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SILVIA BELLUTI

Ricercatore t.d. art. 24 c. 3 lett. B
Dipartimento di Scienze della Vita sede ex-Biologia

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Pubblicazioni

2023 - BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting [Articolo su rivista]
Sorbi, C.; Belluti, S.; Atene, C. G.; Marocchi, F.; Linciano, P.; Roy, N.; Paradiso, E.; Casarini, L.; Ronsisvalle, S.; Zanocco-Marani, T.; Brasili, L.; Lanfrancone, L.; Imbriano, C.; Di Rocco, G.; Franchini, S.
abstract

: The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

2023 - Deciphering the transcriptional network of NF-Y in prostate cancer development and progression [Poster]
Belluti, Silvia; Cuoghi, L.; Mularoni, V.; Forcato, M.; Torricelli, F.; Ciarrocchi, A.; Imbriano, C.
abstract

2023 - Development of Stable Amino-Pyrimidine–Curcumin Analogs: Synthesis, Equilibria in Solution, and Potential Anti-Proliferative Activity [Articolo su rivista]
Mari, Matteo; Boniburini, Matteo; Tosato, Marianna; Rigamonti, Luca; Cuoghi, Laura; Belluti, Silvia; Imbriano, Carol; Avino, Giulia; Asti, Mattia; Ferrari, Erika
abstract

2023 - Discovery of potent pyrrolo-pyrimidine and purine HDAC inhibitors for the treatment of advanced prostate cancer [Articolo su rivista]
Moi, Davide; Bonanni, Davide; Belluti, Silvia; Linciano, Pasquale; Citarella, Andrea; Franchini, Silvia; Sorbi, Claudia; Imbriano, Carol; Pinzi, Luca; Rastelli, Giulio
abstract

2023 - Dysregulation of NF–Y splicing drives metabolic rewiring and aggressiveness in colon cancer [Poster]
Belluti, S.; Mularoni, V.; Campani, Virginia; Rigillo, G.; Cuoghi, L.; Ronzio, M.; Miserocchi, G.; Dolfini, D.; Righi, Valeria; Alessandrini, A.; Zappavigna, V.; Imbriano, C.
abstract

NF-Y is an evolutionarily conserved transcription factor that binds specifically to the CCAAT elements of eukaryotic genes, most of which frequently deregulated in cancer. NF-YA, the regulatory subunit of the NF-Y complex, has two isoforms generated by alternative splicing, NF-YAl and NF-YAs, which differ in the transactivation domain. Transcriptomic data from The Cancer Genome Atlas (TCGA) database highlighted a significant increase in the expression of NF-YAs at the expense of NF-YAl in colorectal cancer (CRC), compared to healthy tissues. Despite this, high NF-YAl levels predict lower patients’ survival and distinguish the mesenchymal molecular subtype CMS4, which is characterized by the worst prognosis. Through the analysis of 3D cellular models, we demonstrated that altered expression of genes related to extracellular matrix and epithelial-mesenchymal transition sustains enhanced migratory and invasive behavior of NF-YAl-transduced cells. Moreover, the integration of metabolomics, bioenergetics and transcriptional analyses demonstrated a direct role for NFYAl in metabolic flexibility of cancer cells that adjust their metabolism in response to environmental changes to potentiate migration. The zebrafish xenograft model confirmed the metastatic potential triggered by NF-YAl in CRC cells. Altogether, our data highlight the transcriptional role of NF-YAl in CRC aggressiveness and suggest splice-switching strategies to hinder NF-YAl-induced metastatic dissemination.

2023 - Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives [Articolo su rivista]
Imbriano, Carol; Moresi, Viviana; Belluti, Silvia; Renzini, Alessandra; Cavioli, Giorgia; Maretti, Eleonora; Molinari, Susanna
abstract

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.

2023 - Histone deacetylase functions and therapeutic implications for adult skeletal muscle metabolism [Articolo su rivista]
Molinari, Susanna; Imbriano, Carol; Moresi, Viviana; Renzini, Alessandra; Belluti, Silvia; Lozanoska-Ochser, Biliana; Gigli, Giuseppe; Cedola, Alessia
abstract

2023 - Nuclear Estrogen Receptors in Prostate Cancer: From Genes to Function [Articolo su rivista]
Belluti, Silvia; Imbriano, Carol; Casarini, Livio
abstract

: Estrogens are almost ubiquitous steroid hormones that are essential for development, metabolism, and reproduction. They exert both genomic and non-genomic action through two nuclear receptors (ERα and ERβ), which are transcription factors with disregulated functions and/or expression in pathological processes. In the 1990s, the discovery of an additional membrane estrogen G-protein-coupled receptor augmented the complexity of this picture. Increasing evidence elucidating the specific molecular mechanisms of action and opposing effects of ERα and Erβ was reported in the context of prostate cancer treatment, where these issues are increasingly investigated. Although new approaches improved the efficacy of clinical therapies thanks to the development of new molecules targeting specifically estrogen receptors and used in combination with immunotherapy, more efforts are needed to overcome the main drawbacks, and resistance events will be a challenge in the coming years. This review summarizes the state-of-the-art on ERα and ERβ mechanisms of action in prostate cancer and promising future therapies.

2023 - The NF-Y splicing signature controls hybrid EMT and ECM-related pathways to promote aggressiveness of colon cancer [Articolo su rivista]
Rigillo, Giovanna; Belluti, Silvia; Campani, Virginia; Ragazzini, Gregorio; Ronzio, Mirko; Miserocchi, Giacomo; Bighi, Beatrice; Cuoghi, Laura; Mularoni, Valentina; Zappavigna, Vincenzo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

: Aberrant splicing events are associated with colorectal cancer (CRC) and provide new opportunities for tumor diagnosis and treatment. The expression of the splice variants of NF-YA, the DNA binding subunit of the transcription factor NF-Y, is deregulated in multiple cancer types compared to healthy tissues. NF-YAs and NF-YAl isoforms differ in the transactivation domain, which may result in distinct transcriptional programs. In this study, we demonstrated that the NF-YAl transcript is higher in aggressive mesenchymal CRCs and predicts shorter patients' survival. In 2D and 3D conditions, CRC cells overexpressing NF-YAl (NF-YAlhigh) exhibit reduced cell proliferation, rapid single cell amoeboid-like migration, and form irregular spheroids with poor cell-to-cell adhesion. Compared to NF-YAshigh, NF-YAlhigh cells show changes in the transcription of genes involved in epithelial-mesenchymal transition, extracellular matrix and cell adhesion. NF-YAl and NF-YAs bind similarly to the promoter of the E-cadherin gene, but oppositely regulate its transcription. The increased metastatic potential of NF-YAlhigh cells in vivo was confirmed in zebrafish xenografts. These results suggest that the NF-YAl splice variant could be a new CRC prognostic factor and that splice-switching strategies may reduce metastatic CRC progression.

2023 - The NF-YA Splicing Signature Controls Aggressiveness of Colon Cancer by Regulating Cell Metabolism and Different Types of Cell Migration [Relazione in Atti di Convegno]
Belluti, Silvia; Mularoni, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-Y isoforms, NFYAs and NF-YAl, differentially control cell proliferation and differentiation. The analysis of patient’s transcriptome profiles from The Cancer Genome Atlas database highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA have a lower overall survival probability. We investigated the role of NF-YA in the metabolism of CRC cells, and the measurement of mitochondrial fuel usage in live cells shows that NF-YAl overexpression enhances the capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Moreover, we demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT, extracellular matrix and adhesion genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NFYAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patients’ stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities.

2022 - Alternative splicing of the transcription factor NF-Y promotes cell migration and invasion in colon cancer [Abstract in Atti di Convegno]
Rigillo, Giovanna; Belluti, Silvia; Campani, Virginia; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

The heterotrimeric transcription factor NF-Y directly controls the expression of genes involved in cellular pathways commonly altered in cancer cells, such as cell cycle, apoptosis and metabolism. Consistently, the binding site for NF-Y is highly enriched in the regulatory regions of genes overexpressed in tumors, and mRNA levels of NF-Y subunits are altered in cancer tissues and cells. In particular, the DNA binding subunit NF-YA is up-regulated in various tumors, among which gastric, lung, breast, ovarian, osteosarcoma and prostate cancers. Moreover, a switch between the two alternatively NF-YA spliced transcripts, NF-YAs and NF-YAl, occurs in tumor tissues compared to normal ones. Colorectal cancer (CRC) is the third most common malignancy worldwide. Four internationally approved consensus molecular subtypes (CMS) represent the best current description of CRC heterogeneity at the gene-expression level: the CMS1 group is characterized by the immune infiltration signature, CMS2 is the canonical epithelial subtype, CMS3 represents the metabolic group, and CMS4 is the mesenchymal one, associated with a worse prognosis and poor response to therapies compared to other subtypes. Here we show that increased levels of NF-YA characterize CRC versus healthy tissues. We identified a significant association between NF-YA isoforms and CRC subtypes: NF-YAs is up-regulated in all CMSs in opposition to NF-YAl, which is down-regulated in all subtypes with the exception of aggressive and metastatic CMS4 group. By using in vitro cell models, we confirmed that NF-YAs is the predominant isoform in CRC cell lines, while NF-YAl levels proportionally increase from epithelial to hybrid and mesenchymal cells. The modulation of NF-YA isoforms in CRC cells significantly affects cancer cell behavior by modulating differently, even oppositely, the transcription of genes associated to extracellular-matrix (ECM) and epithelial-to-mesenchymal transition (EMT). We described different modes of migration and invasion properties for NF-YAs and NF-YAl overexpressing cells by using 2D and 3D culture conditions, time-lapse imaging of CRC cells and intravascular distribution of NF-YAs/l transduced CRC cells in the embryonic zebrafish xenograft model. Altogether, our data highlight the direct role of the longer NF-YA isoform in CRC cell dissemination and suggest its possible use as biomarker for molecular stratification predictive of progressive disease in CRC patients.

2022 - Curcumin-Based β-Diketo Ligands for Ga3+: Thermodynamic Investigation of Potential Metal-Based Drugs [Articolo su rivista]
Mari, Matteo; Carrozza, Debora; Malavasi, Gianluca; Venturi, Ettore; Avino, Giulia; Capponi, Pier Cesare; Iori, Michele; Rubagotti, Sara; Belluti, Silvia; Asti, Mattia; Ferrari, Erika
abstract

Curcumin is known for its therapeutic properties; among these, antioxidant, anti-inflammatory and anti-cancer ones stand out. Besides, curcumin metal complexes have shown widespread application in medicine and can be exploited as lead structures for developing metal-based drugs. Unfortunately, curcumin is poorly bioavailable, mainly due to its instability in physiological conditions; this weakness is tightly connected to the presence of the β-diketo moiety undergoing tautomeric equilibrium. Stability and metal-chelating ability can be tuned by modulating the electronic effects and steric hindrance close to the β-diketo moiety; in addition, formation of a metal complex shifts the tautomeric equilibrium towards the β-keto–enol form and increases stability in biological media. Among the metals used in clinical therapy, gallium nitrate has shown to have significant antitumor activity against non-Hodgkin lymphoma and bladder cancer, thus indicating that gallium-based drugs have potential for further development as antineoplastic agents with improved therapeutic activity. Curcuminoids have demonstrated high affinity for gallium(III), allowing the formation of stable positively charged M:L 1:2 β-diketonate complexes that benefit from the therapeutic activity of both the metal and the ligand. Seven new curcumin derivatives were synthesized and completely characterized. The new derivatives retain the solvent-dependent keto–enol tautomerism, with the prevalence of the diketo form in aqueous solution. Enhanced stability in simulated physiological conditions was observed in comparison to the lead compound curcumin. The presence of Ga3+ anticipates the dissociation of the enolic proton, allowing chelate complex formation, and simultaneously it shifts the tautomeric equilibrium towards the keto–enol form. A complete 1H/13C NMR and UV–Vis study was performed to define the metal-to-ligand stoichiometry ratio and the overall stability constants. In addition, we demonstrated that some of the derivatives have increased antiproliferative activity on colon cancer cells compared to curcumin and antioxidant properties. On the whole, the synthesized curcumin-based molecules may act as new gallium(III) chelators with improved stability with respect to curcumin and could open interesting perspectives for the development of novel therapeutic agents for cancer.

2022 - Histone Marks-Dependent Effect on Alternative Splicing: New Perspectives for Targeted Splicing Modulation in Cancer? [Articolo su rivista]
Imbriano, Carol; Belluti, Silvia
abstract

2022 - The interplay between NF-Y, AR and lipid metabolism regulates tumor aggressiveness in prostate cancer. [Poster]
Belluti, Silvia; Mularoni, Valentina; Imbriano, Carol.
abstract

Prostate cancer (PCa) is the second most frequent cancer in Western men. Computational analyses linked the transcription factor NF-Y to progression from benign to localized PCa, aggressive signatures, response to androgen deprivation therapy (ADT) and metastasis. The NF-YA gene encodes two alternatively spliced transcripts, NF-YAs and NF-YAl. We demonstrated that PCa samples are characterized by increased NF-YA levels, as well as higher NF-YAs/NF-YAl transcriptional ratio. In vitro and in vivo, NF-YA depletion affects PCa tumorigenicity and changes in NF-YA isoforms expression are associated with key clinical and molecular features of aggressive PCa. NF-YAs enhances tumor growth and metastasis, while NF-YAl increases cell motility. Stratification of patients based on NF-YAs expression is predictive of clinical outcome, although a significant decrease in the NF-YAs/NF-YAl ratio characterizes PCa circulating cells. PCa cells depend on male sex hormones for growth and survival, which is the basis of ADT. While ADT is initially effective, patients eventually relapse with castration-resistant prostate cancer (CRPC). One of the cellular processes most affected by androgens is lipid metabolism, whose rewiring contributes to PCa development, progression, metastasis and recurrence. Androgens stimulate de novo lipogenesis and lipid uptake by activating SREBPs, the master transcription factors of cholesterol and fatty acid biosynthesis. SREBPs interact with target promoters in cooperation with NF-Y, which controls de novo lipid biosynthesis pathway. A feedforward mechanism between SREBP and the Androgen Receptor (AR) was also described. We modulated NF-YA in androgen-sensitive healthy and cancer cell lines, performing cellular and molecular studies. RNA-seq analysis highlighted the alteration of lipid and cholesterol metabolic pathways and of the unfolded protein response (UPR) in NF-YAl-overexpressing cells. Bioenergetics metabolic profiling of NF-YA transduced cells confirmed the key role of NF-YAl in PCa cell metabolism. We found a reciprocal regulation of AR and NF-Y that relies in particular on NF-YAl. Our data suggest that NF-Y, and specifically the longer NF-YA isoform, can participate in ADT responsiveness and resistance mediated by metabolic alterations. The characterization of these pathways could be useful in the stratification of PCa patients into sub-categories with different levels of aggressiveness and ADT sensitivity.

2022 - The NF-YA splicing signature controls aggressiveness of colon cancer by regulating different modes of cell migration and cell metabolism. [Poster]
Belluti, Silvia; Rigillo, Giovanna; Mularoni, Valentina; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-YA isoforms, NF-YAs and NF-YAl, differentially control cell proliferation and differentiation. TCGA data highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA levels have a lower overall survival probability. We demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NF-YAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Since metabolic reprogramming is a hallmark of cancer and metastasis, we also investigated the role of NF-YAl in the metabolism of CRC cells. The measure of mitochondrial fuel usage in live cells showed that NF-YAl overexpression enhances the dependency and capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patient stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities.

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Poster]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; REGGIANI BONETTI, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; Reggiani Bonetti, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes.

2021 - Inhibitors of histone deacetylase 6 based on a novel 3-hydroxy-isoxazole zinc binding group [Articolo su rivista]
Linciano, P.; Pinzi, L.; Belluti, S.; Chianese, U.; Benedetti, R.; Moi, D.; Altucci, L.; Franchini, S.; Imbriano, C.; Sorbi, C.; Rastelli, G.
abstract

Histone deacetylase 6 (HDAC6) is an established drug target for cancer treatment. Inhibitors of HDAC6 based on a hydroxamic acid zinc binding group (ZBG) are often associated with undesirable side effects. Herein, we describe the identification of HDAC6 inhibitors based on a completely new 3-hydroxy-isoxazole ZBG. A series of derivatives decorated with different aromatic or heteroaromatic linkers, and various cap groups were synthesised and biologically tested. In vitro tests demonstrated that some compounds are able to inhibit HDAC6 with good potency, the best candidate reaching an IC50 of 700 nM. Such good potency obtained with a completely new ZBG make these compounds particularly attractive. The effect of the most active inhibitors on the acetylation levels of histone H3 and α- tubulin and their anti-proliferative activity of DU145 cells were also investigated. Docking studies were performed to evaluate the binding mode of these new derivatives and discuss structure-activity relationships.

2021 - The transcription factor NF-Y is required for satellite stem cell myogenic progression and skeletal muscle tissue repair [Poster]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Imbriano, Carol
abstract

2021 - The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle [Articolo su rivista]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Ronzio, Mirko; Sauta, Elisabetta; Ciarrocchi, Alessia; Latella, Lucia; Saclier, Marielle; Molinari, Susanna; Vallarola, Antonio; Messina, Graziella; Mantovani, Roberto; Dolfini, Diletta; Imbriano, Carol
abstract

2020 - Transcription Factors in Cancer: When Alternative Splicing Determines Opposite Cell Fates [Articolo su rivista]
Belluti, Silvia; Rigillo, Giovanna; Imbriano, Carol
abstract

Alternative splicing (AS) is a finely regulated mechanism for transcriptome and proteome diversification in eukaryotic cells. Correct balance between AS isoforms takes part in molecular mechanisms that properly define spatiotemporal and tissue specific transcriptional programs in physiological conditions. However, several diseases are associated to or even caused by AS alterations. In particular, multiple AS changes occur in cancer cells and sustain the oncogenic transcriptional program. Transcription factors (TFs) represent a key class of proteins that control gene expression by direct binding to DNA regulatory elements. AS events can generate cancer-associated TF isoforms with altered activity, leading to sustained proliferative signaling, differentiation block and apoptosis resistance, all well-known hallmarks of cancer. In this review, we focus on how AS can produce TFs isoforms with opposite transcriptional activities or antagonistic functions that severely impact on cancer biology. This summary points the attention to the relevance of the analysis of TFs splice variants in cancer, which can allow patients stratification despite the presence of interindividual genetic heterogeneity. Recurrent TFs variants that give advantage to specific cancer types not only open the opportunity to use AS transcripts as clinical biomarkers but also guide the development of new anti-cancer strategies in personalized medicine.

2019 - Potent Anti-Cancer Properties of Phthalimide-Based Curcumin Derivatives on Prostate Tumor Cells [Articolo su rivista]
Belluti, Silvia; Orteca, Giulia; Semeghini, Valentina; Rigillo, Giovanna; Parenti, Francesca; Ferrari, Erika; Imbriano, Carol
abstract

Metastatic castration-resistant prostate cancer is commonly treated with chemotherapy, whose effect is less than satisfactory. This raised the need for novel agents for the treatment of prostate cancer. In the present study, five phthalimide-based curcumin derivatives were synthesized and completely characterized to assess improved stability, pharmacodynamics, and radical scavenging ability. To investigate the potential application in anti-cancer therapy, the anti-proliferative activity of the synthesized molecules was determined on aggressive prostate tumor cells. We demonstrated that the K3F21 derivative has increased potency compared to curcumin, in terms of GI50, anti-proliferative and anti-migrating activities. K3F21 inhibits anchorage-dependent and -independent growth of prostate cancer cells by altering the expression of key genes controlling cell proliferation, such as Cylins D1, B1 and B2, and apoptosis, among which Puma, Noxa, and Bcl-2 family members. Finally, the anti-cancer activity of K3F21 was demonstrated by the analysis of cancer-associated PI3K/AKT, ERK, and p38 signaling pathways.

2019 - Switch of NF-YA splice variants in prostate cancer development and progression [Poster]
Belluti, Silvia; Semeghini, Valentina; Dolfini, Diletta; Rigillo, Giovanna; Imbriano, Carol
abstract

The pioneer transcription factor NF-Y is a heterotrimer composed by NF-YA, -YB and -YC subunits: NF-YA is the limiting subunit and harbors the DNA CCAAT-box binding domain. NF-Y is a master transcriptional regulator that ensures proper cell proliferation, controlling cell cycle, DNA replication, apoptosis and metabolism. Recent analyses pointed out that NF-Y binding site is over-represented in promoters of genes overexpressed during the progression from benign epithelium to Prostate Cancer (PC). Moreover, cell cycle regulation genes have emerged as a signature that can discriminate between metastatic and benign prostate tissues. The analysis of TCGA-RNAseq data highlighted a slight but significant increase of NF-YA levels in PC tumor vs normal tissues, particularly in high Gleason score specimens. Since the NF-YA gene encodes for two splice isoforms, NF-YAl (long) and NF-YAs (short), we generated untransformed and cancer prostate cell lines stably over-expressing NF-YA isoforms, to dissect the functional role of NF-YA in PC development and progression. Our data indicate that NF-YAl and NF-YAs could play different activities in cancer-associated processes, such as clonogenic ability, anchorage-independent growth, 3D cell growth, migration and invasion. In particular, in cancer cells NF-YAl improves invasion ability, while NF-YAs seems to enhance the proliferation of tumor spheroids. These results are consistent with increased NF-YAs/NF-YAl splice ratio observed in the progression from normal to malignant prostatectomy samples. Besides, normal prostate cell lines preferentially express the long NF-YA isoform, while an increase in NF-YAs/NF-YAl ratio can be observed in PC cell lines. These results prompt us to speculate that NF-YAl could participate to metastatization, while NF-YAs could have a major role in tumor growth and colonization. Disclosing this dualism could be crucial to better stratify PC patients and identify new specific anti-cancer treatments.

2019 - The transcription factor NF-Y is required for satellite stem cell proliferation and skeletal muscle tissue repair [Poster]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Imbriano, Carol
abstract

The transcription factor NF-Y, composed by NF-YA, NF-YB and NF-YC subunits, has an important role in the regulation of cellular proliferation and differentiation in different cell types, among which muscle cells. While NF-YA, the DNA binding subunit of NF-Y, is down-regulated in the adult muscle of WT mice, its expression is observed in the mdx mouse, model for Duchenne Muscular Dystrophy, in which a massive regeneration mediated by resident muscle stem cells, namely Satellite Cells (SCs), occurs. With the aim to investigate the role of NF-YA in the SCs proliferation and differentiation, we generated and characterized a conditional knock out mouse model in which NF-YA is deleted in Pax7+ SCs by Tamoxifen induction in adult NF-YAflox/flox:Pax7CreER mice (NF-YA cKO). Cellular and molecular analysis carried out on isolated myofibers and SCs from WT and NF-YA cKO mice highlighted that NF-Y activity is important for the maintenance of SCs homeostasis. NF-YA loss depletes Pax7+ SCs pool and impairs SCs proliferation. Moreover, SCs-mediated regeneration following muscle damage induced by cardiotoxin is delayed in NF-YA cKO. The effect of NF-YA abrogation was also explored in post-natal muscle growth. Immunohistological analysis showed defects in muscle morphology and a decrease in SCs number in 3 weeks aged NF-YA cKO mice, period of major increment of muscle mass  by SCs-mediated myonuclear accretion.  The molecular mechanism underlying the impairment of SCs activity following NF-YA loss was investigated by AdenoCre-induced NF-YA deletion in ex vivo cultured SCs.   Overall, our results highlight a role of NF-Y in muscle regeneration and in SCs fate, whose modulation could be useful to improve stem cell based therapies to treat muscular dystrophies.

2019 - The 1,10-phenanthroline ligand enhances the antiproliferative activity of dna-intercalating thiourea-pd(Ii) and-pt(ii) complexes against cisplatin-sensitive and-resistant human ovarian cancer cell lines [Articolo su rivista]
Marverti, G.; Gozzi, G.; Lauriola, A.; Ponterini, G.; Belluti, S.; Imbriano, C.; Costi, M. P.; D’Arca, D.
abstract

Ovarian cancer is the most lethal gynecological malignancy, often because of the frequent insurgence of chemoresistance to the drugs currently used. Thus, new therapeutical agents are needed. We tested the toxicity of 16 new DNA-intercalating agents to cisplatin (cDDP)-sensitive human ovarian carcinoma cell lines and their resistant counterparts. The compounds were the complexes of Pt(II) or Pd(II) with bipyridyl (bipy) and phenanthrolyl (phen) and with four dierent thiourea ancillary ligands. Within each of the four series of complexes characterized by the same thiourea ligand, the Pd(phen) drugs invariably showed the highest anti-proliferative ecacy. This paralleled both a higher intracellular drug accumulation and a more ecient DNA intercalation than all the other metal-bidentate ligand combinations. The consequent inhibition of topoisomerase II activity led to the greatest inhibition of DNA metabolism, evidenced by the inhibition of the expression of the folate cycle enzymes and a marked perturbation of cell-cycle distribution in both cell lines. These findings indicate that the particular interaction of Pd(II) with phenanthroline confers the best pharmacokinetic and pharmacodynamic properties that make this class of DNA intercalators remarkable inhibitors, even of the resistant cell growth.

2018 - An autoregulatory loop controls the expression of the transcription factor NF-Y [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Basile, Valentina; Rigillo, Giovanna; Salsi, Valentina; Genovese, Filippo; Dolfini, Diletta; Imbriano, Carol
abstract

The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer. Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate how the stability of the complex is severely affected by the absence of one subunit. Our results identified for the first time one of the mechanisms responsible for NF-Y expression, which may be involved in the aberrant expression and activity observed in tumor cells and other pathological conditions.

2016 - Direct non transcriptional role of NF-Y in DNA replication [Articolo su rivista]
Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Ethienne; Mantovani, Roberto; Blow J., Julian; Imbriano, Carol
abstract

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication.

2016 - Direct non transcriptional role of the CCAAT-factor NF-Y in DNA replication [Poster]
Belluti, Silvia; Benatti, Paolo; Benoit, Miotto; Julia, Neusiedler; Diletta, Dolfini; Marjorie, Drac; Etienne, Schwob; Roberto, Mantovani; Julian Blow, J.; Imbriano, Carol
abstract

The heterotrimeric transcription factor NF-Y plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation. The expression levels of the sequence specific subunit NF-YA increase at the onset of S phase, and NF-YA loss in primary and tumor mammalian cells triggers defects in S-phase progression. Here, we show that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Using a Xenopus cell-free system, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication. Spontaneous DNA damage occurs in NF-YA-deficient cells, while NF-YA overexpression reduces the sensitivity to replication stress, suggesting a role for NF-Y in replication stress response and genome maintenance. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific transcription factors required for proper and efficient DNA replication.

2016 - NF-Y loss triggers p53 stabilization and apoptosis in HPV18-positive cells by affecting E6 transcription [Articolo su rivista]
Benatti, Paolo; Basile, Valentina; Dolfini, Diletta; Belluti, Silvia; Tomei, Margherita; Imbriano, Carol
abstract

The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NFYA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response.

2016 - NF-YA splice variants have different roles on muscle differentiation [Articolo su rivista]
Basile, Valentina; Baruffaldi, Fiorenza; Dolfini, Diletta; Belluti, Silvia; Benatti, Paolo; Ricci, Laura; Artusi, Valentina; Tagliafico, Enrico; Mantovani, Roberto; Molinari, Susanna; Imbriano, Carol
abstract

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.

2013 - A non transcriptional role for NF-Y in DNA replication [Poster]
Benatti, Paolo; Belluti, Silvia; Neusiedler, Julia; Basile, Valentina; Blow, J. Julian; Imbriano, Carol
abstract

The heterotrimeric transcription factor NF-Y, composed by NF-YA, NF-YB and NF-YC subunits, is a key transcriptional regulator of cell cycle progression. Using data generated by ENCODE, we identified a striking overlap between loci bound by NF-Y-and ORC2, hinting at a possible role of NF-Y in DNA replication. NF-YA knock-down leads to replication defects and the activation an intra-S checkpoint. We investigated the role of NF-Y in DNA replication by using the Xenopus cell-free system. NF-Y subunits were found to be recruited to chromatin during DNA replication. Both immunodepletion of NF-YA or NF-YB and overexpression of a dominant-negative NF-YA mutant lead to a clear decrease in DNA synthesis. In mammalian cells, NF-Y colocalizes and directly interacts with DNA replication proteins. Nascent strand abundance assay in NF-YA inactivated cells corroborates that NF-Y participates to the DNA replication process. Our data highlight that, in addition to its transcriptional activity in controlling cell proliferation, NF-Y plays a key role in the non transcriptional control of DNA replication.

2013 - A specific role for the splice variants of the transcription factor NF-Y in modulating the transcriptional activity of the myogenic program [Poster]
Basile, Valentina; Baruffaldi, Fiorenza; Belluti, Silvia; Benatti, Paolo; Mantovani, Roberto; Molinari, Susanna; Imbriano, Carol
abstract

Cell proliferation and differentiation programs are highly regulated transcriptional processes essential for myogenesis. The transcription factor NF-Y has been long considered a fundamental player of cell growth by supporting the basal transcription of various cell cycle genes. It is composed by the NF-YB/NF-YC heterodimer and NF-YA, which interacts with the other two subunits and confers the strict sequence specificity to the complex. The NF-YA gene encodes two alternatively splice transcripts (NF-YAs and NF-YAl), which differently regulate cell proliferation and differentiation, as shown in haematopoietic and mouse embryonic stem cells. NF-YAl expression is down-regulated in terminally differentiated muscle cells and in skeletal and cardiac muscle tissues. Its forced expression in muscle cells committed to differentiate impairs their exit from the cell cycle and indirectly interferes with the differentiation program. Here we show that the two NF-YA isoforms play a different role in the transcriptional activity of the myogenic program and may regulate the activity of muscle satellite cells.

2013 - bis-Dehydroxy-Curcumin Triggers Mitochondrial-Associated Cell Death in Human Colon Cancer Cells through ER-Stress Induced Autophagy. [Articolo su rivista]
Basile, Valentina; Belluti, Silvia; Ferrari, Erika; Gozzoli, C; Ganassi, Sonia; Quaglino, Daniela; Saladini, Monica; Imbriano, Carol
abstract

Background: The activation of autophagy has been extensively described as a pro-survival strategy, which helps to keep cells alive following deprivation of nutrients/growth factors and other stressful cellular conditions. In addition to cytoprotective effects, autophagy can accompany cell death. Autophagic vacuoles can be observed before or during cell death, but the role of autophagy in the death process is still controversial. A complex interplay between autophagy and apoptosis has come to light, taking into account that numerous genes, such as p53 and Bcl-2 family members, are shared between these two pathways. Methodology/Principal Findings: In this study we showed a potent and irreversible cytotoxic activity of the stable Curcumin derivative bis-DeHydroxyCurcumin (bDHC) on human colon cancer cells, but not on human normal cells. Autophagy is elicited by bDHC before cell death as demonstrated by increased autophagosome formation -measured by electron microscopy, fluorescent LC3 puncta and LC3 lipidation- and autophagic flux -measured by interfering LC3-II turnover. The accumulation of poly-ubiquitinated proteins and ER-stress occurred upstream of autophagy induction and resulted in cell death. Cell cycle and Western blot analyses highlighted the activation of a mitochondrial-dependent apoptosis, which involves caspase 7, 8, 9 and Cytochrome C release. Using pharmacological inhibitions and RNAi experiments, we showed that ER-stress induced autophagy has a major role in triggering bDHC-cell death. Conclusion/Significance: Our findings describe the mechanism through which bDHC promotes tumor selective inhibition of proliferation, providing unequivocal evidence of the role of autophagy in contrasting the proliferation of colon cancer cells.

2013 - Concurrent inhibition of enzymatic activity and NF-Y-mediated transcription of Topoisomerase-IIα by bis-DemethoxyCurcumin in cancer cells [Articolo su rivista]
Belluti, Silvia; Basile, Valentina; Benatti, Paolo; Ferrari, Erika; Marverti, Gaetano; Imbriano, Carol
abstract

Topoisomerase-IIa (TOP2A) enzyme is essential for cell viability due to its fundamental role in DNA metabolism and in chromatin organization during interphase and mitosis. TOP2A expression is finely regulated at the transcriptional level through the binding of the CCAAT-transcription factor NF-Y to its promoter. Overexpression and/or amplification of TOP2A have been observed in many types of cancers. For this reason, TOP2A is the target of the most widely successful drugs in cancer chemotherapy, such as TOP2A poisons, which stabilize TOP2A-DNA cleavage complexes and create DSBs, leading to chromosome damage and cell death. We previously reported that the Curcumin-derivative bis-DemethoxyCurcumin (bDMC) is an anti-proliferative agent that inhibits cell growth by concomitant G1/S and G2/M arrest. Here we showed that bDMC irreversibly induces DSBs in cancer cells, but not in normal cells, by targeting TOP2A activity and expression. TOP2A ablation by siRNA corroborates its contribution to apoptosis induced by bDMC. Short-term exposure to bDMC induces retention of TOP2A-DNA intermediates, while longer exposure inhibits TOP2A transcription by affecting expression and sub-cellular localization of NF-Y subunits. ChIP analysis highlighted reduced recruitment of NF-Y to TOP2A regulatory regions, concomitantly to histone deacetylation and decreased gene transcription. Our findings suggest that the dual activity of bDMC on TOP2A represents a novel therapeutic strategy to induce persistent apoptosis in cancer cells and identify NF-Y regulation as a promising approach in anti-cancer therapy.

2009 - Curcumin derivatives: molecular basis of their anti-cancer activity. [Articolo su rivista]
Basile, Valentina; Ferrari, Erika; Lazzari, Sandra; Belluti, S.; Pignedoli, Francesca; Imbriano, Carol
abstract

Curcumin, a phenolic compound from the plant Curcuma longa L., has shown a wide-spectrum of chemopreventive, anti-oxidant and anti-tumor properties. Although its promising chemotherapeutic activity, preclinical and clinical studies highlight Curcumin limited therapeutic application due to its instability in physiological conditions. To improve its stability and activity, many derivatives have been synthesized and studied, among which bis-DemethoxyCurcumin (bDMC) and diAcetylCurcumin (DAC). In this report, we show that both bDMC and DAC are more stable than Curcumin in physiological medium. To explore the mechanism of their chemotherapeutic effect, we studied their role in proliferation in the HCT116 human colon cancer cells. We correlated kinetic stability and cellular uptake data to their biological effects. Both bDMC and DAC impair correct spindles formation and induce a p53- and p21(CIP1/WAF1)- independent mitotic arrest, which is more stable and long-lasting for bDMC. A subsequent p53/p21(CIP1/WAF1)- dependent inhibition of G1 to S transition is triggered by Curcumin and DAC as a consequence of the mitotic slippage, preventing postmitotic cells from re-entering the cell cycle. Conversely, the G1/S arrest induced by bDMC is a direct effect of the drug and concomitant to the mitotic block. Finally, we demonstrate that bDMC induces rapid DNA double-strand breaks, moving for its possible development in anti-cancer clinical applications.

2009 - Understanding the molecular mechanisms of Curcumin derivatives chemotherapic activity [Relazione in Atti di Convegno]
Basile, Valentina; Ferrari, Erika; Lazzari, Sandra; Belluti, Silvia; Pignedoli, Francesca; Imbriano, Carol
abstract

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