Glioblastoma, the most aggressive cerebral tumor, is invariably lethal. Glioblastoma cells express several genes typical of normal neural stem cells. One of them, SOX2, is a master gene involved in sustaining self-renewal of several stem cells, in particular neural stem cells. To investigate its role in the aberrant growth of glioblastoma, we silenced SOX2 in freshly derived glioblastoma tumorinitiating cells (TICs). Our results indicate that SOX2 silenced glioblastoma TICs, despite the many mutations they have accumulated, stop proliferating and lose tumorigenicity in immunodeficient mice. SOX2 is then also fundamental for maintenance of the self-renewal capacity of neural stem cells when they have acquired cancer properties. SOX2, or its immediate downstream effectors, would then be an ideal target for glioblastoma therapy.

In this study, cancer cells were isolated from tumor specimens of nine glioblastoma patients. Glioblastoma cells, cultured under suitable culture conditions, displayed markers typical of neural stem cells, were capable of partial multilineage differentiation in vitro, and gave origin to infiltrating tumors when orthotopically injected in NOD/SCID mice. These cells, although resistant to freshly isolated NK cells, were highly susceptible to lysis mediated by both allogeneic and autologous IL-2 (or IL-15)-activated NK cells. Indeed, all stem cellcultured glioblastoma cells analyzed did not express protective amounts of HLA class I molecules, while expressing various ligands of activating NK receptors that triggered optimal NK cell cytotoxicity. Importantly, glioblastoma stem cells expressed high levels of PVR and Nectin-2, the ligands of DNAM-1-activating NK receptor.

Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect.

Epidemiological and preclinical studies propose that metformin, a first-line drug for type-2 diabetes, exerts direct antitumor activity. Although several clinical trials are ongoing, the molecular mechanisms of this effect are unknown.Here we show that chloride intracellular channel-1 (CLIC1) is a direct target of metformin in human glioblastoma cells. Metformin exposure induces antiproliferative effects in cancer stem cell-enriched cultures, isolated from three individual WHO grade IV human glioblastomas. These effects phenocopy metformin-mediated inhibition of a chloride current specifically dependent on CLIC1 functional activity. CLIC1 ion channel is preferentially active during the G1-S transition via transient membrane insertion. Metformin inhibition of CLIC1 activity induces G1 arrest of glioblastoma stem cells. This effect was time-dependent, and prolonged treatments caused antiproliferative effects also for low, clinically significant, metformin concentrations. Furthermore, substitution of Arg29 in the putative CLIC1 pore region impairs metformin modulation of channel activity.The lack of drugs affecting cancer stem cell viability is the main cause of therapy failure and tumor relapse. We identified CLIC1 not only as a modulator of cell cycle progression in human glioblastoma stem cells but also as the main target of metformin's antiproliferative activity, paving the way for novel and needed pharmacological approaches to glioblastoma treatment.

CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. CD38 is a type II transmembrane glycoprotein predominantly expressed on lymphocytes (1) but also present in a number of different cell types, including erythrocytes (2), hematopoietic progenitor cells (1), ␤-pancreatic cells (3), and cerebral (4) and cerebellar (5) neurons. Immunologically, CD38 can be defined as an "orphan receptor" since its binding by specific monoclonal antibodies directed against ectocellular epitopes elicits cellular responses in lymphocytes, including proliferation, activation, and rescue from apoptosis (6, 7). The signal transduction pathways implicated in these events are under study, but phosphorylation/dephosphorylation reactions of target kinases have been already demonstrated (6,8,9). Biochemically, CD38 is a bifunctional ectoenzyme that catalyzes the synthesis of cADPR 1 from NAD ϩ and also its hydrolysis to ADPR (2, 10). Cyclic ADPR is a potent Ca 2ϩ mobilizer from intracellular stores, in invertebrate as well as in mammalian cells (11), and its presence has been described in most mammalian tissues (12).The widespread tissue distribution of the CD38/cADPR system suggests its involvement in the control of pivotal Ca 2ϩ -controlled functions like contraction, secretion, cell proliferation/differentiation, and apoptosis. However, the topological paradox of the ectocellular production of an intracellular Ca 2ϩ mobilizer has raised questions on both the immunological and the biochemical functions of the CD38/cADPR system (13, 14). Cell death has been advocated as a means for local increase in extracellular NAD ϩ concentrations, sufficient to elicit the production of cADPR by the ectoenzyme CD38; in this respect, nanomolar concentrations of NAD ϩ have been detected in plasma (15) and cerebellar interstitial fluid (5) suggesting the theoretical possibility of an extracellular production of cADPR by CD38 "in vivo." In few selected cell systems, i.e. murine B-lymphocytes (10), rat cerebellar granule cells (5), and rat osteoclasts (16), extracellular, exogenously added cADPR was demonstrated to elicit functional responses in intact cells, but most reported effects of cADPR on cellular functions require permeabilization of target cells to ensure binding of the nucleotide to its intracellular receptor(s).Internalization of membrane-bound CD38, as observed in human Namalwa B-lymphocytes upon incubation with NAD ϩ or thiol reagents, is followed by an increase of intracellular cyclase activity (insensitive to protein synthesis inhibitors) and of intracellular cADPR concentration ([cADPR] i ) (17), suggesting that...