Kelli B. Pointer

SIRT1 activates MAO-A in the brain to mediate anxiety and exploratory drive.

SIRT1 is a NAD(+)-dependent deacetylase that governs a number of genetic programs to cope with changes in the nutritional status of cells and organisms. Behavioral responses to food abundance are important for the survival of higher animals. Here we used mice with increased or decreased brain SIRT1 to show that this sirtuin regulates anxiety and exploratory drive by activating transcription of the gene encoding the monoamine oxidase A (MAO-A) to reduce serotonin levels in the brain. Indeed, treating animals with MAO-A inhibitors or selective serotonin reuptake inhibitors (SSRIs) normalized anxiety differences between wild-type and mutant animals. SIRT1 deacetylates the brain-specific helix-loop-helix transcription factor NHLH2 on lysine 49 to increase its activation of the MAO-A promoter. Both common and rare variations in the SIRT1 gene were shown to be associated with risk of anxiety in human population samples. Together these data indicate that SIRT1 mediates levels of anxiety, and this regulation may be adaptive in a changing environment of food availability.

Deviation of innate circadian period from 24 h reduces longevity in mice

The variation of individual life spans, even in highly inbred cohorts of animals and under strictly controlled environmental conditions, is substantial and not well understood. This variation in part could be due to epigenetic variation, which later affects the animal’s physiology and ultimately longevity. Identification of the physiological properties that impact health and life span is crucial for longevity research and the development of anti‐aging therapies. Here, we measured individual circadian and metabolic characteristics in a cohort of inbred F1 hybrid mice and correlated these parameters to their life spans. We found that mice with innate circadian periods close to 24 h (revealed during 30 days of housing in total darkness) enjoyed nearly 20% longer life spans than their littermates, which had shorter or longer innate circadian periods. These findings show that maintenance of a 24‐h intrinsic circadian period is a positive predictor of longevity. Our data suggest that circadian period may be used to predict individual longevity and that processes that control innate circadian period affect aging.

Glioblastoma cancer stem cells: Biomarker and therapeutic advances

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans. It accounts for fifty-two percent of primary brain malignancies in the United States and twenty percent of all primary intracranial tumors. Despite the current standard therapies of maximal safe surgical resection followed by temozolomide and radiotherapy, the median patient survival is still less than 2 years due to inevitable tumor recurrence. Glioblastoma cancer stem cells (GSCs) are a subgroup of tumor cells that are radiation and chemotherapy resistant and likely contribute to rapid tumor recurrence. In order to gain a better understanding of the many GBM-associated mutations, analysis of the GBM cancer genome is on-going; however, innovative strategies to target GSCs and overcome tumor resistance are needed to improve patient survival. Cancer stem cell biology studies reveal basic understandings of GSC resistance patterns and therapeutic responses. Membrane proteomics using phage and yeast display libraries provides a method to identify novel antibodies and surface antigens to better recognize, isolate, and target GSCs. Altogether, basic GBM and GSC genetics and proteomics studies combined with strategies to discover GSC-targeting agents could lead to novel treatments that significantly improve patient survival and quality of life.

WNK1-OSR1 kinase-mediated phospho-activation of Na+-K+-2Cl- cotransporter facilitates glioma migration.

BackgroundThe bumetanide (BMT)-sensitive Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl- content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown.MethodsExpression of NKCC1 and its upstream kinases With-No-K (Lysine) kinase 1 (WNK1) and oxidative stress-responsive kinase-1 (OSR1) in different human glioma cell lines and glioma specimens were detected by western blotting and immunostaining. Live cell imaging and microchemotaxis assay were applied to record glioma cell movements under different treatment conditions. Fluorescence indicators were utilized to measure cell volume, intracellular K+ and Cl- content to reflect the activity of NKCC1 on ion transportation. Small interfering RNA (siRNA)-mediated knockdown of WNK1 or OSR1 was used to explore their roles in regulation of NKCC1 activity in glioma cells. Results of different treatment groups were compared by one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons.ResultsWe show that compared to human neural stem cells and astrocytes, human glioma cells exhibit robust increases in the activation and phosphorylation of NKCC1 and its two upstream regulatory kinases, WNK1 and OSR1. siRNA-mediated knockdown of WNK1 or OSR1 reduces intracellular K+ and Cl- content and RVI in glioma cells by abolishing NKCC1 regulatory phospho-activation. Unexpectedly, TMZ activates the WNK1/OSR1/NKCC1 signaling pathway and enhances glioma migration. Pharmacological inhibition of NKCC1 with its potent inhibitor BMT or siRNA knockdown of WNK1 or OSR1 significantly decreases glioma cell migration after TMZ treatment.ConclusionTogether, our data show a novel role for the WNK1/OSR1/NKCC1 pathway in basal and TMZ-induced glioma migration, and suggest that glioma treatment with TMZ might be improved by drugs that inhibit elements of the WNK1/OSR1/NKCC1 signaling pathway.

Upregulation of NHE1 protein expression enables glioblastoma cells to escape TMZ-mediated toxicity via increased H+ extrusion, cell migration and survival

Sodium-hydrogen exchanger isoform 1 (NHE1) plays a role in survival and migration/invasion of several cancers and is an emerging new therapeutic target. However, the role of NHE1 in glioblastoma and the interaction of NHE1 expression and function in glioblastoma cells with cytotoxic temozolomide (TMZ) therapy remain unknown. In this study, we detected high levels of NHE1 protein only in primary human glioma cells (GC), glioma xenografts and glioblastoma, but not in human neural stem cells or astrocytes. GC exhibited an alkaline resting pHi (7.46±0.04) maintained by robust NHE1-mediated H(+) extrusion. GC treatment with TMZ for 2-24h triggered a transient decrease in pHi, which recovered by 48h and correlated with concurrent upregulation of NHE1 protein expression. NHE1 protein was colocalized with ezrin at lamellipodia and probably involved in GC migration. The TMZ-treated GC exhibited increased migration and invasion, which was attenuated by addition of NHE1 inhibitor HOE-642. Most importantly, NHE1 inhibition prevented prosurvival extracellular signal-regulated kinase activation and accelerated TMZ-induced apoptosis. Taken together, our study provides the first evidence that GC upregulate NHE1 protein to maintain alkaline pHi. Combining TMZ therapy with NHE1 inhibition suppresses GC migration and invasion, and also augments TMZ-induced apoptosis. These findings strongly suggest that NHE1 is an important cytoprotective mechanism in GC and presents a new therapeutic strategy of combining NHE1 inhibition and TMZ chemotherapy.

Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma.

U niversity of California, San Francisco researchers recently reported in Science the mutational profiling of 23 initially low-grade gliomas (LGGs) and associated recurrent tumors and profiled a subset of recurrent tumors in temozolomide-treated patients. Three key findings with potentially important clinical implications for LGG management were demonstrated: (1) LGGs and paired recurrent tumors are highly divergent and often only share a few early mutations, thus partly explaining their differential therapeutic responses; (2) mutant isocitrate dehydrogenase-1 (IDH1) may be critical for LGG formation and is a potential therapeutic target; and (3) temozolomide therapy may contribute to malignant transformation and affect clinical outcome. Johnson et al determined the genetic profiles of LGGs and associated recurrences. Mutations that are shared or exclusive to the initial tumor or recurrences were characterized for each of 23 initial tumors and recurrences found up to 11 years later, and tumor phylogenies were mapped via evolutionary analyses. Overall, the paired tumors shared a significant percentage of mutations exclusive to the initial tumors in 43% of cases. These findings suggest that gliomas and recurrent tumors share early tumorigenic mutational origins but diverge afterward in tumorigenesis. LGG sequencing also revealed that an IDH1 mutation was present and remained unchanged in all paired tumors, highlighting IDH1 as a potentially critical LGG driver mutation. IDH1 mutants produce 2-hydroxyglutarate R-enantiomer, an interesting tumor metabolism product that inhibits histone enzymes and alters gene expression. Recent work highlights a possible LGG-selective therapeutic opportunity because inhibitors of IDH-1 mutant activity selectively reduce tumor growth rate and stimulate glioma differentiation. This work also reported the effects of temozolomide therapy on mutational profiles of recurrent gliomas, especially given that temozolomide use in LGG therapy is controversial. Mutational profiles of paired tumors in 10 temozolomide-treated patients were determined. Recurrent tumors from 6 of the 10 patients exhibited hypermutated phenotypes after temozolomide therapy, carryingmanymoremutations per million base pairs compared with their initial tumors. The hypermutated state is likely caused by the propensity of temozolomide tomutate and compromise DNA mismatch repair pathways. Additionally, the authors characterized the unique hypermutated signature and found significant association with high-grade glioma signaling pathways such as retinoblastoma and protein kinase B–mammalian target of rapamycin signaling (Figure, B). These results suggest that temozolomide therapy may contribute to malignant transformation of LGGs, and further studies are needed to determine whether this alters clinical outcomes.

Small cell carcinoma of the head and neck: An analysis of the National Cancer Database

PURPOSE/OBJECTIVE(S) To evaluate treatment trends and overall survival of patients with small cell carcinoma of the head and neck region. MATERIALS/METHODS Patients from 2004 to 2012 were identified from the National Cancer Database. Patient demographics and overall survival were analyzed. Multivariable analysis was used to identify predictors of survival. RESULTS Among 347,252 head and neck patients a total of 1042 (0.3%) patients with small cell carcinoma were identified. 17% of patients were diagnosed as stage I/II, 61% as stage III/IVA/IVB and 22% as stage IVC disease. The distribution by anatomic site was 9% oral cavity, 12% oropharynx, 35% larynx, 4% hypopharynx, 10% nasopharynx and 30% nasal cavity and paranasal sinuses. The median overall survival by anatomical site was 20.8months for oral cavity, 23.7months for oropharynx, 17.9months for larynx/hypopharynx, 15.1months for nasopharynx and 36.4months for nasal cavity primary tumors. On multivariable analysis across stage, patients with nasal cavity and paranasal sinuses tumors had the best survival and patients with nasopharynx primaries had the worst survival. In stage I/II patients, type of treatment delivered resulted in no overall survival difference (p=0.78). In patients with locally advanced disease, there was no difference in survival between those treated with combined surgery, radiotherapy and chemotherapy compared to those treated only with radiotherapy and chemotherapy (p=0.46). The addition of radiotherapy to chemotherapy in the metastatic setting did not result in improved survival (p=0.14). CONCLUSIONS Small cell carcinoma of the head and neck is a rare malignancy with a poor prognosis. The addition of surgery to radiotherapy and chemotherapy did not improve survival in patients with locally advanced disease.

New Molecular Insights and Potential Therapies for Diffuse Intrinsic Pontine Glioma.

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Abstract B15: Human ether-a-go-go-related-1 gene (hERG) K+ channel as a prognostic marker and therapeutic target for glioblastoma

Purpose: Glioblastoma multiforme (GBM) is the most malignant primary brain tumor, with a median survival of less than two years despite maximal therapy. Human ether a go-go-related-1 gene (hERG) encodes a voltage-dependent K+ channel found overexpressed in GBM cell lines, and linked to aberrant proliferation in other cancers. We analyzed hERG expression in a glioblastoma stem-like cell (GSC)-derived tumor model and a clinically annotated human GBM tissue microarray (TMA) to determine correlation with patient survival. Additionally, since all FDA-approved drugs undergo cardiotoxicity profiling that includes hERG inhibition, GBM patient survival was also analyzed based on whether a patient received hERG inhibitory drugs and their hERG expression levels. Methods: GSC sphere cultures were isolated from patient GBMs by marker-neutral culture in stem cell media, and validated with progenitor markers, multipotent differentiation, and orthotopic xenograft formation by implanting 2x10 5 GSCs into right striata of NOD-SCID mice. Tumor xenografts (10-14 weeks) were verified by MRI and animals were sacrificed when moribund. Histology and immunohistochemistry were performed on paraffin-embedded sections for hERG expression and tumor proliferation. Sphere-forming assays with hERG blockers (E-4031, phenytoin) were performed using two low hERG-expressing and two high hERG-expressing GSC lines to test for drug sensitivity. A GBM TMA of 115 patients linked to clinical data was used to correlate hERG expression with patient survival. Clinical data was analyzed to determine if patient survival was affected by incidental administration of drugs with hERG inhibition activity, and the correlative effect of patient GBM hERG expression levels. Results: hERG expression was upregulated in GBM xenografts with a higher Ki-67 proliferative index. High hERG-expressing GSC lines showed a 50% reduction in sphere formation when treated with known hERG inhibitors at their known hERG channel inactivation IC 50 concentrations (E-4031 7.7nM, phenytoin 100uM). GBM TMA analysis showed significantly worse survival for GBM patients with high hERG expression versus low hERG expression (p= 0.0168). In addition, patients who incidentally received at least one hERG blocker drug had significantly better survival compared to patients who did not receive hERG blockers (p=0.0004). Subgroup analysis showed that GBM patients with high hERG expression who also received a hERG blocker had significantly improved survival (p=0.0495);however, there was no significant difference in survival for low hERG-expressing GBM patients who received hERG blocker drugs (p=0.5482). Conclusions: In this study, we showed that GBM xenografts with higher hERG expression had higher proliferation rates, and treatment with two known hERG inhibitors (phenytoin, E-4031) at IC 50 concentrations for inactivating hERG channel activity also inhibited sphere formation in high hERG expressing GSC lines. Furthermore, TMA analyses showed that patients with high hERG-expressing GBMs who received hERG inhibitory drugs had significantly better survival. This data suggest that already FDA-approved drugs with hERG inhibition activity should be tested in patients with high hERG-expressing GBMs. Citation Format: Kelli B. Pointer, Fang Liu, David K. Jones, Paul A. Clark, Gail A. Robertson, John S. Kuo. Human ether-a-go-go-related-1 gene (hERG) K+ channel as a prognostic marker and therapeutic target for glioblastoma. [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 B15.