Most medulloblastomas are believed to originate from undifferentiated neural stem/progenitor cells (NSCs) present in the external granule layer cells of the cerebellum (15
). The primitive “embryonal” appearance of medulloblastoma cells, as well as their capacity for divergent differentiation, has led to the suggestion that they have a neural stem cell-like phenotype (18
). In addition to arising from stem cells, human medulloblastomas appear to contain stem-like cells required for tumor propagation (59
). Induction of neuronal and glial markers in medulloblastoma cells has been documented as a response to several proposed chemotherapeutic agents (6
), supporting the hypothesis that these tumors show lack of terminal differentiation and suggesting regulation of differentiation status as a promising treatment avenue.
Pathways regulating cerebellar development, such as Hedgehog and Wnt, have been found to be activated by genetic alterations during medulloblastoma tumorigenesis (15
). Both Hedgehog and Wnt are thought to regulate proliferation and differentiation of neural stem cells and may play a similar role in medulloblastoma. However, mutations activating these pathways have been documented in only a modest percentage of human tumors. The receptor gene PTCH
is the member of the Hedgehog pathway most commonly altered in medulloblastoma, but sensitive techniques such as direct sequencing have identified mutations in no more than 10% of cases (14
). Similarly, mutations in CTNNB1
activating the Wnt pathway have been identified in less than 20% of sporadic medulloblastomas (13
). Thus, the mechanism of tumorigenesis for the majority of medulloblastomas is still unknown.
The Myc oncoproteins are also important in medulloblastoma pathogenesis (1
are commonly amplified in the biologically aggressive large cell/anaplastic medulloblastoma subtype (17
). In addition, overexpression of c-Myc mRNA due to gene amplification or other unidentified mechanisms has been associated with worse clinical outcomes (15
). N-Myc also has been implicated in the development of medulloblastoma as a result of Shh pathway activation (30
). However, the c-Myc oncoprotein, or its activated form, v-Myc, is insufficient to cause medulloblastoma when acting alone in NSCs (20
REST/NRSF is a global transcriptional repressor that contains a DNA binding domain and two repressor domains; it silences the transcription of a large number of neuronal differentiation genes by binding to a 23-bp consensus DNA sequence, the RE1 binding site/neuron restrictive silencer (RE1/NRSE), present in these genes' regulatory regions (3
). REST/NRSF is mostly expressed in embryonic stem cells and nonneuronal cells and is rarely expressed in neurons in vivo (3
). However, REST/NRSF is expressed in certain mature neurons in adults (23
), suggesting that it has a complex role that depends on its cellular and physiological environment. Furthermore, there are several isoforms of REST/NRSF, and one such isoform, REST4, functions as a dominant-negative regulator by interfering with REST/NRSF's activity in neurons (56
). Both REST/NRSF and REST4 interact with RILP, a LIM domain protein, for nuclear translocation (56
). For REST/NRSF-dependent promoter repression to occur, REST/NRSF must interact with several cellular cofactors, including Co-REST, N-CoR, mSin3A, and the histone deacetylase complex, and modulate chromatin structure (2
). Furthermore, a small, double-stranded RNA has been found to modulate REST/NRSF activity in the NSCs as well (34
). Our previous work showed that several medulloblastoma cell lines and many human medulloblastomas overexpress REST/NRSF compared with neuronal progenitor cells and fully differentiated neurons (19
). However, neuronal cells constitutively expressing REST/NRSF do not form into tumors and appear to acquire a normal neuronal morphology, except that they manifest axon pathfinding errors (49
). Similarly, our transgenic mice expressing REST/NRSF in neuronal cells appeared to develop normally without tumor formation (unpublished data).
To study what happens when the effects of REST/NRSF are opposed, we constructed a recombinant transcription factor, REST-VP16, in which both the repressor domains of REST/NRSF were replaced with the activation domain of the herpes simplex virus protein VP16 (28
). We found that REST-VP16 operates through RE1/NRSE, competes with endogenous REST/NRSF for DNA binding, and activates cellular REST/NRSF target genes. Furthermore, the high-efficiency expression of REST-VP16 mediated by the adenovirus construct Ad.REST-VP16 in human medulloblastoma cells countered the endogenous REST/NRSF-mediated repression of neuronal promoters and promoted apoptosis through caspase 3 activation, presumably resulting from the simultaneous opposing action of REST/NRSF and REST-VP16 (19
). Because it leads to apoptosis, REST-VP16 also blocks the tumorigenicity of medulloblastoma cells (19
The experiments described here indicate that abnormal expression of REST and Myc in NSCs cooperate to form cerebellar tumors by blocking neuronal differentiation. Although the role of naturally occurring, DNA-binding transcriptional activators in oncogenesis is well established, the role of transcriptional repressors is not. Currently, there is only indirect evidence linking repressors such as evi-1 and CtBP to oncogenesis; a strong link of these repressors with their in vivo gene targets is lacking (9
). The study here also describes such a direct role of a transcriptional repressor in forming medulloblastoma by blocking neuronal differentiation.