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In the tumor microenvironment (TME), ROS production affects survival, progression, and therapy resistance in colorectal cancer (CRC). H2O2-mediated oxidative stress can modulate Wnt/ß-catenin signaling and metabolic reprogramming of the TME. Currently, it is unclear how mild/moderate oxidative stress (eustress) modulates Wnt/ß-catenin/APC and JNK signaling relationships in primary and metastatic CRC cells. In this study, we determined the effects of the H2O2 concentration inducing eustress on isogenic SW480 and SW620 cells, also in combination with JNK inhibition. We assessed cell viability, mitochondrial respiration, glycolysis, and Wnt/ß-catenin/APC/JNK gene and protein expression. Primary CRC cells were more sensitive to H2O2 eustress combined with JNK inhibition, showing a reduction in viability compared to metastatic cells. JNK inhibition under eustress reduced both glycolytic and respiratory capacity in SW620 cells, indicating a greater capacity to adapt to TME. In primary CRC cells, H2O2 alone significantly increased APC, LEF1, LRP6, cMYC and IL8 gene expression, whereas in metastatic CRC cells, this effect occurred after JNK inhibition. In metastatic but not in primary tumor cells, eustress and inhibition of JNK reduced APC, ß-catenin, and pJNK protein. The results showed differential cross-regulation of Wnt/JNK in primary and metastatic tumor cells under environmental eustress conditions. Further studies would be useful to validate these findings and explore their therapeutic potential.

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Chronic Lymphocytic Leukemia (CLL) is the most common form of leukemia in adults, with a highly variable clinical course. Improvement in the knowledge of the molecular pathways behind this disease has led to the development of increasingly specific therapies, such as BCR signaling inhibitors and BCL-2 inhibitors. In this context, the emerging role of microRNAs (miRNAs) in CLL pathophysiology and their possible application in therapy is worth noting. MiRNAs are one of the most important regulatory molecules of gene expression. In CLL, they can act both as oncogenes and tumor suppressor genes, and the deregulation of specific miRNAs has been associated with prognosis, progression, and drug resistance. In this review, we describe the role of the miRNAs that primarily impact the disease, and how these miRNAs could be used as therapeutic tools. Certainly, the use of miRNAs in clinical practice is still limited in CLL. Many issues still need to be solved, particularly regarding their biological and safety profile, even if several studies have suggested their efficacy on the disease, alone or in combination with other drugs.

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Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

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The clinical progression of B cell chronic lymphocytic leukemia (CLL) is associated with immune cell dysfunction and a strong decrease of miR-181b-5p (miR-181b), promoting the death of CLL cells. Here we investigated whether the reduction of miR-181b impairs the immune response in CLL. We demonstrate that activated CD4+ T cells increase miR-181b expression in CLL through CD40-CD40L signaling, which enhances the maturation and activity of cytotoxic T cells and, consequently, the apoptotic response of CLL cells. The cytotoxic response is facilitated by a depletion of the anti-inflammatory cytokine interleukin 10, targeted by miR-181b. In vivo experiments in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice confirmed that miR-181b promotes the apoptotic death of CLL cells only when functional T cells are restored. Overall, our findings suggest that the reinstatement of miR-181b in CLL cells could be an exploitable adjuvant therapeutic option for the treatment of CLL.

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The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.

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Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths in the Western world. Despite progress made with targeted therapies and immune checkpoint inhibitors, the vast majority of patients have to undergo chemotherapy with platinum-based drugs. To increase efficacy and reduce potential side effects, a more comprehensive understanding of the mechanisms of the DNA damage response (DDR) is required. We have shown that overexpressby live cell imaging (Incuyion of the scaffold protein RAN binding protein 9 (RANBP9) is pervasive in NSCLC. More importantly, patients with higher levels of RANBP9 exhibit a worse outcome from treatment with platinum-based drugs. Mechanistically, RANBP9 exists as a target and an enabler of the ataxia telangiectasia mutated (ATM) kinase signaling. Indeed, the depletion of RANBP9 in NSCLC cells abates ATM activation and its downstream targets such as pby live cell imaging (Incuy53 signaling. RANBP9 knockout cells are more sensitive than controls to the inhibition of the ataxia and telangiectasia-related (ATR) kinase but not to ATM inhibition. The absence of RANBP9 renders cells more sensitive to drugs inhibiting the Poly(ADP-ribose)-Polymerase (PARP) resulting in a "BRCAness-like" phenotype. In summary, as a result of increased sensitivity to DNA damaging drugs conferred by its ablation in vitro and in vivo, RANBP9 may be considered as a potential target for the treatment of NSCLC. This article aims to report the results from past and ongoing investigations focused on the role of RANBP9 in the response to DNA damage, particularly in the context of NSCLC. This review concludes with future directions and speculative remarks which will need to be addressed in the coming years.

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Clonal evolution of chronic lymphocytic leukemia (CLL) often follows chemotherapy and is associated with adverse outcome, but also occurs in untreated patients, in which case its predictive role is debated. We investigated whether the selection and expansion of CLL clone(s) precede an aggressive disease shift. We found that clonal evolution occurs in all CLL patients, irrespective of the clinical outcome, but is faster during disease progression. In particular, changes in the frequency of nucleotide variants (NVs) in specific CLL-related genes may represent an indicator of poor clinical outcome.

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Aim: To investigate the genome-wide methylation of genetically characterized colorectal cancer stem cell (CR-CSC) lines. Materials & methods: Eight CR-CSC lines were isolated from primary colorectal cancer (CRC) tissues, cultured and characterized for aneuploidy, mutational status of CRC-related genes and microsatellite instability (MSI). Genome-wide DNA methylation was assessed by MethylationEPIC microarray. Results: We describe a distinctive methylation pattern that is maintained following in vivo passages in immune-compromised mice. We identified an epigenetic CR-CSC signature associated with MSI. We noticed that the preponderance of the differentially methylated positions do not reside at CpG islands, but spread to shelf and open sea regions. Conclusion: Given that CRCs with MSI-high status have a lower metastatic potential, the identification of a MSI-related methylation signature could provide new insights and possible targets into metastatic CRC.

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Aneuploidy and overexpression of hsa-miR-155-5p (miR-155) characterize most solid and hematological malignancies. We recently demonstrated that miR-155 sustains aneuploidy at early stages of in vitro cellular transformation. During in vitro transformation of normal human fibroblast, upregulation of miR-155 downregulates spindle checkpoint proteins as the mitotic checkpoint serine/threonine kinase budding uninhibited by benzimidazoles 1 (BUB1), the centromere protein F (CENPF) and the zw10 kinetochore protein (ZW10), compromising the chromosome alignment at the metaphase plate and leading to aneuploidy in daughter cells. Here we show that the heterogeneous nuclear ribonucleoprotein L (HNRNPL) binds to the polymorphic marker D2S1888 at the 3'UTR of BUB1 gene, impairs the miR-155 targeting, and restores BUB1 expression in chronic lymphocytic leukemia. This mechanism occurs at advanced passages of cell transformation and allows the expansion of more favorable clones. Our findings have revealed, at least in part, the molecular mechanisms behind the chromosomal stabilization of cell lines and the concept that, to survive, tumor cells cannot continuously change their genetic heritage but need to stabilize the most suitable karyotype.

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In this review, we propose that paraganglioma is a fundamentally organized, albeit aberrant, tissue composed of neoplastic vascular and neural cell types that share a common origin from a multipotent mesenchymal-like stem/progenitor cell. This view is consistent with the pseudohypoxic footprint implicated in the molecular pathogenesis of the disease, is in harmony with the neural crest origin of the paraganglia, and is strongly supported by the physiological model of carotid body hyperplasia. Our immunomorphological and molecular studies of head and neck paragangliomas demonstrate in all cases relationships between the vascular and the neural tumor compartments, that share mesenchymal and immature vasculo-neural markers, conserved in derived cell cultures. This immature, multipotent phenotype is supported by constitutive amplification of NOTCH signaling genes and by loss of the microRNA-200s and -34s, which control NOTCH1, ZEB1, and PDGFRA in head and neck paraganglioma cells. Importantly, the neuroepithelial component is distinguished by extreme mitochondrial alterations, associated with collapse of the ΔΨm. Finally, our xenograft models of head and neck paraganglioma demonstrate that mesenchymal-like cells first give rise to a vasculo-angiogenic network, and then self-organize into neuroepithelial-like clusters, a process inhibited by treatment with imatinib.

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Autoimmunity and hematological malignancies are often concomitant in patients. A causal bidirectional relationship exists between them. Loss of immunological tolerance with inappropriate activation of the immune system, likely due to environmental and genetic factors, can represent a breeding ground for the appearance of cancer cells and, on the other hand, blood cancers are characterized by imbalanced immune cell subsets that could support the development of the autoimmune clone. Considerable effort has been made for understanding the proteins that have a relevant role in both processes; however, literature advances demonstrate that microRNAs (miRNAs) surface as the epigenetic regulators of those proteins and control networks linked to both autoimmunity and hematological malignancies. Here we review the most up-to-date findings regarding the miRNA-based molecular mechanisms that underpin autoimmunity and hematological malignancies.

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The hsa-mir-483 gene, located within the IGF2 locus, transcribes for two mature microRNAs, miR-483-5p and miR-483-3p. This gene, whose regulation is mediated by the the CTNNB1/USF1 complex, shows an independent expression from its host gene IGF2. The miR-483-3p affects the Wnt/ß-catenin, the TGF-ß, and the TP53 signaling pathways by targeting several genes as CTNNB1, SMAD4, IGF1, and BBC3. Accordingly, miR-483-3p is associated with various tissues specific physiological properties as insulin and melanin production, as well as with cellular physiological functions such as wounding, differentiation, proliferation, and survival. Deregulation of miR-483-3p is observed in different types of cancer, and its overexpression can inhibit the pro-apoptotic pathway induced by the TP53 target effectors. As a result, the oncogenic characteristics of miR-483-3p are linked to the effect of some of the most relevant cancer-related genes, TP53 and CTNNB1, as well as to one of the most important cancer hallmark: the aberrant glucose metabolism of tumor cells. In this review, we summarize the recent findings regarding the miR-483-3p, to elucidate its functional role in physiological and pathological contexts, focusing overall on its involvement in cancer and in the TP53 pathway.

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Hsa-miR-155-5p (miR-155) is overexpressed in most solid and hematological malignancies. It promotes loss of genomic integrity in cancer cells by targeting genes involved in microsatellite instability and DNA repair; however, the link between miR-155 and aneuploidy has been scarcely investigated. Here we describe a novel mechanism by which miR-155 causes chromosomal instability. Using osteosarcoma cells (U2OS) and normal human dermal fibroblast (HDF), two well-established models for the study of chromosome congression, we demonstrate that miR-155 targets the spindle checkpoint proteins BUB1, CENP-F, and ZW10, thus compromising chromosome alignment at the metaphase plate. In U2OS cells, exogenous miR-155 expression reduced the recruitment of BUB1, CENP-F, and ZW10 to the kinetochores which resulted in defective chromosome congression. In contrast, during in vitro transformation of HDF by enforced expression of SV40 Large T antigen and human telomerase (HDFLT/hTERT), inhibition of miR-155 reduced chromosome congression errors and aneuploidy at early passages. Using live-cell imaging we observed that miR-155 delays progression through mitosis, indicating an activated mitotic spindle checkpoint, which likely fails to reduce aneuploidy. Overall, this study provides insight into a mechanism that generates aneuploidy at early stages of cellular transformation, pointing to a role for miR-155 in chromosomal instability at tumor onset.

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The given and family names of two co-authors were incorrect in the published article. The correct spelling should read as: Sampath Chandra Prasad and Vinagolu K Rajasekhar.

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The ability to reprogram the transcriptional circuitry by remodeling the three-dimensional structure of the genome is exploited by cancer cells to promote tumorigenesis. This reprogramming occurs because of hereditable chromatin chemical modifications and the consequent formation of RNA-protein-DNA complexes that represent the principal actors of the epigenetic phenomena. In this regard, the deregulation of a transcribed non-coding RNA may be both cause and consequence of a cancer-related epigenetic alteration. This review summarizes recent findings that implicate microRNAs in the aberrant epigenetic regulation of cancer cells.

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Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.

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