cells with Notch activation (n = 3 specimens), contrary to expectations based on prior literature. To further investigate this finding, we FACS-isolated these cell populations and characterized them. While both CD133+ (CD133 + /Notch-) and Notch+(CD133-/Notch+) cells fulfill GSC criteria, they differ vastly in their transcriptome, metabolic preferences and differentiation capacity, thus giving rise to histologically distinct tumors. CD133+ GSCs have increased expression of hypoxia-regulated and glycolytic genes, and are able to expand under hypoxia by activating anaerobic glycolysis. In contrast, Notch+ GSCs are unable to utilize anaerobic glycolysis under hypoxia, leading to decreased tumorsphere formation ability. While CD133+ GSCs give rise to histologically homogeneous tumors devoid of large tumor vessels, tumors initiated by Notch+ GSCs are marked by large perfusing vessels enveloped by pericytes. Using a lineage tracing system, we showed that pericytes are derived from Notch+ GSCs. In addition, Notch+ cells are able to give rise to all tumor lineages in vitro and in vivo, including CD133 + /Notch- cells, as opposed to Notch- populations, which have restricted differentiation capacity and do not generate Notch+ lineages. Our findings demonstrate that GSC heterogeneity is a mechanism used by tumors to sustain growth in diverse microenvironmental conditions