Primary Glioblastoma Multiforme Tumors Research Articles

Identification of TMZ resistance-associated histone post-translational modifications in glioblastoma using multi-omics data.

Glioblastoma multiforme (GBM) is among the most aggressive cancers, with current treatments limited in efficacy. A significant hurdle in the treatment of GBM is the resistance to the chemotherapeutic agent temozolomide (TMZ). The methylation status of the MGMT promoter has been implicated as a critical biomarker of response to TMZ. To explore the mechanisms underlying resistance, we developed two TMZ-resistant GBM cell lines through a gradual increase in TMZ exposure. Transcriptome sequencing of TMZ-resistant cell lines revealed that alterations in histone post-translational modifications might be instrumental in conferring TMZ resistance. Subsequently, multi-omics analysis suggests a strong association between histone H3 lysine 9 acetylation (H3K9ac) levels and TMZ resistance. We observed a significant correlation between the expression of H3K9ac and MGMT, particularly in the unmethylated MGMT promoter samples. More importantly, our findings suggest that H3K9ac may enhance MGMT transcription by facilitating the recruitment of the SP1 transcription factor to the MGMT transcription factor binding site. Additionally, by analyzing single-cell transcriptomics data from matched primary and recurrent GBM tumors treated with TMZ, we modeled the molecular shifts occurring upon tumor recurrence. We also noted a reduction in tumor stem cell characteristics, accompanied by an increase in H3K9ac, SP1, and MGMT levels, underscoring the potential role of H3K9ac in tumor relapse following TMZ therapy. The increase in H3K9ac appears to enhance the recruitment of the transcription factor SP1 to its binding sites within the MGMT locus, consequently upregulating MGMT expression and driving TMZ resistance in GBM.

RAD51-Mediated DNA Homologous Recombination Is Independent of PTEN Mutational Status.

Simple SummaryPTEN is an important tumor suppressor that is frequently mutated in malignancy. PTEN mutational loss has been associated with reduced RAD51 expression and homologous recombination deficiency (HRD), however; recent studies have failed to recapitulate these findings. Here, we show that RAD51 expression, foci formation and homologous recombination repair activity are unaltered in normal and tumorigenic PTEN-deficient cells and patient samples. Furthermore, we show that PTEN-deficient tumor cell lines do not synergize with the clinical PARP inhibitor olaparib, underscoring a need to discontinue its use in treating patients with PTEN-deficient tumors that do not otherwise exhibit HRD.PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. Methods: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. Results: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status. Conclusions: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.

Targeting the ABC transporter ABCB5 sensitizes glioblastoma to temozolomide-induced apoptosis through a cell-cycle checkpoint regulation mechanism

Glioblastoma multiforme (GBM) is a malignant brain tumor with a poor prognosis resulting from tumor resistance to anticancer therapy and a high recurrence rate. Compelling evidence suggests that this is driven by subpopulations of cancer stem cells (CSCs) with tumor-initiating potential. ABC subfamily B member 5 (ABCB5) has been identified as a molecular marker for distinct subsets of chemoresistant tumor-initiating cell populations in diverse human malignancies. In the current study, we examined the potential role of ABCB5 in growth and chemoresistance of GBM. We found that ABCB5 is expressed in primary GBM tumors, in which its expression was significantly correlated with the CSC marker protein CD133 and with overall poor survival. Moreover, ABCB5 was also expressed by CD133-positive CSCs in the established human U-87 MG, LN-18, and LN-229 GBM cell lines. Antibody- or shRNA-mediated functional ABCB5 blockade inhibited proliferation and survival of GBM cells and sensitized them to temozolomide (TMZ)-induced apoptosis in vitro Likewise, in in vivo human GBM xenograft experiments with immunodeficient mice, mAb treatment inhibited growth of mutant TP53, WT PTEN LN-229 tumors, and sensitized LN-229 tumors to TMZ therapy. Mechanistically, we demonstrate that ABCB5 blockade inhibits TMZ-induced G2/M arrest and augments TMZ-mediated cell death. Our results identify ABCB5 as a GBM chemoresistance marker and point to the potential utility of targeting ABCB5 to improve current GBM therapies.

Heterogeneous EGFR, CDK4, MDM4, and PDGFRA Gene Expression Profiles in Primary GBM: No Association with Patient Survival.

Background: The prognostic impact of the expression profile of genes recurrently amplified in glioblastoma multiforme (GBM) remains controversial. Methods: We investigated the RNA gene expression profile of epidermal growth factor receptor (EGFR), cyclin-dependent kinase 4 (CDK4), murine doble minute 4 (MDM4), and platelet derived growth factor receptor alpha (PDGFRA) in 83 primary GBM tumors vs. 42 normal brain tissue samples. Interphase FISH (iFISH) analysis for the four genes, together with analysis of intragenic deletions in EGFR and PDGFRA, were evaluated in parallel at the DNA level. As validation cohort, publicly available RNA gene expression data on 293 samples from 10 different GBM patient series were also studied. Results: At the RNA level, CDK4 was the most frequently overexpressed gene (90%) followed by EGFR (58%) and PDGFRA (58%). Chromosome 7 copy number alterations, i.e., trisomy (49%) and polysomy (44%), showed no clear association with EGFR gene expression levels. In turn, intragenic EGFR deletions were found in 39 patients (47%), including EGFRvIII (46%) in association with EGFRvIVa (4%), EGFRvII (2%) or other EGFR deletions (3%) and PDGFRA deletion of exons 8–9 was found in only two tumors (2%). Conclusions: Overall, none of the gene expression profiles and/or intragenic EGFR deletions showed a significant impact on overall survival of GBM supporting the notion that other still unraveled features of the disease might play a more relevant prognostic role in GBM.

Microsurgical resection of a glioblastoma multiforme of the medulla oblongata with intraoperative subcortical stimulation and mapping.

Primary glioblastoma multiforme tumors of the medulla oblongata are rare, especially in the adult population. Perhaps due to this rarity, we are not aware of any previous reports addressing the resection of these tumors or their clinical outcomes.In this surgical video, we present a 43-year-old man with a 1-month history of left-sided paresthesia. The paresthesia initiated in the left hand, along with weakness and reduced fine motor control, and then spread to the entire left side of the body. He had recent weight loss, imbalance, difficulty in swallowing, and hoarseness in his voice. He also had a diminished gag reflex, and significant atrophy of the right side of the tongue with an accompanying deviation of the uvula and fasciculations of the tongue. MRI showed an infiltrative expansile mass within the medulla with peripheral enhancement and central necrosis. In T2/FLAIR sequences, a hyperintense signal extended superiorly into the left inferior aspect of the pons and left inferior cerebellar peduncle and inferiorly into the upper cervical cord.The decision was made to proceed with surgical resection. The patient underwent a midline suboccipital craniotomy with C1 laminectomy for surgical resection of this infiltrative expansile intrinsic mass in the medulla oblongata, with concurrent monitoring of motor and somatosensory evoked potentials and monitoring of lower cranial nerves IX, X, XI, and XII. A gross-total resection of the enhancing portion of the tumor was performed, along with a subtotal resection of the nonenhancing portion. The surgery and postoperative course were uneventful. Histopathology revealed a grade IV astrocytoma. The patient received radiation therapy.In this surgical video, we demonstrate important steps for the microsurgical resection of this challenging glioblastoma multiforme of the medulla oblongata.The video can be found here: https://youtu.be/QHbOVxdxbeU.

Prognostic stratification of adult primary glioblastoma multiforme patients based on their tumor gene amplification profiles.

Several classification systems have been proposed to address genomic heterogeneity of glioblastoma multiforme, but they either showed limited prognostic value and/or are difficult to implement in routine diagnostics. Here we propose a prognostic stratification model for these primary tumors based on tumor gene amplification profiles, that might be easily implemented in routine diagnostics, and potentially improve the patients management. Gene amplification profiles were prospectively evaluated in 80 primary glioblastoma multiforme tumors using single-nucleotide polymorphism arrays and the results obtained validated in publicly available data from 267/347 cases. Gene amplification was detected in 45% of patients, and chromosome 7p11.2 including the EGFR gene, was the most frequently amplified chromosomal region – either alone (18%) or in combination with amplification of DNA sequences in other chromosomal regions (10% of cases). Other frequently amplified DNA sequences included regions in chromosomes 12q(10%), 4q12(7%) and 1q32.1(4%). Based on their gene amplification profiles, glioblastomas were subdivided into: i) tumors with no gene amplification (55%); ii) tumors with chromosome 7p/EGFR gene amplification (with or without amplification of other chromosomal regions) (38%); and iii) glioblastoma multiforme with a single (11%) or multiple (6%) amplified DNA sequences in chromosomal regions other than chromosome 7p. From the prognostic point of view, these amplification profiles showed a significant impact on overall survival of glioblastoma multiforme patients (p>0.001). Based on these gene amplification profiles, a risk-stratification scoring system was built for prognostic stratification of glioblastoma which might be easily implemented in routine diagnostics, and potentially contribute to improved patient management.

P.038 Investigating the role of long non-coding RNAs in glioblastoma multiforme

Background: Malignant gliomas are the most common and deadly brain tumors. Mean survival rate for a patient diagnosed with a glioblastoma multiforme (GBM) remains slightly over one year. Standard of care consists of treatment with temozolomide (TMZ) and radiotherapy. Recent work has highlighted functions of long non-coding RNAs (lncRNAs) in GBM progression and TMZ response even though the information regarding these newly discovered molecules is sparse. The overarching objective of this project was thus to assess the expression of select lncRNAs in GBM tumor samples and in models of TMZ resistance. Methods: A qRT-PCR-based approach was undertaken to measure six lncRNAs in 19 primary GBM samples, four GBM cell lines and in-house developed TMZ-resistant GBM cells. Results: Elevated levels of Hotair and H19 were observed in primary GBM tumors while decreased expression of MEG3 was recorded in the same samples. Interestingly, levels of PANDA increased 3.4-fold in GBM cells resistant to TMZ when compared with their sensitive counterparts. Conclusions: Overall, this work provides evidence of lncRNA deregulation in GBM tumors and reveals a previously unexplored lncRNA potentially involved in TMZ resistance. Modulation of lncRNA targets via RNAi-mediated approaches is envisioned to clarify their function and to strengthen their position as therapeutic options in GBMs.

In-depth immunophenotyping of patients with glioblastoma multiforme: Impact of steroid treatment

ABSTRACTDespite aggressive treatment regimens based on surgery and radiochemotherapy, the prognosis of patients with grade IV glioblastoma multiforme (GBM) remains extremely poor, calling for alternative options such as immunotherapy. Immunological mechanisms including the Natural Killer Group 2 member D (NKG2D) receptor-ligand system play an important role in tumor immune surveillance and targeting the NKG2D system might be beneficial. However, before considering any kind of immunotherapy, a precise characterization of the immune system is important, particularly in GBM patients where conventional therapies with impact on the immune system are frequently co-administered. Here we performed an in-depth immunophenotyping of GBM patients and age-matched healthy controls and analyzed NKG2D ligand expression on primary GBM cells ex vivo. We report that GBM patients have a compromised innate immune system irrespective of steroid (dexamethasone) medication. However, dexamethasone drastically reduced the number of immune cells in the blood of GBM patients. Moreover, higher counts of immune cells influenced by dexamethasone like CD45+ lymphocytes and non-Vδ2 γδ T cells were associated with better overall survival. Higher levels of NKG2D ligands on primary GBM tumor cells were observed in patients who received radiochemotherapy, pointing towards increased immunogenic potential of GBM cells following standard radiochemotherapy. This study sheds light on how steroids and radiochemotherapy affect immune cell parameters of GBM patients, a pre-requisite for the development of new therapeutic strategies targeting the immune system in these patients.

Abstract LB-289: Hypermethylation enforces promoter switch in glioblastoma multiforme tumors

Abstract Complete understanding why a patient is sensitive or resistant to a treatment needs comprehensive whole-genome characterization of the patient's tumor to address underlying molecular mechanism. The developed consortiums, such as The Cancer Genome Atlas (TCGA) and the Encyclopedia of DNA Elements (ENCODE), provide such genomic data at different dimensions (e.g., DNA methylation, RNA gene expression, DNA copy number, and DNA mutation). However, there lacks a patient-specific approach to integration of the multi-dimensional genomic data, which is helpful to provide mechanistic insights for primary or recurrent tumor of an individual patient. Here, we developed a new patient-specific analysis for the genomic data integration and its application to Glioblastoma Multiforme (GBM) tumors discovered a mechanism of epigenetic regulation on drug response of Temozolomide (TMZ). We applied the new patient-specific analysis to 13 recurrent tumors of GBM patients in the TCGA and identified a top gene (#1), CYP19A1, with alternative promoters and intragenic hypermethylation. We detected the gene isoforms of CYP19A1 in the different brain tissues. The whole gene (Exons 1-10) is seldomly expressed in both of the normal brain tissues and low-grade gliomas tumors whereas new gene isoforms are specifically expressed in the primary GBM tumors and recurrent GBM tumors. Among 175 primary GBM tumors, 166 (95%) expressed the isoforms of CYP19A1. In the primary and recurrent GBM tumors, there are 78% (129/166) and 83% (10/12) expressing the isoforms starting from the TSS near to Exon 4 (Ex-4). CYP19A1 has (i) hypermethylated CpGs at the regions of original promoter and the introns upstream of Ex-4, and (ii) alternative exon usage: Exons 4-10 are highly used whereas exons 1-3 are seldomly used. We hypothesize that the hypermethylated CpGs located in the region covering from the promoter (near to TSS) to the intron close to Ex-4 may prevent the start of the TF complex binding to the original promoter and enforce the switch to alternative promoter proximal to Ex-4. The Pol-II ChIP-Seq data of U87 cell line from the ENCODE indicate that the Pol-II alternatively binds to the upstream of Ex-4 because the CpGs close to the original promoters, I.f and I.3, are hypermethylated. We further treated the U87 cell line with 5-AZA to induce demethylation of CpGs. Strikingly, we observed that Ex-2 is highly expressed after demethylation. The results confirmed that the region near to Ex-4 is an alternative promoter for expression of the isoforms of CYP19A1 and the hypermethylation of CpGs controls the usage of Exons 1-10. We tested if the gene expression and new transcript isoforms of CYP19A1 are related to patients’ survival. We checked the gene expression of CYP19A1 in 161 primary tumors of GBM from TCGA, who received radiotherapy, surgery, and chemotherapy of TMZ. The primary tumors of GBM patients with lower CYP19A1 tend to benefit more and survive longer. The statistical significance for the survival analysis based on Cox regression is P < 0.007. We analyzed that the survival statuses of these patients with different gene isoforms. The new isoform starting from Exon 4 to Exon 8 benefits patients’ survival more (628±453), compared to those terminated proximal to Exon 9 (317±152) and those proximal to Exon 10 (367±251). The statistical significance is P = 0.008, Wilcoxon rank sum test. Citation Format: Guangxu Jin, Liang Liu, Xiaobo Zhou. Hypermethylation enforces promoter switch in glioblastoma multiforme tumors. [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 LB-289.

Erratum to: Primary glioblastoma multiforme tumors and recurrence: comparative analysis of the danger signals HMGB1, HSP70, and calreticulin

Purpose Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor. Despite improved multimodal therapies, the tumor recurs in most cases. Diverging patient survival suggests great tumor heterogeneity and different therapy responses. Danger signals such as high-mobility group box protein 1 (HMGB1), heat shock protein 70 (HSP70), and calreticulin (CRT) are biomarker candidates, due to their association with tumor progression versus induction of antitumor immune responses. Overexpression of these danger signals has been reported for various types of tumors; however, their role in GBM is still elusive. A direct comparison of their expression in the primary tumor versus the corresponding relapse is still lacking for most tumor entities. Patients and methods We therefore performed an expression analysis by immunohistochemistry of the danger signals HMGB1, HSP70, and CRT in primary tumors and the corresponding relapses of 9 patients with de novo GBM. Results HMGB1 was highly expressed in primary tumors with a significant reduction in the respective relapse. The extracellular HSP70 expression was significantly increased in the relapse compared to the primary tumor. CRT was generally highly expressed in the primary tumor, with a slight increase in the relapse. Conclusion The combination of a decreased expression of HMGB1, an increased expression of extracellular HSP70, and an increased expression of CRT in the relapse seems to be beneficial for patient survival. HMGB1, extracellular HSP70, and CRT could be taken into concerted consideration as potential biomarkers for the prognosis of patients with GBM.

CBM-03MOLECULAR CHARACTERIZATION OF GLIOMA CIRCULATING TUMOR CELLS (gCTCs) AT SINGLE CELL RESOLUTION

Glioblastoma multiforme (GBM), the most aggressive and most common malignant adult brain tumor, accounts for ∼50% of all gliomas and it is classified histologically as grade IV. Growing evidence for intratumor heterogeneity shows that single-site biopsies or selected profiling of a portion of a resected tumor falls short of revealing the complete genomic landscape of the entire tumor. EGFR amplification occurs in ∼50% of primary GBM tumors, and is frequently associated with the expression of a constitutively active, ligand-independent mutant form of the receptor, EGFRvIII. Glioma circulating tumor cells (gCTCs) have recently been identified in the blood of glioma patients by our research group. In this study, we report that gCTCs can be captured, enumerated and genetically characterized for EGFRvIII. To achieve this goal, we have thus far a) detected gCTCs through a Telomerase promoter–based assay, b) isolated individual gCTCs using a vacuum-assisted capillary microdissection approach, c) performed whole transcriptome amplification and d) analyzed the expression of EGFRviii by qPCR under optimized conditions. Furthermore, our group is conducting an IRB-approved pilot study with the purpose of examining the feasibility of this approach (to support initiation of an expanded study). In this study, we seek to characterize the genetic heterogeneity of individual gCTCs from high grade glioma patients. This approach would have major impact as it will clarify the biologically-relevant question of heterogeneity of gCTCs in a given patient with glioma, predict whether a patient would respond or be resistant to a molecularly targeted therapy, unravel the overall mechanistic underpinnings of a given patient's brain tumor, and lastly, track the biological evolution of the tumor over the patient's treatment course and post-treatment period. Our ultimate goal is to streamline predictive testing and effectively utilize a Neurooncology targeted biomarker to benefit patients.

Investigating a signature of temozolomide resistance in GBM cell lines using metabolomics.

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Current therapeutic approach to treat this malignancy involves a combination of surgery, radiotherapy and chemotherapy with temozolomide. Numerous mechanisms contributing to inherent and acquired resistance to this chemotherapeutic agent have been identified and can lead to treatment failure. This study undertook a metabolomics-based approach to characterize the metabolic profiles observed in temozolomide-sensitive and temozolomide-resistant GBM cell lines as well as in a small sub-set of primary GBM tumors. This approach was also utilized to explore the metabolic changes modulated upon cell treatment with temozolomide and lomeguatrib, an MGMT inhibitor with temozolomide-sensitizing potential. Metabolites previously explored for their potential role in chemoresistance including glucose, citrate and isocitrate demonstrated elevated levels in temozolomide-resistant GBM cells. In addition, a signature of metabolites comprising alanine, choline, creatine and phosphorylcholine was identified as up-regulated in sensitive GBM cell line across different treatments. These results present the metabolic profiles associated with temozolomide response in selected GBM models and propose interesting leads that could be leveraged for the development of therapeutic or diagnostic tools to impact temozolomide response in GBMs.

Increased proportion of FoxP3+ regulatory T cells in tumor infiltrating lymphocytes is associated with tumor recurrence and reduced survival in patients with glioblastoma.

Glioblastoma multiforme (GBM) is an aggressive malignancy associated with profound host immunosuppression mediated in part by FoxP3 expressing regulatory CD4+ T lymphocytes (Tregs) that down-regulate anti-tumor immunity. In order to assess whether FoxP3 was an independent driver differentially expressed in primary versus recurrent GBMs, we stained resected primary and recurrent GBM tumors for CD3, CD4, CD8 and FoxP3 expression using standard immunohistochemistry. Slides were scanned with a high-resolution scanner (ScanScope CS; Aperio), and image analysis software (Aperio ScanScope) was used to enumerate lymphocyte subpopulations allowing for high-throughput analysis and bypassing manual selection bias. As shown in previous studies, enumeration of individual lymphocyte populations did not correlate with clinical outcomes in patients with GBM. However, the CD4+ to regulatory FoxP3+ T cell ratio was diminished in recurrent disease, and increased CD3 and CD8+ to regulatory T cell ratios showed a positive correlation with survival outcomes in primary GBM. These results suggest that while absolute numbers of tumor infiltrating lymphocytes may not be informative for predicting clinical outcomes in patients with GBM, the effective balance of CD3, CD4 and CD8+ T cells to immunosuppressive FoxP3+ regulatory cells may influence clinical outcomes in this patient population.

AI-06 * NON-CANONICAL NF-kB SIGNALING DRIVES THE AGGRESSIVE INVASIVENESS OF GLIOBLASTOMA

The aggressive migration and invasion of glioblastoma multiforme (GBM) cells into healthy brain tissue are major factors contributing to the therapy resistance and poor prognosis of this malignancy. Aberrant activation of NF-kB has been shown to play key roles in the invasiveness and pathogenesis of many cancers, including GBM. Most of these studies have focused on canonical NF-kB signaling, which is mediated by RelA and p50. Activation of the canonical NF-kB pathway is induced by IkB kinase-b (IKKb), whose inhibition has been pursued as a therapeutic approach to attenuate NF-kB activation in cancer with limited success to-date. We have recently shown that the alternative, or non-canonical, NF-kB signaling pathway mediated by RelB, predominates in a very aggressive GBM subtype. Here, we investigate this previously unrecognized role for non-canonical NF-kB signaling in CNS tumor initiation and progression. Using both established and primary GBM tumor lines, we show that in high RelB-expressing GBM cells, loss of RelB inhibits invasion to a greater extent than loss of RelA. Furthermore, RelB expression is sufficient to promote invasion in RelA-deficient GBM cells. Stimulation with Tumor Nectosis Factor Weak Inducer of Apoptosis (TWEAK) preferentially activates non-canonical NF-kB signaling and regulation of Matrix Metalloproteinase 9 (MMP9) expression, resulting in strongly increased invasion. Finally, we show that a key upstream regulator of RelB, NF-kB-inducing kinase (NIK), induces dramatic cell shape changes, increases tumor cell invasion and promotes aggressive orthotopic tumor growth in mouse xenografts. These results not only expand on previously described roles for TWEAK in promoting tumor cell survival, but also demonstrate a potent pro-invasion function for NIK in aggressive GBM and, potentially, other RelB-driven tumors. Notably, oncogenic functions of the non-canonical NF-kB pathway remain poorly elucidated in the CNS. Our data highlight the therapeutic potential of targeting NIK in GBM.

Expression analysis of all protease genes reveals cathepsin K to be overexpressed in glioblastoma.

BackgroundCancer genome and transcriptome analyses advanced our understanding of cancer biology. We performed transcriptome analysis of all known genes of peptidases also called proteases and their endogenous inhibitors in glioblastoma multiforme (GBM), which is one of the most aggressive and deadly types of brain cancers, where unbalanced proteolysis is associated with tumor progression.MethodsComparisons were performed between the transcriptomics of primary GBM tumors and unmatched non-malignant brain tissue, and between GBM cell lines (U87-MG and U373) and a control human astrocyte cell line (NHA). Publicly-available data sets and our own datasets were integrated and normalized using bioinformatics tools to reveal protease and protease inhibitor genes with deregulated expression in both malignant versus non-malignant tissues and cells.ResultsOf the 311 protease genes identified to be differentially expressed in both GBM tissues and cells, 5 genes were highly overexpressed, 2 genes coding for non-peptidase homologues transferrin receptor (TFRC) and G protein-coupled receptor 56 (GPR56), as well as 3 genes coding for the proteases endoplasmic reticulum aminopeptidase 2 (ERAP2), glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and cathepsin K (CTSK), whereas one gene, that of the serine protease carboxypeptidase E (CPE) was strongly reduced in expression. Seventy five protease inhibitor genes were differentially expressed, of which 3 genes were highly overexpressed, the genes coding for stefin B (CSTB), peptidase inhibitor 3 (PI3 also named elafin) and CD74. Seven out of 8 genes (except CSTB) were validated using RT-qPCR in GBM cell lines. CTSK overexpression was validated using RT-qPCR in GBM tissues as well. Cathepsin K immunohistochemical staining and western blotting showed that only proteolytically inactive proforms of cathepsin K were overexpressed in GBM tissues and cells.ConclusionsThe presence of high levels of inactive proforms of cathepsin K in GBM tissues and cells indicate that in GBM the proteolytic/collagenolytic role is not its primary function but it plays rather a different yet unknown role.

Abstract 1213: Comparing protein pathway activation mapping portraits between gliobastoma patient-matched primary tumor, xenografts and neurospheres: implications for precision medicine

Abstract Background: Most current targeted therapeutics are directed against the activated protein kinase-driven signaling pathway architecture. While guiding therapy selection based on protein phosphorylation is biochemically rational given these proteins represent the direct drug targets it is not certain which protein networks/signaling protein targets are the causal driver of any patient's tumor. Thus, the availability of a system that recapitulates the human in vivo setting and allows for credentialing and identification of which activated signaling networks are causally necessary for tumor viability would be extremely important. Methods: To test two such systems and determine how they resemble the cell signaling architecture of the human primary tumor we compared eight snap-frozen laser capture microdissected (LCM) primary glioblastoma multiforme (GBM) samples with tumor-matching LCM mouse xenografts as well as matching neurospheres (NP). Reverse phase protein microarrays were utilized wherein the total level or phosphorylated level of 70 key signal transduction proteins known to be the direct targets for a number of important targeted therapies and implicated in GBM tumor biology were measured. Results: Overall, the signaling architecture of the xenografts more closely resembled the matched primary LCM tumors in this study set. However, unsupervised hierarchical clustering of the data revealed instances where pathway information such as total c-MET, phospho RAF (S259), phospho GSK3αβ (S21/9) and total EGFR was retained across matched LCM primary tumor, xenografts and NP. In addition there were a number of instances where the signaling architecture of the primary human tumor did not resemble the matched NP and xenograft signature. Conclusion: These pilot data indicate that 3D cell culture conditions may effectively recapitulate in vivo signaling network activation in certain instances, although these preliminary results also suggest that xenografts more closely resemble the cell architecture of matching primary GBM tumors. These data highlight the importance and impact of the microenvironment and culture conditions on tumor cell signal transduction network activation. However, two caveats to this approach remain: (a) drug studies in GBM xenografts and NP are limited to those targeting signaling pathways that are faithfully reproduced from the human in vivo setting and (b) the time required to develop a patient matched xenograft model for individualized drug testing may be too long for a fast-progressing disease such as GBM. Citation Format: Claudius Mueller, Ana C. deCarvalho, Tom Mikkelsen, Laila Poisson, Valerie Calvert, Andrew Borgman, David M. Cherba, Mary E. Winn, Emanuel F. Petricoin. Comparing protein pathway activation mapping portraits between gliobastoma patient-matched primary tumor, xenografts and neurospheres: implications for precision medicine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1213. doi:10.1158/1538-7445.AM2014-1213

FoxM1 Inhibition Sensitizes Resistant Glioblastoma Cells to Temozolomide by Downregulating the Expression of DNA-Repair Gene Rad51

Recurrent glioblastoma multiforme (GBM) is characterized by resistance to radiotherapy and chemotherapy and a poor clinical prognosis. In this study, we investigated the role of the oncogenic transcription factor FoxM1 in GBM cells' resistance to alkylator temozolomide (TMZ) and its potential molecular mechanism. FoxM1 expression levels were measured by immunohistochemical analysis in 38 pairs of primary and recurrent GBM tumor samples. Expression levels were also measured in primary recurrent GBM cell lines, and their responses to TMZ were characterized. In a mechanistic study, an siRNA array was used to identify downstream genes, and a chromatin immunoprecipitation assay was used to confirm transcriptional regulation. Recurrent tumors that were TMZ resistant expressed higher levels of FoxM1 than did primary tumors. Recurrent GBM cell lines expressed higher levels of FoxM1 and the DNA damage repair gene Rad51 and were resistant to TMZ. TMZ treatment led to increased FoxM1 and Rad51 expression. FoxM1 knockdown inhibited Rad51 expression and sensitized recurrent GBM cells to TMZ cytotoxicity. FoxM1 directly regulated Rad51 expression through 2 FoxM1-specific binding sites in its promoter. Rad51 reexpression partially rescued TMZ resistance in FoxM1-knockdown recurrent GBM cells. A direct correlation between FoxM1 expression and Rad51 expression was evident in recurrent GBM tumor samples. Targeting the FoxM1-Rad51 axis may be an effective method to reverse TMZ resistance in recurrent GBM.

Real-time dynamic and sequential tracking of tumor propagation and associated immune responses in the CNS microenvironment.

9520 Background: Ex vivo experimental systems are often unable to fully capture complex intercellular communication between tumor cells and surrounding tissues - a critical feature in understanding cancer development and immune evasion. Imaging modality such as bioluminescence lacks the resolution necessary to discern subtle structural differences and heterogeneity in the tumor niche. Microscopic examination of fixed specimens is devoid of the 3-dimensional context or evolution of tumor progression within the same host. Methods: New insights have come from studies involving the use of intravital 2-photon laser scanning microscopy (2P-LSM), which allows deep visualization (>300um) with single-cell resolution (<1um), thus enables direct observation of cellular behavior in intact tissues at a suitable dynamic spatial-time resolution. We study the role of tumor niche in shaping immune repertoire and develop strategies to modify tumor niche to enhance anti-tumor immunity. Results: One example is our study of glioblastoma multiforme (GBM), which contains a cellular hierarchy with a CD133+ sub-population representing self-renewing and tumorigenic GBM stem cells (GSCs). In a xeno-transplant model, GSC was capable of tumor initiation in the mouse brain. To directly test the relative tumorigenic potential of GSCs (CD133+) and non-GSCs (CD133-), we inoculated paired tumor populations from the same primary GBM tumor cells and monitored tumor competition by serial 2P-LSM through implanted cranial windows. Serial 2P-LSM imaging shows that after 35 days, GBM formation was driven exclusively by GSCs but not non-GSCs. To interrogate tumor-associated immune responses, we inoculated syngeneic mouse glioma tumors into C57BL/6 mice. Using this and CNS-inflammatory models, we have begun to undercover the role of perivascular antigen-presenting cells and microglia in guiding the recruitment of CNS-bound lymphocytes. Conclusions: Our data provide the first direct functional evidence that CSCs are responsible for tumor propagation in GBM, and represent an in vivo experimental platform to monitor immunotherapeutic interventions.

A Novel Extracellular Hsp90 Mediated Co-Receptor Function for LRP1 Regulates EphA2 Dependent Glioblastoma Cell Invasion

BackgroundExtracellular Hsp90 protein (eHsp90) potentiates cancer cell motility and invasion through a poorly understood mechanism involving ligand mediated function with its cognate receptor LRP1. Glioblastoma multiforme (GBM) represents one of the most aggressive and lethal brain cancers. The receptor tyrosine kinase EphA2 is overexpressed in the majority of GBM specimens and is a critical mediator of GBM invasiveness through its AKT dependent activation of EphA2 at S897 (P-EphA2S897). We explored whether eHsp90 may confer invasive properties to GBM via regulation of EphA2 mediated signaling.Principal FindingsWe find that eHsp90 signaling is essential for sustaining AKT activation, P-EphA2S897, lamellipodia formation, and concomitant GBM cell motility and invasion. Furthermore, eHsp90 promotes the recruitment of LRP1 to EphA2 in an AKT dependent manner. A finding supported by biochemical methodology and the dual expression of LRP1 and P-EphA2S897 in primary and recurrent GBM tumor specimens. Moreover, hypoxia mediated facilitation of GBM motility and invasion is dependent upon eHsp90-LRP1 signaling. Hypoxia dramatically elevated surface expression of both eHsp90 and LRP1, concomitant with eHsp90 dependent activation of src, AKT, and EphA2.SignificanceWe herein demonstrate a novel crosstalk mechanism involving eHsp90-LRP1 dependent regulation of EphA2 function. We highlight a dual role for eHsp90 in transducing signaling via LRP1, and in facilitating LRP1 co-receptor function for EphA2. Taken together, our results demonstrate activation of the eHsp90-LRP1 signaling axis as an obligate step in the initiation and maintenance of AKT signaling and EphA2 activation, thereby implicating this pathway as an integral component contributing to the aggressive nature of GBM.

Cetuximab Insufficiently Inhibits Glioma Cell Growth Due to Persistent EGFR Downstream Signaling

ABSTRACTOverexpression and/or amplification of the epidermal growth factor receptor (EGFR) is present in 35–45% of primary glioblastoma multiforme tumors and has been correlated with a poor prognosis. In this study, we investigated the effect of cetuximab and intracellular signaling pathways downstream of EGFR, important for cell survival and proliferation. We show insufficient EGFR downregulation and competition with endogenous EGFR ligands upon cetuximab treatment. Dose–response experiments showed inhibition of EGFR phosphorylation without affecting two of the prominent downstream signaling pathways. Our results indicate that amplification and/or overexpression of EGFR is an unsatisfactory predictor for response to cetuximab.