Aberrant activation of the Hedgehog (Hh) pathway leads to the development of several tumors, including medulloblastoma (MB), the most common pediatric brain malignancy. Hh inhibitors acting on GLI1, the final effector of Hh signaling, offer a valuable opportunity to overcome the pitfalls of the existing therapies to treat Hh-driven cancers. In this study, the toxicity, delivery, biodistribution, and anticancer efficacy of Glabrescione B (GlaB), a selective GLI1 inhibitor, were investigated in preclinical models of Hh-dependent MB. To overcome its poor water solubility, GlaB was formulated with a self-assembling amphiphilic polymer forming micelles, called mPEG5kDa-cholane. mPEG5kDa-cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG5kDa-cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG5kDa-cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG5kDa-cholane micelles was delivered through the blood-brain barrier and drastically inhibited tumor growth in both allograft and orthotopic models of Hh-dependent MB. Our findings reveal that mPEG5kDa-cholane/GlaB is a good candidate for the treatment of Hh-driven tumors and provide relevant implications for the translation of GlaB into clinical practice.

The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.

SHH Medulloblastoma (SHH-MB) is a pediatric brain tumor characterized by an inappropriate activation of the developmental Hedgehog (Hh) signaling. SHH-MB patients treated with the FDA-approved vismodegib, an Hh inhibitor that targets the transmembrane activator Smoothened (Smo), have shown the rapid development of drug resistance and tumor relapse due to novel Smo mutations. Moreover, a subset of patients did not respond to vismodegib because mutations were localized downstream of Smo. Thus, targeting downstream Hh components is now considered a preferable approach. We show here that selective inhibition of the downstream Hh effectors HDAC1 and HDAC2 robustly counteracts SHH-MB growth in mouse models. These two deacetylases are upregulated in tumor and their knockdown inhibits Hh signaling and decreases tumor growth. We demonstrate that mocetinostat (MGCD0103), a selective HDAC1/HDAC2 inhibitor, is a potent Hh inhibitor and that its effect is linked to Gli1 acetylation at K518. Of note, we demonstrate that administration of mocetinostat to mouse models of SHH-MB drastically reduces tumor growth, by reducing proliferation and increasing apoptosis of tumor cells and prolongs mouse survival rate. Collectively, these data demonstrate the preclinical efficacy of targeting the downstream HDAC1/2-Gli1 acetylation in the treatment of SHH-MB.

Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is a central player of Hh signalling, a pathway crucial for development and deregulated in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh signalling, our understanding of the mechanism regulating this key event remains limited. Here, we show that the Itch/β-arrestin2 complex binds SuFu and induces its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations occurring in medulloblastoma patients are insensitive to Itch activity, thus leading to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our findings uncover mechanisms controlling the tumour suppressive functions of SuFu and reveal that their alterations are implicated in medulloblastoma tumorigenesis.

Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule cell progenitors (GCPs) and its misregulation is linked to various disorders, including cerebellar cancer medulloblastoma. The effects of Shh pathway are mediated by the Gli family of transcription factors, which controls the expression of a number of target genes, including Gli1. Here, we identify Mastermind-like 1 (Maml1) as a novel regulator of the Shh signaling since it interacts with Gli proteins, working as a potent transcriptional coactivator. Notably, Maml1 silencing results in a significant reduction of Gli target genes expression, with a negative impact on cell growth of NIH3T3 and Patched1-/- mouse embryonic fibroblasts (MEFs), bearing a constitutively active Shh signaling. Remarkably, Shh pathway activity results severely compromised both in MEFs and GCPs deriving from Maml1-/- mice with an impairment of GCPs proliferation and cerebellum development. Therefore Maml1-/- phenotype mimics aspects of Shh pathway deficiency, suggesting an intrinsic requirement for Maml1 in cerebellum development. The present study shows a new role for Maml1 as a component of Shh signaling, which plays a crucial role in both development and tumorigenesis.

Inherited or somatic mutations in the MRE11, RAD50 and NBN genes increase the incidence of tumours, including medulloblastoma (MB). On the other hand, MRE11, RAD50 and NBS1 protein components of the MRN complex are often overexpressed and sometimes essential in cancer. In order to solve the apparent conundrum about the oncosuppressive or oncopromoting role of the MRN complex, we explored the functions of NBS1 in an MB-prone animal model.

Silymarin is the standardized extract from the fruits of Silybum marianum (L.) Gaertn., a well-known hepatoprotectant and antioxidant. Recently, bioactive compounds of silymarin, i.e., silybins and their 2,3-dehydro derivatives, have been shown to exert anticancer activities, yet with unclear mechanisms. This study combines in silico and in vitro methods to reveal the potential interactions of optically pure silybins and dehydrosilybins with novel protein targets. The shape and chemical similarity with approved drugs were evaluated in silico, and the potential for interaction with the Hedgehog pathway receptor Smoothened (SMO) and BRAF kinase was confirmed by molecular docking. In vitro studies on SMO and BRAF V600E kinase activity and in BRAF V600E A-375 human melanoma cell lines were further performed to examine their effects on these proteins and cancer cell lines and to corroborate computational predictions. Our in silico results direct to new potential targets of silymarin constituents as dual inhibitors of BRAF and SMO, two major targets in anticancer therapy. The experimental studies confirm that BRAF kinase and SMO may be involved in mechanisms of anticancer activities, demonstrating dose-dependent profiles, with dehydrosilybins showing stronger effects than silybins. The results of this work outline the dual SMO/BRAF effect of flavonolignans from Silybum marianum with potential clinical significance. Our approach can be applied to other natural products to reveal their potential targets and mechanism of action.

Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes.

: Pharmacological Hedgehog (Hh) pathway inhibition has emerged as a valuable anticancer strategy. A number of small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector glioma-associated oncogene 1 (Gli1) has been designed and developed. In a recent study, we exploited the high versatility of the natural isoflavone scaffold for targeting the Hh signaling pathway at multiple levels showing that the simultaneous targeting of Smo and Gli1 provided synergistic Hh pathway inhibition stronger than single administration. This approach seems to effectively overcome the drug resistance, particularly at the level of Smo. Here, we combined the pharmacophores targeting Smo and Gli1 into a single and individual isoflavone, compound 22, which inhibits the Hh pathway at both upstream and downstream level. We demonstrate that this multitarget agent suppresses medulloblastoma growth in vitro and in vivo through antagonism of Smo and Gli1, which is a novel mechanism of action in Hh inhibition.

Glioblastoma multiforme (GB) is the most malignant primary brain tumor in humans, with an overall survival of approximatively 15 months. The molecular heterogeneity of GB, as well as its rapid progression, invasiveness and the occurrence of drug-resistant cancer stem cells, limits the efficacy of the current treatments. In order to develop an innovative therapeutic strategy, it is mandatory to identify and characterize new molecular players responsible for the GB malignant phenotype. In this study, the RNA-binding ubiquitin ligase MEX3A was selected from a gene expression analysis performed on publicly available datasets, to assess its biological and still-unknown activity in GB tumorigenesis. We find that MEX3A is strongly up-regulated in GB specimens, and this correlates with very low protein levels of RIG-I, a tumor suppressor involved in differentiation, apoptosis and innate immune response. We demonstrate that MEX3A binds RIG-I and induces its ubiquitylation and proteasome-dependent degradation. Further, the genetic depletion of MEX3A leads to an increase of RIG-I protein levels and results in the suppression of GB cell growth. Our findings unveil a novel molecular mechanism involved in GB tumorigenesis and suggest MEX3A and RIG-I as promising therapeutic targets in GB.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated βTrCP, the substrate-receptor subunit of the SCFβTrCP ubiquitin ligase, and promotes βTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors.