Glioblastoma Primary Tumors Research Articles

MiR-454 inhibited cell proliferation of human glioblastoma cells by suppressing PDK1 expression.

It has been well documented that aberrant expression of microRNAs is associated with carcinogenesis of glioblastoma (GBM), however the underlying mechanisms are not clear. In this present study, we aimed to clarify the biological function of miR-454 in GBM. MiR-454 was identified to be significantly down-regulated in GBM primary tumors and cell lines. Overexpression of miR-454 in GBM cells resulted in arresting cells at G0/G1 phase and thus inhibiting cell proliferation. Bioinformatic analysis predicted 3-phosphoinositide-dependent protein kinase-1 (PDK1) as a target of miR-454 which acted as a tumor promoter gene. Increased miR-454 significantly repressed PDK1 expression, and then regulating cell proliferation and cell cycle regulators, down-regulation of Cyclin D1 and p-pRb and p21 was up-regulated. Taken together, our study has revealed miR-454 as a tumor suppressor in GBM.

Abstract 4141: Tumor cell enrichment is critical for assessing cell signaling pathways in glioblastoma multiforme.

Abstract Introduction & Methods: Glioblastoma tumor cells are generally assumed to be mixed with few distinct non-tumorigenic cell types and few cells of the microenvironment in general, owing to their location in the brain and the high tumor cell density compared to normal brain parenchyma. However, even small cell populations can have a significant impact on the abundance of phosphorylated proteins. We have analyzed the level and/or phosphorylation of 133 proteins in laser capture microdissected glioblastoma primary tumors (n=39) and compared the results to non-tumor cell enriched, whole tissue lysates. Results: A large portion (44 percent) of endpoints were significantly different between matched tumor cell enriched and non-enriched samples - even when limiting the investigation to tumor samples with at least 90 percent tumor content. EGFR protein levels, which is highly expressed in tumor cells compared to normal brain parenchyma, increased in enriched and non-enriched samples following EGFR mutation, while EGFR phosphorylation was only significantly increased in tumor cell enriched samples. In contrast, PTEN, which has a higher expression in normal brain compared to tumor cells, was only found to be reduced in tumor cell enriched samples from patients with either PTEN mutation or loss in PTEN copy number, with no difference seen in non-enriched samples. Patient stratification by either phosphorylated EGFR, Akt, mTOR, Stat1, VEGFR2 or Bcl-2 based on non-tumor cell enriched samples was dramatically different from tumor cell enriched samples, showing significant misclassification for up to 40 percent of patients even in high tumor content samples. Conclusions: This study demonstrates the critical necessity for selected tumor cell population procurement when investigating protein levels and phosphorylation in glioblastoma. Only tumor cell enriched samples correctly reflected expected tumor biology in the face of EGFR and PTEN mutations. Moreover, tumor cell enrichment had a dramatic effect on the stratification of clinical patient samples that would form the basis for targeted therapy selection. Citation Format: Claudius Mueller, Ana C. deCarvalho, Tom Mikkelsen, Valerie Calvert, Virginia Espina, Lance A. Liotta, Emanuel F. Petricoin III. Tumor cell enrichment is critical for assessing cell signaling pathways in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4141. doi:10.1158/1538-7445.AM2013-4141

Lipid phosphate phosphatase-3 regulates tumor growth via β-catenin and Cyclin-D1 signaling

BackgroundThe acquisition of proliferative and invasive phenotypes is considered a hallmark of neoplastic transformation; however, the underlying mechanisms are less well known. Lipid phosphate phosphatase-3 (LPP3) not only catalyzes the dephosphorylation of the bioactive lipid sphingosine-1-phosphate (S1P) to generate sphingosine but also may regulate embryonic development and angiogenesis via the Wnt pathway. The goal of this study was to determine the role of LPP3 in tumor cells.ResultsWe observed increased expression of LPP3 in glioblastoma primary tumors and in U87 and U118 glioblastoma cell lines. We demonstrate that LPP3-knockdown inhibited both U87 and U118 glioblastoma cell proliferation in culture and tumor growth in xenograft assays. Biochemical experiments provided evidence that LPP3-knockdown reduced β-catenin, CYCLIN-D1, and CD133 expression, with a concomitant increase in phosphorylated β-catenin. In a converse experiment, the forced expression of LPP3 in human colon tumor (SW480) cells potentiated tumor growth via increased β-catenin stability and CYCLIN-D1 synthesis. In contrast, elevated expression of LPP3 had no tumorigenic effects on primary cells.ConclusionsThese results demonstrate for the first time an unexpected role of LPP3 in regulating glioblastoma progression by amplifying β-catenin and CYCLIN-D1 activities.

Abstract 4235: Notch ligand-dependent cancer stem cell niche in glioblastoma

Abstract Recent data shows that endothelial cells function as a stem cell niche to promote CD133-positive cancer stem-like cells (CSLCs) self-renewal in glioblastoma (GBM). However, the mechanism by which endothelial cells function as a stem cell niche is largely unknown. To test if endothelial cells and differentiated tumor cells could function as a niche by providing Notch ligands to CSLCs, first we co-cultured GBM neurospheres with primary human endothelial cells (PHECs) which express Notch ligand Jagged/Delta. We found that PHECs promote GBM neurosphere growth, CD133 expression and clonogenicity in vitro, whereas knocking down Notch ligand expression in PHECs abrogates PHEC-induced GBM growth. We also found that the CD133-negative GBM cells express a higher level of Jagged/Delta and glial differentiation marker GFAP compared with CD133-positive population. When GBM neurospheres were forced to differentiate and grow as mono-layer attached cells, CD133-positive population was reduced, whereas expression of GFAP (glial marker), GalC (oligodendrocyte marker), Tuj1 (neuronal marker) and Notch ligands were induced. Furthermore, treatment of GBM neurospheres with a Jagged peptide increases GBM growth in a dosage dependent fashion. In addition, Notch ligands are highly expressed in the blood vessels of GBM primary tumors and intracranial xenografts in mouse, and CSLCs accumulate around the blood vessels within the tumors. Finally, we found that knocking down Notch ligands in PHECs inhibits GBM xenograft propagation in mice through reducing CD133-positive population. In summary, we show that Notch activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells replicating the classical process of lateral inhibition and by stromal niche signals, suggesting that targeting both CSLCs and their niche may provide a profound strategy to deplete GBM CSLCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4235.

Ex vivo activation and expansion of γδ T cells for immunotherapy of glioblastoma

2553 Background: Activated γδ T cells will efficiently kill established glioblastoma (GBM) cell lines in vitro. However, their potential for cell therapy has been limited by their relatively small numbers in the peripheral blood and their sensitivity to activation induced cell death (AICD) during ex vivo expansion. Our laboratory developed a method whereby γδ T cells, when stimulated by IFN-γ, IL- 12, and anti-CD2, acquire resistance to AICD and can be expanded in culture with anti-CD3 and IL-2. The γδ T cells expanded by this method are highly cytotoxic to GBM. We have now sought to adapt the laboratory method for human use. Methods: The laboratory method and selection procedure were modified to employ pharmaceutical-grade reagents. Peripheral blood mononuclear cells (1.0 × 106/ml) were stimulated overnight with media containing cGMP grade human serum, IFN-γ, IL-12, and anti-CD2. Media containing human serum, IL-2 and anti-CD3 was then added (1 v/v) and refreshed 1 v/v 3× weekly. The cultures were harvested after two weeks followed by depletion of CD4+ and CD8+ T cells, thereby enriching γδ T cells. Cytotoxicity of expanded of γδ T cells was evaluated against GBM primary tumor cultures, established GBM cell lines, and cultured astrocytes using a commercial flow cytometric method. Results: After two weeks in culture, γδ T cells expanded up to 1000 fold (usually 200–400 fold), consistent with results from our previous research-scale method. Positive selection yielded a γδ T cell product with >80% purity and >70% recovery. The cell products exhibited incremental cytotoxicity against several primary GBM cultures and established GBM cell lines. Astrocytoma cells were not killed. Conclusions: Apoptosis-resistant γδ T cells can be expanded and selected using clinically approvable reagents and in numbers sufficient for immunotherapy of malignant brain tumors. Initial data show that expanded γδ T cells retain cytotoxicity against the GBM primary cultures and spare normal astrocytes. No significant financial relationships to disclose.