Giorgio Corte

SOX2 silencing in glioblastoma tumor-initiating cells causes stop of proliferation and loss of tumorigenicity.

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 tumor‐initiating 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. STEM CELLS 2009;27:40–48

NK Cells Recognize and Kill Human Glioblastoma Cells with Stem Cell-Like Properties

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 cell-cultured 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.

Implication of OTX2 in pigment epithelium determination and neural retina differentiation

The expression pattern of Otx2, a homeobox-containing gene, was analyzed from the beginning of eye morphogenesis until neural retina differentiation in chick embryos. Early on, Otx2 expression was diffuse throughout the optic vesicles but became restricted to their dorsal part when the vesicles contacted the surface ectoderm. As the optic cup forms,Otx2 was expressed only in the outer layer, which gives rise to the pigment epithelium. This early Otx2expression pattern was complementary to that of PAX2, which localizes to the ventral half of the developing eye and optic stalk.Otx2 expression was always observed in the pigment epithelium at all stages analyzed but was extended to scattered cells located in the central portion of the neural retina around stage 22. The number of cells expressing Otx2 transcripts increased with time, following a central to peripheral gradient. Bromodeoxyuridine labeling in combination with immunohistochemistry with anti-OTX2 antiserum and different cell-specific markers were used to determine that OTX2-positive cells are postmitotic neuroblasts undergoing differentiation into several, if not all, of the distinct cell types present in the chick retina. These data indicate thatOtx2 might have a double role in eye development. First, it might be necessary for the early specification and subsequent functioning of the pigment epithelium. Later, OTX2 expression might be involved in retina neurogenesis, defining a differentiation feature common to the distinct retinal cell classes.

Comparative Analysis of DNA Repair in Stem and Nonstem Glioma Cell Cultures

It has been reported that cancer stem cells may contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. We have examined DNA repair in five stem and nonstem glioma cell lines. The population doubling time was significantly increased in stem compared with nonstem cells, and enhanced activation of Chk1 and Chk2 kinases was observed in untreated CD133+ compared with CD133− cells. Neither DNA base excision or single-strand break repair nor resolution of pH2AX nuclear foci were increased in CD133+ compared with CD133− cells. We conclude that glioma stem cells display elongated cell cycle and enhanced basal activation of checkpoint proteins that might contribute to their radioresistance, whereas enhanced DNA repair is not a common feature of these cells. (Mol Cancer Res 2009;7(3):383–92)

Expression of CXC chemokine receptors 1-5 and their ligands in human glioma tissues : Role of CXCR4 and SDF1 in glioma cell proliferation and migration

Chemokines have been involved in cellular processes associated to malignant transformation such as proliferation, migration and angiogenesis. The expression of five CXC chemokine receptors and their main ligands was analysed by RT-PCR in 31 human astrocytic neoplasms. The mRNAs for all the receptors analysed were identified in a high percentage of tumours, while their ligands showed lower expression. CXCR4 and SDF1 were the most frequently mRNA identified (29/31 and 13/31 of the gliomas studied, respectively). Thus, we further analysed the cell localization of CXCR4 and SDF1 in immunohistochemistry experiments. We show a marked co-localization of CXCR4 and SDF1 in tumour cells, mainly evident in psudolpalisade and microcystic degeneration areas and in the vascular endothelium. In addition, hSDF1alpha induced a significant increase of DNA synthesis in primary human glioblastoma cell cultures and chemotaxis in a glioblastoma cell line. These results provide evidence of the expression of multiple CXC chemokines and their receptors in brain tumours and that in particular CXCR4 and SDF1 sustain proliferation and migration of glioma cells to promote malignant progression.

Otx dose-dependent integrated control of antero-posterior and dorso-ventral patterning of midbrain

Organizing centers emit signaling molecules that specify different neuronal cell types at precise positions along the anterior–posterior (A–P) and dorsal–ventral (D–V) axes of neural tube during development. Here we report that reduction in Otx proteins near the alar–basal plate boundary (ABB) of murine midbrain resulted in a dorsal shift of Shh expression, and reduction in Otx proteins at the midbrain–hindbrain boundary (MHB) resulted in an anterior expansion of the Fgf8 domain. Our data thus indicate that an Otx dose-dependent repressive effect coordinates proper positioning of Shh and Fgf8 expression. Furthermore, this control is effective for conferring proper cell identity in the floor-plate region of midbrain and does not require an Otx2-specific property. We propose that this mechanism may provide both A–P and D–V positional information to neuronal precursors located within the midbrain.

EMX2 protein in the developing mouse brain and olfactory area.

The distribution of EMX2, the protein product of the homeobox gene Emx2, was analyzed in the developing mouse CNS by means of a polyclonal antibody we raised against it. The protein is present in the rostral brain, the olfactory area and a set of scattered cells lying between the nasal pits and the telencephalon. In the cortical neuroepithelium EMX2 is expressed all along the rostro-caudal axis in a graded distribution with a caudal-medial maximum and a rostral-lateral minimum. Anti-EMX2 immunoreactivity is also detectable in Cajal-Retzius cells as well as in apical dendrites of marginal neurons of the cortical plate. We also observe that the EMX2 and EMX1 homeoproteins display complementary expression patterns in olfactory bulbs and amygdaloid complex. Here, they demarcate different neuronal populations, involved in processing olfactory information coming from the vomero-nasal organ and from the main olfactory epithelium, respectively. EMX2 is also detectable in mesencephalic structures, such as the optic tectum and tegmentum. The graded distribution of EMX2 along antero-posterior and medial-lateral axes of the primitive cortex prefigures a role of this protein in the subdivision of the cortex in cytoarchitectonic regions and possibly functional areas, whereas its presence in Cajal-Retzius cells suggests a role in the process of cortical lamination.

Emx2 Promotes Symmetric Cell Divisions and a Multipotential Fate in Precursors from the Cerebral Cortex

Distinct sets of precursor cells generate the mammalian cerebral cortex. During neurogenesis most precursors are specified to generate a single cell type and only few are multipotent. The cell-intrinsic molecular determinants of these distinct lineages are not known. Here we describe that retroviral transduction of the transcription factor Emx2 in precursors from the cerebral cortex results in a significant increase of large clones that are generated mostly by symmetric cell divisions and contain multiple cell types, comprising neurons and glial cells. Thus, Emx2 is the first cell-intrinsic determinant able to instruct CNS precursors towards a multipotential fate. To evaluate the role of endogenous Emx2 in cortical precursors, we examined cell division in Emx2-/- mice. These analyses further supported the role of endogenous Emx2 in the regulation of symmetric cell divisions in the developing cortex.

Different Response of Human Glioma Tumor-initiating Cells to Epidermal Growth Factor Receptor Kinase Inhibitors

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

OTX2 homeoprotein in the developing central nervous system and migratory cells of the olfactory area

We analyzed the distribution of OTX2 during mouse development. OTX2 is a homeoprotein encoded by Otx2, a vertebrate homeobox gene expressed in the developing brain and anterior head regions. The protein is already detectable in pre-streak embryos, in nuclei of embryonic ectoderm or epiblast and primitive endoderm or hypoblast. Its distribution is uniform along the entire epiblast, while showing an antero-posterior gradient along the hypoblast at the time when primitive streak first forms. Between embryonic day 7 (E7) and E7.5 there is a progressive confinement of the protein to the anterior ectoderm corresponding to the forming headfold. At E7.5-E7.8, the protein is mainly confined in this region but is still present, though at lower level, in more posterior ectoderm. Starting from day 8 of development it is essentially confined to anterior neuroectoderm corresponding to presumptive fore- and midbrain. Its subsequent distribution in forebrain, midbrain, developing isthmo-cerebellum and posterior central nervous system is analyzed in detail. Of particular interest is the presence of OTX2 in nuclei of cells of the olfactory system starting from its origin in the olfactory placode. OTX2 protein is present in some cells of the olfactory epithelium, in both the major olfactory epithelium and the vomero-nasal organ, and in scattered migratory cells present in the mesenchyme outside it. These cells surround the axon bundles of the olfactory nerve along its path from the olfactory epithelium in the nasal cavities to the olfactory bulb in rostral telencephalon and include both ensheathing glial cells and luteinizing hormone-releasing hormone (LHRH)-positive cells.