Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies

Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum-free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen method

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies

Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum-free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen metho

EZH2-Regulated DAB2IP Is a Medulloblastoma Tumor Suppressor and a Positive Marker for Survival

Purpose: Medulloblastoma is the most common malignant brain tumor in children. Despite recent improvements, the molecular mechanisms driving medulloblastoma are not fully understood and further elucidation could provide cues to improve outcome prediction and therapeutic approaches. Experimental Design: Here, we conducted a meta-analysis of mouse and human medulloblastoma gene expression data sets, to identify potential medulloblastoma tumor suppressor genes. Results: We identified DAB2IP, a member of the RAS-GTPase-activating protein family (RAS GAP), and showed that DAB2IP expression is repressed in medulloblastoma by EZH2-induced trimethylation. Moreover, we observed that reduced DAB2IP expression correlates significantly with a poor overall survival of patients with medulloblastoma, independent of metastatic stage. Finally, we showed that ectopic DAB2IP expression enhances stress-induced apoptosis in medulloblastoma cells and that reduced expression of DAB2IP in medulloblastoma cells conveys resistance to irradiation-induced cell death. Conclusion: These results suggest that repression of DAB2IP may at least partly protect medulloblastoma cells from apoptotic cell death. Moreover, DAB2IP may represent a molecular marker to distinguish patients with medulloblastoma at high risk from those with a longer survival prognosis. Clin Cancer Res; 18(15); 4048-58. (C)2012 AAC

Pre-B-cell leukemia homeobox interacting protein 1 is overexpressed in astrocytoma and promotes tumor cell growth and migration

Glial brain tumors cause considerable mortality and morbidity in children and adults. Innovative targets for therapy are needed to improve survival and reduce long-term sequelae. The aim of this study was to find a candidate tumor-promoting protein, abundantly expressed in tumor cells but not in normal brain tissues, as a potential target for therapy. In silico proteomics and genomics, immunohistochemistry, and immunofluorescence microscopy validation were performed. RNA interference was used to ascertain the functional role of the overexpressed candidate target protein. In silico proteomics and genomics revealed pre-B-cell leukemia homeobox (PBX) interacting protein 1 (PBXIP1) overexpression in adult and childhood high-grade glioma and ependymoma compared with normal brain. PBXIP1 is a PBX-family interacting microtubule-binding protein with a putative role in migration and proliferation of cancer cells. Immunohistochemical studies in glial tumors validated PBXIP1 expression in astrocytoma and ependymoma but not in oligodendroglioma. RNAi-mediated PBXIP1-knockdown in glioblastoma cell lines strongly reduced proliferation and migration and induced morphological changes, indicating that PBXIP1 knockdown decreases glioma cell viability and motility through rearrangements of the actin cytoskeleton. Furthermore, expression of PBXIP1 was observed in radial glia and astrocytic progenitor cells in human fetal tissues, suggesting that PBXIP1 is an astroglial progenitor cell marker during human embryonic development. PBXIP1 is a novel protein overexpressed in astrocytoma and ependymoma, involved in tumor cell proliferation and migration, that warrants further exploration as a novel therapeutic target in these tumor

Western blot for detection of P-gp, MRP1, and BCRP1 in pHGG cultures.

<p>MW represents approximate molecular weight of these proteins, as indicated at the right. The pHGG lanes were loaded with 20 µg of protein, the lanes with positive controls were loaded with 5 µg of protein.</p

Classical drugs used in this study.

<p>Mechanism of action, concentrations, and ABC efflux transporter substrate specificity for classical therapeutics.</p

Tumor and patient characteristics corresponding with the primary cell cultures.

<p>f: female, m: male, AA: anaplastic astrocytoma, GBM; glioblastoma, GC GBM: giant cell glioblastoma multiforme, AO: anaplastic oligodendroglioma, DA2: diffuse fibrillary astrocytoma, GTR: gross total resection, STR: subtotal resection, PR: partial resection. Chemotherapy was administered adjuvant to resection and/or radiotherapy in all treated patients.</p>*<p>hypofractionated radiotherapy 15 fractions of 3 Gy,</p>**<p>survivor, n.a.: does not apply.</p

Immunohistochemical staining of ABC-transporters in pHGG sections.

<p>Expression of P-gp (A) and BCRP1 (C) is located to the endothelial cells of the tumor vasculature. Whereas MRP1 (B) expression is visualized mainly in the cytoplasm of tumor cells as well as in the vasculature.</p

a. Cell survival among primary pHGG cultures exposed to classical chemotherapeutic drugs. b. Cell survival among primary pHGG cultures exposed to novel drugs.

<p>a. Cell survival among primary pHGG cultures exposed to classical chemotherapeutic drugs. b. Cell survival among primary pHGG cultures exposed to novel drugs.</p

Novel drugs used in this study.

<p>Drug targets, concentrations, and ABC efflux transporter substrate specificity for novel, targeted drugs.</p