Cancers are driven by a population of cells with the stem cell properties of self-renewal and unlimited growth. As a subpopulation within the tumor mass, these cells are believed to constitute a tumor cell reservoir. Pathways controlling the renewal of normal stem cells are deregulated in cancer. The polycomb group gene Bmi1, which is required for neural stem cell self-renewal and also controls anti-oxidant defense in neurons, is upregulated in several cancers, including medulloblastoma. We have found that Bmi1 is consistently and highly expressed in GBM. Downregulation of Bmi1 by shRNAs induced a differentiation phenotype and reduced expression of the stem cell markers Sox2 and Nestin. Interestingly, expression of glycogen synthase kinase 3 beta (GSK3β), which was found to be consistently expressed in primary GBM, also declined. This suggests a functional link between Bmi1 and GSK3β. Interference with GSK3β activity by siRNA, the specific inhibitor SB216763, or lithium chloride (LiCl) induced tumor cell differentiation. In addition, tumor cell apoptosis was enhanced, the formation of neurospheres was impaired, and clonogenicity reduced in a dose-dependent manner. GBM cell lines consist mainly of CD133-negative (CD133-) cells. Interestingly, ex vivo cells from primary tumor biopsies allowed the identification of a CD133- subpopulation of cells that express stem cell markers and are depleted by inactivation of GSK3β. Drugs that inhibit GSK3, including the psychiatric drug LiCl, may deplete the GBM stem cell reservoir independently of CD133 status.

Purpose: High-grade gliomas are difficult to treat due to their location behind the blood-brain barrier and to inherent radioresistance and chemoresistance. Experimental Design: Because tumorigenesis is considered a multistep process of accumulating mutations affecting distinct signaling pathways, combinations of compounds, which inhibit nonoverlapping pathways, are being explored to improve treatment of gliomas. Histone deacetylase inhibitors (HDI) have proven antitumor activity by blocking cell proliferation, promoting differentiation, and inducing tumor cell apoptosis. Results: In this report, we show that the HDIs trichostatin A, sodium butyrate, and low nanomolar doses of LAQ824 combined with the glycolysis inhibitor 2-deoxy-D-glucose induce strong apoptosis in cancer cell lines of brain, breast, and cervix in a p53-independent manner. HDIs upregulate p21, which is blocked by concomitant administration of 2-deoxy-D-glucose. Conclusions: We propose simultaneous blockade of histone deacetylation and glycolysis as a novel therapeutic strategy for several major cancers.Glioblastoma multiforme (GBM) are the most frequent human brain tumors. These aggressive, highly invasive, and neurologically destructive tumors are among the deadliest of human cancers, with a median survival ranging from 9 to 12 months (1). Despite treatment efforts including new technological advances in neurosurgery, radiation therapy, and clinical trials with novel therapeutic agents (2), median survival has not changed significantly over the past two decades. Meanwhile, cancer drug development has been moving from conventional cytotoxic chemotherapeutics to more sophisticated drugs exploiting biological mechanism of tumorigenesis (3).In high-grade gliomas, several genes and pathways are altered due to a severe mutator phenotype that leads to the accumulation of mutations in critical regulatory genes. Mutations of PTEN, RB, p16/p14, p53 (4), and receptor tyrosine kinase (5 -7) result in up-regulation of pathways that promote tumor growth, invasion, and resistance to apoptotic stimuli (8).Besides classic mutations, epigenetic silencing of tumor suppressor genes frequently leads to dysregulation of signaling pathways promoting tumorigenesis. Histone acetyltransferase and histone deacetylase (HDAC) catalyze the acetylation and deacetylation of lysine residues in the tails of histone proteins (e.g., lysine residue in histones 3 and 4), regulating the affinity of the protein transcriptional complexes to the DNA (9). Thus, the recruitment of histone acetyltransferase and HDAC is considered as a key element in the dynamic regulation of genes involved in cellular proliferation and differentiation during normal development and carcinogenesis (10). HDAC inhibitors (HDI) induce re-expression of silenced tumor suppressor genes (11,12). HDIs induce differentiation and promote apoptosis in transformed and cancerous but not in normal cells (11,13). HDIs have been classified in different classes: shortchain fatty acid (as sodium butirrate, valproic...

The oncogenic epidermal growth factor receptor (EGFR) pathway triggers downstream phosphatidylinositol 3-kinase (PI3K)/RAS-mediated signaling cascades. In transgenic mice, glioblastoma cannot develop on single but only on simultaneous activation of the EGFR signaling mediators RAS and AKT. However, complete blockade of EGFR activation does not result in apoptosis in human glioblastoma cells, suggesting additional cross-talk between downstream pathways. Based on these observations, we investigated combination therapies using protein kinase inhibitors against EGFR, platelet-derived growth factor receptor, and mammalian target of rapamycin, assessing glioblastoma cell survival. Clinically relevant doses of AEE788, Gleevec (imatinib), and RAD001 (everolimus), alone or in combinations, did not induce glioblastoma cell apoptosis. In contrast, simultaneous inactivation of the EGFR downstream targets mitogen-activated protein/ extracellular signal-regulated kinase (ERK) kinase and PI3K by U0126 and wortmannin triggered rapid tumor cell death. Blocking EGFR with AEE788 in combination with sublethal concentrations of the microtubule stabilizer patupilone also induced apoptosis and reduced cell proliferation in glioblastoma cells, accompanied by reduced AKT and ERK activity. These data underline the critical role of the PI3K/AKT and the RAS/RAF/mitogenactivated protein/ERK kinase/ERK signaling cascades in the cell-intrinsic survival program of sensitive glioblastoma cell lines. We conclude that drug combinations, which down-regulate both ERK and protein kinase B/AKT activity, may prove effective in overcoming cell resistance in a subgroup of glioblastoma. [Mol Cancer Ther 2007; 6(2):773 -81]

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