Medulloblastoma is the most common malignant pediatric brain tumor with an average 5-year survival of 50 percent. It is a form of primitive neuroectodermal tumor that is believed to arise due to the abnormal arrest of neuroectodermal stem cells to a large extent in their neuronal differentiation pathway. However, the mechanism of this process remains elusive. For the first time, a transcription factor (REST or NRSF) has been discovered that represses transcription of most, if not all, neuronal genes by binding to a specific DNA sequence present in their regulatory regions. REST protein is found in a functional form in almost all cell types including neuroblasts, and is not found in differentiated neurons. Thus, REST appears to be one of the earliest and most critical negative regulators of neuronal differentiation. We found that REST is functionally present and can repress expression of neuronal promoters in these cells. The level of REST-dependent repression correlates inversely with the level of neuronal differentiation. This raised the hypothesis that these medulloblastoma cells are arrested at a pre-differentiation state, and that the presence of functional REST may be responsible for arresting these tumor cells during differentiation. To counter REST activity, we constructed two negative mutants, REST1-VP16 and REST2-VP16 (REST-VP16), by replacing different repressor domains of REST with the strong activation domain of the viral activator, VP16. REST-VP16 bind to the same DNA binding site as REST, but function as activators, instead of repressors. Expression of REST-VP16 in medulloblastoma cells was found to block REST-dependent repression, activate neuronal promoters from transfected plasmids, activate cellular neuronal genes that are characteristic markers for terminal neuronal differentiation, and arrest these cells at G1 phase of the cell cycle within 48 h. Therefore, these experiments raised the hypothesis that countering REST function through the expression of REST-VP16 in medulloblastoma cells would modulate their biology. The objectives of the present project are to express REST-VP16 in medulloblastoma cells by stable transfection, and in medulloblastoma xenografts in athymic mice by adenoviral vectors, and to examine their impact on the transformed/neoplastic properties of these cells and tumors. It will also be tested if anti-REST antibodies can be used to detect REST in medulloblastoma tumor sections, and whether the various neuronal genes expressed in response to REST-VP16 could be used as medulloblastoma markers. Thus, the present project has the potential of discovering REST as a novel target for therapeutic intervention of medulloblastoma.
| Kamal, Mohamed M; Sathyan, Pratheesh; Singh, Sanjay K et al. (2012) REST regulates oncogenic properties of glioblastoma stem cells. Stem Cells 30:405-14 |
| Singh, Sanjay K; Veo, Bethany L; Kagalwala, Mohamedi N et al. (2012) Dynamic status of REST in the mouse ESC pluripotency network. PLoS One 7:e43659 |
| Gopalakrishnan, Vidya; Bie, Bihua; Sinnappah-Kang, Neeta D et al. (2010) Myoblast-derived neuronal cells form glutamatergic neurons in the mouse cerebellum. Stem Cells 28:1839-47 |
| Kagalwala, M N; Singh, S K; Majumder, S (2008) Stemness is only a state of the cell. Cold Spring Harb Symp Quant Biol 73:227-34 |
| Singh, Sanjay K; Kagalwala, Mohamedi N; Parker-Thornburg, Jan et al. (2008) REST maintains self-renewal and pluripotency of embryonic stem cells. Nature 453:223-7 |
| Majumder, Sadhan (2006) REST in good times and bad: roles in tumor suppressor and oncogenic activities. Cell Cycle 5:1929-35 |
| Su, Xiaohua; Gopalakrishnan, Vidya; Stearns, Duncan et al. (2006) Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation. Mol Cell Biol 26:1666-78 |
| Su, Xiaohua; Kameoka, Sei; Lentz, Susan et al. (2004) Activation of REST/NRSF target genes in neural stem cells is sufficient to cause neuronal differentiation. Mol Cell Biol 24:8018-25 |
| Watanabe, Yumi; Kameoka, Sei; Gopalakrishnan, Vidya et al. (2004) Conversion of myoblasts to physiologically active neuronal phenotype. Genes Dev 18:889-900 |
| Immaneni, A; Lawinger, P; Zhao, Z et al. (2000) REST-VP16 activates multiple neuronal differentiation genes in human NT2 cells. Nucleic Acids Res 28:3403-10 |
