Isabella Screpanti

Concerted microRNA control of Hedgehog signalling in cerebellar neuronal progenitor and tumour cells

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.

Alternative splicing of the ErbB-4 cytoplasmic domain and its regulation by hedgehog signaling identify distinct medulloblastoma subsets

Medulloblastoma (MB) results from aberrant development of cerebellar neurons in which altered hedgehog (Hh) signalling plays a major role. We investigated the possible influence of Hh signalling on ErbB-receptor expression in MB, in particular that of the ErbB-4 CYT-1 and CYT-2 isoforms generated by alternative splicing of the cytoplasmic domain. ErbB-4 expression was downregulated in Hh-induced MBs from Patched-1+/− mice. Hh signalling (reflected by enhanced expression of the Gli1 transcription factor) inhibited ErbB-4 expression in mouse cerebellar granule progenitors and human MB cells. Analysis of 26 human primary MBs revealed a subset of 11 tumors characterized by low Gli1 levels, upregulated ErbB-4 expression and increased CYT-1:CYT-2 ratios. Interestingly, CYT-1 and Gli1 levels were inversely correlated. ErbB-4 CYT-1 and CYT-2 had different phenotypic effects in cultured MB cells: in response to neuregulin treatment, CYT-2 overexpression inhibited proliferation whereas CYT-1, which includes a phosphatidylinositol 3-kinase (PI3K)-binding site that is missing in CYT-2, enhanced resistance to starvation- and etoposide-induced apoptosis by activating PI3K/Akt signalling. CYT-1:CYT-2 ratios displayed correlation with tumor histotype and ErbB-2 levels, which are established prognostic indices for MB. These findings demonstrate that low-level Hh signalling in human MB is associated with the selective maintenance of high ErbB-4 CYT-1 expression, an alteration that exerts tumor-promoting effects.

PCAF ubiquitin ligase activity inhibits Hedgehog/Gli1 signaling in p53-dependent response to genotoxic stress

The Hedgehog (Hh) signaling regulates tissue development, and its aberrant activation is a leading cause of malignancies, including medulloblastoma (Mb). Hh-dependent tumorigenesis often occurs in synergy with other mechanisms, such as loss of p53, the master regulator of the DNA damage response. To date, little is known about mechanisms connecting DNA-damaging events to morphogen-dependent processes. Here, we show that genotoxic stress triggers a cascade of signals, culminating with inhibition of the activity of Gli1, the final transcriptional effector of Hh signaling. This inhibition is dependent on the p53-mediated elevation of the acetyltransferase p300/CBP-associated factor (PCAF). Notably, we identify PCAF as a novel E3 ubiquitin ligase of Gli1. Indeed PCAF, but not a mutant with a deletion of its ubiquitination domain, represses Hh signaling in response to DNA damage by promoting Gli1 ubiquitination and its proteasome-dependent degradation. Restoring Gli1 levels rescues the growth arrest and apoptosis effect triggered by genotoxic drugs. Consistently, DNA-damaging agents fail to inhibit Gli1 activity in the absence of either p53 or PCAF. Finally, Mb samples from p53-null mice display low levels of PCAF and upregulation of Gli1 in vivo, suggesting PCAF as potential therapeutic target in Hh-dependent tumors. Together, our data define a mechanism of inactivation of a morphogenic signaling in response to genotoxic stress and unveil a p53/PCAF/Gli1 circuitry centered on PCAF that limits Gli1-enhanced mitogenic and prosurvival response.

Proteomic analysis of human sonic hedgehog (SHH) medulloblastoma stem-like cells

Human medulloblastoma (MB) is a malignant brain tumor that comprises four distinct molecular subgroups including the Sonic Hedgehog (SHH)-MB group. A leading cause of the SHH subgroup is an aberrant activation of the SHH pathway, a developmental signaling that regulates postnatal development of the cerebellum by promoting the mitotic expansion of granule neural precursors (GNPs) in the external granule layer (EGL). The abnormal SHH signaling pathway drives not only SHH-MB but also its cancer stem-like cells (SLCs), which represent a fraction of the tumor cell population that maintain cancer growth and have been associated with high grade tumors. Here, we report the first proteomic analysis of human SHH-MB SLCs before and after Retinoic Acid (RA)-induced differentiation. A total of 994 nLC-MS buckets were statistically analysed returning 68 modulated proteins between SLCs and their differentiated counterparts. Heat Shock Protein 70 (Hsp70) was one of the proteins that characterized the protein profile of SLCs. By means of Ingenuity Pathway Analysis (IPA), Genomatix analysis and extending the network obtained using the differentially expressed proteins we found a correlation between Hsp70 and the NF-κB complex. A key driver of the SHH-MB group is cMET whose downstream proliferation/survival signalling is indeed via PI3K/Akt/NF-κB. We confirmed the results of the proteomic analysis by western blot, underlining that a P-p65/NF-κB activatory complex is highly expressed in SLCs. Taking together these results we define a new protein feature of SHH-MB SLCs.

Loss of miR-107, miR-181c and miR-29a-3p Promote Activation of Notch2 Signaling in Pediatric High-Grade Gliomas (pHGGs)

The mechanisms by which microRNAs control pediatric high-grade gliomas (pHGGs) have yet to be fully elucidated. Our studies of patient-derived pHGG tissues and of the pHGG cell line KNS42 revealed down-regulation in these tumors of three microRNAs, specifically miR-107, miR-181c, and miR-29a-3p. This down-regulation increases the proliferation of KNS42 cells by de-repressing expression of the Notch2 receptor (Notch2), a validated target of miR-107 and miR-181c and a putative target of miR-29a-3p. Inhibition (either pharmacologic or genetic) of Notch2 or re-expression of the implicated microRNAs (all three combined but also individually) significantly reduced KNS42 cell proliferation. These findings suggest that Notch2 pathway activation plays a critical role in pHGGs growth and reveal a direct epigenetic mechanism that controls Notch2 expression, which could potentially be targeted by novel forms of therapy for these childhood tumors characterized by high-morbidity and high-mortality.