Nancy Lemke

GRP78 is overexpressed in glioblastomas and regulates glioma cell growth and apoptosis

We characterized the expression and function of the endoplasmic reticulum protein GRP78 in glial tumors. GRP78 is highly expressed in glioblastomas but not in oligodendrogliomas, and its expression is inversely correlated with median patient survival. Overexpression of GRP78 in glioma cells decreases caspase 7 activation and renders the cells resistant to etoposide- and cisplatin-induced apoptosis, whereas silencing of GRP78 decreases cell growth and sensitizes glioma cells to etoposide, cisplatin, and gamma-radiation. Thus, GRP78 contributes to the increased apoptosis resistance and growth of glioma cells and may provide a target for enhancing the therapeutic responsiveness of these tumors.

Cilengitide induces autophagy-mediated cell death in glioma cells.

We studied the effect of the integrin inhibitor cilengitide in glioma cells. Cilengitide induced cell detachment and decreased cell viability, and induction of autophagy followed by cell apoptosis. In addition, cilengitide decreased the cell renewal of glioma stem-like cells (GSCs). Inhibition of autophagy decreased the cytotoxic effect of cilengitide. Pretreatment of glioma cells and GSCs with cilengitide prior to γ-irradiation resulted in a larger increase in autophagy and a more significant decrease in cell survival. We found that cilengitide induced autophagy collectively in glioma cells, xenografts, and GSCs, which contributed to its cytotoxic effects and sensitized these cells to γ-radiation.

TRAIL conjugated to nanoparticles exhibits increased anti-tumor activities in glioma cells and glioma stem cells in vitro and in vivo

Glioblastomas (GBM) are characterized by resistance to chemotherapy and radiotherapy, and therefore, alternative therapeutic approaches are needed. TRAIL induces apoptosis in cancer but not in normal cells and is considered to be a promising anti-tumor agent. However, its short in vivo half-life and lack of efficient administration modes are serious impediments to its therapeutic efficacy. Nanoparticles (NP) have been used as effective delivery tools for various anticancer drugs. TRAIL was conjugated to magnetic ferric oxide NP by binding the TRAIL primary amino groups to activated double bonds on the surface of the NP. The effect of NP-TRAIL was examined on the apoptosis of glioma cells and self-renewal of glioma stem cells (GSCs). In addition, the ability of the NP-TRAIL to track U251 cell-derived glioma xenografts and to affect cell apoptosis, tumor volume, and survival among xenografted rats was also examined. Conjugation of TRAIL to NP increased its apoptotic activity against different human glioma cells and GSCs, as compared with free recombinant TRAIL. Combined treatment with NP-TRAIL and γ-radiation or bortezomib sensitized TRAIL-resistant GSCs to NP-TRAIL. Using rhodamine-labeled NP and U251 glioma cell-derived xenografts, we demonstrated that the NP-TRAIL were found in the tumor site and induced a significant increase in glioma cell apoptosis, a decrease in tumor volume, and increased animal survival. In summary, conjugation of TRAIL to NP increased its apoptotic activity both in vitro and in vivo. Therefore, NP-TRAIL represents a targeted anticancer agent with more efficient action for the treatment of GBM and the eradication of GSCs.

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Loss of Sparc in p53-null Astrocytes Promotes Macrophage Activation and Phagocytosis Resulting in Decreased Tumor Size and Tumor Cell Survival

Both the induction of SPARC expression and the loss of the p53 tumor suppressor gene are changes that occur early in glioma development. Both SPARC and p53 regulate glioma cell survival by inverse effects on apoptotic signaling. Therefore, during glioma formation, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival. This study determined whether the loss of Sparc in astrocytes that are null for p53 would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. In vitro, the loss of Sparc in p53‐null astrocytes resulted in an increase in cell proliferation, but a loss of tumorigenicity. At 7 days after intracranial implantation, Sparc‐null tumors had decreased tumor cell survival, proliferation and reduced tumor size. The loss of Sparc promoted microglia/macrophage activation and phagocytosis of tumor cells. Our results indicate that the loss of p53 by deletion/mutation in the early stages of glioma formation may cooperate with the induction of SPARC to potentiate cancer cell survival and escape from immune surveillance.

Abstract 427: Sox2 modulation of cancer stem cell behavior in GBM

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background and Objective: Glioblastoma (GBM) is the most aggressive primary brain tumor. GBM cells are phenotypically plastic and can present cancer stem cell (CSC) properties, such as the ability to self-renew and differentiate into the cells comprising the bulk of the tumor, phenocopying the parental tumor in mouse xenografts. GBM CSCs express neural stem cell (NSC) markers, including Sox2, a HMG-box transcription factor that regulates the expression of genes associated with self-renewal and differentiation. Culturing dissociated GBM tumors in serum-containing medium typically results in loss of the CSC phenotype and downregulation of Sox2, while serum-free neurosphere medium (NM) is used for selection and propagation of CSCs. We have previously identified a GBM specimen (GS1) which, contrary to the norm, retained Sox2 expression in low passage serum culture, along with the ability to dedifferentiate into neurospheres in vitro, and to form tumors in rodents (deCarvalho et al., Stem Cells 2010, 28:181-190). Here we investigate the endogenous expression of Sox2 in GBM biopsies, xenograft tumors, and cultured cells, and test the hypothesis that Sox2 may be a driver of the cancer stem cell phenotype in GBMs. Experimental Approaches: Endogenous expression of Sox2, downstream targets, and cell lineage markers, was determined by immunohistochemistry. miRNAs targeting Sox2 were stably expressed in GBM cells. The effect of Sox2 knockdown on clonogenicity, global gene expression, proliferation in vitro, tumor formation and morphology was determined. Results: Sox2 expression was heterogeneous within and among the 26 GBM surgical specimens tested, ranging from 1% to over 80% positive nuclei. Robust Sox2 expression was observed in over 80% of 56 GBM orthotopic xenografts analyzed. Sox2 positive cells co-expressed mesenchymal (vimentin), neural (nestin), and astrocytic (GFAP) markers. Knocking down Sox2 expression significantly impaired the ability of GS1 cells to form neurospheres, without affecting cell proliferation, significantly altered the expression of known target genes, such as Nestin, and genes in various signaling pathways, and affected xenograft tumor formation. Sox2 protein levels in GBMs were further regulated by post-transcriptional mechanisms. Conclusions: Sox2 is part of the signature gene set for the proneural GBM subclass. Our results show that Sox2 protein expression is ubiquitous in GBM tumors and xenografts, independent of patient survival or treatment status. Sox2 is a master regulator of embryonic and neural stem cell phenotype, and our data contributes to the view that it may have an important role in the plasticity of GBM cells in acquiring CSC phenotype, with possible implications for the pathology of GBMs. 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 427. doi:1538-7445.AM2012-427