Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.

MicroRNAs (miRNA) are crucial post-transcriptional regulators of gene expression and control cell differentiation and proliferation. However, little is known about their targeting of specific developmental pathways. Hedgehog (Hh) signalling controls cerebellar granule cell progenitor development and a subversion of this pathway leads to neoplastic transformation into medulloblastoma (MB). Using a miRNA high-throughput profile screening, we identify here a downregulated miRNA signature in human MBs with high Hh signalling. Specifically, we identify miR-125b and miR-326 as suppressors of the pathway activator Smoothened together with miR-324-5p, which also targets the downstream transcription factor Gli1. Downregulation of these miRNAs allows high levels of Hh-dependent gene expression leading to tumour cell proliferation. Interestingly, the downregulation of miR-324-5p is genetically determined by MB-associated deletion of chromosome 17p. We also report that whereas miRNA expression is downregulated in cerebellar neuronal progenitors, it increases alongside differentiation, thereby allowing cell maturation and growth inhibition. These findings identify a novel regulatory circuitry of the Hh signalling and suggest that misregulation of specific miRNAs, leading to its aberrant activation, sustain cancer development.

Hedgehog (Hh) pathway has a pivotal function in development and tumorigenesis, processes sustained by stem cells (SCs). The transcription factor Nanog controls stemness acting as a key determinant of both embryonic SC self-renewal and differentiated somatic cells reprogramming to pluripotency, in concert with the loss of the oncosuppressor p53. How Nanog is regulated by microenvironmental signals in postnatal SC niches has been poorly investigated. Here, we show that Nanog is highly expressed in SCs from postnatal cerebellum and medulloblastoma, and acts as a critical mediator of Hh-driven self-renewal. Indeed, the downstream effectors of Hh activity, Gli1 and Gli2, bind to Nanog-specific cis-regulatory sequences both in mouse and human SCs. Loss of p53, a key event promoting cell stemness, activates Hh signalling, thereby contributing to Nanog upregulation. Conversely, Hh downregulates p53 but does not require p53 to control Nanog. Our data reveal a mechanism for the function of Hh in the control of stemness that represents a crucial component of an integrated circuitry determining cell fate decision and involved in the maintenance of cancer SCs. The EMBO Journal (2010) 29, 2646-2658. doi:10.1038/emboj.2010.131; Published online 25 June 201

Infant high grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histological review, methylation profiling, custom panel and genome/exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an 'intrinsic' spectrum of disease specific to the infant population. These included those with targetable MAP-kinase alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n=31), NTRK1/2/3 (n=21), ROS1 (n=9) and MET (n=4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly supports the concept that infant gliomas require a change in diagnostic practice and management.

Hedgehog signaling is essential for tissue development and stemness, and its deregulation has been observed in many tumors. Aberrant activation of Hedgehog signaling is the result of genetic mutations of pathway components or other Smo-dependent or independent mechanisms, all triggering the downstream effector Gli1. For this reason, understanding the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, representing a critical goal in tumor biology. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify Glabrescione B as the first small molecule binding to Gli1 zinc finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, Glabrescione B inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. The identification of the structural requirements of Gli1/DNA interaction highlights their relevance for pharmacologic interference of Gli signaling.