Glycoproteomic analysis of glioblastoma stem cell differentiation.

Cancer stem cells are responsible for tumor formation through self-renewal and differentiation into multiple cell types and thus represent a new therapeutic target for tumors. Glycoproteins play a critical role in determining the fates of stem cells such as self-renewal, proliferation, and differentiation. Here we applied a multilectin affinity chromatography and quantitative glycoproteomics approach to analyze alterations of glycoproteins relevant to the differentiation of a glioblastoma-derived stem cell line HSR-GBM1. Three lectins including concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) were used to capture glycoproteins, followed by LC-MS/MS analysis. A total of 73 and 79 high-confidence (FDR < 0.01) glycoproteins were identified from the undifferentiated and differentiated cells, respectively. Label-free quantitation resulted in the discovery of 18 differentially expressed glycoproteins, wherein 9 proteins are localized in the lysosome. All of these lysosomal glycoproteins were up-regulated after differentiation, where their principal function was hydrolysis of glycosyl residues. Protein-protein interaction and functional analyses revealed the active involvement of lysosomes during the process of glioblastoma stem cell differentiation. This work provides glycoprotein markers to characterize differentiation status of glioblastoma stem cells that may be useful in stem-cell therapy of glioblastoma.

NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts.

Cancer stem cells (CSCs) are thought to be critical for the engraftment and long-term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem-like cells with higher NOTCH activity. We therefore used low-passage and established GBM-derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by gamma-secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug-impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem-like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem-like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas.

KIT expression and methylation in medulloblastoma and PNET cell lines and tumors.

The stem cell factor/kit tyrosine kinase receptor pathway is related to tumor growth and progression in several cancers including Ewing sarcoma, a peripheral PNET (pPNET). Identifying additional groups of tumors that may use the pathway is important as they might be responsive to imatinib mesylate treatment. MB and central PNET (cPNET) are embryonal tumors of the CNS that share similar undifferentiated morphology with Ewing sarcomas and display aggressive clinical behavior. cPNET outcome is significantly lower than MB outcome, even for localized tumors treated with high-risk MB therapy. The elucidation of signaling pathways involved in MB and cPNET pathogenesis, and the discovery of new therapeutic targets is necessary to improve the treatment of these neoplasms. We analyzed KIT expression in 2 MB, one pPNET, one cPNET and 2 rhabdomyosarcoma (RMS) cell lines. Also, in 13 tumor samples (12 MB and one cPNET), we found KIT overexpression in the most aggressive cell lines (metastatic MB and pPNET). Hypermethylation of KIT was clear in the RMS non-expressing cell lines. Among MB tumors, we could see variable levels of KIT expression; a subset of them (25%) might be related in its growth pattern to KIT up-regulation. No methylated KIT was detected in the tumors expressing the lowest levels of KIT. Our results point to methylation as an epigenetic regulatory mechanism for KIT inhibition only in the KIT non-expressing RMS cell lines, and neither in the rest of the cell lines nor in the tumor samples.

Neuroprotection and enhancement of remyelination by estradiol and dexamethasone in cocultures of rat DRG neurons and Schwann cells.

A simple and reproducible demyelination-remyelination system was developed with cocultures of rat dorsal root ganglia (DRG) neurons and Schwann cells, and the effects of steroid hormones were examined in this system. Addition of forskolin to the cocultures induced demyelination and lower levels of myelin protein P0 expression were seen in immunoblots after eight days of treatment. Removal of forskolin caused remyelination and the amount of P0 expression was partially recovered. States of demyelination and remyelination were further confirmed by morphological examination of myelin internodes, such as incorporation of fluorescently-labeled fatty acid and immunostaining of P0 and myelin associated glycoprotein. Ultrastructural analyses of the cocultures showed the presence of myeloid bodies and peeling of myelin lamellae during the demyelination process. Using this demyelination-remyelination system, regulatory roles of steroid hormones during demyelination and remyelination were studied. Immunoblots of P0 and incorporation of fluorescently-labeled fatty acid demonstrated that treatments of the cocultures with dexamethasone and estradiol not only reduced the extent of demyelination but also enhanced remyelination. These results suggest that steroid hormones have roles as neuroprotective agents against demyelination and augmentative agents for remyelination.

Steroid hormone signaling between Schwann cells and neurons regulates the rate of myelin synthesis.

Fluorescence digital imaging microscopy was used to develop a method that allows the continuous monitoring and quantitative measurement of a single myelin internode throughout its development. Using this technique, steroid hormones such as progesterone and dexamethasone were shown to reduce the time required for the initiation and to regulate the rate of myelin synthesis. Progesterone was capable of increasing the rate of myelin synthesis in Schwann cell/neuronal co-cultures in a dose-dependent manner. RT-PCR and in situ hydridization studies revealed that the mRNAs for P450scc and 3beta-hydroxysteroid dehydrogenase, the enzymes involved in progesterone biosynthesis, were induced at the onset of myelin synthesis. The progesterone receptor protein translocated into the nucleus of the neurons during myelin synthesis, suggesting that progesterone could also be affecting neuronal gene expression. Changes in gene expression caused by progesterone are being examined to identify additional factors that may control myelin formation.

Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells.

One important function of endothelial cells in glioblastoma multiforme (GBM) is to create a niche that helps promote self-renewal of cancer stem-like cells (CSLC). However, the underlying molecular mechanism for this endothelial function is not known. Since activation of NOTCH signaling has been found to be required for propagation of GBM CSLCs, we hypothesized that the GBM endothelium may provide the source of NOTCH ligands. Here, we report a corroboration of this concept with a demonstration that NOTCH ligands are expressed in endothelial cells adjacent to NESTIN and NOTCH receptor-positive cancer cells in primary GBMs. Coculturing human brain microvascular endothelial cells (hBMEC) or NOTCH ligand with GBM neurospheres promoted GBM cell growth and increased CSLC self-renewal. Notably, RNAi-mediated knockdown of NOTCH ligands in hBMECs abrogated their ability to induce CSLC self-renewal and GBM tumor growth, both in vitro and in vivo. Thus, our findings establish that NOTCH activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells and their surrounding endothelial cells in the tumor microenvironment, suggesting that targeting both CSLCs and their niche may provide a novel strategy to deplete CSLCs and improve GBM treatment.

Regulatory role of cytochrome P450scc and pregnenolone in myelination by rat Schwann cells.

To investigate the production of steroid hormones by Schwann cells and to examine the regulation of steroid hormone production during myelination, cultures of rat Schwann cells were differentiated into their myelinating phenotype in the absence of neurons with dibutyryl cAMP (db-cAMP). During this process, the expression of P450scc (involved in steroid biosynthesis) was elevated at both the mRNA and protein levels as evident in RT-PCR, Western blots, and immunostaining. Labeling of the cells with [14C] acetate revealed enhanced production of pregnenolone during differentiation into the myelinating phenotype. Disruption of P450scc's activity with an inhibitor diminished the extent of differentiation into the myelinating phenotype as levels of mRNA and protein expression of myelin protein zero (P0) declined. However, the effect was reversed with the addition of pregnenolone. Furthermore, when the differentiating cultures were treated with pregnenolone, mRNA expression of P0 was upregulated, suggesting the stimulation of the differentiation process. Together, these results provide evidence for Schwann cells as a major producer of steroid hormones and pregnenolone production by P450scc as an important regulatory step during myelination.