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.

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.

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.

Hedgehog signalling (Hh) is a developmental conserved pathway strongly involved in cancers when deregulated. This important pathway is orchestrated by numerous regulators, transduces through distinct routes and is finely tuned at multiple levels. In this regard, ubiquitylation processes stand as essential for controlling Hh pathway output. Although this post-translational modification governs proteins turnover, it is also implicated in non-proteolytic events, thereby regulating the most important cellular functions. The HECT E3 ligase Itch, well known to control immune response, is emerging to have a pivotal role in tumorigenesis. By illustrating Itch specificities on Hh signalling key components, here we review the role of this HECT E3 ubiquitin ligase in suppressing Hh-dependent tumours and explore its potential as promising target for innovative therapeutic approaches.

Background: Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration of the second motor neuron. The phenotype ranges from very severe to very mild forms. All patients have the homozygous loss of the SMN1 gene and a variable number of SMN2 (generally 2–4 copies), inversely related to the severity. The amazing results of the available treatments have made compelling the need of prognostic biomarkers to predict the progression trajectories of patients. Besides the SMN2 products, few other biomarkers have been evaluated so far, including some miRs.Methods: We performed whole miRNome analysis of muscle samples of patients and controls (14 biopsies and 9 cultures). The levels of muscle differentially expressed miRs were evaluated in serum samples (51 patients and 37 controls) and integrated with SMN2 copies, SMN2 full-length transcript levels in blood and age (SMA-score).Results: Over 100 miRs were differentially expressed in SMA muscle; 3 of them (hsa-miR-181a-5p, -324-5p, -451a; SMA-miRs) were significantly upregulated in the serum of patients. The severity predicted by the SMA-score was related to that of the clinical classification at a correlation coefficient of 0.87 (p<10^-5).Conclusions: miRNome analyses suggest the primary involvement of skeletal muscle in SMA pathogenesis. The SMA-miRs are likely actively released in the blood flow; their function and target cells require to be elucidated. The accuracy of the SMA-score needs to be verified in replicative studies: if confirmed, its use could be crucial for the routine prognostic assessment, also in presymptomatic patients.Funding: Telethon Italia (grant #GGP12116).

The Hedgehog (HH) pathway governs cell proliferation and patterning during embryonic development and is involved in regeneration, homeostasis and stem cell maintenance in adult tissues. The activity of this signaling is finely modulated at multiple levels and its dysregulation contributes to the onset of several human cancers. Ubiquitylation is a coordinated post-translational modification that controls a wide range of cellular functions and signaling transduction pathways. It is mediated by a sequential enzymatic network, in which ubiquitin ligases (E3) and deubiquitylase (DUBs) proteins are the main actors. The dynamic balance of the activity of these enzymes dictates the abundance and the fate of cellular proteins, thus affecting both physiological and pathological processes. Several E3 ligases regulating the stability and activity of the key components of the HH pathway have been identified. Further, DUBs have emerged as novel players in HH signaling transduction, resulting as attractive and promising drug targets. Here, we review the HH-associated DUBs, discussing the consequences of deubiquitylation on the maintenance of the HH pathway activity and its implication in tumorigenesis. We also report the recent progress in the development of selective inhibitors for the DUBs here reviewed, with potential applications for the treatment of HH-related tumors.