Poor Prognosis Of Glioblastoma Research Articles

Abstract 5337: USP26 regulates TGF-β signaling by deubiquitinating and stabilizing SMAD7

Abstract The amplitude of transforming growth factor-β (TGF-β) signal is tightly regulated to ensure appropriate physiological responses. As part of negative feedback loop SMAD7, a direct transcriptional target of downstream TGF-β signaling, acts as a scaffold to recruit the E3 ligase SMURF2 to target the TGF-β receptor complex for ubiquitin mediated degradation. Here we identify the deubiquitinating enzyme USP26 as a novel integral component of this negative feedback loop. We demonstrate that TGF-β rapidly enhances the expression of USP26 and reinforces SMAD7 stability by limiting the ubiquitin mediated turnover of SMAD7. Conversely, knockdown of USP26 rapidly degrades SMAD7 resulting in TGF-β receptor stabilization and enhanced levels of p-SMAD2. Clinically, loss of USP26 correlates with high TGF-β activity and confers poor prognosis in glioblastoma. Our data identify USP26 as a novel negative regulator of the TGF-β pathway and suggests that loss of USP26 expression may be an important factor in glioblastoma pathogenesis. Citation Format: Pieter J.A. Eichhorn, Sarah Lui, Prasanna Iyengar, Patrick Jaynes. USP26 regulates TGF-β signaling by deubiquitinating and stabilizing SMAD7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5337. doi:10.1158/1538-7445.AM2017-5337

Antrodia cinnamomea sensitizes radio-/chemo-therapy of cancer stem-like cells by modulating microRNA expression

Ethnopharmacological relevanceThe discovery of many tissue-specific cancer stem cells (CSCs) continues to attract scientific attention. These CSCs are considered to be associated with chemo- and radio-resistance, and consequently, failure of conventional anticancer therapies. The recent demonstration of several microRNAs as enhancers of tumorigenicity via modulation of epithelial-mesenchymal transition and cancer stemness, makes them putative novel therapeutic target in oncology. Antrodia cinnamomea is a Chinese traditional medicine with several biological functions including anti-inflammation, antioxidant, and cancer prevention. However, the anti-CSC capability of A. Cinnamomea is not clear yet. Aim of the studyTo investigate the inhibitory effect of A. cinnamomea mycelium and extract on CSCs derived from various human cancer cell lines using our in-house therapeutics and human genome-wide miRNA screening panels. Materials and methodsA broad range of human cancer cell lines, including the acute monocytic leukemia (THP-1), glioblastoma multiforme (GBM 8401), lung carcinoma (A549), breast adenocarcinoma (MDA-MB-231), hepatoblastoma (HepG2), colorectal adenocarcinoma (SW620), and foreskin fibroblast (HS68), were exposed to A. cinnamomea in this study. CD133+ CSCs generated from the cell lines were characterized and isolated by flow cytometry, effect of chemo- and radiotherapy was assessed using the MTT assay, while the RT-PCR and human genome wide qRT-PCR determined the differential gene expression patterns. A comparative analysis of the anticancer effect of A. cinnamomea and Cisplatin, Taxol, or irradiation was also performed. ResultsOur results indicated that A. cinnamomea mycelium and its ethyl acetate extracts showed anti-proliferation effects against all types of CSCs, especially the lung, breast, and head and neck squamous cell carcinoma CSCs. Furthermore, CSCs treatment with A. cinnamomea combined with irradiation or chemotherapeutics demonstrated significant anti-cancer effect. We also established an association between the CSC-inhibitory effect of A. cinnamomea and significant downregulation of several microRNAs and cancer stemness expression levels in brain and breast CSCs. More importantly, higher CD133 expression is associated with poor prognosis in glioblastoma and breast cancer patients. ConclusionHerein, we demonstrate the putative role of A. cinnamomea as an effective ethnopharmacologic therapeutic agent for cancer treatment.

Riluzole: a potential therapeutic intervention in human brain tumor stem-like cells.

A small subpopulation of tumor stem-like cells has the capacity to initiate tumors and mediate radio- and chemoresistance in diverse cancers hence also in glioblastoma (GBM). It has been reported that this capacity of tumor initiation in the brain is mainly dependent on the body’s nutrient supply. This population of so-called brain tumor initiating or brain tumor stem-like cells (BTSCs) is able to extract nutrients like glucose with a higher affinity. Riluzole, a drug approved for treating amyotrophic lateral sclerosis (ALS), was reported to possess anticancer properties, affecting the glutamate metabolism. We report that riluzole treatment inhibits the growth of brain tumor stem-like cells enriched cultures isolated from two human glioblastomas. The effects of riluzole on these cells were associated with an inhibition of a poor prognostic indicator: glucose transporter 3 (GLUT3). A decrease in GLUT3 is associated with a decrease in the p-Akt/HIF1α pathway. Further, downregulation of the DNA (Cytosine-5-)-methyltransferase 1 (DNMT1) gene that causes hypermethylation of various tumor-suppressor genes and leads to a poor prognosis in GBM, was detected. Two hallmarks of cancer cells—proliferation and cell death—were positively influenced by riluzole treatment. Finally, we observed that riluzole reduced the tumor growth in in vivo CAM assay, suggesting it could be a possible synergistic drug for the treatment of glioblastoma.

USP26 regulates TGF‐β signaling by deubiquitinating and stabilizing SMAD7

The amplitude of transforming growth factor‐β (TGF‐β) signal is tightly regulated to ensure appropriate physiological responses. As part of negative feedback loop SMAD7, a direct transcriptional target of downstream TGF‐β signaling acts as a scaffold to recruit the E3 ligase SMURF2 to target the TGF‐β receptor complex for ubiquitin‐mediated degradation. Here, we identify the deubiquitinating enzyme USP26 as a novel integral component of this negative feedback loop. We demonstrate that TGF‐β rapidly enhances the expression of USP26 and reinforces SMAD7 stability by limiting the ubiquitin‐mediated turnover of SMAD7. Conversely, knockdown of USP26 rapidly degrades SMAD7 resulting in TGF‐β receptor stabilization and enhanced levels of p‐SMAD2. Clinically, loss of USP26 correlates with high TGF‐β activity and confers poor prognosis in glioblastoma. Our data identify USP26 as a novel negative regulator of the TGF‐β pathway and suggest that loss of USP26 expression may be an important factor in glioblastoma pathogenesis.

P09.24 The germline mutation PIK3R1Met326Ile correlates with the levels of cysteine<->rich protein 61 and poor prognosis of glioblastoma

P09.24 The germline mutation PIK3R1Met326Ile correlates with the levels of cysteine<->rich protein 61 and poor prognosis of glioblastoma

Tbx2 confers poor prognosis in glioblastoma and promotes temozolomide resistance with change of mitochondrial dynamics

Tbx2 is a cancer-related protein that was found to be overexpressed in several human malignancies. The present study aims to investigate the clinical significance and biological role of Tbx2 in human astrocytoma. We examined its protein expression in 102 cases of astrocytoma tissues using immunohistochemical staining. Negative Tbx2 staining was observed in normal astrocytes, and positive nuclear staining was found in 41 out of 102 astrocytoma specimens. The rate of Tbx2 overexpression in pylocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma multiform (GBM) were 0%, 26.1%, 40%, and 52%, respectively. Tbx2 overexpression correlated with poor prognosis in patients with astrocytoma or GBM. Tbx2 plasmid transfection was performed in A172 cells, and Tbx2 siRNA knockdown was carried out in U251 cells. Cell Counting Kit-8, cell cycle analysis, and matrigel invasion assay showed that Tbx2 overexpression upregulated cell proliferation, G1-S transition, and invasion, with corresponding change of cyclin D1, p21, and MMP 2 and 9. Importantly, we demonstrated that Tbx2 reduced apoptosis and conferred resistance to temozolomide in GBM cell lines. Further experiments showed that Tbx2 could regulate mitochondrial fission/fusion balance. Western blot showed that Tbx2 overexpression reduced caspase 3 cleavage, while it induced Bcl-2 and p-Drp1 upregulation. In conclusion, our results indicated that Tbx2 might serve as an indicator for poor prognosis and also be useful as an important therapeutic in human GBM, which inhibits apoptosis through regulation of mitochondrial function.

Genome-wide ChIP-seq analysis of EZH2-mediated H3K27me3 target gene profile highlights differences between low- and high-grade astrocytic tumors.

Enhancer of zeste homolog-2(EZH2) is a key epigenetic regulator that functions as oncogene and also known for inducing altered trimethylation of histone at lysine-27 (H3K27me3) mark in various tumors. However, H3K27me3 targets and their precise relationship with gene expression are largely unknown in astrocytic tumors. In this study, we checked EZH2 messenger RNA and protein expression in 90 astrocytic tumors of different grades using quantitative PCR and immunohistochemistry, respectively. Further, genome-wide ChIP-seq analysis for H3K27me3 modification was also performed on 11 glioblastomas (GBMs) and 2 diffuse astrocytoma (DA) samples. Our results showed EZH2 to be highly overexpressed in astrocytic tumors with a significant positive correlation with grade. Interestingly, ChIP-seq mapping revealed distinct differences in genes and pathways targeted by these H3K27me3 modifications between GBM versus DA. Neuroactive ligand receptor pathway was found most enriched in GBM (P = 9.4 × 10-25), whereas DA were found to be enriched in metabolic pathways. Also, GBM showed a higher enrichment of H3K27me3 targets reported in embryonic stem cells and glioma stem cells as compared with DAs. Our results show majority of these H3K27me3 target genes were downregulated, not only due to H3K27me3 modification but also due to concomitant DNA methylation. Further, H3K27me3 modification-associated gene silencing was not restricted to promoter but also present in gene body and transcription start site regions. To the best of our knowledge, this is the first high-resolution genome-wide mapping of H3K27me3 modification in adult astrocytic primary tissue samples of human, highlighting the differences between grades. Interestingly, we identified SLC25A23 as important target of H3K27me3 modification, which was downregulated in GBM and its low expression was associated with poor prognosis in GBMs.

Underexpression of HOXA11 Is Associated with Treatment Resistance and Poor Prognosis in Glioblastoma

PurposeHomeobox (HOX) genes are essential developmental regulators that should normally be in the silenced state in an adult brain. The aberrant expression of HOX genes has been associated with the prognosis of many cancer types, including glioblastoma (GBM). This study examined the identity and role of HOX genes affecting GBM prognosis and treatment resistance.Materials and MethodsThe full series of HOX genes of five pairs of initial and recurrent human GBM samples were screened by microarray analysis to determine the most plausible candidate responsible for GBM prognosis. Another 20 newly diagnosed GBM samples were used for prognostic validation. In vitro experiments were performed to confirm the role of HOX in treatment resistance. Mediators involved in HOX gene regulation were searched using differentially expressed gene analysis, gene set enrichment tests, and network analysis.ResultsThe underexpression of HOXA11 was identified as a consistent signature for a poor prognosis among the HOX genes. The overall survival of the GBM patients indicated a significantly favorable prognosis in patients with high HOXA11 expression (31±15.3 months) compared to the prognoses in thosewith low HOXA11 expression (18±7.3 months, p=0.03). When HOXA11 was suppressed in the GBM cell lines, the anticancer effect of radiotherapy and/or temozolomide declined. In addition, five candidate mediators (TGFBR2, CRIM1, TXNIP, DPYSL2, and CRMP1) that may confer an oncologic effect after HOXA11 suppression were identified.ConclusionThe treatment resistance induced by the underexpression of HOXA11 can contribute to a poor prognosis in GBM. Further investigation will be needed to confirm the value of HOXA11 as a potential target for overcoming the treatment resistance by developing chemo- or radiosensitizers.

Abstract 4470: ST3GAL1-associated transcriptomic program portends poor prognosis in glioblastoma

Abstract Cell surface sialylation has been associated with tumor cell invasiveness in several cancers. Patients with grade IV glioblastoma multiforme (GBM) often show a median survival period of fifteen months, even with the current standard-of-care treatment. Among the reasons for this poor prognosis lies in the cellular and molecular heterogeneity of tumor cells. The Cancer Genome Atlas efforts have shown that histologically identical GBM tumors can be divided into four molecular subtypes based on gene expression, with each subtype corresponding to unique genomic aberrations and clinical outcome. These findings highlight the limitation of relying solely on morphological approaches to diagnose and subsequently treat patients. We conducted a lectin screen with the goal of identifying candidates that stained normal and tumor cells differentially. We identified a sialyltransferase, ST3Gal1 as a mediator of the binding pattern of one such lectin, Peanut Agglutinin. We demonstrate that ST3Gal1 promotes tumor cell invasiveness and enriches for self-renewal potential in the mesenchymal tumor subclass of patients, typified by highly aggressive and recurrent profiles. Depletion of ST3Gal1 extends tumor latency and prolongs survival in mice. Importantly, an enrichment of the ST3GAL1 transcriptomic program portends poor prognosis in glioma patients. Moving forward, since there are no mutations or changes in copy number of ST3GAL1, we explored epigenetic regulation as a possible mechanism. By tapping into data from the NIH Roadmap Epigenome, and transcriptomic analysis of our patient tumor-derived cells, we identified a histone clustering signature that correlates with survival outcome and disease progression. We are currently investigating one such histone modifier, lysine (K)-specific demethylase 1A (LSD1) and its role at modifying ST3GAL1 transcription and activity. Citation Format: Yuk Kien Chong, Edwin Sandanaraj, Lynnette Koh, Moogaambikai Thangaveloo, Melanie SY Tan, Geraldene Koh, Tan Boon Toh, Grace GY Lim, Joanna Holbrook, Oi Lian Kon, Mahendran Nadarajah, Ivan Ng, Wai Hoe Ng, Nguan Soon Tan, Kah Leong Lim, Carol S. Tang, Beng Ti Ang. ST3GAL1-associated transcriptomic program portends poor prognosis in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4470.

Unique spectral markers discern recurrent Glioblastoma cells from heterogeneous parent population.

An inability to discern resistant cells from bulk tumour cell population contributes to poor prognosis in Glioblastoma. Here, we compared parent and recurrent cells generated from patient derived primary cultures and cell lines to identify their unique molecular hallmarks. Although morphologically similar, parent and recurrent cells from different samples showed variable biological properties like proliferation and radiation resistance. However, total RNA-sequencing revealed transcriptional landscape unique to parent and recurrent populations. These data suggest that global molecular differences but not individual biological phenotype could differentiate parent and recurrent cells. We demonstrate that Raman Spectroscopy a label-free, non-invasive technique, yields global information about biochemical milieu of recurrent and parent cells thus, classifying them into distinct clusters based on Principal-Component-Analysis and Principal-Component-Linear-Discriminant-Analysis. Additionally, higher lipid related spectral peaks were observed in recurrent population. Importantly, Raman spectroscopic analysis could further classify an independent set of naïve primary glioblastoma tumour tissues into non-responder and responder groups. Interestingly, spectral features from the non-responder patient samples show a considerable overlap with the in-vitro generated recurrent cells suggesting their similar biological behaviour. This feasibility study necessitates analysis of a larger cohort of naïve primary glioblastoma samples to fully envisage clinical utility of Raman spectroscopy in predicting therapeutic response.

Glioblastoma in the Canton of Zurich, Switzerland revisited: 2005 to 2009.

A population-based analysis of patients with glioma diagnosed between 1980 and 1994 in the Canton of Zurich in Switzerland confirmed the overall poor prognosis of glioblastoma. To explore changes in outcome, registry data were reevaluated for patients diagnosed between 2005 and 2009. Patients with glioblastoma who were diagnosed between 2005 and 2009 were identified by the Zurich and Zug Cancer Registry. The prognostic significance of epidemiological and clinical data, isocitrate dehydrogenase 1 (IDH1)(R132H) mutation status, and O6 methylguanine DNA methyltransferase (MGMT) promoter methylation status was analyzed using the Kaplan-Meier method and the Cox proportional hazards model. A total of 264 patients with glioblastoma were identified, for an annual incidence of 3.9 compared with the previous incidence of 3.7. The mean age of the patients at the time of diagnosis was 59.5 years in the current cohort compared with 61.3 years previously. The overall survival (OS) rate was 46.4% at 1 year, 22.5% at 2 years, and 14.4% at 3 years in the current study compared with 17.7% at 1 year, 3.3% at 2 years, and 1.2% at 3 years as reported previously. The median OS for all patients with glioblastoma was 11.5 months compared with 4.9 months in the former patient population. The median OS was 1.9 months for best supportive care, 6.2 months for radiotherapy alone, 6.7 months for temozolomide alone, and 17.0 months for radiotherapy plus temozolomide. Multivariate analysis revealed age, Karnofsky performance score, extent of tumor resection, first-line treatment, year of diagnosis, and MGMT promoter methylation status were associated with survival in patients with IDH1(R132H) -nonmutant glioblastoma. The OS of patients newly diagnosed with glioblastoma in the Canton of Zurich in Switzerland markedly improved from 1980 through 1994 to 2005 through 2009. Cancer 2016;122:2206-15. © 2016 American Cancer Society.

Association between cytoplasmic CRABP2, altered retinoic acid signaling, and poor prognosis in glioblastoma.

Retinoic acid (RA), a metabolite of vitamin A, is required for the regulation of growth and development. Aberrant expression of molecules involved in RA signaling has been reported in various cancer types including glioblastoma multiforme (GBM). Cellular retinoic acid-binding protein 2 (CRABP2) has previously been shown to play a key role in the transport of RA to retinoic acid receptors (RARs) to activate their transcription regulatory activity. Here, we demonstrate that CRABP2 is predominantly located in the cytoplasm of GBM tumors. Cytoplasmic, but not nuclear, CRABP2 levels in GBM tumors are associated with poor patient survival. Treatment of malignant glioma cell lines with RA results in a dose-dependent increase in accumulation of CRABP2 in the cytoplasm. CRABP2 knockdown reduces proliferation rates of malignant glioma cells, and enhances RA-induced RAR activation. Levels of CRYAB, a small heat shock protein with anti-apoptotic activity, and GFAP, an astrocyte-specific intermediate filament protein, are greatly reduced in CRABP2-depleted cells. Restoration of CRYAB expression partially but significantly reversed the effect of CRABP2 depletion on RAR activation. Our combined in vivo and in vitro data indicate that: (i) CRABP2 is an important determinant of clinical outcome in GBM patients, and (ii) the mechanism of action of CRABP2 in GBM involves sequestration of RA in the cytoplasm and activation of an anti-apoptotic pathway, thereby enhancing proliferation and preventing RA-mediated cell death and differentiation. We propose that reducing CRABP2 levels may enhance the therapeutic index of RA in GBM patients.

Concurrent Expression of VEGF-C and Neuropilin-2 Is Correlated with Poor Prognosis in Glioblastoma.

Vascular endothelial growth factor-C (VEGF-C) is a secreted growth factor involved in many oncogenic processes, and neuropilin-2 (NRP2) is essential for neuronal guidance as a well-acknowledged co-receptor of VEGF receptors. The overexpression of NRP2 has been reported in many types of cancers, but the significance of NRP2 in glioblastoma is not elucidated. To investigate the clinical significance of VEGF-C and NRP2 in glioblastoma, we detected their expression in 86 cases of glioblastoma with immunohistochemistry and categorized our cohort into high- and low-expression groups according to the immunohistochemistry score, which was the product of the score of staining intensity multiplied by the score reflecting the percentage of positive cells. The proportion of glioblastoma with high VEGF-C expression was 34.9% (30/86), and that with high NRP2 expression was 37.2% (32/86). The proportion of glioblastoma with high expression of both VEGF-C and NRP2 was 15.1% (13/86). Moreover, the proportion of cases with high VEGF-C and low NRP2 was 19.7% (17/86), while the proportion of cases with low VEGF-C and high NRP2 was 22.1% (19/86). The high expression of both VEGF-C and NRP2 was associated with poorer survival rate (P = 0.023), and can be identified as an independent prognostic factor in glioblastoma (P = 0.030). Moreover, cases with low NRP2 expression are easier for complete tumor resection (P = 0.038). In conclusion, the concurrent high expression of VEGF-C and NRP2 is predictive of the unfavorable prognosis in glioblastoma, indicating that the VEGF-C-NRP2 signaling pathway is a potential and promising drug target in glioblastoma therapy.

PKCδ activated by c-MET enhances infiltration of human glioblastoma cells through NOTCH2 signaling

Poor prognosis of glioblastoma (GBM) is attributable to the propensity of tumor cells to infiltrate into the brain parenchyma. Protein kinase C (PKC) isozymes are highly expressed or aberrantly activated in GBM. However, how this signaling node translates to GBM cell invasiveness remains unknown. Here, we report that among PKC isoforms, PKCδ is strongly associated with infiltration of GBM cells. Notably, PKCδ enhanced Tyr418 phosphorylation of the non-receptor tyrosine kinase SRC, which in turn activated STAT3 and subsequent NOTCH2 signaling, ultimately leading to GBM cell invasiveness. Furthermore, we showed that PKCδ was aberrantly activated in GBM cells by c-MET, a receptor tyrosine kinase hyperactivated in GBM. In agreement, inhibition either component in the c-MET/PKCδ/SRC/STAT3 signaling axis effectively blocked the NOTCH2 signaling and invasiveness of GBM cells. Taken together, our findings shed a light on the signaling mechanisms behind the constitutive activation of PKCδ signaling in GBM.

Abstract C26: Development of selective MELK kinase inhibitors for breast cancer treatment

Abstract Breast cancer is the second most common cancer in the world and the most frequent cancer among women. Despite the progress in developing breast cancer therapies, approximately, 15% of all breast cancers are diagnosed as triple negative breast cancer (TNBC) and due to the lack of estrogen and progesterone receptors this subgroup of patients remains difficult to treat with hormonal therapies. Additionally, therapies targeting HER2, such as Herceptin, are also inefficient against TNBC. In recent years, maternal embryonic leucine zipper kinase (MELK) has been identified as a novel oncogenic target that is highly expressed in several types of solid cancers: breast (especially triple negative breast cancer), colon, ovary, lung, and brain and present at low levels in normal tissues. MELK overexpression in patient tumors strongly correlates with poor prognosis in glioblastoma and breast cancer. siRNA mediated knockdown of MELK kinase significantly inhibits growth of tumor cell lines both in vitro and in vivo. Therefore, MELK kinase is emerging as a novel and interesting target with significant potential for therapeutic intervention in cancer. MELK is an atypical member of the AMPK family of serine-threonine kinases that been implicated has been implicated in stem cell renewal, cell cycle progression, cytokinesis, mRNA splicing and apoptosis. Its activity is correlated with its phosphorylation level, is cell cycle dependent, and maximal during mitosis although direct upstream regulators of MELK kinase activity are unknown. Despite the fact that the exact function is currently under investigation, selective targeting of MELK may be an effective cancer treatment strategy in a wide range of solid tumors. In this study, we are reporting development of a series of selective MELK kinase inhibitors. Synthesized compounds exert excellent selectivity and potency in MELK inhibition in a low nanomolar range. Therapeutic effect of the compounds was investigated in the panel of breast cancer cell lines with different genetic background as well as with different MELK kinase levels; it was shown that for some cell lines compounds induced cell death with nanomolar ED50 values. The compound's effect on the proliferation and in the colony formation assay was also investigated. Taken altogether, the presented data supports our rationale of using MELK kinase inhibitors as a novel approach for the cancer therapy. Citation Format: Piotr Kowalczyk, Paulina Węgrzyn, Monika Prokopowicz, Martyna Knop, Karolina Mazur, Katarzyna Dziedzic, Karolina Gluza, Martyna Knop, Katarzyna Dziedzic, Karolina Mazur, Adam Radzimierski, Claude Commandeur, Magdalena Zawadzka, Kristjan Bloudoff, Fred Vaillancourt, Nick Larsen, John Wang, Dom Reynolds, Daisuke Ito, Jian Zou, Michelle Aicher, Pete Smith, Ping Zhu, Krzysztof Brzózka. Development of selective MELK kinase inhibitors for breast cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C26.

Homeobox C9 suppresses Beclin1-mediated autophagy in glioblastoma by directly inhibiting the transcription of death-associated protein kinase 1.

The transcription factor homeobox C9 (HOXC9) plays a crucial role in developmental regulatory systems, where it determines the specific positional identities of cells along the anteroposterior axis. The expression of HOXC9 has been found to be dysregulated in some cancers such as lung cancer, breast cancer, and neuroblastoma. Here, we report for the first time that HOXC9 is a novel autophagy regulator and reveal its oncogenic role in cell survival and its usefulness as a prognostic marker in glioblastoma patients. Kaplan-Meier analysis was performed to evaluate the possible prognostic value of HOXC9 in glioblastoma. Growth curve assays, subcutaneous, and orthotopic implantations were used to analyze cell viability and tumor formation, respectively. Luciferase and chromatin immunoprecipitation assays were employed to explore the mechanisms involved in the association between HOXC9 and its downstream effector, death-associated protein kinase 1 (DAPK1). High expression of HOXC9 was found to be an indicator of a poor prognosis in glioblastoma. HOXC9 knockdown resulted in a significant reduction of cell viability, migration, invasion, and tumorigenicity and a marked increase in autophagy. During the autophagy process, HOXC9 inhibited DAPK1 transcription by directly binding to its promoter. The downregulation of HOXC9 releases its transcriptional inhibition of DAPK1, resulting in the activation of the DAPK1-Beclin1 pathway, which induces autophagy in glioblastoma cells. Collectively, our data indicate that HOXC9 is an oncogene in glioblastoma. We have revealed its role in the control of autophagy, and we suggest that HOXC9 is a novel and promising therapeutic target.

EPID-16PATTERNS OF CARE AND OUTCOME IN GLIOBLASTOMA PATIENTS IN THE CANTON OF ZURICH: A POPULATION-BASED STUDY (2005-2009)

A population-based analysis of patterns of care and outcome in glioma patients diagnosed 1980-1994 in the Canton of Zurich, Switzerland, has confirmed the overall poor prognosis of glioblastoma. To explore changes in outcome over the last decades, the registry data were re-evaluated for patients diagnosed in the time frame 2005-2009. Patients diagnosed with glioblastoma in the Canton of Zurich from 2005-2009 were identified by the Cancer Registry Zurich. Clinical and epidemiological data, as well as molecular markers were assessed, and analyzed using the Kaplan-Meier method and the Cox proportional hazards model. In the current database, there were 264 patients with glioblastoma, including 256 primary glioblastomas with an annual incidence of 3.98 compared to 3.55 in the former patient group. Median age at diagnosis was 60.0 years recently as opposed to 61.3 years previously. Overall survival (OS) for all glioblastoma patients was 41.7% at 1 year, 22.7% at 2 years and 13.1% at 3 years in the present study while previously the OS was significantly lower, namely 17.7% at 1 year, 3.3% at 2 years and 1.2% at 3 years, respectively. Median OS for primary glioblastomas was 11.0 months for the period ending 2009 versus 4.7 months for the patient population of 1980-1994. In the present study, by treatment, the median OS for best supportive care, radiotherapy alone, temozolomide alone or radiotherapy plus temozolomide was 2.0, 6.0, 6.0 or 17.0 months, respectively. Multivariate analysis revealed age, KPS, extent of resection, adjuvant treatment regimens, year of diagnosis, IDH-1 mutation status and MGMT promoter methylation status significantly associated with survival. The OS of patients in the Canton of Zurich with newly diagnosed glioblastoma has markedly improved from the period of 1980-1994 to 2005-2009.

IDH1 mutation detection by droplet digital PCR in glioma.

Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation.

Abstract 2249: TERT promoter mutation as poor prognosis marker in glioblastoma via sphere formation and self- renewal capacity

Abstract Telomerase reverse transcriptase (TERT) activity was increased allowing the maintenance of telomere length in cancers. TERT promoter mutation has been reported in a variety of cancers, especially in glioblastoma (GBM) with high frequency. In present study, we screened TERT promoter mutation in 25 GBMs and analyzed the results with their respective genetic profile and its renewal capacity by in vitro sphere formation. TERT promoter mutation (C228T, C250T) was found in 16 of 25 GBMs (64.0%) and was associated with male (p < 0.01). They showed inverse correlations with IDH1 and ATRX mutations and a positive correlation with EGFR amplification though it did not show significance. GBM with sphere formation harbored higher frequency of TERT promoter mutation (91.6%) than those without sphere formation (27.3%; p = 0.01), which suggest that TERT mutation promotes self-renewal capacity of GBM. GBM with TERT mutations showed a shorter survival than those without TERT mutation in univariate analysis (median, 83.6 vs. 123.8 weeks; p = 0.045). These results suggested that TERT promoter mutation affect poor prognosis of GBM via promoting self- renewal capacity. Citation Format: Jae-Ho Lee, Hyunsu Lee, Seon Rang Woo, Kyeung-Min Joo, KeonUk Park. TERT promoter mutation as poor prognosis marker in glioblastoma via sphere formation and self- renewal capacity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2249. doi:10.1158/1538-7445.AM2015-2249

Introduction: brain tumor immunotherapy.

The poor prognosis of glioblastoma (GBM) mandates new therapies. With FDA approval of several immunotherapy agents over the past few years, much attention and resources have been directed towards new immune based approaches and therapies in all cancer types. Immunotherapy for gliomas has been an area of immense interest. In this special issue of Journal of Neuro-Oncology, we have gathered renowned experts in the various disciplines of immunotherapy to discuss the current state, obstacles thus far encountered, and newly emerging issues of immune-based therapies in neuro-oncology.