NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts.

Cancer stem cells (CSCs) are thought to be critical for the engraftment and long-term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem-like cells with higher NOTCH activity. We therefore used low-passage and established GBM-derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by gamma-secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug-impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem-like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem-like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas.

Insulin-like growth factor-I receptor expression regulates neuroblastoma metastasis to bone.

Neuroblastoma is a pediatric tumor that preferentially metastasizes to bone. Patients with bone metastases have a mortality rate >93%, indicating a need for novel treatment targets. Our laboratory has shown that type I insulin-like growth factor receptor (IGF-IR) expression and activation regulate neuroblastoma cell proliferation, motility, invasion, and survival, and that expression of the IGF-IR correlates with neuroblastoma tumorigenicity. Bone expresses large amounts of IGF ligands, and the IGF system is required for normal bone physiology. The current study addresses the role of the IGF system in neuroblastoma metastasis to bone. Upon reaching the bone marrow through the circulation, neuroblastoma cells must dock at the bone marrow endothelium, extravasate into the bone microenvironment, and destroy bone tissue to allow for tumor growth. This report examines the effects of high IGF-IR expression on neuroblastoma cell interaction with bone. The current data show that neuroblastoma cells with high IGF-IR expression, either endogenously or through transfection, adhere to human bone marrow endothelial cells and subsequently migrate toward both IGF-I and human bone stromal cells. High IGF-IR-expressing neuroblastoma cells adhere tightly to bone stromal cells, flatten, and extend processes. When neuroblastoma cells are injected directly into the tibiae of mice, those cells with increased IGF-IR form both osteolytic lesions within the tibiae and secondary tumors within other sites. These results support the hypothesis that IGF-IR expression in neuroblastoma cells increases tumor cell interaction with the bone microenvironment, resulting in greater formation of metastases.

The Adolescent Brain Cognitive Development (ABCD) study: Imaging acquisition across 21 sites.

The ABCD study is recruiting and following the brain development and health of over 10,000 9-10 year olds through adolescence. The imaging component of the study was developed by the ABCD Data Analysis and Informatics Center (DAIC) and the ABCD Imaging Acquisition Workgroup. Imaging methods and assessments were selected, optimized and harmonized across all 21 sites to measure brain structure and function relevant to adolescent development and addiction. This article provides an overview of the imaging procedures of the ABCD study, the basis for their selection and preliminary quality assurance and results that provide evidence for the feasibility and age-appropriateness of procedures and generalizability of findings to the existent literature.

Reduced brain activation during inhibitory control in children with COMT Val/Val genotype.

INTRODUCTION: Behavioral undercontrol is a well-established risk factor for substance use disorder, identifiable at an early age well before the onset of substance use. However, the biological mechanistic structure underlying the behavioral undercontrol/substance use relationship is not well understood. The enzyme catechol O-methyltransferase (COMT) catabolizes dopamine and norepinephrine in the prefrontal cortex and striatum, brain regions involved in behavioral control. The goal of this work was to investigate the association between genetic variation in COMT functioning and fronto-striatal brain functioning during successful inhibitory control, a critical aspect of behavioral control. METHODS: Participants were 65 (22 female) 7-12 year olds who were genotyped for the functional COMT Val158Met (rs4680) single-nucleotide polymorphism and underwent functional magnetic resonance imaging while performing a go/no-go task. The majority of the sample (80%) had at least one parent with a history of alcohol use disorder and were thus at heightened risk for substance use disorders. RESULTS: There was a significant main effect of genotype on brain activation in left and right putamen during successful versus failed inhibition and in right inferior frontal gyrus/insula during successful inhibition versus baseline. Follow-up tests revealed that Met homozygotes had greater activation in each region relative to Val homozygotes. CONCLUSIONS: These results are relevant for understanding how specific genes influence brain functioning related to underlying risk factors for substance use disorders and other disinhibitory psychopathologies.

N-Myc overexpression leads to decreased beta1 integrin expression and increased apoptosis in human neuroblastoma cells.

Neuroblastoma is a childhood tumor thought to arise through improper differentiation of neural crest cells. Increased N-Myc expression in neuroblastoma indicates highly malignant disease and poor patient prognosis. N-myc enhances cell growth, insulin-like growth factor type I receptor (IGF-IR) expression, and tumorigenicity in combination with Bcl-2. Despite these effects, N-Myc overexpression in SHEP neuroblastoma cells (SHEP/N-Myc cells) increases serum-withdrawal and mannitol-induced apoptosis. Although we have previously shown a protective effect of IGF-I in SHEP cells, in SHEP/N-Myc cells IGF-I rescue from mannitol-induced apoptosis is prevented. N-Myc overexpression has little effect on IGF-IR signaling pathways, but results in increased Akt phosphorylation when Bcl-2 is coexpressed. A loss of integrin-mediated adhesion promotes apoptosis in many systems. SHEP/N-Myc cells have dramatically less beta1 integrin expression than control cells, consistent with previous reports. beta1 integrin expression is decreased in more tumorigenic neuroblastoma cells lines, including IMR32 and SH-SY5Y cells. Reintroduction of beta1 integrin into the N-Myc-overexpressing cells prevents mannitol-mediated apoptosis. We speculate that N-Myc repression of beta1 integrin expression leads to a less differentiated phenotype, resulting in increased growth and tumorigenesis if properly supported or apoptosis if deprived of growth sustaining molecules.

Development of impulse control circuitry in children of alcoholics.

BACKGROUND: Difficulty with impulse control is heightened in children with a family history of alcohol use disorders and is a risk factor for later substance problems. Cross-sectional functional magnetic resonance imaging studies have shown altered impulse control processing in adolescents with a positive family history, yet developmental trajectories have yet to be examined. METHODS: Longitudinal functional magnetic resonance imaging was conducted in children of alcoholic families (family history positive [FH+]; n = 43) and children of control families (family history negative [FH-]; n = 30) starting at ages 7-12 years. Participants performed a go/no-go task during functional magnetic resonance imaging at intervals of 1-2 years, with two to four scans performed per subject. We implemented a repeated-measures linear model fit across all subjects to conduct a whole-brain search for developmental differences between groups. RESULTS: Performance improved with age in both groups, and there were no performance differences between groups. Significant between-group differences in linear age-related activation changes were found in the right caudate, middle cingulate, and middle frontal gyrus. Post hoc analyses revealed significant activation decreases with age in the caudate and middle frontal gyrus for FH- subjects and a significant increase with age in middle cingulate activation for FH+ subjects. Group differences were evident at age 7-12 years, even in alcohol- and drug-naïve participants, with FH+ subjects showing significantly blunted activation at baseline compared with FH- subjects. CONCLUSIONS: Differences in response inhibition circuitry are visible in FH+ individuals during childhood; these differences continue into adolescence, displaying trajectories that are inconsistent with development of normal response inhibition. These patterns precede problem drinking and may be a contributing factor for subsequent substance use problems.

Sensory neurons and schwann cells respond to oxidative stress by increasing antioxidant defense mechanisms.

Elevated blood glucose is a key initiator of mechanisms leading to diabetic neuropathy. Increases in glucose induce acute mitochondrial oxidative stress in dorsal root ganglion (DRG) neurons, the sensory neurons normally affected in diabetic neuropathy, whereas Schwann cells are largely unaffected. We propose that activation of an antioxidant response in DRG neurons would prevent glucose-induced injury. In this study, mild oxidative stress (1 microM H2O2) leads to the activation of the transcription factor Nrf2 and expression of antioxidant (phase II) enzymes. DRG neurons are thus protected from subsequent hyperglycemia-induced injury, as determined by activation of caspase 3 and the TUNEL assay. Schwann cells display high basal antioxidant enzyme expression and respond to hyperglycemia and mild oxidative stress via further increases in these enzymes. The botanical compounds resveratrol and sulforaphane activate the antioxidant response in DRG neurons. Other drugs that protect DRG neurons and block mitochondrial superoxide, identified in a compound screen, have differential ability to activate the antioxidant response. Multiple cellular targets exist for the prevention of hyperglycemic oxidative stress in DRG neurons, and these form the basis for new therapeutic strategies against diabetic neuropathy.

Integrin-linked kinase complexes with caveolin-1 in human neuroblastoma cells.

Integrin-linked kinase (ILK) and caveolin-1 (cav-1) are implicated in the pathogenesis of cancer. Overexpression of ILK leads to altered expression of cell cycle regulators, a decreased level of cell adhesion to the extracellular matrix, a decreased level of apoptosis, in vitro phosphorylation of Akt, and tumor formation in nude mice. Conversely, cav-1 expression is frequently downregulated in many forms of cancer. We examined whether ILK and cav-1 interact in SHEP human neuroblastoma cells because ILK is present in caveolae-enriched membranes and contains a putative cav-binding domain. SHEP cells were stably transfected with vector, wild-type ILK (ILK-wt), kinase-deficient ILK (ILK-kd), or mutant cav-binding domain ILK (ILK-mutCavbd). Control SHEP cells and ILK transfectants express high levels of ILK and cav-1. Immunoprecipitation with anti-cav-1 co-immunoprecipitates a 59 kDa protein that is immunoreactive with the anti-ILK antibody, and this interaction is partially prevented in cells expressing ILK-mutCavbd. Cav-1 and ILK partially colocalize in SHEP cells, also supporting these data. Last, affinity chromatography with a biotinylated cav-scaffolding domain peptide precipitates ILK-wt but not ILK-mutCavbd. These data suggest that the cav-binding domain of ILK and the cav-scaffolding domain of cav-1 mediate complex formation in human neuroblastoma cells.