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AimsThe goal of this study was to determine whether NADPH oxidase (NOX)-produced reactive oxygen species enhances brain tumor growth of glioblastoma (GBM) under hypoxic conditions and during radiation treatment.ResultsExogenous ROS promoted brain tumor growth in gliomasphere cultures that expressed functional PTEN, but not in tumors that were PTEN deficient. Hypoxia induced the production of endogenous cytoplasmic ROS and tumor cell growth via activation of NOX. NOX activation resulted in oxidation of PTEN and downstream Akt activation. Radiation also promoted ROS production via NOX which, in turn, resulted in cellular protection that could be abrogated by knockdown of the key NOX component, p22. Knockdown of p22 also inhibited tumor growth and enhanced the efficacy of radiation in PTEN-expressing GBM cells.InnovationWhile other studies have implicated NOX function in GBM models, these studies demonstrate NOX activation and function under physiological hypoxia and following radiation in GBM, two conditions that are seen in patients. NOX plays an important role in a PTEN-expressing GBM model system, but not in PTEN-non-functional systems and provide a potential, patient-specific therapeutic opportunity.ConclusionsThis study provides a strong basis for pursuing NOX inhibition in PTEN-expressing GBM cells as a possible adjunct to radiation therapy.

Lynch syndrome (LS), is an autosomal dominant disorder predisposing patients to multiple cancers, predominantly colorectal (CRC) and endometrial, and is implicated in 2-4% of all CRC cases. LS is characterized by mutations of four mismatch repair (MMR) genes which code for proteins responsible for recognizing and repairing DNA lesions occurring through multiple mechanisms including oxidative stress (OS). Increased OS can cause DNA mutations and is considered carcinogenic. Due to reduced MMR activity, LS patients have an increased risk of cancer as a result of a decreased ability to recognize and repair DNA lesions caused by OS. Due to its carcinogenic properties, reducing the level of OS may reduce the risk of cancer. Nutritional Nrf2 activators have been shown to reduce the risk of carcinogenesis in the general population through activation of the endogenous antioxidant system. Common nutritional Nrf2 activators include sulforaphane, curcumin, DATS, quercetin, resveratrol, and EGCG. Since LS patients are more susceptible to carcinogenesis caused by OS, it is hypothesized that nutritional Nrf2 activators may have the potential to reduce the risk of cancer in those with LS by modulating OS and inflammation. The purpose of this paper is to review the available evidence in support of this statement.

Gliomas are the most malignant and aggressive form of brain tumors, and account for the majority of brain cancer-related deaths. Malignant gliomas, including glioblastoma are treated with radiation and temozolomide, with only a minor benefit in survival time. A number of advances have been made in understanding glioma biology, including the discovery of molecular heterogeneity between tumors from different patients as well as within tumors from the same patient. This chapter summarizes some of the biological hallmarks including pseudopalisading necrosis, microvascular hyperplasia and angiogenesis, local invasion, altered metabolism, the immunosuppressive microenvironment and heterogeneity, and cancer stem-like cells. We also discuss the molecular hallmarks of glioblastoma, which include 1p/19q codeletion, IDH, EGFR, p53, PTEN/Akt pathway, Rb, Ras/MAPK pathway, extrachromosomal DNA, MGMT, TERT, and ATRX. This chapter is meant to serve as a primer to glioblastoma and provides a brief overview of a very complex disease.

Fatty acids are well known as important constituents for the synthesis of membrane lipids and as sources of cellular energy in the CNS. However, fatty acids can also act as vital second messenger molecules in the nervous system and regulate the activity of many proteins affecting cell growth and survival. Here, we show that an essential dietary fatty acid, Decosahexaenoic acid, (DHA), can enhance stem cell function in vitro and in vivo. We found that this effect is not due to an increase in the overall proliferation rate of all neural progenitors, but is due to an increase in the number of multipotent stem cells that leads to greater levels of subventricular zone (SVZ) neurogenesis with restoration of olfactory function in aged mice. These effects were likely mediated through increased EGF-receptor sensitivity, a conversion of EGRFR+ progenitors back into an EGRFR+/GFAP+ stem cell state, and the activation of the PI3K/AKT signaling pathway, which is a critical pathway in many NSC cell functions including cell growth and survival. Together these data demonstrate that neural stem cells in the aged and quiescent neurogenic niche of the mouse SVZ retain their ability to self-renew and contribute to neurogenesis when apparently rejuvenated by DHA and PI3K/AKT pathway activation. DHA stimulation of this signaling enhances the number of multipotent stem cells and neurogenesis in young and aged rodent and human stem cells and hence may have implications for the manipulation of neural stem cells for brain repair.Significance StatementWe have identified potentially important effects of DHA on the stem cell population which may be unique to the SVZ stem cell niche. Our studies demonstrate that DHA can promote the production of neural stem cells, possibly via a non-proliferative mechanism stimulated by EGF receptor activation, and prolongs their viability. Aging animals undergo an apparent loss in SVZ stem cells and an associated decline in olfactory bulb function. We find that dietary DHA supplementation at least partially restores stem cell numbers, olfactory bulb neurogenesis and olfactory discrimination and memory in aged mice, demonstrating a capacity for rejuvenation is retained despite age-related changes to the niche, which has significant implications for ameliorating cognitive decline in aging and for endogenous brain repair.

Background There is considerable interest in defining the metabolic abnormalities of IDH mutant tumors to exploit for therapy. While most studies have attempted to discern function by using cell lines transduced with exogenous IDH mutant enzyme, in this study, we perform unbiased metabolomics to discover metabolic differences between a cohort of patient-derived IDH1 mutant and IDH wildtype gliomaspheres. Methods Using both our own microarray and the TCGA datasets, we performed KEGG analysis to define pathways differentially enriched in IDH1 mutant and IDH wildtype cells and tumors. Liquid chromatography coupled to mass spectrometry analysis with labeled glucose and deoxycytidine tracers was used to determine differences in overall cellular metabolism and nucleotide synthesis. Radiation-induced DNA damage and repair capacity was assessed using a comet assay. Differences between endogenous IDH1 mutant metabolism and that of IDH wildtype cells transduced with the IDH1 (R132H) mutation were also investigated. Results Our KEGG analysis revealed that IDH wildtype cells were enriched for pathways involved in de novo nucleotide synthesis, while IDH1 mutant cells were enriched for pathways involved in DNA repair. LC-MS analysis with fully labeled 13C-glucose revealed distinct labeling patterns between IDH1 mutant and wildtype cells. Additional LC-MS tracing experiments confirmed increased de novo nucleotide synthesis in IDH wildtype cells relative to IDH1 mutant cells. Endogenous IDH1 mutant cultures incurred less DNA damage than IDH wildtype cultures and sustained better overall growth following X-ray radiation. Overexpression of mutant IDH1 in a wildtype line did not reproduce the range of metabolic differences observed in lines expressing endogenous mutations, but resulted in depletion of glutamine and TCA cycle intermediates, an increase in DNA damage following radiation, and a rise in intracellular ROS. Conclusions These results demonstrate that IDH1 mutant and IDH wildtype cells are easily distinguishable metabolically by analyzing expression profiles and glucose consumption. Our results also highlight important differences in nucleotide synthesis utilization and DNA repair capacity that could be exploited for therapy. Altogether, this study demonstrates that IDH1 mutant gliomas are a distinct subclass of glioma with a less malignant, but also therapy-resistant, metabolic profile that will likely require distinct modes of therapy. Electronic supplementary material The online version of this article (10.1186/s40170-018-0177-4) contains supplementary material, which is available to authorized users.

Author(s): Laks, Dan R; Oses-Prieto, Juan A; Alvarado, Alvaro G; Nakashima, Jonathan; Chand, Shreya; Azzam, Daniel B; Gholkar, Ankur A; Sperry, Jantzen; Ludwig, Kirsten; Condro, Michael C; Nazarian, Serli; Cardenas, Anjelica; Shih, Michelle YS; Damoiseaux, Robert; France, Bryan; Orozco, Nicholas; Visnyei, Koppany; Crisman, Thomas J; Gao, Fuying; Torres, Jorge Z; Coppola, Giovanni; Burlingame, Alma L; Kornblum, Harley I | Abstract: Background:Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy. Methods:To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures. Results:An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization. Conclusions:These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.

Gliomas are the most malignant and aggressive form of brain tumors, and account for the majority of brain cancer related deaths. Malignant gliomas, including glioblastoma are treated with radiation and temozolomide, with only a minor benefit in survival time. A number of advances have been made in understanding glioma biology, including the discovery of cancer stem cells, termed glioma stem cells (GSC). Some of these advances include the delineation of molecular hetereogeneity both between tumors from different patients as well as within tumors from the same patient. Such research highlights the importance of identifying and validating molecular markers in glioma. This review, intended as a practical resource for both clinical and basic investigators, summarizes some of the more well-known molecular markers (MGMT, 1p/19q, IDH, EGFR, p53, PI3K, Rb, and RAF), discusses how they are identified, and what, if any, clinical relevance they many have, in addition to discussing some of the specific biology for these markers. Additionally, we discuss identification methods for studying putative GSC’s (CD133, CD15, A2B5, Nestin, ALDH1, Proteasome activity, ABC transporters, and Label-retention). While much research has been done on these markers, there is still a significant amount that we do not yet understand, which may account for some conflicting reports in the literature. Furthermore, it is unlikely that the investigator will be able to utilize one single marker to prospectively identify and isolate GSC from all, or possibly, any gliomas.

p190RhoGAP (p190) is a negative regulator of RhoGTPases and a putative tumor suppressor, whose mechanism of tumor suppression is poorly defined. Ectopic expression of p190 induces various morphological phenotypes, including multinucleation, dendrite-like formation, and chromatin condensation, suggesting an involvement in apoptosis. We examined the possibility that p190 can function as a tumor suppressor by regulating induction of apoptosis. We show that the predominant phenotype of p190 overexpression in a variety of cell lines is apoptosis, which is mediated through p190's regulation of Rho and caspases. The secondary phenotypes, multinucleation and dendrite-like formation, are determined by transformation status, not cell lineage, and appear to be intermediate phenotypes in the p190-induced apoptotic pathway. Finally, we show that p190 levels can regulate the apoptotic response of breast cancer cell lines to docetaxel through its regulation of Rho. Together, these findings suggest that one mechanism by which p190 can mediate its tumor-suppressive function is through regulation of Rho-activated cell death pathways and that this function can be exploited to optimize the action of cytoskeletal-based chemotherapeutics, such as the taxanes.

Manfred Hintermair, Klaus Sarimski Entwicklung horgeschadigter Kinder im Vorschulalter – Stand der Forschung, empirische Analysen und padagogische Empfehlungen Heidelberg. Median-Verlag, 2016. 192 S., € 29,90, ISBN 978-3-941146-65-57

The tumor suppressor protein Phosphatase and Tensin Homolog (PTEN) is a negative regulator of the Akt pathway which promotes proliferation, migration, and self-renewal. PTEN is mutated or deleted in ~40% of glioblastoma (GBM) resulting in increased Akt activity; however almost all GBM's have increased Akt activity, regardless of PTEN status. This raises the possibility that GBM may inactivate PTEN through means other than mutation or deletion. Treatment with exogenous reactive oxygen species (ROS), like hydrogen peroxide, inactivates PTEN through oxidation in the phosphatase domain. The low oxygen environment of tumors (1%O2) increases ROS levels as compared to normal body tissue (4% O2), and tissue culture conditions (20% O2). Recent data indicate that low to moderate levels of ROS are required for cell maintenance and stem cell renewal while moderate increases in ROS may promote migration, invasion, and proliferation. Indeed, our lab recently found that normal neural stem cells have higher ROS levels, which is required for stem cell renewal. Moreover, data from our lab and others, have found that growing cells under physiological oxygen levels increases ROS and promotes renewal and proliferation. This suggests that the low-oxygen environment of tumors may promote proliferation and tumorigenesis through post-translational modification of proteins, like PTEN. NADPH oxidase (NOX) is a membrane bound complex of proteins that produces a significant amount of endogenous intracellular ROS. While this protein was originally identified in macrophages as being important for immune surveillance, new data suggests that the family of NOX proteins (1-4) has many other biological functions in a variety of different cell types. New data has found that the constitutively active NOX4 acts as an oncogene in several different cancer types by promoting migration, invasion, and proliferation. Within the brain, NOX2 promotes ROS-induced stem cell renewal in normal neural stem cells. Additionally, increased NOX2 expression in GBM patients correlates with shorter survival, while patients with low NOX2 expression generally live longer. Together these data suggest that, through NOX2, the low-oxygen environment of GBM increases ROS levels that oxidize and inhibit PTEN. This novel post-translational modification may increase proliferation and promote tumorigenicity through activation of Akt in cells with intact PTEN. We examined endogenous ROS levels in patient-derived GBM samples and found that increased ROS correlated with faster proliferating cells. Furthermore, low levels of O2 (1%) increased proliferation and Akt activation in a PTEN dependent manner. Both PTEN null and knockdown cells showed no increase in either proliferation or Akt activation over cells grown at room air (20%). Moreover, pharmacological inhibition of Akt abrogated ROS-induced proliferation, suggesting this mechanism functions through the PTEN/Akt pathway. This response was due directly to ROS production, as treatment with antioxidants decreased both ROS production and proliferation at low oxygen and room air. Additionally, we found that growth at 1% O2 led to the oxidation and inhibition of PTEN as seen by Akt phosphorylation. Treatment with apocynin, a NOX2 inhibitor decreased ROS production, proliferation, and Akt phosphorylation, suggesting that ROS-induced proliferation may be mediated through the NOX2 protein. This data not only show PTEN oxidation and inhibition under physiological conditions for the first time, it suggests that the low-oxygen tumor microenvironment may alter PTEN positive GBM to up-regulate the Akt pathway and promote tumorigenesis. Citation Format: Kirsten Ludwig, Harley Kornblum. NOX2 generated reactive oxygen species promotes glioblastoma growth through inhibition of PTEN. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B31.

Radiation is a mainstay of treatment for glioblastoma (GBM). While all the effects of radiation are not known, it is thought to induce cell death by increasing the production of reactive oxygen species (ROS). High amounts of ROS are known to induce cell death, however recent data suggests that a moderate increase in ROS can promote proliferation, migration, tumorigenesis, and stem cell maintenance. We have found that in primary patient-derived gliomasphere lines both exogenous and endogenous ROS oxidizes PTEN, resulting in Akt activation and increased proliferation. Under hypoxic conditions, endogenous ROS is produced primarily by a complex called NADPH oxidase (NOX), whose formation results in PTEN oxidation and downstream activation of Akt and cell proliferation. Prior studies demonstrate that radiation itself can induce activation of Akt, however the mechanism is still unclear. In the present study, we tested the hypothesis that NOX-produced ROS plays a role in the response to radiation in GBM. We find that following radiation, NOX4 expression and activation increases, and chemical or genetic inhibition of this complex decreases radiation induced ROS, and rescues PTEN oxidation as well as Akt activation. Most importantly, NOX inhibition decreases cell viability and survival following radiation, suggesting that activation of this complex helps promote survival following radiation. While NOX inhibition did decrease ROS levels in PTEN null lines, no significant difference in survival or Akt activation was found, suggesting that NOX protection functions through the inactivation of PTEN. Finally, NOX-produced ROS occurs primarily in the cytosol, as no significant decrease in mitochondrial ROS levels were detected following NOX inhibition. Taken together, these data indicate that NOX may be a viable target to promote the sensitization of GBM to radiation, especially in PTEN-intact tumors.

The cytokinetic furrow is organized by the RhoA GTPase, which recruits actin and myosin II to the furrow and drives contractility. Here, we show that the RhoA GTPase-activting protein (GAP) p190RhoGAP-A (also known as ARHGAP35) has a role in cytokinesis and is involved in regulating levels of RhoA-GTP and contractility. Cells depleted of p190RhoGAP-A accumulate high levels of RhoA-GTP and markers of high RhoA activity in the furrow, resulting in failure of the cytokinetic furrow to progress to abscission. The loss of p190RhoGAP-A can be rescued by a low dose of the myosin II inhibitor blebbistatin, suggesting that cells fail cytokinesis because they have too much myosin activity. p190RhoGAP-A binds the cytokinetic organizer anillin, and mutants of p190RhoGAP-A that are unable to bind anillin or unable to inactivate RhoA fail to rescue cytokinesis defects in p190RhoGAP-A-depleted cells. Taken together, these data demonstrate that a complex of p190RhoGAP-A and anillin modulates RhoA-GTP levels in the cytokinetic furrow to ensure progression of cytokinesis.

TMIC-16. NOX GENERATED REACTIVE OXYGEN SPECIES PROMOTES GLIOBLASTOMA GROWTH AND SURVIVAL THROUGH INACTIVATION OF PTEN Kirsten Ludwig and Harley Kornblum; University of California, Los Angeles, Los Angeles, CA, USA PTEN is inactivated in 40% of glioblastoma (GBM), however almost all GBM’s have increased Akt activity, regardless of PTEN status. Treatment with exogenous reactive oxygen species (ROS) inactivates PTEN through oxidation, suggesting that a similar event may occur in the hypoxic tumor environment. Recent data indicate that low levels of ROS are required for cell maintenance and stem cell renewal while moderate increases may promote tumorigenesis. Much of the endogenous ROS is produced by NADPH oxidase (NOX), amembraneboundcomplexofproteins,activatedunderhypoxic conditions. This raises the possibility that the tumor microenvironment may inactivate PTEN through means other than mutation or deletion. We investigated the role of NOX generated ROS in primary cultures of patient-derived GBM cells across the spectrum of GBM subtypes. Endogenous ROS levels correlated with proliferation; those with higher ROS had higher proliferation rates. Placement of cells in low oxygen levels (1%) enhanced proliferation, an effect that was reversed by treatment with antioxidants and the NOX inhibitor Apocynin, supporting the hypothesis that NOX-induced ROS promotes GBM growth. The effects of hypoxia-induced NOX activation were dependent upon PTEN, as PTEN knockdown or PTEN null cells exhibited no enhanced growthwhenplaced inhypoxicconditions.Furthermore,pharmacologicblockade of AKT prevented enhanced growth in hypoxia. A role for PTEN was further demonstrated by the finding that it was oxidatively inactivated in GBM cells grown under hypoxic conditions. Finally, pharmacological inhibition of NOX rescued radiation-induced activation of Akt resulting in increased radio-sensitivity. Taken together, these data support the hypothesis that the relativelyhypoxic tumorenvironment activatesNOX, resulting in functional inactivation of PTEN andenhanced GBMproliferation andsurvival. Thesefindings represent a novel opportunity for therapeutic intervention in a subset of GBM. Neuro-Oncology 17:v221–v225, 2015. doi:10.1093/neuonc/nov236.16 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.