Anastasia Murat

Stem Cell–Related “Self-Renewal” Signature and High Epidermal Growth Factor Receptor Expression Associated With Resistance to Concomitant Chemoradiotherapy in Glioblastoma

PURPOSEnGlioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide.nnnPATIENTS AND METHODSnGene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy.nnnRESULTSnAn expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a self-renewal signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response.nnnCONCLUSIONnThis study provides first clinical evidence for the implication of a glioma stem cell or self-renewal phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Modulation of angiogenic and inflammatory response in glioblastoma by hypoxia

Glioblastoma are rapidly proliferating brain tumors in which hypoxia is readily recognizable, as indicated by focal or extensive necrosis and vascular proliferation, two independent diagnostic criteria for glioblastoma. Gene expression profiling of glioblastoma revealed a gene expression signature associated with hypoxia-regulated genes. The correlated gene set emerging from unsupervised analysis comprised known hypoxia-inducible genes involved in angiogenesis and inflammation such as VEGF and BIRC3, respectively. The relationship between hypoxia-modulated angiogenic genes and inflammatory genes was associated with outcome in our cohort of glioblastoma patients treated within prospective clinical trials of combined chemoradiotherapy. The hypoxia regulation of several new genes comprised in this cluster including ZNF395, TNFAIP3, and TREM1 was experimentally confirmed in glioma cell lines and primary monocytes exposed to hypoxia in vitro. Interestingly, the cluster seems to characterize differential response of tumor cells, stromal cells and the macrophage/microglia compartment to hypoxic conditions. Most genes classically associated with the inflammatory compartment are part of the NF-kappaB signaling pathway including TNFAIP3 and BIRC3 that have been shown to be involved in resistance to chemotherapy. Our results associate hypoxia-driven tumor response with inflammation in glioblastoma, hence underlining the importance of tumor-host interaction involving the inflammatory compartment.

Calcineurin Blockade Prevents Cardiac Mitogen-activated Protein Kinase Activation and Hypertrophy in Renovascular Hypertension

Chronic stimulation of the renin-angiotensin system induces an elevation of blood pressure and the development of cardiac hypertrophy via the actions of its effector, angiotensin II. In cardiomyocytes, mitogen-activated protein kinases as well as protein kinase C isoforms have been shown to be important in the transduction of trophic signals. The Ca2+/calmodulin-dependent phosphatase calcineurin has also been suggested to play a role in cardiac growth. In the present report, we investigate possible cross-talks between calcineurin, protein kinase C, and mitogen-activated protein kinase pathways in controlling angiotensin II-induced hypertrophy. Angiotensin II-stimulated cardiomyocytes and mice with angiotensin II-dependent renovascular hypertension were treated with the calcineurin inhibitor cyclosporin A. Calcineurin, protein kinase C, and mitogen-activated protein kinase activations were determined. We show that cyclosporin A blocks angiotensin II-induced mitogen-activated protein kinase activation in cultured primary cardiomyocytes and in the heart of hypertensive mice. Cyclosporin A also inhibits specific protein kinase C isoforms. In vivo, cyclosporin A prevents the development of cardiac hypertrophy, and this effect appears to be independent of hemodynamic changes. These data suggest cross-talks between the calcineurin pathway, the protein kinase C, and the mitogen-activated protein kinase signaling cascades in transducing angiotensin II-mediated stimuli in cardiomyocytes and could provide the basis for an integrated model of cardiac hypertrophy.

Elevated levels of MIC-1/GDF15 in the cerebrospinal fluid of patients are associated with glioblastoma and worse outcome

For patients with brain tumors identification of diagnostic and prognostic markers in easy accessible biological material, such as plasma or cerebrospinal fluid (CSF), would greatly facilitate patient management. MIC‐1/GDF15 (growth differentiation factor 15) is a secreted protein of the TGF‐beta superfamily and emerged as a candidate marker exhibiting increasing mRNA expression during malignant progression of glioma. Determination of MIC‐1/GDF15 protein levels by ELISA in the CSF of a cohort of 94 patients with intracranial tumors including gliomas, meningioma and metastasis revealed significantly increased concentrations in glioblastoma patients (median, 229 pg/ml) when compared with control cohort of patients treated for non‐neoplastic diseases (median below limit of detection of 156 pg/ml, p < 0.0001, Mann–Whitney test). However, plasma MIC‐1/GDF15 levels were not elevated in the matching plasma samples from these patients. Most interestingly, patients with glioblastoma and increased CSF MIC‐1/GDF15 had a shorter survival (p = 0.007, log‐rank test). In conclusion, MIC‐1/GDF15 protein measured in the CSF may have diagnostic and prognostic value in patients with intracranial tumors.

The Wnt inhibitory factor 1 (WIF1) is targeted in glioblastoma and has a tumor suppressing function potentially by induction of senescence

Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.

Epigenetic Deregulation of DNA Repair and Its Potential for Therapy

Epigenetic silencing of essential components of DNA repair pathways is a common event in many tumor types, and comprise O6-methylguanine-DNA methyltransferase (MGMT), human mut L homolog 1 (hMLH1), Werner syndrome gene (WRN), breast cancer susceptibility gene 1 (BRCA1), and genes of the Fanconi anemia pathway. Most interestingly, some of these alterations become the Achilles heel of the affected tumors upon treatment with certain classes of anticancer agents. That is, patients whose tumors carry such defects can be stratified for respective therapy rendering some classic DNA damaging agents, such as alkylators or DNA crosslinking agents, into “targeted therapies.” Here we review some of the affected repair pathways that, when inactivated, sensitize the tumors to specific drugs and are thus exploitable for individualized therapy. (Clin Cancer Res 2009;15(16):5026–31)

Abstract 2117: The Wnt inhibitory factor 1 (WIF-1) has tumor suppressing functions in glioblastoma potentially by inducing cellular senescence

Glioblastoma multiforme is the most aggressive form of human glioma and despite recent progress in therapy the prognosis remains dismal with a median survival of 15 months. Expression based prediction of gene alterations identified Wnt inhibitory factor I (WIF1) as a new candidate tumor suppressor gene involved in glioblastoma. WIF1 encodes a secreted Wnt antagonist and it was strongly down-regulated in most glioblastoma as compared to normal brain, implying deregulation of Wnt signaling. Silencing of the WIF1 gene was found to be mediated by deletion and WIF1 promoter hypermethylation. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose dependent decrease of Wnt pathway activity. To further dissect the biological effects of WIF1 re-expression, we established WIF1 overexpressing glioblastoma cell lines. We observed that WIF1 re-expression inhibited cell proliferation in vitro and strongly reduced anchorage independent growth. The ability of forming colonies in soft agar was reduced to less than 11% of the control. Moreover, the expression of WIF1 was able to completely abolish tumorigenicity in a respective xenograft model in nude mice. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype characterized by the appearance of enlarged flattened and multinucleated cells positive for the presence of β-galactosidase, a late marker of senescence. These results provide evidence that WIF1 has tumor suppressing properties in glioblastoma, hence, the implication of a deregulated Wnt pathway may render glioblastoma sensitive to Wnt signaling inhibitors, potentially by diverting the tumor cells into a senescence-like state. 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 2117. doi:10.1158/1538-7445.AM2011-2117