Background: Facioscapulohumeral muscular dystrophy (FSHD) is associated with an epigenetic defect on 4qter. Two clinically indistinguishable forms of FSHD are known, FSHD1 and FSHD2. FSHD1 is caused by contraction of the highly polymorphic D4Z4 macrosatellite repeat array on chromosome 4q35. FSHD2 is caused by pathogenic mutations of the SMCHD1 gene. Both genetic defects lead to D4Z4 DNA hypomethylation. In the presence of a polymorphic polyadenylation signal (PAS), DNA hypomethylation leads to inappropriate expression of the D4Z4-encoded DUX4 transcription factor in skeletal muscle. Currently, hypomethylation is not diagnostic per se because of the interference of non-pathogenic arrays and the lack of information about the presence of DUX4-PAS. Methods: We investigated, by bisulfite sequencing, the DNA methylation levels of the region distal to the D4Z4 array selectively in PAS-positive alleles. Results: Comparison of FSHD1, FSHD2 and Control subjects showed a highly significant difference of methylation levels in all CpGs tested. Importantly, using a cohort of 112 samples, one of these CpGs (CpG6) is able to discriminate the affected individuals with a sensitivity of 0.95 supporting this assay potential for FSHD diagnosis. Moreover, our study showed a relationship between PAS-specific methylation and severity of the disease. Conclusions: These data point to the CpGs distal to the D4Z4 array as a critical region reflecting multiple factors affecting the epigenetics of FSHD. Additionally, methylation analysis of this region allows the establishment of a rapid and sensitive tool for FSHD diagnosis.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that estrogens counteract the differentiation impairment of FSHD myoblasts without affecting cell proliferation or survival. Estrogen effects are mediated by estrogen receptor β (ERβ), which reduces chromatin occupancy and transcriptional activity of double homeobox 4 (DUX4), a protein whose aberrant expression has been implicated in FSHD pathogenesis. During myoblast differentiation, we observed that the levels and activity of DUX4 increased progressively and were associated with its enhanced recruitment in the nucleus. ERβ interfered with this recruitment by relocalizing DUX4 in the cytoplasm. This work identifies estrogens as a potential disease modifier that underlie sex-related differences in FSHD by protecting against myoblast differentiation impairments in this disease.

Restoration of wild-type p53 tumor suppressor function has emerged as an attractive anticancer strategy. Therapeutics targeting the two p53-negative regulators, MDM2 and MDM4, have been developed, but most agents selectively target the ability of only one of these molecules to interact with p53, leaving the other free to operate. Therefore, we developed a method that targets the activity of MDM2 and MDM4 simultaneously based on recent studies indicating that formation of MDM2/MDM4 heterodimer complexes are required for efficient inactivation of p53 function. Using computational and mutagenesis analyses of the heterodimer binding interface, we identified a peptide that mimics the MDM4 C-terminus, competes with endogenous MDM4 for MDM2 binding, and activates p53 function. This peptide induces p53-dependent apoptosis in vitro and reduces tumor growth in vivo. Interestingly, interfering with the MDM2/MDM4 heterodimer specifically activates a p53-dependent oxidative stress response. Consistently, distinct subcellular pools of MDM2/MDM4 complexes were differentially sensitive to the peptide; nuclear MDM2/MDM4 complexes were particularly highly susceptible to the peptide-displacement activity. Taken together, these data identify the MDM2/MDM4 interaction interface as a valuable molecular target for therapeutic reactivation of p53 oncosuppressive function. Cancer Res; 75(21); 4560–72. ©2015 AACR.

The non-orthotopic expression of olfactory receptors (ORs) includes the male reproductive system, and in particular spermatozoa; their active ligands could be essential to sperm chemotaxis and chemical sperm–oocyte communication. OR51E2 expression has been previously reported on sperm cells’ surface. It has been demonstrated in different cellular models that olfactory receptor 51E2 (OR51E2) binds volatile short-chain fatty acids (SCFAs) as specific ligands. In the present research, we make use of Western blot, confocal microscopy colocalization analysis, and the calcium-release assay to demonstrate the activation of sperm cells through OR51E2 upon SCFAs stimulus. Moreover, we perform a novel modified swim-up assay to study the involvement of OR51E2/SCFAs in sperm migration. Taking advantage of computer-assisted sperm analysis (CASA system), we determine the kinematics parameters of sperm cells migrating towards SCFAs-enriched medium, revealing that these ligands are able to promote a more linear sperm-cell orientation. Finally, we obtain SCFAs by mass spectrometry in cervico-vaginal mucus and show for the first time that a direct incubation between cervical mucus and sperm cells could promote their activation. This study can shed light on the possible function of chemosensory receptors in successful reproduction activity, laying the foundation for the development of new strategies for the treatment of infertile individuals.

(1) Background: PTTG1 sustains the invasiveness of several cancer types. We previously reported that in seminomas, PTTG1 was detected in the peripheral area of the tumor and in the leading infiltrative edge. Here, we investigate the PTTG1 role on the invasive properties of seminoma. (2) Methods: three seminoma cell lines were used as in vitro model. PTTG1 levels and localization were investigated by biochemical and immunofluorescence analyses. Wound-healing, Matrigel invasion assays, and zymography were applied to study migratory and invasive capability of the cell lines. RNA interference and overexpression experiments were performed to address the PTTG1 role in seminoma invasiveness. PTTG1 and its target MMP-2 were analyzed in human testicular tumors using the Atlas database. (3) Results: PTTG1 was highly and differentially expressed in the seminoma cell lines. Nuclear PTTG1 was positively correlated to the aggressive phenotype. Its modulation confirms these results. Atlas database analysis revealed that PTTG1 was localized in the nucleus in seminoma compared with non-seminoma tumors, and that MMP-2 levels were significantly higher in seminomas. (4) Conclusions: nuclear PTTG1 promotes invasiveness of seminoma cell lines. Atlas database supported these results. These data lead to the hypothesis that nuclear PTTG1 is an eligible prognostic factor in seminomas.

Introduction: Restoration of the p53 tumor suppressor function is an attractive anticancer strategy. Despite the development of several therapeutics targeting the two main p53 negative regulators, MDM2 and MDM4, no one has yet reached clinical application. In the past, several efforts have been employed to develop more specific and efficient compounds that can improve and/or overcome some of the features related to small molecule compounds (SMC). Peptides and peptidomimetics are emerging as attractive molecules given their increased selectivity, reduced toxicity and reduced tendency to develop tumor-resistance compared to SMC. Area c overed: This article reviews publications and patents (publicly available up to April 2016) for peptides and derivatives aimed to reactivate the oncosuppressive function of p53, with a particular focus on inhibitors of MDM2/MDM4. Emphasis is placed on the efficacy of these compounds compared to the p53-reactivating small molecules developed so far. Expert o pinion: A number of promising peptides for p53 reactivation in cancer therapy have been developed. These compounds appear to possess improved features compared to SMC, especially for their ability to simultaneously target the MDM2/MDM4 inhibitors, and their increased specificity.