Laura Antonucci

Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation.

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.

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.

The coactivator CRTC1 promotes cell proliferation and transformation via AP-1

Regulation of gene expression in response to mitogenic stimuli is a critical aspect underlying many forms of human cancers. The AP-1 complex mediates the transcriptional response to mitogens, and its deregulation causes developmental defects and tumors. We report that the coactivator CRTC1 cyclic AMP response element-binding protein (CREB)-regulated transcription coactivator 1 is a potent and indispensable modulator of AP-1 function. After exposure of cells to the AP-1 agonist 12-O-tetradecanoylphorbol-13-acetate (TPA), CRTC1 is recruited to AP-1 target gene promoters and associates with c-Jun and c-Fos to activate transcription. CRTC1 consistently synergizes with the proto-oncogene c-Jun to promote cellular growth, whereas AP-1–dependent proliferation is abrogated in CRTC1-deficient cells. Remarkably, we demonstrate that CRTC1-Maml2 oncoprotein, which causes mucoepidermoid carcinomas, binds and activates both c-Jun and c-Fos. Consequently, ablation of AP-1 function disrupts the cellular transformation and proliferation mediated by this oncogene. Together, these data illustrate a novel mechanism required to couple mitogenic signals to the AP-1 gene regulatory program.

Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

The morphogenic Hedgehog (Hh) signaling regulates postnatal cerebellar development and its aberrant activation leads to medulloblastoma. The transcription factors Gli1 and Gli2 are the activators of Hh pathway and their function is finely controlled by different covalent modifications, such as phosphorylation and ubiquitination. We show here that Gli2 is endogenously acetylated and that this modification represents a key regulatory step for Hedgehog signaling. The histone acetyltransferase (HAT) coactivator p300, but not other HATs, acetylates Gli2 at the conserved lysine K757 thus inhibiting Hh target gene expression. By generating a specific anti acetyl-Gli2(Lys757) antisera we demonstrated that Gli2 acetylation is readily detectable at endogenous levels and is attenuated by Hh agonists. Moreover, Gli2 K757R mutant activity is higher than wild type Gli2 and is no longer enhanced by Hh agonists, indicating that acetylation represents an additional level of control for signal dependent activation. Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling. Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin. Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes.

Non-canonical Hedgehog/AMPK-Mediated Control of Polyamine Metabolism Supports Neuronal and Medulloblastoma Cell Growth

Developmental Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs), and its aberrant activation is a leading cause of medulloblastoma. We show here that Hedgehog promotes polyamine biosynthesis in GCPs by engaging a non-canonical axis leading to the translation of ornithine decarboxylase (ODC). This process is governed by AMPK, which phosphorylates threonine 173 of the zinc finger protein CNBP in response to Hedgehog activation. Phosphorylated CNBP increases its association with Sufu, followed by CNBP stabilization, ODC translation, and polyamine biosynthesis. Notably, CNBP, ODC, and polyamines are elevated in Hedgehog-dependent medulloblastoma, and genetic or pharmacological inhibition of this axis efficiently blocks Hedgehog-dependent proliferation of medulloblastoma cells in vitro and in vivo. Together, these data illustrate an auxiliary mechanism of metabolic control by a morphogenic pathway with relevant implications in development and cancer.

The energy sensor AMPK regulates Hedgehog signaling in human cells through a unique Gli1 metabolic checkpoint

Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs) and its aberrant activation is a leading cause of Medulloblastoma, the most frequent pediatric brain tumor. We show here that the energy sensor AMPK inhibits Hh signaling by phosphorylating a single residue of human Gli1 that is not conserved in other species. Studies with selective agonists and genetic deletion have revealed that AMPK activation inhibits canonical Hh signaling in human, but not in mouse cells. Indeed we show that AMPK phosphorylates Gli1 at the unique residue Ser408, which is conserved only in primates but not in other species. Once phosphorylated, Gli1 is targeted for proteasomal degradation. Notably, we show that selective AMPK activation inhibits Gli1-driven proliferation and that this effect is linked to Ser408 phosphorylation, which represents a key metabolic checkpoint for Hh signaling. Collectively, this data unveil a novel mechanism of inhibition of Gli1 function, which is exclusive for human cells and may be exploited for the treatment of Medulloblastoma or other Gli1 driven tumors.

CNBP regulates wing development in Drosophila melanogaster by promoting IRES-dependent translation of dMyc

CCHC-type zinc finger nucleic acid binding protein (CNBP) is a small conserved protein, which plays a key role in development and disease. Studies in animal models have shown that the absence of CNBP results in severe developmental defects that have been mostly attributed to its ability to regulate c-myc mRNA expression. Functionally, CNBP binds single-stranded nucleic acids and acts as a molecular chaperone, thus regulating both transcription and translation. In this work we report that in Drosophila melanogaster, CNBP is an essential gene, whose absence causes early embryonic lethality. In contrast to what observed in other species, ablation of CNBP does not affect dMyc mRNA expression, whereas the protein levels are markedly reduced. We demonstrate for the first time that dCNBP regulates dMyc translation through an IRES-dependent mechanism, and that knockdown of dCNBP in the wing territory causes a general reduction of wing size, in keeping with the reported role of dMyc in this region. Consistently, reintroduction of dMyc in CNBP-deficient wing imaginal discs rescues the wing size, further supporting a key role of the CNBP-Myc axis in this context. Collectively, these data show a previously uncharacterized mechanism, whereby, by regulating dMyc IRES-dependent translation, CNBP controls Drosophila wing development. These results may have relevant implications in other species and in pathophysiological conditions.

Non-Alcoholic Fatty Liver Disease and Nutritional Implications: Special Focus on Copper

Non-alcoholic fatty liver disease (NAFLD) is characterized by excess lipids in hepatocytes, due to excessive fatty acid influx from adipose tissue, de novo hepatic lipogenesis, in addition to excessive dietary fat and carbohydrate intake. Chronic hepatic lipid overload induces mitochondrial oxidative stress and cellular damage leading the development of NAFLD into a more severe liver disease condition, non-alcoholic steato-hepatitis (NASH). In turn, this can progress to cirrhosis and hepatocellular carcinoma (HCC). Among others, copper is one of the main bio-metals required for the preponderance of the enzymes involved in physiological redox reactions, which primarily occurs during mitochondrial respiration. Thus, copper homeostasis could be considered a target point for counteracting the progression of NAFLD. Accordingly, many diseases are correlated to unbalanced copper levels and, actually, some clinical trials are examining the use of copper chelating agents. Currently, no pharmacological interventions are approved for NAFLD, but nutritional and lifestyle modifications are always recommended. Fittingly, antioxidant food agents recognized to improve NAFLD and its complications have been described in the literature to bind copper. Therefore, this review describes the role of nutrition in the development and progression of NAFLD with a particular focus on copper and copper-binding antioxidant compounds against NAFLD.

Copper/MYC/CTR1 interplay: a dangerous relationship in hepatocellular carcinoma

Free serum copper correlates with tumor incidence and progression of human cancers, including hepatocellular carcinoma (HCC). Copper extracellular uptake is provided by the transporter CTR1, whose expression is regulated to avoid excessive intracellular copper entry. Inadequate copper serum concentration is involved in the pathogenesis of Non Alcoholic Fatty Liver Disease (NAFLD), which is becoming a major cause of liver damage progression and HCC incidence. Finally, MYC is over-expressed in most of HCCs and is a critical regulator of cellular growth, tumor invasion and metastasis. The purpose of our study was to understand if higher serum copper concentrations might be involved in the progression of NAFLD-cirrhosis toward-HCC. We investigated whether high exogenous copper levels sensitize liver cells to transformation and if it exists an interplay between copper-related proteins and MYC oncogene. NAFLD-cirrhotic patients were characterized by a statistical significant enhancement of serum copper levels, even more evident in HCC patients. We demonstrated that high extracellular copper concentrations increase cell growth, migration, and invasion of liver cancer cells by modulating MYC/CTR1 axis. We highlighted that MYC binds a specific region of the CTR1 promoter, regulating its transcription. Accordingly, CTR1 and MYC proteins expression were progressively up-regulated in liver tissues from NAFLD-cirrhotic to HCC patients. This work provides novel insights on the molecular mechanisms by which copper may favor the progression from cirrhosis to cancer. The Cu/MYC/CTR1 interplay opens a window to refine HCC diagnosis and design new combined therapies.

β-arrestin1-mediated acetylation of Gli1 regulates Hedgehog/Gli signaling and modulates self-renewal of SHH medulloblastoma cancer stem cells

BackgroundAberrant Sonic Hedgehog/Gli (Hh/Gli) signaling pathway is a critical regulator of Sonic hedgehog medulloblastoma (SHH-MB). Cancer stem cells (CSCs), thought to be largely responsible for tumor initiation, maintenance, dissemination and relapse, have been identified in SHH-MB. Since we previously demonstrated that Hh/Gli signaling controls CSCs features in SHH-MB and that in these tumors miR-326 is down regulated, here we investigated whether there is a functional link between Hh/Gli signaling and miR-326.MethodsWe evaluated β-arrestin1 (Arrb1) and its intragenic miR-326 levels in CSCs derived from SHH-MB. Subsequently, we modulated the expression of Arrb1 and miR-326 in CSCs in order to gain insight into their biological role. We also analyzed the mechanism by which Arrb1 and miR-326 control Hh/Gli signaling and self-renewal, using luciferase and protein immunoprecipitation assays.ResultsLow levels of Arrb1 and miR-326 represent a feature of CSCs derived from SHH-MB. We observed that re-expression of Arrb1 and miR-326 inhibits Hh/Gli signaling pathway at multiple levels, which cause impaired proliferation and self-renewal, accompanied by down regulation of Nanog levels. In detail, miR-326 negatively regulates two components of the Hh/Gli pathway the receptor Smoothened (Smo) and the transcription factor Gli2, whereas Arrb1 suppresses the transcriptional activity of Gli1, by potentiating its p300-mediated acetylation.ConclusionsOur results identify a new molecular mechanism involving miR-326 and Arrb1 as regulators of SHH-MB CSCs. Specifically, low levels of Arrb1 and miR-326 trigger and maintain Hh/Gli signaling and self-renewal.