Global Impairment of Immediate-Early Genes Expression in Rett Syndrome Models and Patients Linked to Myelination Defects.

Rett syndrome (RTT) is a severe neurodevelopmental disease caused almost exclusively by mutations to the MeCP2 gene. This disease may be regarded as a synaptopathy, with impairments affecting synaptic plasticity, inhibitory and excitatory transmission and network excitability. The complete understanding of the mechanisms behind how the transcription factor MeCP2 so profoundly affects the mammalian brain are yet to be determined. What is known, is that MeCP2 involvement in activity-dependent expression programs is a critical link between this protein and proper neuronal activity, which allows the correct maturation of connections in the brain. By using RNA-sequencing analysis, we found several immediate-early genes (IEGs, key mediators of activity-dependent responses) directly bound by MeCP2 at the chromatin level and upregulated in the hippocampus and prefrontal cortex of the Mecp2-KO mouse. Quantification of the IEGs response to stimulus both in vivo and in vitro detected an aberrant expression pattern in MeCP2-deficient neurons. Furthermore, altered IEGs levels were found in RTT patient's peripheral blood and brain regions of post-mortem samples, correlating with impaired expression of downstream myelination-related genes. Altogether, these data indicate that proper IEGs expression is crucial for correct synaptic development and that MeCP2 has a key role in the regulation of IEGs.

Comparative Proteomic Profiling of Secreted Extracellular Vesicles from Breast Fibroadenoma and Malignant Lesions: A Pilot Study.

Extracellular vesicles (EVs) shuttle proteins, RNA, DNA, and lipids crucial for cell-to-cell communication. Recent findings have highlighted that EVs, by virtue of their cargo, may also contribute to breast cancer (BC) growth and metastatic dissemination. Indeed, EVs are gaining great interest as non-invasive cancer biomarkers. However, little is known about the biological and physical properties of EVs from malignant BC lesions, and even less is understood about EVs from non-malignant lesions, such as breast fibroadenoma (FAD), which are clinically managed using conservative approaches. Thus, for this pilot study, we attempted to purify and explore the proteomic profiles of EVs from benign breast lesions, HER2+ BCs, triple-negative BCs (TNBCs), and continuous BC cell lines (i.e., BT-549, MCF-10A, and MDA-MB-231), combining experimental and semi-quantitative approaches. Of note, proteome-wide analyses showed 49 common proteins across EVs harvested from FAD, HER2+ BCs, TNBCs, and model BC lines. This is the first feasibility study evaluating the physicochemical composition and proteome of EVs from benign breast cells and primary and immortalized BC cells. Our preliminary results hold promise for possible implications in precision medicine for BC.

miR-216a Acts as a Negative Regulator of Breast Cancer by Modulating Stemness Properties and Tumor Microenvironment.

Breast cancer is the most frequent malignancy in females in terms of both incidence and mortality. Underlying the high mortality rate is the presence of cancer stem cells, which divide indefinitely and are resistant to conventional chemotherapies, so causing tumor relapse. In the present study, we identify miR-216a-5p as a downregulated microRNA in breast cancer stem cells vs. the differentiated counterpart. We demonstrate that overexpression of miR-216a-5p impairs stemness markers, mammosphere formation, ALDH activity, and the level of Toll-like receptor 4 (TLR4), which plays a significant role in breast cancer progression and metastasis by leading to the release of pro-inflammatory molecules, such as interleukin 6 (IL-6). Indeed, miR-216a regulates the crosstalk between cancer cells and the cells of the microenvironment, in particular cancer-associated fibroblasts (CAFs), through regulation of the TLR4/IL6 pathway. Thus, miR-216a has an important role in the regulation of stem phenotype, decreasing stem-like properties and affecting the cross-talk between cancer cells and the tumor microenvironment.

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-ß-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

The Role of Exo-miRNAs in Cancer: A Focus on Therapeutic and Diagnostic Applications.

Exosomes are extracellular vesicles released into biological fluids where they act as carriers of various molecules, including proteins, lipids, and RNAs, between cells, modulating or perturbing specific physiological processes. Recently, it has been suggested that tumoral cells release excessive amounts of exosomes that, through their cargo, promote tumor progression, stimulating growth, angiogenesis, metastasis, insensitivity to chemotherapy, and immune evasion. Increasing evidence highlights exosomal microRNAs (exo-miRNAs) as important players in tumorigenesis. MicroRNA (miRNA) are a class of small non-coding RNA able to regulate gene expression, targeting multiple mRNAs and inducing translational repression and/or mRNA degradation. Exo-miRNAs are highly stable and easily detectable in biological fluids, and for these reasons, miRNAs are potential cancer biomarkers useful diagnostically and prognostically. Furthermore, since exosomes are natural delivery systems between cells, they can be appropriately modified to carry therapeutic miRNAs to specific recipient cells. Here we summarize the main functions of exo-miRNAs and their possible role for diagnostic and therapeutic applications.

PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment.

PRUNE is a member of the DHH (Asp-His-His) phosphoesterase protein superfamily of molecules important for cell motility, and implicated in cancer progression. Here we investigated multiple families from Oman, India, Iran and Italy with individuals affected by a new autosomal recessive neurodevelopmental and degenerative disorder in which the cardinal features include primary microcephaly and profound global developmental delay. Our genetic studies identified biallelic mutations of PRUNE1 as responsible. Our functional assays of disease-associated variant alleles revealed impaired microtubule polymerization, as well as cell migration and proliferation properties, of mutant PRUNE. Additionally, our studies also highlight a potential new role for PRUNE during microtubule polymerization, which is essential for the cytoskeletal rearrangements that occur during cellular division and proliferation. Together these studies define PRUNE as a molecule fundamental for normal human cortical development and define cellular and clinical consequences associated with PRUNE mutation.

Exosomal microRNAs synergistically trigger stromal fibroblasts in breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC progression is sustained by recruitment of a strong tumor microenvironment (TME) mainly composed of cancer-associated fibroblasts (CAFs) able to endorse tumor hallmarks. Increasing evidences demonstrate that exosomes mediate the crosstalk between cancer cells and the TME. We examined TNBC-derived exosomes and their microRNA (miRNA) cargo in activation of normal fibroblasts (NFs) toward CAFs. We demonstrated that TNBC cell-derived exosomes increased NF collagen contraction and migration alongside CAF molecular markers. Exosome-activated fibroblasts promoted the invasion potential of normal breast epithelial cells, as assessed by an organotypic co-culture assay that resembled the in vivo context. We also investigated TNBC cell-derived exosome cargo in activating NFs to CAFs by performing small RNA sequencing. We found that the synergistic action of miR-185-5p, miR-652-5p, and miR-1246 boosted fibroblast migration and contraction, promoting specific CAF subspecialization toward a pro-migratory functional state. These data highlight the role of breast cancer cells in re-education of the TME and their contribution to tumor evolution.

Erratum: Exosomal microRNAs synergistically trigger stromal fibroblasts in breast cancer.

[This corrects the article DOI: 10.1016/j.omtn.2022.02.013.].

miR-34c-3p targets CDK1 a synthetic lethality partner of KRAS in non-small cell lung cancer.

Lung cancer is still the leading cause of death by cancer worldwide despite advances both in its detection and therapy. Multiple oncogenic driver alterations have been discovered, opening the prospective for new potential therapeutic targets. Among them, KRAS mutations represent the most frequent oncogene aberrations in non-small cell lung cancer (NSCLC) patients with a negative prognostic impact, but effective therapies targeting KRAS are not well characterized yet. Here, we demonstrate that the microRNA miR-34c-3p is a positive prognostic factor in KRAS-mutated NSCLC patients. Firstly, looking at the TGCA dataset, we found that high miR-34c-3p expression correlated with longer survival of KRAS-mutated NSCLC patients. In vitro assays on immortalized and patient-derived primary NSCLC cells revealed that miR-34c-3p overexpression increased apoptosis and lowered proliferation rate in KRASmut cells. Computational analysis and in vitro assays identified CDK1, one of the most promising lethal targets for KRAS-mutant cancer, as a target of miR-34c-3p. Moreover, the combination of CDK1 inhibition (mediated by RO3306) and miR-34c-3p overexpression resulted in an additive effect on the viability of KRASmut-expressing cells. Altogether, our findings demonstrate that miR-34c-3p is a novel biomarker that may allow tailored treatment for KRAS-mutated NSCLC patients.

Modulating the Crosstalk between the Tumor and the Microenvironment Using SiRNA: A Flexible Strategy for Breast Cancer Treatment.

Tumorigenesis is a complex and multistep process in which sequential mutations in oncogenes and tumor-suppressor genes result in enhanced proliferation and apoptosis escape. Over the past decades, several studies have provided evidence that tumors are more than merely a mass of malignant cancer cells, with the tumor microenvironment (TME) also contributing to cancer progression. For this reason, the focus of cancer research in recent years has shifted from the malignant cancer cell itself to the TME and its interactions. Since the TME actively participates in tumor progression, therapeutic strategies targeting it have created great interest. In this context, much attention has been paid to the potential application of small interfering RNA (siRNA), a class of non-coding RNA that has the ability to downregulate the expression of target genes in a sequence-specific way. This is paving the way for a novel therapeutic approach for the treatment of several diseases, including cancer. In this review, we describe recent efforts in developing siRNA therapeutics for the treatment of breast cancer, with particular emphasis on TME regulation. We focus on studies that adapt siRNA design to reprogram/re-educate the TME and eradicate the interplay between cancer cells and TME.

Targeting Ephrin Receptor Tyrosine Kinase A2 with a Selective Aptamer for Glioblastoma Stem Cells.

Despite the benefits associated with radiotherapy and chemotherapy for glioblastoma (GBM) treatment, most patients experience a relapse following initial therapy. Recurrent or progressive GBM usually does not respond anymore to standard therapy, and this is associated with poor patient outcome. GBM stem cells (GSCs) are a subset of cells resistant to radiotherapy and chemotherapy and play a role in tumor recurrence. The targeting of GSCs and the identification of novel markers are crucial issues in the development of innovative strategies for GBM eradication. By differential cell SELEX (systematic evolution of ligands by exponential enrichment), we have recently described two RNA aptamers, that is, the 40L sequence and its truncated form A40s, able to bind the cell surface of human GSCs. Both aptamers were selective for stem-like growing GBM cells and are rapidly internalized into target cells. In this study, we demonstrate that their binding to cells is mediated by direct recognition of the ephrin type-A receptor 2 (EphA2). Functionally, the two aptamers were able to inhibit cell growth, stemness, and migration of GSCs. Furthermore, A40s was able to cross the blood-brain barrier (BBB) and was stable in serum in in vitro experiments. These results suggest that 40L and A40s represent innovative potential therapeutic tools for GBM.