Brian Vaillant

The transcriptional coactivator TAZ regulates mesenchymal differentiation in malignant glioma

Recent molecular classification of glioblastoma (GBM) has shown that patients with a mesenchymal (MES) gene expression signature exhibit poor overall survival and treatment resistance. Using regulatory network analysis of available expression microarray data sets of GBM, including The Cancer Genome Atlas (TCGA), we identified the transcriptional coactivator with PDZ-binding motif (TAZ), to be highly associated with the MES network. TAZ expression was lower in proneural (PN) GBMs and lower-grade gliomas, which correlated with CpG island hypermethylation of the TAZ promoter compared with MES GBMs. Silencing of TAZ in MES glioma stem cells (GSCs) decreased expression of MES markers, invasion, self-renewal, and tumor formation. Conversely, overexpression of TAZ in PN GSCs as well as murine neural stem cells (NSCs) induced MES marker expression and aberrant osteoblastic and chondrocytic differentiation in a TEAD-dependent fashion. Using chromatin immunoprecipitation (ChIP), we show that TAZ is directly recruited to a majority of MES gene promoters in a complex with TEAD2. The coexpression of TAZ, but not a mutated form of TAZ that lacks TEAD binding, with platelet-derived growth factor-B (PDGF-B) resulted in high-grade tumors with MES features in a murine model of glioma. Our studies uncover a direct role for TAZ and TEAD in driving the MES differentiation of malignant glioma.

A prognostic gene expression signature in infratentorial ependymoma

Patients with ependymoma exhibit a wide range of clinical outcomes that are currently unexplained by clinical or histological factors. Little is known regarding molecular biomarkers that could predict clinical behavior. Since recent data suggest that these tumors display biological characteristics according to their location (cerebral vs. infratentorial vs. spinal cord), rather than explore a broad spectrum of ependymoma, we focused on molecular alterations in ependymomas arising in the infratentorial compartment. Unsupervised clustering of available gene expression microarray data revealed two major subgroups of infratentorial ependymoma. Group 1 tumors over expressed genes that were associated with mesenchyme, Group 2 tumors showed no distinct gene ontologies. To assess the prognostic significance of these gene expression subgroups, real-time reverse transcriptase polymerase chain reaction assays were performed on genes defining the subgroups in a training set. This resulted in a 10-gene prognostic signature. Multivariate analysis showed that the 10-gene signature was an independent predictor of recurrence-free survival after adjusting for clinical factors. Evaluation of an external dataset describing subgroups of infratentorial ependymomas showed concordance of subgroup definition, including validation of the mesenchymal subclass. Importantly, the 10-gene signature was validated as a predictor of recurrence-free survival in this dataset. Taken together, the results indicate a link between clinical outcome and biologically identified subsets of infratentorial ependymoma and offer the potential for prognostic testing to estimate clinical aggressiveness in these tumors.

ELTD1, a potential new biomarker for gliomas

BACKGROUND Glioblastoma multiforme (GBM), a high-grade glioma, is characterized by being diffuse, invasive, and highly angiogenic and has a very poor prognosis. Identification of new biomarkers could help in the further diagnosis of GBM. OBJECTIVE To identify ELTD1 (epidermal growth factor, latrophilin, and 7 transmembrane domain-containing protein 1 on chromosome 1) as a putative glioma-associated marker via a bioinformatic method. METHODS We used advanced data mining and a novel bioinformatics method to predict ELTD1 as a potential novel biomarker that is associated with gliomas. Validation was done with immunohistochemistry, which was used to detect levels of ELTD1 in human high-grade gliomas and rat F98 glioma tumors. In vivo levels of ELTD1 in rat F98 gliomas were assessed using molecular magnetic resonance imaging. RESULTS ELTD1 was found to be significantly higher (P = .03) in high-grade gliomas (50 patients) compared with low-grade gliomas (21 patients) and compared well with traditional immunohistochemistry markers including vascular endothelial growth factor, glucose transporter 1, carbonic anhydrase IX, and hypoxia-inducible factor 1α. ELTD1 gene expression indicates an association with grade, survival across grade, and an increase in the mesenchymal subtype. Significantly high (P < .001) in vivo levels of ELTD1 were additionally found in F98 tumors compared with normal brain tissue. CONCLUSION Results of this study strongly suggests that associative analysis was able to accurately identify ELTD1 as a putative glioma-associated biomarker. The detection of ELTD1 was also validated in both rodent and human gliomas and may serve as an additional biomarker for gliomas in preclinical and clinical diagnosis of gliomas.

Antineutrophil cytoplasmic antibody-associated active scleritis

OBJECTIVE To determine whether antineutrophil cytoplasmic antibody (ANCA) testing provides prognostic information in evaluating scleritis. METHODS Retrospective medical record review of patients evaluated at a tertiary care center from January 1, 1995, to June 30, 2006, was performed to compare clinical features, treatments, and associated systemic disease in patients who test positive for ANCA vs patients whose ANCA tests are negative. RESULTS Among 78 patients identified, 14 tested positive for ANCA. Patients with positive ANCA test results were more likely to have an associated systemic disorder (10 of 14 or 71%) than were patients who tested negative for ANCA (26 of 64 or 41%; P = .04), and the disorder was more likely to have been diagnosed as a result of scleritis work-up (2 of 10 or 20% vs 19 of 26 or 73%; P = .007). Patients with positive ANCA test results had significantly more ocular complications (21 of 14 or 86% vs 20 of 64 or 31%; P < .001), including keratopathy (5 of 14 or 36% vs 6 of 64 or 9%; P = .02), visual acuity of 20/50 or worse (8 of 14 or 57% vs 11 of 64 or 17%; P = .001), and vascular pannus (3 of 14 or 21% vs 1 of 64 or 2%; P = .02). Aggressive therapy, such as chronic systemic corticosteroids (9 of 14 or 64% vs 9 of 64 or 14%; P < .001) and alkylator therapy (8 of 14 or 57% vs 7 of 64 or 11%; P < .001), was more likely to be recommended for patients who tested positive for ANCA. CONCLUSIONS A substantial subset of patients with scleritis are also positive for ANCA. These patients are more likely to have severe ocular disease and undiagnosed primary vasculitic disease, thereby requiring more aggressive therapy. An ANCA test may be useful in the evaluation and treatment of patients with scleritis.

Experimental validation of 5 in-silico predicted glioma biomarkers

BACKGROUND Glioblastoma multiforme (GBM) is a high-grade glioma with poor prognosis. Identification of new biomarkers specific to GBM could help in disease diagnosis. We have developed and validated a bioinformatics method to predict proteins likely to be suitable as glioma biomarkers via a global microarray meta-analysis to identify uncharacterized genes consistently coexpressed with known glioma-associated genes. METHODS A novel bioinformatics method was implemented called global microarray meta-analysis, using approximately 16,000 microarray experiments to identify uncharacterized genes consistently coexpressed with known glioma-associated genes. These novel biomarkers were validated as proteins highly expressed in human gliomas varying in tumor grades using immunohistochemistry. Glioma gene databases were used to assess delineation of expression of these markers in varying glioma grades and subtypes of GBM. RESULTS We have identified 5 potential biomarkers-spondin1, Plexin-B2, SLIT3, fibulin-1, and LINGO1-that were validated as proteins highly expressed on the surface of human gliomas using immunohistochemistry. Expression of spondin1, Plexin-B2, and SLIT3 was significantly higher (P < .01) in high-grade gliomas than in low-grade gliomas. These biomarkers were significant discriminators in grade IV gliomas compared with either grade III or II tumors and also distinguished between GBM subclasses. CONCLUSIONS This study strongly suggests that this type of bioinformatics approach has high translational potential to rapidly discern which poorly characterized proteins may be of clinical relevance.

Aberrant mesenchymal differentiation of glioma stem-like cells: implications for therapeutic targeting

Differentiation has been proposed as a therapeutic strategy for glioblastoma (GBM) in part due to observations of stem-like cells in GBM that have been shown to undergo terminal differentiation in response to growth factor withdrawal and BMP activation. However, the effects of long term exposure to serum culture conditions on glioma sphere cultures/glioma stem-like cells (GSCs) have not been examined. Here we show that GSCs retained both neurosphere formation and tumor initiation abilities after short or long term serum exposure. Under these conditions, GSCs expressed both neural lineage and stem cell markers, highlighting the aberrant pseudo-differentiation state. GSCs maintained under adherent serum cultured conditions continued to proliferate and initiate tumor formation with efficiencies similar to GSCs maintained under proliferating (neurosphere) conditions. Proneural (PN) GSCs under serum exposure showed an induction of mesenchymal (MES) gene expression signatures. Our data indicate that exposure to serum containing media result in aberrant differentiation (e.g. toward MES lineage) and activation of alternative oncogenic pathways in GSCs.

Abstract 3314: Phenotypic plasticity in glioma stem cells mediated by NFκB signaling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Here we report the identification of two major subtypes of glioma stem cells (GSCs) that closely mirror previously described glioblastoma (GBM) groups, namely proneural (PN) and mesenchymal (MES), based on gene expression patterns. Using flow cytometry, we found that PN GSCs express both CD15/SSEA1, a marker for neural stem cells, as well as CD44, a MES marker. In contrast MES GSCs were CD15low/CD44high. MES GSCs and CD44high subpopulations in PN GSCs showed increased expression of transcription factors previously reported to induce the MES phenotype (STAT3, C/EBP-α, and TAZ), and were -resistant to α-irradiation. By distilling cytokines that can induce MES differentiation, we found that TNF-α found in patient-derived microglial conditioned media or conditioned media from MES GSCs caused a PN-to-MES transition through activation of NFκB resulting in radio-resistance. We found that NFκB mediates MES transition indirectly by inducing the transcriptional circuitry of STAT3, C/EBP-α, and TAZ. Blockade of NFκB activation using minocycline abrogated the MES phenotype (both induced and constitutive), reduced tumor growth, and rendered GSCs radio-sensitive. Taken together, our findings reveal that phenotypic and surface marker plasticity is common in GSCs, and that the NFκB driven PN-to-MES transition provides an escape mechanism for GSCs to evade cytotoxic treatment. Based on our studies, we propose that minocycline, a commonly used antibiotic, could target GSCs and should be further evaluated in preclinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3314. doi:1538-7445.AM2012-3314

Abstract LB-114: Molecular and lineage analysis of glioblastoma stem cells identifies clinically relevant models of glioblatoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The Cancer Genome Atlas project described a robust gene expression-based molecular classification of GBM. However, the functional and biological significance of the molecular subclasses are being determined. In this study, we hypothesize that Glioma Stem Cells (GSCs) isolated from individual patient tumor samples will recapitulate the molecular characteristics of tumor samples and provide a relevant model for functional analysis of the molecular subclasses. Thus we conducted a comprehensive analysis of 26 GSC lines with expression array, RPPA, and a series of neural lineage markers. Analysis of the expression data classified the 26 GSC lines into four subtypes ( Classical, Proneural 1, Proneural 2 and Mesenchymal ,) closely similar to the TCGA subclasses with a distinct profile for each subtype (c-Myc, Cyclin D2 for Classical ; Olig2, NKX2–2, Notch-1, Notch-3 for Proneural 2 ; BMP4, DCX, p16INK4a, ID2 for Proneural 1 ; CD44, CAV1, TGFBR2 for Mesenchymal ). Further analysis showed that GSC subtypes exhibit divergent patterns of signaling pathway activation. The major pathways activated in 4 subtypes were Notch pathway in Proneural 2 , Wnt/β-Catenin in Proneural 2 and Classical , while FGF/VEGF and TGF-β in Mesenchymal. In vitro treatment with SB 431542, a TGF-βR inhibitor, showed that Mesenchymal subtype is more sensitive than other subtypes. More importantly, lineage analysis of GSCs subtypes show that Proneural and Classical GSCs differentially express lineage markers for neural stem/progenitor cells and were responsive to differentiating agent retinoic acid. In addition, GSC subtypes exhibit distinct biological behaviors in self-renewal capacity, proliferation, invasiveness, angiogenic potential, response to growth factor stimulus, and differentiation in vitro and in vivo with Classical subtype being more proliferative and Mesenchymal subtype being more angiogenic and invasive. In conclusion, our comprehensive analysis showed that GSCs reflect patient tumor subclass and these subtypes showed distinct regulatory pathway activation, lineage profile and biological behaviors. Thus GSC is biologically and molecularly a more relevant model system for preclinical studies of therapeutic intervention and to expand our molecular understanding of human GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-114. doi:10.1158/1538-7445.AM2011-LB-114

Abstract 978: A regulatory role for TAZ in mesenchymal differentiation of glioblastoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Gene expression profiling studies have revealed that the mesenchymal signature (MES) is a predictor for poor survival and treatment resistance in glioblastoma (GBM) patients, whereas patients with a proneural (PN) signature perform better in the clinic. We found that the HIPPO pathway transcription co-factor TAZ, but not YAP is epigenetically silenced by CpG island hypermethylation in lower grade/PN tumors whereas its promoter is hypomethylated in GBM/MES tumors. Treatment of a demethylation agent (2′-deoxy-5-azacytidine) increased TAZ mRNA levels in glioma stem cells lacking this protein. Using bioinformatic approaches we found TAZ to be significantly connected to the MES gene signature network that was distinct from recently reported regulators of MES transition in GBM, viz., STAT3 and CEBPβ. Approximately 60% of the in silico inferred targets were confirmed to be direct targets of TAZ by ChIP-PCR. Silencing TAZ in GSCs decreased invasion and blocked tumorigenesis when implanted intracranially into mice. Conversely, overexpression of TAZ in GSCs that lack this protein resulted in increased MES gene expression, suppressed PN gene expression, increased invasion, differentiation toward an osteogenic and chondrocytic lineage and reduced tumor latency in the xenograft model. In primary NSCs, forced TAZ resulted in increased expression of mesenchymal markers such as fibronectin, and smooth muscle actin, whereas astrocytic or neuronal differentiation was suppressed. All aforementioned phenotypes were abolished when overexpressing a single point mutant version of TAZ (S51A) that lacks binding to its bona fide transcription partner, TEAD. Finally overexpression of TAZ in a PGDF-RCAS transgenic mouse model caused increased tumor grade and incidence and shift toward a MES phenotype. Our study identifies TAZ as important master regulator of PN to MES transition in gliomas and presents as an attractive therapeutic target for this devastating disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 978. doi:10.1158/1538-7445.AM2011-978

Abstract B27: TAZ as a regulator of mesenchymal transformation and clinical aggressiveness in gliomas

Background: Glioblastoma (GBM) is a highly malignant and infiltrative brain tumor. Our laboratory has performed extensive microarray studies on GBM and has shown that a subset of these tumors have a gene expression signature characterized by those associated with the mesenchyme. Tumors which overexpress these genes (mesenchymal tumors) are associated with poor outcome and resistance to treatment. In an effort to identify the molecular mechanisms by which this mesenchymal shift occurs, we have identified TAZ (transcriptional coactivator with PDZ-binding motif) as a transcription co-activator whose expression level is tightly associated with the mesenchymal change (elevated TAZ is positively associated with higher expression of key mesenchymal genes). High expression of TAZ also correlated with higher grade glioma as well as poorer patient outcome. These data lead to our main hypothesis, that TAZ activation is critically important in the mesenchymal transition and aggressive clinical behavior in GBM. Results: To further discern TAZ9s role in GBM, we stably silenced or overexpressed this gene in glioma stem cell lines depending on their basal levels of TAZ. Loss of TAZ did not affect proliferation, but did decrease invasion and dramatically decreased tumor formation when injected intracranially into severe combined immunodeficiency (SCID) mice. Five of 5 mice injected with control stem cell cultures developed tumors, while only 2 of 10 TAZ-knockdown cultures resulted in tumor formation. Furthermore, the 2 tumors that formed were of lower histologic grade compared to controls. Cells expressing a constitutively active form of TAZ were injected into SCID mice and 5 of the 5 mice injected with the overexpression clone died less than 60 days post-injection, while the mice injected with the vector control (low TAZ levels) survived an average of 120 days post-injection. Overexpression of TAZ increased invasion, and induced osteogenesis as evidenced by alizarin red staining, but did not alter proliferation. Further TAZ activation regulates the expression of signature mesenchymal genes, as shown by Affymetrix profiling of TAZ-transfected and TAZ-knockdown constructs. These data support the hypothesis that TAZ is a central regulator of mesenchymal gene expression in glioma. To further characterize TAZ function in human glioma, we examined the expression of its binding partners (TEAD1-4), as well as a downstream target (FN1) in human tumor specimens. Western analyses showed that TEAD4 was specifically upregulated in grade IV/GBM compared to lower grade gliomas, and TAZ/TEAD4 expression tightly paralleled FN1 expression, a marker of mesenchymal phenotype. Inspection of the TAZ promoter region revealed a CpG island in the promoter region that was methylated in most lower grade tumors (grade II/III gliomas), but not in grade IV GBMs implying that methylation may be one means by which TAZ is regulated and appears to be epigenetically silenced in tumors with a more favorable outcome. To confirm this, we found that treatment of TAZ-methylated glioma stem cell lines with a demethylation agent resulted in an increase in TAZ expression in both the mRNA and protein levels. Discussion: We have identified TAZ as a critical regulator in the mesenchymal transition in gliomas. Since mesenchymal differentiation is associated with gliomagenesis and tumor aggressiveness, strategies to target TAZ and its downstream targets may be warranted in alternative treatment options for patients. This study may also provide a rationale for developing inhibitors which directly target TAZ. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B27.