Aldo Pagano

45A non-coding RNA overexpression induces modifications to the neuroblastoma cell cytoskeleton, leading to a cascade of reactions that interfere with proliferation control, cell migration, and tumorigenic potential. Through real-time RT-PCR, Western blotting, and immunofluorescence analysis, we investigated the different expression and/or localization of GTSE1, MCAK, Aurora B, and p53 and the altered organization of tubulin in different NB cell models stably overexpressing or downregulating 45A ncRNA. The proper regulation of these proteins’ expression and function is fundamental in cytoskeleton organization, as their impairment leads to chromosomal instability. We demonstrate that 45A ncRNA not only directly regulates the expression of the aforementioned proteins but can also affect GTSE1 subcellular localization: in 45A-overexpressing cells, the protein is accumulated in nuclei, while 45A downregulation leads to significant GTSE1 cytoplasm relocation and simultaneous p53 cytoplasmatic sequestration. This shuttling of the oncosuppressor reduces the apoptotic potential of 45A-downregulating cells, explaining the observed resistance to toxoids. Furthermore, 45A overexpression leads to an increased number of abnormal spindles, thus promoting chromosomal instability and possibly explaining the increased tumorigenic potential exhibited by 45A-overexpressing cells. These data highlight the role of 45A ncRNA in the functional regulation of several proteins involved in microtubule dynamics, supporting its possible relevance in prognosis.

Human breast cancer (HBC) is the most common and often lethal malignancy in women. Canine mammary tumors (CMTs) share significant molecular and clinical characteristics with HBC, which makes dogs a valuable spontaneous model for the study of HBC. HBC chemotherapy treatment relies mainly on carboplatin, which is effective but, in turn, highly toxic. Here we tested enrofloxacin, a Minichromosomal Maintenance Complex Component (MCM2) inhibitor, for its ability to increase tumor cell sensitivity to platinum-based drugs, thus suggesting a potential synergistic therapeutic strategy. CMT samples were used to establish primary cell cultures. Cells were treated with carboplatin, enrofloxacin, and their combination at different concentrations. Cytotoxic and antiproliferative effects were assessed using xCELLigence and MTT assays. Single-drug treatments exert limited effects on cell proliferation, while enrofloxacin significantly enhances carboplatin efficacy, leading to a complete growth arrest within 48 h. The MTT assay confirms a strong synergistic effect of the two drugs, whereas the Dose Reduction Index analysis indicates that carboplatin could be decreased without losing effectiveness. These findings suggest that combined therapy could represent a more effective and less toxic option for HBC and CMTs. This work also strengthens the possible use of the canine model for cancer studies within a One Health framework.

Neoplastic transformation of mammalian cells involves intricate interactions between genetic, epigenetic and architecture modifications of the nucleus. Neuroblastoma is a malignant pediatric tumor with high biological and clinical heterogeneity representing a challenging model of study. We aimed to explore the changes in genome architecture and epigenetics being associated with neuroblastoma malignancy. We employed the neuroblastoma cell line SKNBE2 overexpressing the ncRNA NDM29 to differentiate from highly malignant into neuron-like cells. By 3D confocal microscopy, we explored the nuclear architecture (volume, elongation, compactness, and chromatin density). Using super-resolution microscopy (STED) and histone H3 immunolabelling we assessed the epigenetic rearrangement, and by enzyme-linked immunoassay the global DNA methylation. Then we assessed the mRNA expression of the main epigenetic modifying enzymes by quantitative PCR, and the expression of NF-κB-regulated genes by cDNA microarray. Compared to malignant NB cells, the NDM29-overexpressing cells, assuming a neuron-like phenotype, exhibited smaller and more elongated nuclei, redistribution of H3K9-acetylated and -methylated chromatin domains and DNA hypermethylation. In line with these results, in neuron-like cells the acetyltransferase KAT2A and the DNA methyltransferase DNMT1 were up-regulated, while most of NF-κB-regulated genes were down-regulated. Our findings reveal modifications of the nuclear structure and epigenome during neuroblastoma retro-transformation induced by NDM29 overexpression, with impacts on gene expression. These results offer potential insights into better understanding the mechanism of neuroblastoma malignancy in terms of chromatin rearrangements, opening exciting prospects for prognostic and therapeutic approaches with a focus on the nuclear level.

Obesity is associated with fatty liver disease. Available therapies show modest efficacy, and nutraceuticals with good effectiveness and safety are largely investigated. We focused on five natural compounds, three plant phenolic compounds (carvacrol, rosmarinic acid, silybin), and two thyroid hormones (T2: 3,5-diiodo-l-thyronine; T3: 3,5,3’-triiodo-L-thyronine) as comparison, to assess their beneficial effects on two cellular models of hepatosteatosis and adipogenesis. All compounds ameliorated the lipid accumulation and oxidative stress in both models, but with different potencies. The peroxisome proliferator-activated receptors (PPARs) are pivotal controllers of adipogenesis and lipid metabolism. For the main isoforms, PPARγ and PPARa, we assessed their possible binding to the compounds by molecular docking calculations, and their expression pattern by real-time PCR. All compounds bind both PPARs with different affinity, while not all compounds affect their expression. The results may clarify the distinctive molecular mechanisms underlying the action of the five compounds in the different cell models with possible applications to treat obesity.

Chromatin exhibits non-random distribution within the nucleus being arranged into discrete domains that are spatially organized throughout the nuclear space. Both the spatial distribution and structural rearrangement of chromatin domains in the nucleus depend on epigenetic modifications of DNA and/or histones and structural elements such as the nuclear envelope. These components collectively contribute to the organization and rearrangement of chromatin domains, thereby influencing genome architecture and functional regulation. This study develops an innovative, user-friendly, ImageJ-based plugin, called IsoConcentraChromJ, aimed quantitatively delineating the spatial distribution of chromatin regions in concentric patterns. The IsoConcentraChromJ can be applied to quantitative chromatin analysis in both two- and three-dimensional spaces. After DNA and histone staining with fluorescent probes, high-resolution images of nuclei have been obtained using advanced fluorescence microscopy approaches, including confocal and stimulated emission depletion (STED) microscopy. IsoConcentraChromJ workflow comprises the following sequential steps: nucleus segmentation, thresholding, masking, normalization, and trisection with specified ratios for either 2D or 3D acquisitions. The effectiveness of the IsoConcentraChromJ has been validated and demonstrated using experimental datasets consisting in nuclei images of pre-adipocytes and mature adipocytes, encompassing both 2D and 3D imaging. The outcomes allow to characterize the nuclear architecture by calculating the ratios between specific concentric nuclear areas/volumes of acetylated chromatin with respect to total acetylated chromatin and/or total DNA. The novel IsoConcentrapChromJ plugin could represent a valuable resource for researchers investigating the rearrangement of chromatin architecture driven by epigenetic mechanisms using nuclear images obtained by different fluorescence microscopy methods.

Neuroblastoma is the most frequent extracranial tumor in neonatal patients. Engineering SKNBE2 Myc-N amplified Neuroblastoma cell lines we generated individual clones expressing NDM29 non-coding-RNA at different levels. Using these cell models we documented a specific role of NDM29 in tumor cell differentiation leading to the identification of novel genes to be pharmacologically targeted for an efficient control of tumor malignancy (CA9 and MCM2 genes). Further studies taking advantage of the same models suggested the possible co-existence of different cell sub-populations in the apparently homogeneous cell culture with possible different roles in tumor maintenance and growth. Here we test this hypothesis by performing single-cell sequencing analysis and identified different sub-populations on the base of their gene expression patterns. The characterization of their specific profiles of membrane markers allowed an efficient separation of the individual populations by FACS analysis. Our preliminary results show the co-existance of cells with a marked neural phenotype with other two populations one VEGF secreting and the other VEGF recipient/responder. These preliminary findings suggest further detailed investigations of possible paracrine loops that might be at the base of tumor cell nodule growth and maintenance.Citation Format: Michele Tomanelli, Davide Ceresa, Adriana Bajetto, Paola Modesto, Paolo Malatesta, Tullio Florio, Aldo Pagano. Identification of cell sub-populations in Neuroblastoma cell lines with single-cell-sequencing analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 147. Neuroblastoma is the most frequent extracranial tumor in neonatal patients. Engineering SKNBE2 Myc-N amplified Neuroblastoma cell lines we generated individual clones expressing NDM29 non-coding-RNA at different levels. Using these cell models we documented a specific role of NDM29 in tumor cell differentiation leading to the identification of novel genes to be pharmacologically targeted for an efficient control of tumor malignancy (CA9 and MCM2 genes). Further studies taking advantage of the same models suggested the possible co-existence of different cell sub-populations in the apparently homogeneous cell culture with possible different roles in tumor maintenance and growth. Here we test this hypothesis by performing single-cell sequencing analysis and identified different sub-populations on the base of their gene expression patterns. The characterization of their specific profiles of membrane markers allowed an efficient separation of the individual populations by FACS analysis. Our preliminary results show the co-existance of cells with a marked neural phenotype with other two populations one VEGF secreting and the other VEGF recipient/responder. These preliminary findings suggest further detailed investigations of possible paracrine loops that might be at the base of tumor cell nodule growth and maintenance.Citation Format: Michele Tomanelli, Davide Ceresa, Adriana Bajetto, Paola Modesto, Paolo Malatesta, Tullio Florio, Aldo Pagano. Identification of cell sub-populations in Neuroblastoma cell lines with single-cell-sequencing analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 147.

Intracranial primary tumors (IPTs) are aggressive forms of malignancies that cause high mortality in both humans and domestic animals. Meningiomas are frequent adult IPTs in humans, dogs, and cats, and both benign and malignant forms cause a decrease in life quality and survival. Surgery is the primary therapeutic approach to treat meningiomas, but, in many cases, it is not resolutive. The chemotherapy and targeted therapy used to treat meningiomas also display low efficacy and many side effects. Therefore, it is essential to find novel pharmacological approaches to increase the spectrum of therapeutic options for meningiomas. This review analyzes the similarities between human and domestic animal (dogs and cats) meningiomas by evaluating the molecular and histological characteristics, diagnosis criteria, and treatment options and highlighting possible research areas to identify novel targets and pharmacological approaches, which are useful for the diagnosis and therapy of this neoplasia to be used in human and veterinary medicine.

Meningiomas are mainly benign brain tumors, although about 20% of histologically benign cases are clinically aggressive and recur after resection. We hypothesize that meningioma brain invasiveness and recurrence may be related to the presence of cancer stem cells and their high responsiveness to the CXCL12-CXCR4/CXCR7 chemokine axis. The aim of this study was to isolate meningioma stem cells from human samples, characterize them for biological features related to malignant behavior, and to identify the role of CXCR4/CXCR7 in these processes. Meningioma stem cells were isolated from patient-derived primary cultures in stem cell-permissive conditions, and characterized for phenotype, self-renewal, proliferation and migration rates, vasculogenic mimicry, and in vivo tumorigenesis, in comparison with differentiated meningioma cells and stem-like cells isolated from normal meninges. These cell populations were challenged with CXCL12 and CXCL11 and receptor antagonists to define the chemokine role in stem cell-related functions. Stem-like cells isolated from meningioma cultures display higher proliferation and migration rates, vasculogenic mimicry, as compared to meningioma non-stem cells or cells isolated from normal meninges and were the only tumorigenic population in vivo. In meningioma cells, these stem-like functions were under the control of the CXCR4/CXCR7 chemokine axis. We report a role for CXCL11 and CXCL12 in the control of malignant features in stem-like cells isolated from human meningioma, providing a possible basis for the aggressive clinical behavior observed in subsets of these tumors. CXCR4/CXCR7 antagonists might represent a useful approach for meningioma at high risk of recurrence and malignant progression.