Hartinger R; Lederer EM; Schena E; Lattanzi G; Djabali K
In: Cells, vol. 12, iss. 10, pp. 1350, 2023.
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease that causes premature aging symptoms, such as vascular diseases, lipodystrophy, loss of bone mineral density, and alopecia. HGPS is mostly linked to a heterozygous and de novo mutation in the LMNA gene (c.1824 C > T; p.G608G), resulting in the production of a truncated prelamin A protein called "progerin". Progerin accumulation causes nuclear dysfunction, premature senescence, and apoptosis. Here, we examined the effects of baricitinib (Bar), an FDA-approved JAK/STAT inhibitor, and a combination of Bar and lonafarnib (FTI) treatment on adipogenesis using skin-derived precursors (SKPs). We analyzed the effect of these treatments on the differentiation potential of SKPs isolated from pre-established human primary fibroblast cultures. Compared to mock-treated HGPS SKPs, Bar and Bar + FTI treatments improved the differentiation of HGPS SKPs into adipocytes and lipid droplet formation. Similarly, Bar and Bar + FTI treatments improved the differentiation of SKPs derived from patients with two other lipodystrophic diseases: familial partial lipodystrophy type 2 (FPLD2) and mandibuloacral dysplasia type B (MADB). Overall, the results show that Bar treatment improves adipogenesis and lipid droplet formation in HGPS, FPLD2, and MADB, indicating that Bar + FTI treatment might further ameliorate HGPS pathologies compared to lonafarnib treatment alone.
Frittoli E; Palamidessi A; Iannelli F; Zanardi F; Villa S; Barzaghi L; Abdo H; Cancila V; Beznoussenko GV; Della Chiara G; Pagani M; Malinverno C; Bhattacharya D; Pisati F; Yu W; Galimberti V; Bonizzi G; Martini E; Mironov AA; Gioia U; Ascione F; Li Q; Havas K; Magni S; Lavagnino Z; Pennacchio FA; Maiuri P; Caponi S; Mattarelli M; Martino S; d'Adda di Fagagna F; Rossi C; Lucioni M; Tancredi R; Pedrazzoli P; Vecchione A; Petrini C; Ferrari F; Lanzuolo C; Bertalot G; Nader G; Foiani M; Piel M; Cerbino R; Giavazzi F; Tripodo C; Scita G
In: Nature materials, vol. 22, iss. 5, pp. 644-655, 2023.
The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.
De Giuseppe R; Di Napoli I; Tomasinelli CE; Vincenti A; Biino G; Sommella E; Ferron L; Campiglia P; Ferrara F; Casali PM; Cena H
In: Journal of nutrition health & aging, vol. 27, iss. 6, no 463-471, 2023.
Background: Camelina sativa oil is one of the richest dietary sources of omega-3, with polyunsaturated fatty acids amounts of over 50%, linolenic acid content of around 40-45%, and linoleic acid of about 15%. Moreover, this oil is a valuable source of antioxidants which provide oxidative stability. All those features raise interest in considering Camelina oil as an alternative and sustainable oil source providing stable omega-3-rich emulsions for functional food production. Objectives: The present study aimed to investigate the effects of Camelina oil-enriched crackers on serum omega-3 concentration, inflammatory markers and serum lipid profile. Design: Randomized placebo-controlled pilot trial. Setting: Research and Development Center (Complife Italia s.r.l.). Participants: Sixty-six free-living older volunteers (aged≥65 years). Intervention: Older adults were enrolled and randomly assigned to one of two groups: the camelina group or the placebo group. Subjects consumed daily 35 g of crackers (Camelina enriched crackers or placebo ones) twice daily for 12 weeks. Measurements: Serum polyunsaturated fatty acid profile, inflammatory status and serum lipid panel parameters were recorded pre and post-intervention. Results: In the camelina group, alpha-linolenic acid serum concentration was significantly higher (p<0.01) compared to the placebo group at the end of the study. Concerning inflammatory plasma markers, a significant mean pro-inflammatory interleukin-18 plasma concentration decrease in the placebo group compared to the camelina one was observed (p<0.05). No significant differences in other mean inflammatory markers concentrations post-intervention were noted in either group. Lastly, examining the change in lipid profile, it is noteworthy that a higher reduction of total cholesterol, low-density lipoprotein and triglycerides in the camelina group post-intervention, despite the lack of statistical significance. Conclusion: Camelina oil significantly elevated the serum alpha-linolenic acid concentration with no significant changes in inflammatory markers and lipid profile. Trial registration: ClinicalTrials.gov NCT04965948.
Lauriola A; Davalli P; Marverti G; Santi S; Caporali A; D'Arca D
In: Cancers-Basel, vol. 15, iss. 11, pp. 3009, 2023.
Immunotherapy is a cancer treatment that exploits the capacity of the body's immune system to prevent, control, and remove cancer. Immunotherapy has revolutionized cancer treatment and significantly improved patient outcomes for several tumor types. However, most patients have not benefited from such therapies yet. Within the field of cancer immunotherapy, an expansion of the combination strategy that targets independent cellular pathways that can work synergistically is predicted. Here, we review some consequences of tumor cell death and increased immune system engagement in the modulation of oxidative stress and ubiquitin ligase pathways. We also indicate combinations of cancer immunotherapies and immunomodulatory targets. Additionally, we discuss imaging techniques, which are crucial for monitoring tumor responses during treatment and the immunotherapy side effects. Finally, the major outstanding questions are also presented, and directions for future research are described.
Gambarotto L; Metti S; Corpetti M; Baraldo M; Sabatelli P; Castagnaro S; Cescon M; Blaauw B; Bonaldo P
In: Autophagy, Forthcoming.
COL6 (collagen type VI)-related myopathies (COL6-RM) are a distinct group of inherited muscle disorders caused by mutations of COL6 genes and characterized by early-onset muscle weakness, for which no cure is available yet. Key pathophysiological features of COL6-deficient muscles involve impaired macroautophagy/autophagy, mitochondrial dysfunction, neuromuscular junction fragmentation and myofiber apoptosis. Targeting autophagy by dietary means elicited beneficial effects in both col6a1 null (col6a1-/-) mice and COL6-RM patients. We previously demonstrated that one-month per os administration of the nutraceutical spermidine reactivates autophagy and ameliorates myofiber defects in col6a1-/- mice but does not elicit functional improvement. Here we show that a 100-day-long spermidine regimen is able to rescue muscle strength in col6a1-/- mice, with also a beneficial impact on mitochondria and neuromuscular junction integrity, without any noticeable side effects. Altogether, these data provide a rationale for the application of spermidine in prospective clinical trials for COL6-RM.Abbreviations: AChR: acetylcholine receptor; BTX: bungarotoxin; CNF: centrally nucleated fibers; Colch: colchicine; COL6: collagen type VI; COL6-RM: COL6-related myopathies; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NMJ: neuromuscular junction; Spd: spermidine; SQSTM1/p62: sequestosome 1; TA: tibialis anterior; TOMM20: translocase of outer mitochondrial membrane 20; TUNEL: terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling.
Maraventano G; Ticli G; Cazzalini O; Stivala LA; Ramos-Gonzalez M; Rodríguez JL; Prosperi E
In: Molecules, vol. 28, iss. 11, pp. 4326, 2023.
An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). Although several methods are available for the biochemical analysis of this molecule, its determination at the single cell level may provide significant advantages when investigating the influence of cell heterogeneity and cell type in the DNA damage response. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detection with the glycoprotein avidin has also been proposed because of a structural similarity between its natural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliability and sensitivity is not clear. In this study, we compared the immunofluorescence determination of 8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugated with the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different cell types by treatment with potassium bromate (KBrO3), a chemical inducer of reactive oxygen species (ROS). By using increasing concentrations of KBrO3, as well as different reaction conditions, our results indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greater than that attained with avidin-AF488. These findings suggest that immunofluorescence techniques are best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage.
Choudhary R; Niska-Blakie J; Adhil M; Liberi G; Achar YJ; Giannattasio M; Foiani M
In: Cell reports, vol. 42, iss. 7, pp. 112747, 2023.
Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetics, genomics, and transmission electron microscopy, we find that Rrm3hPif1 and Sen1hSenataxin helicases assist termination at TERs; Sen1 specifically acts at telomeres. rrm3 and sen1 genetically interact and fail to terminate replication, exhibiting fragility at termination zones (TERs) and telomeres. sen1rrm3 accumulates RNA-DNA hybrids and X-shaped gapped or reversed converging forks at TERs; sen1, but not rrm3, builds up RNA polymerase II (RNPII) at TERs and telomeres. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of Top1 and Top2 when forks encounter transcription head on or codirectionally, respectively, thus preventing the slowing down of DNA and RNA polymerases. Hence Rrm3 and Sen1 are indispensable to generate permissive topological conditions for replication termination.
Gioia U; Tavella S; Martínez-Orellana P; Cicio G; Colliva A; Ceccon M; Cabrini M; Henriques AC; Fumagalli V; Paldino A; Presot E; Rajasekharan S; Iacomino N; Pisati F; Matti V; Sepe S; Conte MI; Barozzi S; Lavagnino Z; Carletti T; Volpe MC; Cavalcante P; Iannacone M; Rampazzo C; Bussani R; Tripodo C; Zacchigna S; Marcello A; d'Adda di Fagagna F
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs' biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.
Zhu G; Khalid F; Zhang D; Cao Z; Maity P; Kestler HA; Orioli D; Scharffetter-Kochanek K; Iben S
In: Cells, vol. 12, iss. 14, pp. 1877, 2023.
Mutations in a broad variety of genes can provoke the severe childhood disorder trichothiodystrophy (TTD) that is classified as a DNA repair disease or a transcription syndrome of RNA polymerase II. In an attempt to identify the common underlying pathomechanism of TTD we performed a knockout/knockdown of the two unrelated TTD factors TTDN1 and RNF113A and investigated the consequences on ribosomal biogenesis and performance. Interestingly, interference with these TTD factors created a nearly uniform impact on RNA polymerase I transcription with downregulation of UBF, disturbed rRNA processing and reduction of the backbone of the small ribosomal subunit rRNA 18S. This was accompanied by a reduced quality of decoding in protein translation and the accumulation of misfolded and carbonylated proteins, indicating a loss of protein homeostasis (proteostasis). As the loss of proteostasis by the ribosome has been identified in the other forms of TTD, here we postulate that ribosomal dysfunction is a common underlying pathomechanism of TTD.
Salucci S; Aramini B; Bartoletti-Stella A; Versari I; Martinelli G; Blalock WL; Stella F; Faenza I
In: Cancers-Basel, vol. 15, iss. 12, pp. 3245, 2023.
Lung cancer (LC) is the second most common neoplasm in men and the third most common in women. In the last decade, LC therapies have undergone significant improvements with the advent of immunotherapy. However, the effectiveness of the available treatments remains insufficient due to the presence of therapy-resistant cancer cells. For decades, chemotherapy and radiotherapy have dominated the treatment strategy for LC; however, relapses occur rapidly and result in poor survival. Malignant lung tumors are classified as either small- or non-small-cell lung carcinoma (SCLC and NSCLC). Despite improvements in the treatment of LC in recent decades, the benefits of surgery, radiotherapy, and chemotherapy are limited, although they have improved the prognosis of LC despite the persistent low survival rate due to distant metastasis in the late stage. The identification of novel prognostic molecular markers is crucial to understand the underlying mechanisms of LC initiation and progression. The potential role of phosphatidylinositol in tumor growth and the metastatic process has recently been suggested by some researchers. Phosphatidylinositols are lipid molecules and key players in the inositol signaling pathway that have a pivotal role in cell cycle regulation, proliferation, differentiation, membrane trafficking, and gene expression. In this review, we discuss the current understanding of phosphoinositide-specific phospholipase enzymes and their emerging roles in LC.
Dominik N; Magri S; Currò R; Abati E; Facchini S; Corbetta M; MacPherson H; Di Bella D; Sarto E; Stevanovski I; Chintalaphani SR; Akcimen F; Manini A; Vegezzi E; Quartesan I; Montgomery KA; Pirota V; Crespan E; Perini C; Grupelli GP; Tomaselli PJ; Marques W; Genomics England Research Consortium; Shaw J; Polke J; Salsano E; Fenu S; Pareyson D; Pisciotta C; Tofaris GK; Nemeth AH; Ealing J; Radunovic A; Kearney S; Kumar KR; Vucic S; Kennerson M; Reilly MM; Houlden H; Deveson I; Tucci A; Taroni F; Cortese A
Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis Journal Article Forthcoming
In: Brain, Forthcoming.
Cerebellar Ataxia, Neuropathy and Vestibular Areflexia Syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing (WGS) data from nearly 10,000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n=6 from 5 families), AAGGC (n=2 from 1 family), AGAGG (n=1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, here we show a pathogenic role for large AAAGG repeat configuration expansions (n=5). Long read sequencing was used to fully characterise the entire repeat sequence and revealed a pure AGGGC expansion in six patients, whereas the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs seem to have arisen from a common haplotype and are predicted to form highly stable G quadruplexes, which have been previously demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions, since the AAAGG motif when very large (>500 repeats) or in the presence of AAGGG interruptions. Accurate sizing and full sequencing of the satellite repeat with long read is recommended in clinically selected cases, in order to achieve an accurate molecular diagnosis and counsel patients and their families.
Abdel-Shafy EA; Melak T; MacIntyre DA; Zadra G; Zerbini LF; Piazza S; Cacciatore S
In: Bioinformatics advances, vol. 3, iss. 1, 2023.
Computational analysis and interpretation of metabolomic profiling data remains a major challenge in translational research. Exploring metabolic biomarkers and dysregulated metabolic pathways associated with a patient phenotype could offer new opportunities for targeted therapeutic intervention. Metabolite clustering based on structural similarity has the potential to uncover common underpinnings of biological processes. To address this need, we have developed the MetChem package. MetChem is a quick and simple tool that allows to classify metabolites in structurally related modules, thus revealing their functional information.
Ferrigno A; Campagnoli LIM; Barbieri A; Marchesi N; Pascale A; Croce AC; Vairetti M; Di Pasqua LG
In: International journal of molecular sciences, vol. 24, iss. 12, pp. 9808, 2023.
The endogenous antioxidant defense plays a big part in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), a common metabolic disorder that can lead to serious complications such as cirrhosis and cancer. HuR, an RNA-binding protein of the ELAV family, controls, among others, the stability of MnSOD and HO-1 mRNA. These two enzymes protect the liver cells from oxidative damage caused by excessive fat accumulation. Our aim was to investigate the expression of HuR and its targets in a methionine-choline deficient (MCD) model of NAFLD. To this aim, we fed male Wistar rats with an MCD diet for 3 and 6 weeks to induce NAFLD; then, we evaluated the expression of HuR, MnSOD, and HO-1. The MCD diet induced fat accumulation, hepatic injury, oxidative stress, and mitochondrial dysfunction. A HuR downregulation was also observed in association with a reduced expression of MnSOD and HO-1. Moreover, the changes in the expression of HuR and its targets were significantly correlated with oxidative stress and mitochondrial injury. Since HuR plays a protective role against oxidative stress, targeting this protein could be a therapeutic strategy to both prevent and counteract NAFLD.
Attardo GM; Benoit JB; Michalkova V; Kondragunta A; Baumann AA; Weiss BL; Malacrida A; Scolari F; Aksoy S
In: iScience, vol. 26, iss. 7, pp. 107108, 2023.
Lipid metabolism is critical for insect reproduction, especially for species that invest heavily in the early developmental stages of their offspring. The role of symbiotic bacteria during this process is understudied but likely essential. We examined the role of lipid metabolism during the interaction between the viviparous tsetse fly (Glossina morsitans morsitans) and its obligate endosymbiotic bacteria (Wigglesworthia glossinidia) during tsetse pregnancy. We observed increased CTP:phosphocholine cytidylyltransferase (cct1) expression during pregnancy, which is critical for phosphatidylcholine biosynthesis in the Kennedy pathway. Experimental removal of Wigglesworthia impaired lipid metabolism via disruption of the Kennedy pathway, yielding obese mothers whose developing progeny starve. Functional validation via experimental cct1 suppression revealed a phenotype similar to females lacking obligate Wigglesworthia symbionts. These results indicate that, in Glossina, symbiont-derived factors, likely B vitamins, are critical for the proper function of both lipid biosynthesis and lipolysis to maintain tsetse fly fecundity.
Chetta P; Sriram R; Zadra G
In: Cancers - Basel, vol. 15, iss. 13, pp. 3473, 2023.
Advanced prostate cancer represents the fifth leading cause of cancer death in men worldwide. Although androgen-receptor signaling is the major driver of the disease, evidence is accumulating that disease progression is supported by substantial metabolic changes. Alterations in de novo lipogenesis and fatty acid catabolism are consistently reported during prostate cancer development and progression in association with androgen-receptor signaling. Therefore, the term "lipogenic phenotype" is frequently used to describe the complex metabolic rewiring that occurs in prostate cancer. However, a new scenario has emerged in which lactate may play a major role. Alterations in oncogenes/tumor suppressors, androgen signaling, hypoxic conditions, and cells in the tumor microenvironment can promote aerobic glycolysis in prostate cancer cells and the release of lactate in the tumor microenvironment, favoring immune evasion and metastasis. As prostate cancer is composed of metabolically heterogenous cells, glycolytic prostate cancer cells or cancer-associated fibroblasts can also secrete lactate and create "symbiotic" interactions with oxidative prostate cancer cells via lactate shuttling to sustain disease progression. Here, we discuss the multifaceted role of lactate in prostate cancer progression, taking into account the influence of the systemic metabolic and gut microbiota. We call special attention to the clinical opportunities of imaging lactate accumulation for patient stratification and targeting lactate metabolism.
Lodola C; Secchi M; Sinigiani V; De Palma A; Rossi R; Perico D; Mauri PL; Maga G
In: International journal of molecular sciences, vol. 24, iss. 6, pp. 5784, 2023.
The nucleocapsid protein Np of SARS-CoV-2 is involved in the replication, transcription, and packaging of the viral genome, but it also plays a role in the modulation of the host cell innate immunity and inflammation response. Ectopic expression of Np alone was able to induce significant changes in the proteome of human cells. The cellular RNA helicase DDX1 was among the proteins whose levels were increased by Np expression. DDX1 and its related helicase DDX3X were found to physically interact with Np and to increase 2- to 4-fold its affinity for double-stranded RNA in a helicase-independent manner. Conversely, Np inhibited the RNA helicase activity of both proteins. These functional interactions among Np and DDX1 and DDX3X highlight novel possible roles played by these host RNA helicases in the viral life cycle.
Immunoelectron Microscopy Methods Journal Article
In: Methods in molecular biology, vol. 2655, pp. 201-210, 2023.
"Immunoelectron microscopy" defines a group of techniques developed for visualizing where components of cells or tissues are localized, by means of a transmission electron microscope (TEM) at a subcellular resolution. The method is based on antigen recognition by primary antibodies and subsequent visualization of recognized structures by means of electron-opaque gold granules, which are easily visible in TEM images. The potentially high resolution of this method relies on the very small size of the colloidal gold label, which consists of granules ranging from 1 to 60 nm in diameter, mostly used in the 5-15 nm sizes.
Lecca M; Bedeschi MF; Izzi C; Dordoni C; Rinaldi B; Peluso F; Caraffi SG; Prefumo F; Signorelli M; Zanzucchi M; Bione S; Ghigna C; Sassi S; Novelli A; Valente EM; Superti-Furga A; Garavelli L; Errichiello E
In: Clinical genetics, vol. 104, iss. 2, pp. 230, 2023.
Spondylocostal dysostosis (SCD), a condition characterized by multiple segmentation defects of the vertebrae and rib malformations, is caused by bi-allelic variants in one of the genes involved in the Notch signaling pathway that tunes the ""segmentation clock"" of somitogenesis: DLL3, HES7, LFNG, MESP2, RIPPLY2, and TBX6. To date, seven individuals with LFNG variants have been reported in the literature. In this study we describe two newborns and one fetus with SCD, who were found by trio-based exome sequencing (trio-ES) to carry homozygous (c.822-5C>T) or compound heterozygous (c.[863dup];[1063G>A]) and (c.[521G>T];[890T>G]) variants in LFNG. Notably, the c.822-5C>T change, affecting the polypyrimidine tract of intron 5, is the first non-coding variant reported in LFNG. This study further refines the clinical and molecular features of spondylocostal dysostosis 3 and adds to the numerous investigations supporting the usefulness of trio-ES approach in prenatal and neonatal settings.
Contadini C; Cirotti C; Carbone A; Norouzi M; Cianciusi A; Crespan E; Perini C; Maga G; Barilà D; Musumeci F; Schenone S
In: Pharmaceuticals - Basel, vol. 16, iss. 7, pp. 958, 2023.
Src is a non-receptor tyrosine kinase (TK) whose involvement in cancer, including glioblastoma (GBM), has been extensively demonstrated. In this context, we started from our in-house library of pyrazolo[3,4-d]pyrimidines that are active as Src and/or Bcr-Abl TK inhibitors and performed a lead optimization study to discover a new generation derivative that is suitable for Src kinase targeting. We synthesized a library of 19 compounds, 2a-s. Among these, compound 2a (SI388) was identified as the most potent Src inhibitor. Based on the cell-free results, we investigated the effect of SI388 in 2D and 3D GBM cellular models. Interestingly, SI388 significantly inhibits Src kinase, and therefore affects cell viability, tumorigenicity and enhances cancer cell sensitivity to ionizing radiation.
Jones CY; Williams CL; Moreno SP; Morris DK; Mondello C; Karlseder J; Bertuch AA
Hyperextended telomeres promote formation of C-circle DNA in telomerase positive human cells Journal Article Forthcoming
In: Journal of biological chemistry, Forthcoming.
Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). Currently, the primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. We investigated C-circle formation in the human cen3tel cell line, a long-telomere, telomerase+ (LTT+) cell line with progressively hyper-elongated telomeres (up to ∼100 kb). cECTR signal was observed in 2D gels and C-circle assays but not t-circle assays, which also detect cECTRs. Telomerase activity and C-circle signal were not separable in the analysis of clonal populations, consistent with C-circle production occurring within telomerase+ cells. We observed similar cECTR results in two other LTT+ cell lines, HeLa1.3 (∼23 kb telomeres) and HeLaE1 (∼50 kb telomeres). In LTT+ cells, telomerase activity did not directly impact C-circle signal; instead, C-circle signal correlated with telomere length. LTT+ cell lines were less sensitive to hydroxyurea than ALT+ cell lines, suggesting that ALT status is a stronger contributor to replication stress levels than telomere length. Additionally, the DNA repair-associated protein FANCM did not suppress C-circles in LTT+ cells as it does in ALT+ cells. Thus, C-circle formation may be driven by telomere length, independently of telomerase and replication stress, highlighting limitations of C-circles as a stand-alone ALT biomarker.
Faris P; Negri S; Faris D; Scolari F; Montagna D; Moccia F
In: Current medicinal chemistry, vol. 30, iss. 40, pp. 4506-4532, 2023.
Hydrogen sulfide (H2S) is an endogenous gaseous molecule present in all living organisms and has been traditionally studied for its toxicity. Interestingly, increased understanding of H2S effects in organ physiology has recently shown its relevance as a signalling molecule, with potentially important implications in variety of clinical disorders, including cancer. H2S is primarily produced in mammalian cells under various enzymatic pathways. A developing focus of H2S is a blooming hotspot that studies chemical, biological mechanisms, and therapeutic effects of H2S. Herein, we describe the physiological and biochemical properties of H2S, the enzymatic pathways leading to its endogenous production and its catabolic routes. In addition, we discuss the role of currently known H2S-releasing agents, or H2S donors, including their potential as therapeutic tools. Then we illustrate the mechanisms known to support the pleiotropic effects of H2S, with a particular focus on persulfhydration, which plays a key role in H2S-mediating signalling pathways. We then address the paradoxical role played by H2S in tumour biology and discuss the potential of exploiting H2S levels as novel cancer biomarkers and diagnostic tools. Finally, we describe the most recent preclinical applications focused on assessing the anti-cancer impact of most common H2S-releasing compounds. While the evidence in favour of H2S as an alternative cancer therapy in the field of translational medicine is yet to be clearly provided, application of H2S is emerging as potent anticancer therapies in preclinical trails.
Croce AC; Ferrigno A; Palladini G; Mannucci B; Vairetti M; Di Pasqua LG
In: Molecules, vol. 28, iss. 9, pp. 3818, 2023.
The autofluorescence of specific fatty acids, retinoids, and bilirubin in crude serum can reflect changes in liver functional engagement in maintaining systemic metabolic homeostasis. The role of these fluorophores as intrinsic biomarkers of pharmacological actions has been investigated here in rats administered with obeticholic acid (OCA), a Farnesoid-X Receptor (FXR) agonist, proven to counteract the increase of serum bilirubin in hepatic ischemia/reperfusion (I/R) injury. Fluorescence spectroscopy has been applied to an assay serum collected from rats submitted to liver I/R (60/60 min ± OCA administration). The I/R group showed changes in the amplitude and profiles of emission spectra excited at 310 or 366 nm, indicating remarkable alterations in the retinoid and fluorescing fatty acid balance, with a particular increase in arachidonic acid. The I/R group also showed an increase in bilirubin AF, detected in the excitation spectra recorded at 570 nm. OCA greatly reversed the effects observed in the I/R group, confirmed by the biochemical analysis of bilirubin and fatty acids. These results are consistent with a relationship between OCA anti-inflammatory effects and the acknowledged roles of fatty acids as precursors of signaling agents mediating damaging responses to harmful stimuli, supporting serum autofluorescence analysis as a possible direct, real-time, cost-effective tool for pharmacological investigations.
Di Martino A; Cescon M; D'Agostino C; Schilardi F; Sabatelli P; Merlini L; Faldini C
Collagen VI in the Musculoskeletal System Journal Article
In: International journal of molecular sciences, vol. 24, iss. 6, pp. 5095, 2023.
Collagen VI exerts several functions in the tissues in which it is expressed, including mechanical roles, cytoprotective functions with the inhibition of apoptosis and oxidative damage, and the promotion of tumor growth and progression by the regulation of cell differentiation and autophagic mechanisms. Mutations in the genes encoding collagen VI main chains, COL6A1, COL6A2 and COL6A3, are responsible for a spectrum of congenital muscular disorders, namely Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM), which show a variable combination of muscle wasting and weakness, joint contractures, distal laxity, and respiratory compromise. No effective therapeutic strategy is available so far for these diseases; moreover, the effects of collagen VI mutations on other tissues is poorly investigated. The aim of this review is to outline the role of collagen VI in the musculoskeletal system and to give an update about the tissue-specific functions revealed by studies on animal models and from patients' derived samples in order to fill the knowledge gap between scientists and the clinicians who daily manage patients affected by collagen VI-related myopathies.
Croce AC; Scolari F
In: Biology-Basel, vol. 12, iss. 3, pp. 433, 2023.
Melanin is present in various biological substrates where it may participate in several processes, from innate immunity to the still-unsolved opposite roles in antioxidant protection, including photoprotection and the related ability to interact with light. Melanin-light interaction has also been an important source of inspiration for the development of innovative bioengineering applications. These are based on melanin's light-energy-absorption ability of its chemically and structurally complex components and precursors, and on the improvement in analytical and diagnostic procedures in biomedicine. In this regard, here, we characterized the fluorescence spectral properties of melanin and of its precursor L-tyrosine in an aqueous solution during spontaneous melanization. Besides the confirmation of the typical fluorescence-emission signature of melanin and L-tyrosine, we provide additional insights on both emission and excitation spectra recorded during melanization. On these bases, we performed a subsequent characterization on the aqueous extracts from two different melanin-containing biological substrates, namely hairs from a domestic black cat and eggs from the Asian tiger mosquito. The results from the mild extraction procedure, purposely applied to obtain only the soluble components, combined with fluorescence spectral analysis are expected to promote further investigation of the melanization processes, particularly in insects.
Fontana B; Gallerani G; Salamon I; Pace I; Roncarati R; Ferracin M
ARID1A in cancer: Friend or foe? Journal Article
In: Frontiers in Oncology, vol. 13, pp. 1136248, 2023.
ARID1A belongs to a class of chromatin regulatory proteins that function by maintaining accessibility at most promoters and enhancers, thereby regulating gene expression. The high frequency of ARID1A alterations in human cancers has highlighted its significance in tumorigenesis. The precise role of ARID1A in cancer is highly variable since ARID1A alterations can have a tumor suppressive or oncogenic role, depending on the tumor type and context. ARID1A is mutated in about 10% of all tumor types including endometrial, bladder, gastric, liver, biliopancreatic cancer, some ovarian cancer subtypes, and the extremely aggressive cancers of unknown primary. Its loss is generally associated with disease progression more often than onset. In some cancers, ARID1A loss is associated with worse prognostic features, thus supporting a major tumor suppressive role. However, some exceptions have been reported. Thus, the association of ARID1A genetic alterations with patient prognosis is controversial. However, ARID1A loss of function is considered conducive for the use of inhibitory drugs which are based on synthetic lethality mechanisms. In this review we summarize the current knowledge on the role of ARID1A as tumor suppressor or oncogene in different tumor types and discuss the strategies for treating ARID1A mutated cancers.
Milosevic E; Stanisavljevic N; Boskovic S; Stamenkovic N; Novkovic M; Bavelloni A; Cenni V; Kojic S; Jasnic J
Antitumor activity of natural pigment violacein against osteosarcoma and rhabdomyosarcoma cell lines Journal Article Forthcoming
In: Journal of cancer research and clinical oncology, Forthcoming.
Purpose: Sarcomas are rare and heterogenic tumors with unclear etiology. They develop in bone and connective tissue, mainly in pediatric patients. To increase efficacy of current therapeutic options, natural products showing selective toxicity to tumor cells are extensively investigated. Here, we evaluated antitumor activity of bacterial pigment violacein in osteosarcoma (OS) and rhabdomyosarcoma (RMS) cell lines. Methods: The toxicity of violacein was assessed in vitro and in vivo, using MTT assay and FET test. The effect of violacein on cell migration was monitored by wound healing assay, cell death by flow cytometry, uptake of violacein by fluorescence microscopy, generation of reactive oxygen species (ROS) by DCFH-DA assay and lipid peroxidation by TBARS assay. Results: Violacein IC50 values for OS and RMS cells were in a range from 0.35 to 0.88 µM. Its selectivity toward malignant phenotype was confirmed on non-cancer V79-4 cells, and it was safe in vivo, for zebrafish embryos in doses up to 1 µM. Violacein induced apoptosis and affected the migratory potential of OS and RMS cells. It was found on the surfaces of tested cells. Regarding the mechanism of action, violacein acted on OS and RMS cells independently of oxidative signaling, as judged by no increase in intracellular ROS generation and no lipid peroxidation. Conclusion: Our study provided further evidence that reinforces the potential of violacein as an anticancer agent and candidate to consider for improvement of the effectiveness of traditional OS and RMS therapies.
Piazzi M; Bavelloni A; Salucci S; Faenza I; Blalock WL
In: Genes, vol. 14, iss. 7, pp. 1386, 2023.
The advent of next generation sequencing (NGS) has fostered a shift in basic analytic strategies of a gene expression analysis in diverse pathologies for the purposes of research, pharmacology, and personalized medicine. What was once highly focused research on individual signaling pathways or pathway members has, from the time of gene expression arrays, become a global analysis of gene expression that has aided in identifying novel pathway interactions, the discovery of new therapeutic targets, and the establishment of disease-associated profiles for assessing progression, stratification, or a therapeutic response. But there are significant caveats to this analysis that do not allow for the construction of the full picture. The lack of timely updates to publicly available databases and the "hit and miss" deposition of scientific data to these databases relegate a large amount of potentially important data to "garbage", begging the question, "how much are we really missing?" This brief perspective aims to highlight some of the limitations that RNA binding/modifying proteins and RNA processing impose on our current usage of NGS technologies as relating to cancer and how not fully appreciating the limitations of current NGS technology may negatively affect therapeutic strategies in the long run.
Di Matteo A; Belloni E; Pradella D; Chiaravalli AM; Pini GM; Bugatti M; Alfieri R; Barzan C; Franganillo Tena E; Bione S; Terenzani E; Sessa F; Wyatt CDR; Vermi W; Ghigna C
In: International journal of molecular sciences, vol. 24, iss. 9, pp. 8102, 2023.
Angiogenesis is crucial for cancer progression. While several anti-angiogenic drugs are in use for cancer treatment, their clinical benefits are unsatisfactory. Thus, a deeper understanding of the mechanisms sustaining cancer vessel growth is fundamental to identify novel biomarkers and therapeutic targets. Alternative splicing (AS) is an essential modifier of human proteome diversity. Nevertheless, AS contribution to tumor vasculature development is poorly known. The Neuro-Oncological Ventral Antigen 2 (NOVA2) is a critical AS regulator of angiogenesis and vascular development. NOVA2 is upregulated in tumor endothelial cells (ECs) of different cancers, thus representing a potential driver of tumor blood vessel aberrancies. Here, we identified novel AS transcripts generated upon NOVA2 upregulation in ECs, suggesting a pervasive role of NOVA2 in vascular biology. In addition, we report that NOVA2 is also upregulated in ECs of gastric cancer (GC), and its expression correlates with poor overall survival of GC patients. Finally, we found that the AS of the Rap Guanine Nucleotide Exchange Factor 6 (RapGEF6), a newly identified NOVA2 target, is altered in GC patients and associated with NOVA2 expression, tumor angiogenesis, and poor patient outcome. Our findings provide a better understanding of GC biology and suggest that AS might be exploited to identify novel biomarkers and therapeutics for anti-angiogenic GC treatments.
Starace M; Pampaloni F; Bruni F; Quadrelli F; Cedirian S; Baraldi C; Misciali C; Di Martino A; Sabatelli P; Merlini L; Piraccini BM
In: International journal of molecular sciences, vol. 24, iss. 7, pp. 6678, 2023.
Collagen VI-related myopathies are characterized by severe muscle involvement and skin involvement (keratosis pilaris and impaired healing with the development of abnormal scars, especially keloids). Scalp involvement and hair loss have not been reported among cutaneous changes associated with collagen VI mutations. The aim of this study is to describe the clinical, trichoscopic, and histological findings of the scalp changes in patients affected by COL VI mutations and to estimate their prevalence. Patients with Ullrich congenital muscular dystrophy were enrolled and underwent clinical and trichoscopic examinations and a scalp biopsy for histopathology. Five patients were enrolled, and all complained of hair loss and scalp itching. One patient showed yellow interfollicular scales with erythema and dilated, branched vessels, and the histological findings were suggestive of scalp psoriasis. Two patients presented with scarring alopecia patches on the vertex area, and they were histologically diagnosed with folliculitis decalvans. The last two patients presented with scaling and hair thinning, but they were both diagnosed with folliculitis and perifolliculitis. Ten more patients answered to a "scalp involvement questionnaire", and six of them confirmed to have or have had scalp disorders and/or itching. Scalp involvement can be associated with COL VI mutations and should be investigated.
Khalid F; Phan T; Qiang M; Maity P; Lasser T; Wiese S; Penzo M; Alupei M; Orioli D; Scharffetter-Kochanek K; Iben S
In: Human molecular genetics, vol. 32, iss. 7, pp. 1102-1113, 2023.
TFIIH is a complex essential for transcription of protein-coding genes by RNA polymerase II, DNA repair of UV-lesions and transcription of rRNA by RNA polymerase I. Mutations in TFIIH cause the cancer prone DNA-repair disorder xeroderma pigmentosum (XP) and the developmental and premature aging disorders trichothiodystrophy (TTD) and Cockayne syndrome (CS). 50% of TTD cases are caused by TFIIH mutations. Using TFIIH mutant patient cells from TTD and XP subjects we can show that the stress-sensitivity of the proteome is reduced in TTD, but not in XP. Using three different methods to investigate the accuracy of protein synthesis by the ribosome, we demonstrate that translational fidelity of the ribosomes of TTD, but not XP cells, is decreased. The process of ribosomal synthesis and maturation is affected in TTD cells and can lead to instable ribosomes. Isolated ribosomes from TTD patients show an elevated error rate when challenged with oxidized mRNA, explaining the oxidative hypersensitivity of TTD cells. Treatment of TTD cells with N-acetyl cysteine normalized the increased translational error-rate and restored translational fidelity. Here we describe a pathomechanism that might be relevant for our understanding of impaired development and aging-associated neurodegeneration.
Musolf AM; Haarman AEG; Luben RN; Ong JS; Patasova K; Trapero RH; Marsh J; Jain I; Jain R; Wang PZ; Lewis DD; Tedja MS; Iglesias AI; Li H; Cowan CS; Consortium for Refractive Error; Myopia (CREAM); Biino G; Klein AP; Duggal P; Mackey DA; Hayward C; Haller T; Metspalu A; Wedenoja J; Parssinen O; Cheng CY; Saw SM; Stambolian D; Hysi PG; Khawaja AP; Vitart V; Hammond CJ; van Duijn CM; Verhoeven VJM; Klaver CCW; Bailey-Wilson JE.
In: Communications biology, vol. 6, iss. 1, pp. 6, 2023.
Refractive error, measured here as mean spherical equivalent (SER), is a complex eye condition caused by both genetic and environmental factors. Individuals with strong positive or negative values of SER require spectacles or other approaches for vision correction. Common genetic risk factors have been identified by genome-wide association studies (GWAS), but a great part of the refractive error heritability is still missing. Some of this heritability may be explained by rare variants (minor allele frequency [MAF] ≤ 0.01.). We performed multiple gene-based association tests of mean Spherical Equivalent with rare variants in exome array data from the Consortium for Refractive Error and Myopia (CREAM). The dataset consisted of over 27,000 total subjects from five cohorts of Indo-European and Eastern Asian ethnicity. We identified 129 unique genes associated with refractive error, many of which were replicated in multiple cohorts. Our best novel candidates included the retina expressed PDCD6IP, the circadian rhythm gene PER3, and P4HTM, which affects eye morphology. Future work will include functional studies and validation. Identification of genes contributing to refractive error and future understanding of their function may lead to better treatment and prevention of refractive errors, which themselves are important risk factors for various blinding conditions.
Storci G; Barbato F; Ricci F; Tazzari PL; De Matteis S; Tomassini E; Dicataldo M; Laprovitera N; Arpinati M; Ursi M; Maffini E; Campanini E; Dan E; Manfroi S; Santi S; Ferracin M; Bonafe M; Bonifazi F
In: Frontiers in immunology, vol. 13, 2023.
Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Rabbit anti-T lymphocyte globulin (ATLG) in addition to calcineurin inhibitors and antimetabolites is a suitable strategy to prevent GVHD in several transplant settings. Randomized studies already demonstrated its efficacy in terms of GVHD prevention, although the effect on relapse remains the major concern for a wider use. Tailoring of ATLG dose on host characteristics is expected to minimize its side effects (immunological reconstitution, relapse, and infections). Here, day -6 to day +15 pharmacokinetics of active ATLG serum level was first assayed in an explorative cohort of 23 patients by testing the ability of the polyclonal serum to bind antigens on human leukocytes. Significantly lower levels of serum active ATLG were found in the patients who developed GVHD (ATLG_AUCCD45: 241.52 ± 152.16 vs. 766.63 +/- 283.52 (μg*day)/ml, p = 1.46e-5). Consistent results were obtained when the ATLG binding capacity was assessed on CD3+ and CD3+/CD4+ T lymphocytes (ATLG_AUCCD3: 335.83 ± 208.15 vs. 903.54 ± 378.78 (μg*day)/ml, p = 1.92e-4; ATLG_AUCCD4: 317.75 ± 170.70 vs. 910.54 ± 353.35 (μg*day)/ml, p = 3.78e-5. Concomitantly, at pre-infusion time points, increased concentrations of CD69+ extracellular vesicles (EVs) were found in patients who developed GVHD (mean fold 9.01 ± 1.33; p = 2.12e-5). Consistent results were obtained in a validation cohort of 12 additional ATLG-treated HSCT patients. Serum CD69+ EVs were mainly represented in the nano (i.e. 100 nm in diameter) EV compartment and expressed the leukocyte marker CD45, the EV markers CD9 and CD63, and CD103, a marker of tissue-resident memory T cells. The latter are expected to set up a host pro-inflammatory cell compartment that can survive in the recipient for years after conditioning regimen and contribute to GVHD pathogenesis. In summary, high levels of CD69+ EVs are significantly correlated with an increased risk of GVHD, and they may be proposed as a tool to tailor ATLG dose for personalized GVHD prevention.
Sgritta M; Vignoli B; Pimpinella D; Griguoli M; Santi S; Bialowas A; Wiera G; Zacchi P; Malerba F; Marchetti C; Canossa M; Cherubini E
In: iScience, vol. 26, iss. 1, pp. 105728, 2023.
In Neurodevelopmental Disorders, alterations of synaptic plasticity may trigger structural changes in neuronal circuits involved in cognitive functions. This hypothesis was tested in mice carrying the human R451C mutation of Nlgn3 gene (NLG3R451C KI), found in some families with autistic children. To this aim, the spike time dependent plasticity (STDP) protocol was applied to immature GABAergic Mossy Fibers (MF)-CA3 connections in hippocampal slices from NLG3R451C KI mice. These animals failed to exhibit STD-LTP, an effect that persisted in adulthood when these synapses became glutamatergic. Similar results were obtained in mice lacking the Nlgn3 gene (NLG3 KO mice), suggesting a loss of function. The loss of STD-LTP was associated with a premature shift of GABA from the depolarizing to the hyperpolarizing direction, a reduced BDNF availability and TrkB phosphorylation at potentiated synapses. These effects may constitute a general mechanism underlying cognitive deficits in those forms of Autism caused by synaptic dysfunctions.
Mazzini G; Danova M
In: Marco Biggiogera Carlo Pellicciari, Manuela Malatesta (Ed.): vol. 2556, pp. 1-25, Humana New York, NY, Histochemistry of Single Molecules Methods and Protocols, 2023.
For over half a century, fluorescence has been the milestone of most of the quantitative approaches in various fields from chemistry and biochemistry to microscopy. This latter also evolved into cytometry, thanks to the development of fluorescence techniques. The dyes of classical cytochemistry were replaced by fluorochromes, and the pioneer microphotometry was replaced by microfluorometry. The latter has great advantages in terms of simplicity, sensitivity, and accuracy. The extensive research and availability of new fluorochromes as well as the technological evolution contributed to the success of microfluorometry. The development of flow cytometry in the 1960s gave a giant boost to cell analysis and in particular to the clinical diagnostics. The synergy between flow cytometry and the subsequent development of monoclonal antibodies allowed the setup of multiparametric analytical panels that are today popular and irreplaceable in many clinical and research laboratories. Multiparametric analysis has required the application of an increasing number of fluorochromes, but their simultaneous use creates problems of mutual contamination, hence the need to develop new fluorescent probes. Semiconductor and nanotechnology research enabled the development of new probes called nanocrystals or quantum dots, which offered great advantages to the multiparametric analysis: in fact, thanks to their spectrofluorometric peculiarities, dozens of quantum dots may be simultaneously used without appreciable crosstalk between them. New analytical horizons in cytometry seem to be associated with a new concept of analysis that replaces fluorescence toward new markers with (non-radiative) isotopes of heavy metals. Thus, the mass flow cytometry was born, which seems to guarantee the simultaneous compensation-free analysis of up to 100 markers on a single sample aliquot.
Ma C; Zhou Y; Fanelli GN; Stopsack KH; Fiorentino M; Zadra G; Mucci LA; Loda M; Tyekucheva S; Penney KL
In: Molecular cancer research, vol. 21, iss. 3, pp. 253-260, 2023.
Prostate cancer has a heterogeneous prognosis. Most previous studies have focused on the identification of prognostic biomarkers in the prostate cancer tumor. However, it is increasingly recognized that the tumor microenvironment contributes to prostate cancer aggressiveness and progression. We therefore examined whole transcriptome expression of the prostate stroma and associations with aggressive and lethal prostate cancer. We performed RNA sequencing (Illumina TruSeq Exome Capture) of 272 tumor-adjacent and 120 benign-adjacent macrodissected prostate stromal samples from 293 men with prostate cancer from the Health Professionals Follow-up Study and Physicians' Health Study. We performed differential expression analysis comparing gene expression and pathways by Gleason score and lethal outcome. We also tested a previously developed stromal gene signature of Gleason score in these datasets. Comparing high- with low-Gleason score cancers, 26 genes (P < 0.001) and 12 pathways (FDR < 0.20) were significantly differentially expressed in tumor-adjacent stroma, including pathways related to stroma composition remodeling and DNA repair, with 73 genes and 65 pathways significant in benign-adjacent stroma. Comparing lethal with nonlethal prostate cancer, 11 genes were differentially expressed in tumor-adjacent and 15 genes in benign-adjacent stroma, and pathways involved in inflammatory response were differentially enriched in both tumor and benign-adjacent stroma. In addition, our previously identified Gleason stromal gene signature was validated to be associated with Gleason score in these data. Implications: Our study uncovers stroma-specific genes and pathways that are differentially enriched with high Gleason score and lethal prostate cancer, demonstrating that the molecular investigation of the tumor microenvironment can provide additional information about prostate cancer prognosis.
Salucci S; Bavelloni A; Stella AB; Fabbri F; Vannini I; Piazzi M; Volkava K; Scotlandi K; Martinelli G; Faenza I; Blalock W
In: Nutrients, vol. 15, iss. 3, pp. 740, 2023.
Approximately 7% of cancers arising in children and 1% of those arising in adults are soft tissue sarcomas (STS). Of these malignancies, rhabdomyosarcoma (RMS) is the most common. RMS survival rates using current therapeutic protocols have remained largely unchanged in the past decade. Thus, it is imperative that the main molecular drivers in RMS tumorigenesis are defined so that more precise, effective, and less toxic therapies can be designed. Curcumin, a common herbal supplement derived from plants of the Curcuma longa species, has an exceptionally low dietary biotoxicity profile and has demonstrated anti-tumorigenic benefits in vitro. In this study, the anti-tumorigenic activity of curcumin was assessed in rhabdomyosarcoma cell lines and used to identify the major pathways responsible for curcumin's anti-tumorigenic effects. Curcumin treatment resulted in cell cycle arrest, inhibited cell migration and colony forming potential, and induced apoptotic cell death. Proteome profiler array analysis demonstrated that curcumin treatment primarily influenced flux through the AKT-mammalian target of rapamycin (mTOR), signal transducer and activator of transcription (STAT), AMP-dependent kinase (AMPK), and p53 associated pathways in a rhabdomyosarcoma subtype-specific manner. Thus, the strategic, combinational therapeutic targeting of these pathways may present the best option to treat this group of tumors.
Ronco R; Perini C; Currò R; Dominik N; Facchini S; Gennari A; Simone R; Stuart S; Nagy S; Vegezzi E; Quartesan I; El-Saddig A; Lavin T; Tucci A; Szymura A; Novis De Farias LE; Gary A; Delfeld M; Kandikatla P; Niu N; Tawde S; Shaw J; Polke J; Reilly MM; Wood NW; Crespan E; Gomez C; Chen JYH; Schmahmann JD; Gosal D; Houlden H; Das S; Cortese A
In: NEUROLOGY, vol. 100, iss. 5, pp. e543-e554, 2023.
Background and objective: Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease characterized by adult-onset and slowly progressive sensory neuropathy, cerebellar dysfunction, and vestibular impairment. In most cases, the disease is caused by biallelic (AAGGG)n repeat expansions in the second intron of the replication factor complex subunit 1 (RFC1). However, a small number of cases with typical CANVAS do not carry the common biallelic repeat expansion. The objective of this study was to expand the genotypic spectrum of CANVAS by identifying sequence variants in RFC1-coding region associated with this condition. Methods: Fifteen individuals diagnosed with CANVAS and carrying only 1 heterozygous (AAGGG)n expansion in RFC1 underwent whole-genome sequencing or whole-exome sequencing to test for the presence of a second variant in RFC1 or other unrelated gene. To assess the effect of truncating variants on RFC1 expression, we tested the level of RFC1 transcript and protein on patients' derived cell lines. Results: We identified 7 patients from 5 unrelated families with clinically defined CANVAS carrying a heterozygous (AAGGG)n expansion together with a second truncating variant in trans in RFC1, which included the following: c.1267C>T (p.Arg423Ter), c.1739_1740del (p.Lys580SerfsTer9), c.2191del (p.Gly731GlufsTer6), and c.2876del (p.Pro959GlnfsTer24). Patient fibroblasts containing the c.1267C>T (p.Arg423Ter) or c.2876del (p.Pro959GlnfsTer24) variants demonstrated nonsense-mediated mRNA decay and reduced RFC1 transcript and protein. Discussion: Our report expands the genotype spectrum of RFC1 disease. Full RFC1 sequencing is recommended in cases affected by typical CANVAS and carrying monoallelic (AAGGG)n expansions. In addition, it sheds further light on the pathogenesis of RFC1 CANVAS because it supports the existence of a loss-of-function mechanism underlying this complex neurodegenerative condition.
Poggialini F; Vagaggini C; Brai A; Pasqualini C; Crespan E; Maga G; Perini C; Cabella N; Botta L; Musumeci F; Frosini M; Schenone S; Dreassi E
In: Pharmaceutics, vol. 15, iss. 2, pp. 453, 2023.
The therapeutic use of tyrosine kinase inhibitors (TKIs) represents one of the successful strategies for the treatment of glioblastoma (GBM). Pyrazolo[3,4-d]pyrimidines have already been reported as promising small molecules active as c-Src/Abl dual inhibitors. Herein, we present a series of pyrazolo[3,4-d]pyrimidine derivatives, selected from our in-house library, to identify a promising candidate active against GBM. The inhibitory activity against c-Src and Abl was investigated, and the antiproliferative profile against four GBM cell lines was studied. For the most active compounds endowed with antiproliferative efficacy in the low-micromolar range, the effects toward nontumoral, healthy cell lines (fibroblasts FIBRO 2-93 and keratinocytes HaCaT) was investigated. Lastly, the in silico and in vitro ADME properties of all compounds were also assessed. Among the tested compounds, the promising inhibitory activity against c-Src and Abl (Ki 3.14 µM and 0.44 µM, respectively), the irreversible, apoptotic-mediated death toward U-87, LN18, LN229, and DBTRG GBM cell lines (IC50 6.8 µM, 10.8 µM, 6.9 µM, and 8.5 µM, respectively), the significant reduction in GBM cell migration, the safe profile toward FIBRO 2-93 and HaCaT healthy cell lines (CC50 91.7 µM and 126.5 µM, respectively), the high metabolic stability, and the excellent passive permeability across gastrointestinal and blood-brain barriers led us to select compound 5 for further in vivo assays.
Falcinelli M; Dell'Omo G; Grassi E; Mariella E; Leto SM; Scardellato S; Lorenzato A; Arena S; Bertotti A; Trusolino L; Bardelli A; d'Adda di Fagagna F
In: Cell death and disease, vol. 14, iss. 2, pp. 96, 2023.
Telomere maintenance is necessary to maintain cancer cell unlimited viability. However, the mechanisms maintaining telomere length in colorectal cancer (CRC) have not been extensively investigated. Telomere maintenance mechanisms (TMM) include the re-expression of telomerase or alternative lengthening of telomeres (ALT). ALT is genetically associated with somatic alterations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes. Cells displaying ALT present distinctive features including C-circles made of telomeric DNA, long and heterogenous telomeric tracts, and telomeric DNA co-localized with promyelocytic leukemia (PML) bodies forming so-called ALT-associated PML bodies (APBs). Here, we identified mutations in ATRX and/or DAXX genes in an extensive collection of CRC samples including 119 patient-derived organoids (PDOs) and 232 established CRC cell lines. C-circles measured in CRC PDOs and cell lines showed low levels overall. We also observed that CRC PDOs and cell lines did not display a significant accumulation of APBs or long telomeres with no appreciable differences between wild-type and mutated ATRX/DAXX samples. Overall, our extensive analyses indicate that CRC is not prone to engage ALT, even when carrying genetic lesions in ATRX and/or DAXX, and support the notion that ATRX/DAXX genomic footprints are not reliable predictors of ALT.
Cardone N; Moula M; Baelde RJ; Biquand A; Villanova M; Metay C; Fiorillo C; Baratto S; Merlini L; Sabatelli P; Romero NB; Relaix F; Authier FJ; Taglietti V; Savarese M; de Winter J; Ottenheijm C; Richard I; Malfatti E.
In: Acta neuropathologica communications, vol. 11, iss. 1, pp. 48, 2023.
Congenital titinopathies are an emerging group of a potentially severe form of congenital myopathies caused by biallelic mutations in titin, encoding the largest existing human protein involved in the formation and stability of sarcomeres. In this study we describe a patient with a congenital myopathy characterized by multiple contractures, a rigid spine, non progressive muscular weakness, and a novel homozygous TTN pathogenic variant in a metatranscript-only exon: the c.36400A > T, p.Lys12134*. Muscle biopsies showed increased internalized nuclei, variability in fiber size, mild fibrosis, type 1 fiber predominance, and a slight increase in the number of satellite cells. RNA studies revealed the retention of intron 170 and 171 in the open reading frame, and immunoflourescence and western blot studies, a normal titin content. Single fiber functional studies showed a slight decrease in absolute maximal force and a cross-sectional area with no decreases in tension, suggesting that weakness is not sarcomere-based but due to hypotrophy. Passive properties of single fibers were not affected, but the observed increased calcium sensitivity of force generation might contribute to the contractural phenotype and rigid spine of the patient. Our findings provide evidence for a pathogenic, causative role of a metatranscript-only titin variant in a long survivor congenital titinopathy patient with distal arthrogryposis and rigid spine.