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Tommaso ZANOCCO MARANI

Ricercatore Universitario
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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Pubblicazioni

2023 - A Novel In Vivo Active Pemphigus Model Targeting Desmoglein1 and Desmoglein3: A Tool Representing All Pemphigus Variants [Articolo su rivista]
Lotti, Roberta; Atene, Claudio Giacinto; Zanfi, Emma Dorotea; Bertesi, Matteo; Pincelli, Carlo; Zanocco-Marani, Tommaso
abstract

: Background: Pemphigus is a life-threatening blistering autoimmune disease. Several forms, characterized by the presence of autoantibodies against different autoantigens, have been described. In Pemphigus Vulgaris (PV), autoantibodies target the cadherin Desmoglein 3 (DSG3), while in Pemphigus foliaceous (PF) autoantibodies target the cadherin Desmoglein 1 (DSG1). Another variant, mucocutaneous Pemphigus, is characterized by the presence of IgG against both DSG1 and DSG3. Moreover, other forms of Pemphigus characterized by the presence of autoantibodies against other autoantigens have been described. With regard to animal models, one can distinguish between passive models, where pathological IgG are transferred into neonatal mice, and active models, where B cells deriving from animals immunized against a specific autoantigen are transferred into immunodeficient mice that develop the disease. Active models recreate PV and a form of Pemphigus characterized by the presence of IgG against the cadherin Desmocollin 3 (DSC3). Further approaches allow to collect sera or B/T cells from mice immunized against a specific antigen to evaluate the mechanisms underlying the onset of the disease. Objective: To develop and characterize a new active model of Pemphigus where mice express auto antibodies against either DSG1 alone, or DSG1 and DSG3, thereby recapitulating PF and mucocutaneous Pemphigus, respectively. In addition to the existing models, with the active models reported in this work, it will be possible to recapitulate and mimic the main forms of pemphigus in adult mice, thus allowing a better understanding of the disease in the long term, including the benefit/risk ratio of new therapies. Results: The new DSG1 and the DSG1/DSG3 mixed models were developed as proposed. Immunized animals, and subsequently, animals that received splenocytes from the immunized donors produce a high concentration of circulating antibodies against the specific antigens. The severity of the disease was assessed by evaluating the PV score, evidencing that the DSG1/DSG3 mixed model exhibits the most severe symptoms among those analyzed. Alopecia, erosions, and blistering were observed in the skin of DSG1, DSG3 and DSG1/DSG3 models, while lesions in the mucosa were observed only in DSG3 and DSG1/DSG3 animals. The effectiveness of the corticosteroid Methyl-Prednisolone was evaluated in the DSG1 and DSG1/DSG3 models, that showed only partial responsiveness.

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 - The Capacity of Magnesium to Induce Osteoclast Differentiation Is Greatly Enhanced by the Presence of Zoledronate [Articolo su rivista]
Ricchiuto, Silvia; Palumbo, Rossella; Lami, Francesca; Gavioli, Francesca; Caselli, Lorenzo; Montanari, Monica; Zappavigna, Vincenzo; Anesi, Alexandre; Zanocco-Marani, Tommaso; Grande, Alexis
abstract

Simple Summary A number of skeletal disorders, all characterized by a metabolic or neoplastic loss of bone tissue, are cured with drugs called Bisphosphonates (BPs), which exert their therapeutic effect by suppressing cells named osteoclasts, normally mediating bone resorption. Unfortunately, these drugs can also provoke a dangerous side effect known as osteonecrosis of the jaw (ONJ), a bone infection localized in the oral cavity and characterized by gingival ulceration, sometimes accompanied by suppuration and pain. This condition, occasionally arising spontaneously, is more often started by a tooth extraction. The reduced number of osteoclasts, determined by BPs, is thought to favor the bacterial invasion of healthy bone and the incapacity to eliminate infected bone, that are in turn responsible for the appearance of ONJ. Here we show that Magnesium, used for decades as dietary supplement, can invert the effect of BPs, transforming them, through a sort of paradox effect, into powerful activators of osteoclast production. These results suggest that Magnesium might be used in a topical approach aimed to cure or prevent ONJ. Notably, the capacity of Magnesium to activate osteoclast production was even observed in absence of BPs, suggesting its application also in ONJ forms caused by agents distinct to BPs.Abstract Bisphosphonates (BPs) are successfully used to cure a number of diseases characterized by a metabolic reduction in bone density, such as Osteoporosis, or a neoplastic destruction of bone tissue, such as multiple myeloma and bone metastases. These drugs exert their therapeutic effect by causing a systemic osteoclast depletion that, in turn, is responsible for reduced bone resorption. Unfortunately, in addition to their beneficial activity, BPs can also determine a frightening side effect known as osteonecrosis of the jaw (ONJ). It is generally believed that the inability of osteoclasts to dispose of inflamed/necrotic bone represents the main physiopathological aspect of ONJ. In principle, a therapeutic strategy able to elicit a local re-activation of osteoclast production could counteract ONJ and promote the healing of its lesions. Using an experimental model of Vitamin D3-dependent osteoclastogenesis, we have previously demonstrated that Magnesium is a powerful inducer of osteoclast differentiation. Here we show that, surprisingly, this effect is greatly enhanced by the presence of Zoledronate, chosen for our study because it is the most effective and dangerous of the BPs. This finding allows us to hypothesize that Magnesium might play an important role in the topical therapy of ONJ.

2022 - In Vitro, Ex Vivo and In Vivo Models for the Study of Pemphigus [Articolo su rivista]
Lotti, Roberta; Atene, CLAUDIO GIACINTO; Zanfi Emma, Dorotea; Bertesi, Matteo; Zanocco-Marani, Tommaso
abstract

Pemphigus is a life-threatening autoimmune disease. Several phenotypic variants are part of this family of bullous disorders. The disease is mainly mediated by pathogenic autoantibodies, but is also directed against two desmosomal adhesion proteins, desmoglein 1 (DSG1) and 3 (DSG3), which are expressed in the skin and mucosae. By binding to their antigens, autoantibodies induce the separation of keratinocytes, in a process known as acantholysis. The two main Pemphigus variants are Pemphigus vulgaris and foliaceus. Several models of Pemphigus have been described: in vitro, ex vivo and in vivo, passive or active mouse models. Although no model is ideal, different models display specific characteristics that are useful for testing different hypotheses regarding the initiation of Pemphigus, or to evaluate the efficacy of experimental therapies. Different disease models also allow us to evaluate the pathogenicity of specific Pemphigus autoantibodies, or to investigate the role of previously not described autoantigens. The aim of this review is to provide an overview of Pemphigus disease models, with the main focus being on active models and their potential to reproduce different disease subgroups, based on the involvement of different autoantigens.

2021 - Magnesium favors the capacity of vitamin d3 to induce the monocyte differentiation of u937 cells [Articolo su rivista]
Parenti, S.; Sandoni, L.; Montanari, M.; Zanocco-Marani, T.; Anesi, A.; Iotti, S.; Manfredini, R.; Frassineti, C.; Davalli, P.; Grande, A.
abstract

The hematopoietic U937 cells are able to differentiate into monocytes, macrophages, or osteoclasts when stimulated, respectively, with vitamin D3 (VD3), phorbol 12-myristate 13-acetate (PMA) or PMA plus VD3. We have previously demonstrated that magnesium (Mg) strongly potentiates the osteoclastic differentiation of U937 cells. In this study, we investigated whether such an effect may be ascribed to a capacity of Mg to modulate the monocyte differentiation of U937 cells and/or to an ability of Mg and VD3 to act directly and independently on the early phases of the osteoclastic differentiation. To address this issue, we subjected U937 cells to an individual and combined treatment with Mg and VD3 and then we analyzed, by flow cytometry and quantitative real-time polymerase chain reaction, the expression of a number of genes related to the early phases of the differentiation pathways under consideration. The results obtained indicated that Mg favors the monocyte differentiation of U937 cells induced by VD3 and at the same time, Mg contrasts the inhibitory effect that VD3 exerts on the osteoclastic differentiation in the absence of PMA. The crucial and articulated role played by Mg in diverse pathways of the osteoclastic differentiation of U973 cells is emphasized.

2021 - Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts [Articolo su rivista]
Bertesi, M.; Fantini, S.; Alecci, C.; Lotti, R.; Martello, A.; Parenti, S.; Carretta, C.; Marconi, A.; Grande, A.; Pincelli, C.; Zanocco Marani, T.
abstract

The mRNA-destabilizing protein tristetraprolin (TTP), encoded by the ZFP36 gene, is known to be able to end inflammatory responses by directly targeting and destabilizing mRNAs encoding pro-inflammatory cytokines. We analyzed its role in psoriasis, a disease characterized by chronic inflammation. We observed that TTP is downregulated in fibroblasts deriving from psoriasis patients compared to those deriving from healthy individuals and that psoriatic fibroblasts exhibit abnormal inflammasome activity compared to their physiological counterpart. This phenomenon depends on TTP downregulation. In fact, following restoration, TTP is capable of directly targeting for degradation NLRP3 mRNA, thereby drastically decreasing inflammasome activation. Moreover, we provide evidence that ZFP36 undergoes methylation in psoriasis, by virtue of the presence of long stretches of CpG dinucleotides both in the promoter and the coding region. Besides confirming that a perturbation of TTP expression might underlie the pathogenesis of psoriasis, we suggest that deregulated inflammasome activity might play a role in the disease alongside deregulated cytokine expression.

2021 - Regulatory Role Played by the mRNA Binding Protein Tristetraprolin in the Skin and its Involvement in Different Diseases [Articolo su rivista]
Lotti, Roberta; Zanocco-Marani, Tommaso
abstract

The mRNA binding protein Tristetraprolin (TTP), encoded by the ZFP36 gene, plays a fundamental regulatory role in a wide variety of cellular processes by means of its widespread expression in different tissues, and of its ability to post transcriptionally regulate the stability, and therefore the expression, of multiple specific target mRNAs. Because of these features, TTP expression and activity are strictly regulated, and malfunctions of such mechanisms underlie different pathologies. Here we recapitulate the role of TTP in the skin, and its involvement in different conditions, with special reference to psoriasis and cancer.

2020 - Cadherins down-regulation: towards a better understanding of their relevance in colorectal cancer [Articolo su rivista]
Losi, Lorena; ZANOCCO MARANI, Tommaso; Grande, Alexis
abstract

The down-regulation of cadherin expression in colorectal cancer (CRC) has been widely studied. However, existing data on cadherin expression are highly variable and its relevance to CRC development has not been completely established. This review examines published studies on cadherins whose down-regulation has been already demonstrated in CRC, trying to establish a relationship with promoter methylation, the capacity to influence the Wnt / CTNNB1 (catenin beta 1, beta-catenin) signalling pathway and the clinical implications for disease outcome. Moreover, it also analyses factors that may explain data variability and highlights the importance of considering the altered subcellular localization of the examined cadherins. The results of this survey reveal that thirty of one hundred existing cadherins appear to be down-regulated in CRC. Among these, ten are cadherins, sixteen are protocadherins, equally divided between clustered and non clustered, and four are cadherin - related. These findings suggest that, to better define the role played by cadherin down-regulation in CRC pathogenesis, the expression of multiple rather than individual cadherins should be taken into account and further functional studies are necessary to clarify the relative ability of individual cadherins to inhibit CTNNB1 therefore acting as tumor suppressors.

2020 - Depletion of Trichoplein (TpMs) Causes Chromosome Mis-Segregation, DNA Damage and Chromosome Instability in Cancer Cells [Articolo su rivista]
Lauriola, Angela; Martello, Andrea; Fantini, Sebastian; Marverti, Gaetano; Zanocco-Marani, Tommaso; Davalli, Pierpaola; Guardavaccaro, Daniele; Mai, Sabine; Caporali, Andrea; D’Arca, Domenico
abstract

Mitotic perturbations frequently lead to chromosome mis-segregation that generates genome instability, thereby triggering tumor onset and/or progression. Error-free mitosis depends on fidelity-monitoring systems that ensure the temporal and spatial coordination of chromosome segregation. Recent investigations are focused on mitotic DNA damage response (DDR) and chromosome mis-segregations with the aim of developing more efficient anti-cancer therapies. We previously demonstrated that trichoplein keratin filament binding protein (TpMs) exhibits hallmarks of a tumor suppressor gene in cancer-derived cells and human tumors. Here, we show that silencing of TpMs expression results in chromosome mis-segregation, DNA damage and chromosomal instability. TpMs interacts with Mad2, and TpMs depletion results in decreased levels of Mad2 and Cyclin B1 proteins. All the genetic alterations observed are consistent with both defective activation of the spindle assembly checkpoint and mitotic progression. Thus, low levels of TpMs found in certain human tumors may contribute to cellular transformation by promoting genomic instability.

2020 - Wnt/CTNNB1 signal transduction pathway inhibits the expression of ZFP36 in squamous cell carcinoma, by inducing transcriptional repressors SNAI1, SLUG and TWIST [Articolo su rivista]
Zanfi, E. D.; Fantini, S.; Lotti, R.; Bertesi, M.; Marconi, A.; Grande, A.; Manfredini, R.; Pincelli, C.; Zanocco-Marani, T.
abstract

The Wnt/CTNNB1 pathway is often deregulated in epithelial tumors. The ZFP36 gene, encoding the mRNA binding protein Tristetraprolin (TTP), is downregulated in several cancers, where it has been described to behave as a tumor suppressor. By this report, we show that Wnt/CTNNB1 pathway is constitutively activated, and ZFP36 expression is downregulated in Squamous Cell Carcinoma (SCC) cell lines compared to normal keratinocytes. Moreover, we suggest that the decrease of ZFP36 expression might depend on the activity of transcriptional repressors SNAI1, SLUG and TWIST, whose expression is induced by Wnt/CTNNB1, highlighting a potential regulatory mechanism underlying ZFP36 downregulation in epithelial cancers.

2019 - Development of a Desmocollin-3 Active Mouse Model Recapitulating Human Atypical Pemphigus [Articolo su rivista]
Lotti, Roberta; Atene, Claudio Giacinto; Marconi, Alessandra; Di Rocco, Giulia; Reggiani Bonetti, L; Zanocco Marani, Tommaso; Pincelli, Carlo
abstract

Pemphigus vulgaris (PV) is a life-threatening mucocutaneous autoimmune blistering disease. It is often associated with autoantibodies to the desmosomal adhesion proteins Desmoglein 3 (DSG3) and Desmoglein 1 (DSG1). Recently, auto-antigens, such as desmocollins and others have been described in PV and in atypical pemphigus forms such as Pemphigus Herpetiformis (PH), Pemphigus Vegetans (PVeg), and Paraneoplastic Pemphigus (PP). Desmocollins belong to a cadherin subfamily that provides structure to the desmosomes and play an important role in cell-to-cell adhesion. In order to verify the pathogenic activity of anti-Desmocollin 3 (DSC3) antibodies, we developed an active disease model of pemphigus expressing anti-DSC3 autoantibodies or antiDSC3 and anti-DSG3 antibodies. This approach included the adoptive transfer of DSC3 and/or DSG3 lymphocytes to Rag2(-/-) immunodeficient mice that express DSC3 and DSG3. Our results show that the presence of anti-DSC3 auto-antibodies is sufficient to determine the appearance of a pathological phenotype relatable to pemphigus, but with features not completely super-imposable to those observed in the DSG3 active model, suggesting that the DSC3 active model might mimic the atypical pemphigus. Moreover, the presence of both anti-DSC3 and anti-DSG3 antibodies determines a more severe phenotype and a slower response to prednisolone. In conclusion, we have developed an adult DSC3 pemphigus mouse model that differs from the DSG3 model and supports the concept that antigens other than desmogleins may be responsible for different phenotypes in human pemphigus.

2019 - Loss of expression of μ-protocadherin and protocadherin-24 in sporadic and hereditary nonpolyposis colorectal cancers [Articolo su rivista]
Losi, Lorena; Lancellotti, Cesare; Parenti, Sandra; Scurani, Letizia; Zanocco-Marani, Tommaso; Buffoli, Federico; Grassia, Roberto; Ferrari, Sergio; Grande, Alexis
abstract

Colorectal cancer (CRC) is a neoplastic disease in which normal mucosa undergoes a process of malignant transformation due to the progressive accumulation of molecular alterations affecting proto-oncogenes and oncosuppressor genes. Some of these modifications exert their carcinogenic potential by promoting a constitutive activation of the β-catenin signaling proliferation pathway, and when present, loss of cadherin expression also significantly contributes to the same effect. Using a combined approach of molecular and immunohistochemical analysis, we have previously demonstrated that most sporadic CRCs exhibit a down-regulated expression of a cadherin, named μ-protocadherin, that is generally observed in association with a higher proliferation rate and a worse prognosis. The aim of this report was to perform a comparative immunohistochemical assessment of μ-protocadherin and a similar cadherin, named protocadherin-24, in sporadic CRC and hereditary nonpolyposis colorectal cancer. The data obtained put in evidence that double-negative CRCs, lacking both the analyzed protocadherins, are more represented among sporadic tumors, whereas double-positive CRCs, maintaining their expression, exhibit an opposite trend. As expected, loss of protocadherin expression was accompanied by nuclear localization of β-catenin and increased positivity of the Ki-67 proliferation marker. This finding is consistent with the different clinical evolution of the 2 considered CRC sets according to which patients with hereditary nonpolyposis colorectal cancer experience a better prognosis as compared with those affected by a sporadic CRC.

2019 - Physiological expression of miR-130a during differentiation of CD34+ human hematopoietic stem cells results in the inhibition of monocyte differentiation [Articolo su rivista]
Mammoli, F.; Parenti, S.; Lomiento, M.; Gemelli, C.; Atene, C. G.; Grande, A.; Corradini, R.; Manicardi, Agnese; Fantini, S.; Zanocco-Marani, T.; Ferrari, S.
abstract

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10, IRF8, KLF4, MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML.

2018 - KLF4 mediates the effect of 5-ASA on the b-catenin pathway in colon cancer cells [Articolo su rivista]
Parenti, Sandra; Montorsi, Lucia; Fantini, Sebastian; Mammoli, Fabiana; Gemelli, Claudia; Atene, Claudio Giacinto; Losi, Lorena; Frassineti, Chiara; Calabretta, Bruno; Tagliafico, Enrico; Ferrari, Sergio; Zanocco-Marani, Tommaso; Grande, Alexis
abstract

Mesalazine (5-ASA) is an aminosalicylate anti-inflammatory drug capable of inducing m-protocadherin, a protein expressed by colorectal epithelial cells that is downregulated upon malignant transformation. Treatment with 5-ASA restores m-protocadherin expression and promotes the sequestration of b-catenin to the plasma membrane. Here, we show that 5-ASA–induced m-protocadherin expression is directly regulated by the KLF4 transcription factor. In addition, we suggest the existence of a dual mechanism whereby 5-ASA–mediated b-catenin inhibition is caused by m-protocadherin–dependent sequestration of b-catenin to the plasma membrane and by the direct binding of KLF4 to b-catenin. In addition, we found that 5-ASA treatment suppresses the expression of miR-130a and miR-135b, which target KLF4 mRNA, raising the possibility that this mechanism is involved in the increased expression of KLF4 induced by 5-ASA.

2016 - Expression of μ-protocadherin is negatively regulated by the activation of the β-catenin signaling pathway in normal and cancer colorectal enterocytes [Articolo su rivista]
Montorsi, Lucia; Parenti, Sandra; Losi, Lorena; Ferrarini, F; Gemelli, Claudia; Rossi, A; Manco, Gianrocco; Ferrari, Sergio; Calabretta, Bruno; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; Grande, Alexis
abstract

Mu-protocadherin (MUCDHL) is an adhesion molecule predominantly expressed by colorectal epithelial cells which is markedly downregulated upon malignant transformation. Notably, treatment of colorectal cancer (CRC) cells with mesalazine lead to increased expression of MUCDHL, and is associated with sequestration of β-catenin on the plasma membrane and inhibition of its transcriptional activity. To better characterize the causal relationship between β-catenin and MUCDHL expression, we performed various experiments in which CRC cell lines and normal colonic organoids were subjected to culture conditions inhibiting (FH535 treatment, transcription factor 7-like 2 siRNA inactivation, Wnt withdrawal) or stimulating (LiCl treatment) β-catenin activity. We show here that expression of MUCDHL is negatively regulated by functional activation of the β-catenin signaling pathway. This finding was observed in cell culture systems representing conditions of physiological stimulation and upon constitutive activation of β-catenin in CRC. The ability of MUCDHL to sequester and inhibit β-catenin appears to provide a positive feedback enforcing the effect of β-catenin inhibitors rather than serving as the primary mechanism responsible for β-catenin inhibition. Moreover, MUCDHL might have a role as biomarker in the development of CRC chemoprevention drugs endowed with β-catenin inhibitory activity.

2016 - Loss of zfp36 expression in colorectal cancer correlates to wnt/ ß-catenin activity and enhances epithelial-to-mesenchymal transition through upregulation of zeb1, sox9 and macc1 [Articolo su rivista]
Montorsi, Lucia; Guizzetti, Filippo; Alecci, Claudia; Caporali, Andrea; Martello, Andrea; Giacinto Atene, Claudio; Parenti, Sandra; Pizzini, Silvia; Zanovello, Paola; Bortoluzzi, Stefania; Ferrari, Sergio; Grande, Alexis; ZANOCCO MARANI, Tommaso
abstract

The mRNA-destabilizing protein ZFP36 has been previously described as a tumor suppressor whose expression is lost during colorectal cancer development. In order to evaluate its role in this disease, we restored ZFP36 expression in different cell contexts, showing that the presence of this protein impairs the epithelial-to-mesenchymal transition (EMT) and induces a higher susceptibility to anoikis. Consistently, we found that ZFP36 inhibits the expression of three key transcription factors involved in EMT: ZEB1, MACC1 and SOX9. Finally, we observed for the first time that its expression negatively correlates with the activity of Wnt/β-catenin pathway, which is constitutively activated in colorectal cancer. This evidence provides a clue on the mechanism leading to the loss of ZFP36 in CRC.

2015 - ZFP36 stabilizes RIP1 via degradation of XIAP and cIAP2 thereby promoting ripoptosome assembly [Articolo su rivista]
Selmi, Tommaso; Alecci, Claudia; Dell' Aquila, Miriam; Montorsi, Lucia; Martello, Andrea; Guizzetti, Filippo; Volpi, Nicola; Parenti, Sandra; Ferrari, Sergio; Salomoni, Paolo; Grande, Alexis; ZANOCCO MARANI, Tommaso
abstract

BACKGROUND: ZFP36 is an mRNA binding protein that exerts anti-tumor activity in glioblastoma by triggering cell death, associated to an increase in the stability of the kinase RIP1. METHODS: We used cell death assays, size exclusion chromatography, Co-Immunoprecipitation, shRNA lentivectors and glioma neural stem cells to determine the effects of ZFP36 on the assembly of a death complex containing RIP1 and on the induction of necroptosis. RESULTS: Here we demonstrate that ZFP36 promotes the assembly of the death complex called Ripoptosome and induces RIP1-dependent death. This involves the depletion of the ubiquitine ligases cIAP2 and XIAP and leads to the association of RIP1 to caspase-8 and FADD. Moreover, we show that ZFP36 controls RIP1 levels in glioma neural stem cell lines. CONCLUSIONS: We provide a molecular mechanism for the tumor suppressor role of ZFP36, and the first evidence for Ripoptosome assembly following ZFP36 expression. These findings suggest that ZFP36 plays an important role in RIP1-dependent cell death in conditions where IAPs are depleted.

2014 - MafB is a downstream target of the IL-10/STAT3 signaling pathway, involved in the regulation of macrophage de-activation [Articolo su rivista]
Gemelli, C.; Zanocco Marani, T.; Bicciato, S.; Mazza, E. M. C.; Boraschi, D.; Salsi, V.; Zappavigna, V.; Parenti, S.; Selmi, T.; Tagliafico, E.; Ferrari, S.; Grande, A.
abstract

In spite of the numerous reports implicating MafB transcription factor in the molecular control of monocyte-macrophage differentiation, the precise genetic program underlying this activity has been, to date, poorly understood. To clarify this issue, we planned a number of experiments that were mainly conducted on human primary macrophages. In this regard, a preliminary gene function study, based on MafB inactivation and over-expression, indicated MMP9 and IL-. 7R genes as possible targets of the investigated transcription factor. Bioinformatics analysis of their promoter regions disclosed the presence of several putative MARE elements and a combined approach of EMSA and luciferase assay subsequently demonstrated that expression of both genes is indeed activated by MafB through a direct transcription mechanism. Additional investigation, performed with similar procedures to elucidate the biological relevance of our observation, revealed that MafB is a downstream target of the IL-10/STAT3 signaling pathway, normally inducing the macrophage de-activation process. Taken together our data support the existence of a signaling cascade by which stimulation of macrophages with the IL-10 cytokine determines a sequential activation of STAT3 and MafB transcription factors, in turn leading to an up-regulated expression of MMP9 and IL-. 7R genes. © 2014 Elsevier B.V.

2013 - The Orosomucoid1 protein is involved in the vitamin D – mediated macrophage de-activation process [Articolo su rivista]
Gemelli, Claudia; Martello, Andrea; Montanari, Monica; ZANOCCO MARANI, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis
abstract

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 - VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

2012 - ZFP36 expression impairs glioblastoma cell lines viability and invasiveness by targeting multiple signal transduction pathways. [Articolo su rivista]
Selmi, Tommaso; Martello, Andrea; Vignudelli, Tatiana; Ferrari, Erika; Grande, Alexis; Gemelli, Claudia; Salomoni, P; Ferrari, Sergio; ZANOCCO MARANI, Tommaso
abstract

RNA binding proteins belonging to the TIS11/TTP gene family regulate the stability of multiple targets. Their inactivation or deregulated expression has recently been related to cancer, and it has been suggested that they are capable of displaying tumor suppressor activities. Here we describe three new targets of ZFP36 (PIM-1, PIM-3 and XIAP) and show by different approaches that its ectopic expression is capable of impairing glioblastoma cell lines viability and invasiveness by interfering with different transduction pathways. Moreover, we provide evidence that compounds capable of inducing the expression of TIS11/TTP genes determine a comparable biological effect on the same cell contexts.

2011 - Alpha – 1 – acid glycoprotein-A is a new VDR transcriptional target involved in monocyte differentiation and activation processes. [Poster]
Gemelli, Claudia; Martello, Andrea; Montanari, Monica; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; ZANOCCO MARANI, Tommaso; Ferrari, Sergio; Grande, Alexis
abstract

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3--VDR--ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

2010 - TIS11/TTP gene family: It's never too late for tumor suppressors. [Articolo su rivista]
ZANOCCO MARANI, Tommaso
abstract

Review on TIS11 gene family.

2010 - ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with Stat5b Pathway. [Articolo su rivista]
Vignudelli, Tatiana; Selmi, Tommaso; A., Martello; Parenti, Sandra; Grande, Alexis; Gemelli, Claudia; ZANOCCO MARANI, Tommaso; Ferrari, Sergio
abstract

ZFP36L1 is a member of a family of CCCH tandem zinc finger proteins (TTP family) able to bind to AU-rich elements in the 3'-untranslated region of mRNAs, thereby triggering their degradation. The present study suggests that such mechanism is used during hematopoiesis to regulate differentiation by post-transcriptionally modulating the expression of specific target genes. In particular, it demonstrates that ZFP36L1 negatively regulates erythroid differentiation by directly binding the 3' untranslated region of Stat5b encoding mRNA. Stat5b down-regulation obtained by ZFP36L1 overexpression results, in human hematopoietic progenitors, in a drastic decrease of erythroid colonies formation. These observations have been confirmed by silencing experiments targeting Stat5b and by treating hematopoietic stem/progenitor cells with drugs able to induce ZFP36L1 expression. Moreover, this study shows that different members of ZFP36L1 family act redundantly, since cooverexpression of ZFP36L1 and family member ZFP36 determines a cumulative effect on Stat5b down-regulation. This work describes a mechanism underlying ZFP36L1 capability to regulate hematopoietic differentiation and suggests a new target for the therapy of hematopoietic diseases involving Stat5b/JAK2 pathway, such as chronic myeloproliferative disorders.

2009 - TFE3 transcription factor regulates the expression of MAFB during macrophage differentiation [Articolo su rivista]
ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Parenti, Sandra; Gemelli, Claudia; Condorelli, F; Martello, A; Selmi, Tommaso; Grande, Alexis; Ferrari, Sergio
abstract

Transcription Factor for Immunoglobulin Heavy-Chain Enhancer 3 (Tfe3) is a transactivator of metabolic genes that are regulated through an EBox located in their promoters. It is involved in physiological processes such as osteoclast and macrophage differentiation, as well as in pathological processes such as translocations underlying different cancer diseases. MAFB is a basic region/leucine zipper transcription factor that affects transcription by binding specific DNA regions known as MARE. It plays a pivotal role in regulating lineage-specific hematopoiesis by repressing transcription of erythroid specific genes in myeloid cells and enhancing expression of macrophage and megakaryocytic genes. Here we have shown MAFB to be highly induced in human hematopoietic cells undergoing macrophage differentiation following Tfe3 ectopic expression, and to be down regulated, compared to the controls, in the same cell population following Phorbol Esters (PMA) dependent differentiation coupled to Tfe3 gene silencing. Electrophoretic mobility shift assays identified a Tfe3-binding site (EBox) in the MAFB promoter region that is conserved in different mammalian species. MAFB promoter was transactivated by co-expression of Tfe3 in reporter gene assays while deletion or mutation of the MAFB EBox prevented transactivation by Tfe3. Both of these genes were previously included in the group of transcription factors able to drive macrophage differentiation. The observation that MAFB belongs to the Tfe3 regulon suggests the existence of a pathway where these two gene families act synergistically to determine differentiation.

2008 - The vitamin D3/Hox-A10 pathway supports MafB function during the monocyte differentiation of human CD34+ hemopoietic progenitors [Articolo su rivista]
Gemelli, Claudia; Orlandi, Claudia; ZANOCCO MARANI, Tommaso; Martello, A; Vignudelli, Tatiana; Ferrari, Francesco; Montanari, Monica; Parenti, Sandra; Testa, Anna; Grande, Alexis; Ferrari, Sergio
abstract

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D3 receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D3/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.

2006 - IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA [Abstract in Atti di Convegno]
Tenedini, Elena; Tagliafico, Enrico; Manfredini, Rossella; Ferrari, Francesco; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Tatiana Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Ferrari, Sergio
abstract

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to All-Trans Retinoic-Acid (ATRA) and Vitamin-D3 (VD) and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature of 11 genes, among them is particularly intriguing the presence of Meis1 and ID3, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients known to have poor prognostic significance.

2006 - Identification of a molecular signature predictive of sensitivity to differentiation induction in acute myeloid leukemia [Articolo su rivista]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Grande, Alexis; Ferrari, F.; Roncaglia, Enrica; Bicciato, Silvio; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Gemelli, Claudia; Montanari, Monica; Vignudelli, Tatiana; ZANOCCO MARANI, Tommaso; Parenti, Sandra; Paolucci, Paolo; Martinelli, G.; Piccaluga, P. P.; Baccarani, M.; Specchia, G.; Torelli, U.; Ferrari, Sergio
abstract

Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.

2006 - Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors. [Articolo su rivista]
ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Gemelli, Claudia; Pirondi, Sara; Testa, Anna; Montanari, Monica; Parenti, Sandra; Tenedini, Elena; Grande, Alexis; Ferrari, Sergio
abstract

The MItf-Tfe family of basic helix–loop–helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage.

2006 - Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells [Articolo su rivista]
Gemelli, Claudia; Montanari, Monica; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Siena, Michela; Zini, Roberta; Salati, Simona; Tagliafico, Enrico; Manfredini, Rossella; Specchia, G; Grande, Alexis; Ferrari, Sergio
abstract

Upregulation of specific transcription factors is a generally accepted mechanism to explain the commitment of hematopoietic stem cells along precise maturation lineages. Based on this premise, transduction of primary hematopoietic stem/progenitor cells with viral vectors containing the investigated transcription factors appears as a suitable experimental model to identify such regulators. Although MafB transcription factor is believed to play a role in the regulation of monocytic commitment, no demonstration is, to date, available supporting this function in normal human hematopoiesis. To address this issue, we retrovirally transduced cord blood CD34+ hematopoietic progenitors with a MafB cDNA. Immunophenotypic and morphological analysis of transduced cells demonstrated the induction of a remarkable monomacrophage differentiation. Microarray analysis confirmed these findings and disclosed the upregulation of macrophage-related transcription factors belonging to the AP-1, MAF, PPAR and MiT families. Altogether our data allow to conclude that MafB is a key regulator of human monocytopoiesis.

2005 - Correlation between differentiation plasticity and mRNA expression profiling of CD34+-derived CD14- and CD14+ human normal myeloid precursors [Articolo su rivista]
Montanari, Monica; Gemelli, Claudia; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, T; Siena, M; Zini, Roberta; Salati, Simona; Chiossi, G; Tagliafico, Enrico; Manfredini, Rossella; Grande, Alexis; Ferrari, Sergio
abstract

In spite of their apparently restricted differentiation potentiality, hematopoietic precursors are plastic cells able to trans-differentiate from a maturation lineage to another. To better characterize this differentiation plasticity, we purified CD14- and CD14+ myeloid precursors generated by 'in vitro' culture of human CD34+ hematopoietic progenitors. Morphological analysis of the investigated cell populations indicated that, as expected, they consisted of granulocyte and monocyte precursors, respectively. Treatment with differentiation inducers revealed that CD14- cells were bipotent granulo-monocyte precursors, while CD14+ cells appeared univocally committed to a terminal macrophage maturation. Flow cytometry analysis demonstrated that the conversion of granulocyte precursors to the mono-macrophage maturation lineage occurs through a differentiation transition in which the granulocyte-related myeloperoxidase enzyme and the monocyte-specific CD14 antigen are co-expressed. Expression profiling evidenced that the observed trans-differentiation process was accompanied by a remarkable upregulation of the monocyte-related MafB transcription factor.

2005 - IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA [Abstract in Atti di Convegno]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Ferrari, Sergio; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Specchia, Giorgina; Martinelli, Giovanni; Baccarani, Michele; Piccaluga, Pier Paolo; Torelli, Umberto; Ferrari, Sergio
abstract

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to ATRA and VD and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients with citogenetically and molecularly defined lesions that are known to have poor prognostic significance.

2005 - The kinetic status of hematopoietic stem cell subpopulations underlies a differential expression of genes involved in self-renewal, commitment, and engraftment. [Articolo su rivista]
Manfredini, Rossella; Zini, Roberta; Salati, Simona; Siena, M; Tenedini, Elena; Tagliafico, Enrico; Montanari, Monica; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, T; Grande, A; Fogli, M; Rossi, L; Fagioli, Me; Catani, L; Lemoli, Rm; Ferrari, Sergio
abstract

The gene expression profile of CD34(-) hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage-negative (Lin(-)) CD34(-), Lin(-)CD34(+), and Lin(+)CD34(+) cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34(-) cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and growth-arrest genes. According to the expression of their receptors and transducers, interleukin (IL)-10 and IL-17 showed an inhibitory effect on the clonogenic activity of CD34(-) cells. Conversely, CD34(+) cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34(-) cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self-renewal and lineage commitment. The preferential expression in CD34(+) cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34(+) cells. Thus, the different kinetic status of CD34(-) and CD34(+) cells, detailed by molecular and functional analysis, significantly influences their biological behavior

2003 - Development of an IL-6 antagonist peptide that induces apoptosis in IL-6 dependent 7TD1 cells. [Articolo su rivista]
Manfredini, Rossella; Tenedini, Elena; M., Siena; Tagliafico, Enrico; Montanari, Monica; Grande, Alexis; ZANOCCO MARANI, Tommaso; C., Poligani; Zini, Roberta; A., Bergamaschi; DE RIENZO, Francesca; DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Ferrari, Sergio
abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of proliferation and differentiation of hematopoietic cells and in the pathogenesis of many diseases, including multiple myeloma. This study pursues a way to interfere with IL-6 pathway in an attempt to modulate its biological activity. Here we describe the rational design and biological evaluation of peptides able to antagonize the murine IL-6 activity by interfering with IL-6 Receptor alpha in 7TD1 cells, a IL-6-dependent B-cell line. Of the peptide tested, only Guess 4a is capable of interfering with IL-6 transducing pathway, therefore inducing growth arrest and apoptosis of 7TD1 cells.

2003 - Italian family with two independent mutations:3358T/A in BRCA1 and 8756delA in BRCA2 genes. [Articolo su rivista]
Cortesi, L.; Turchetti, D.; Bertoni, Carlo Maria; ZANOCCO MARANI, Tommaso; Silvestri, C.; Vinceti, Marco; Federico, Massimo; Silingardi, Vittorio; Ferrari, Sergio
abstract

Hereditary breast/ovarian cancer is a well-characterized clinical entity, largely attributed to the inheritance of BRCA1 or BRCA2 mutations. Among general population, the mutation's frequency of these genes is very low; therefore, the identification of two independent mutations in the same family is a rare event. This study reports the presence of two mutations, one in the BRCA1 and the second in the BRCA2 gene in an Italian Caucasian kindred. This family is composed of more than 250 individuals, spanning through five generations, among which endogamy was a common phenomenon. Considering the tumor spectrum, this family is characterized by a high incidence of different types of cancer. In our study, we considered only three out of seven family units for BRCA1 and BRCA2 analysis. In one of the family units, we found independent mutations of both BRCA genes. The BRCA1 mutation on exon 11 (3358TA) was identified originally in the index case and subsequently in 18 members of this family, whereas the same mutation was not detected in a related family member with male breast cancer. The male breast cancer patient led to the identification, through mutational analysis, of a new BRCA2 mutation (8756delA). This BRCA2 mutation was also found in the male breast cancer patient's daughter. The discovery of the BRCA2 mutation allowed us to alert the patient's daughter who, otherwise, could be falsely reassured since she had a negative BRCA1 test.

2003 - Requirement of the coiled-coil domains of p92(c-Fes) for nuclear localization in myeloid cells upon induction of differentiation [Articolo su rivista]
Tagliafico, Enrico; M., Siena; ZANOCCO MARANI, Tommaso; Manfredini, Rossella; Tenedini, Elena; M., Montanari; Grande, Alexis; Ferrari, Sergio
abstract

The nonreceptor tyrosine kinase Fes is implicated in myeloid cells differentiation. It has been observed that its localization can be cytoplasmic, perinuclear, or nuclear. To further characterize this point, we studied Fes subcellular localization in myeloid cell lines (HL60 and K562) and in COS1 cells. Fes was observed in both the nucleus and the cytoplasm of HL60, K562 cells over-expressing Fes and only in the cytoplasm of COS1 cells, suggesting that nuclear localization is cell context dependent. Moreover, in myeloid cells, the treatment with differentiation-inducing agents such as retinoic acid, phorbol esters and vitamin D, is followed by an increase of the oligomeric form of Fes in the nucleus. In fact, oligomerization seems to be necessary for translocation to occur, since Fes mutants missing the coiled-coil domains are not able to form oligomers and fail to localize in the nucleus. The active form of Fes is tyrosine phosphorylated; however, phosphorylation is not required for Fes to localize in the nucleus, since tyrosine kinase inhibitors do not block the translocation process.

2002 - Gene Expression profile of Vitamin D3 treated HL60 cells shows a phenotypic but not a complete functional conversion to monocytes [Abstract in Atti di Convegno]
Tenedini, Elena; Bergamaschi, A; Manfredini, Rossella; Percudani, R; Siena, M; ZANOCCO MARANI, Tommaso; Grande, Alexis; Montanari, Monica; Gemelli, Claudia; Torelli, U; Ferrari, Sergio; Tagliafico, Enrico
abstract

Acute Myeloid leukemia blast cells are characterized by their inability to proceed spontaneously toward terminal differentiation. To tackle this problem we have studied the changes occurring in the gene expression profile during the differentiation of HL60 cells treated with VD using the Affymetrix GeneChip technology and we have compared the molecular phenotype of VD induced cells to that of CD14+ pheripheral monocytes.

2002 - Gene expression Profile of Human Myeloid Cells [Abstract in Atti di Convegno]
Siena, M; Manfredini, R; Bergamaschi, A; Tenedini, Elena; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; Montanari, Monica; Gemelli, Claudia; Grande, Alexis; Ferrari, Sergio
abstract

Array technologies have made it possible to monitor simultaneously the expression pattern of thousands of genes. Working on normal human hempoietic stem cells it is possible to evaluate their gene expression profile, changes in gene expression occurring in their early commitment phase and to compare the gene expression profiling with normallly differentiated myeloid cells, i.e. granulocytes and monocytes.

2002 - Gene expression profile of vitamin D3 treated HL60 cells shows an incomplete molecular phenotypic conversion to monocytes [Articolo su rivista]
Tagliafico, Enrico; Tenedini, Elena; A., Bergamaschi; ZANOCCO MARANI, Tommaso; R., Percudani; M., Siena; Manfredini, Rossella; Grande, Alexis; M., Montanari; C., Gemelli; U., Torelli; Ferrari, Sergio
abstract

By high density oligonucleotide microarrays we have studied the expression profile of proliferating and VD treated HL60 cells and the molecular phenotype of VD monocytes and that of CD14+ peripheral monocytes has been compared. The results indicate that important changes in functional categories of the differentially expressed genes underlie the differentiation transition from myeloblasts to monocytes. This differential gene expression pattern leads to an increased expression of mRNAs involved in surface and external activities since many of the VD induced genes belong to ligand binding, receptors, cell surface antigens, defense/immunity and adhesion molecules functional categories. results also indicate that the molecular phenotypes of monocytes and VD induced cells diverge for a small but significant set of defense related genes. Particularly, class II MHC genes are not expressed in these cells. Furthermore, the high levels of expression of these genes induced by serum treatment of monocytes are decreased by VD.

2002 - Physiological levels of 1 alpha, 25 di-hydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors [Articolo su rivista]
Grande, Alexis; M., Montanari; Tagliafico, Enrico; Manfredini, Rossella; ZANOCCO MARANI, Tommaso; M., Siena; Tenedini, Elena; A., Gallinelli; Ferrari, Sergio
abstract

Although supraphysiological levels of 1alpha, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo- and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid-culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38-hematopoietic stem cells, the down-regulation of CD34 antigen, and the increase of monocyte-committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.

2002 - Requirement of the coiled coil domains of p92c-Fes for nuclear translocation in myeloid cells upon induction of differentiation [Abstract in Atti di Convegno]
ZANOCCO MARANI, Tommaso; Siena, M; Tagliafico, Enrico; Manfredini, Rossella; Tenedini, Elena; Montanari, Monica; Grande, Alexis; Gemelli, Claudia; Ferrari, Sergio
abstract

The non-receptor tyrosine kinase Fes is expressed in hematopoietic progenitors, differentiated myeloid cells and other cell types, such as vascular endothelial cells and neuroblastoma cell lines. To further clarify this point we performed confocal microscopy and western blot experiments on myeloid cell lines and COS1 cells. In myeloid cells the treatment with differentiation inducing agents such as ATRA, PMA and VD is followed by an increase of Fes abundance in the nuclear compartment. The active form of Fes is phosphorylated on residue 713 and is present into the nucleus while treated cells with the tyrosine kinase inhibitor Genistein clearly showed that phosphorylation is not a required event in order to Fes to translocate to the nucleus.

2001 - A functionally active RAR alpha nuclear receptor is expressed in retinoic acid non responsive early myeloblastic cell lines [Articolo su rivista]
Grande, Alexis; M., Montanari; Manfredini, Rossella; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; F., Trevisan; Ligabue, Giulia; M., Siena; Ferrari, Stefano; Ferrari, Sergio
abstract

Although ail-trans retinoic acid (ATRA) can restore the differentiation capacity of leukemic promyelocytes, early leukemic myeloblasts are conversely not responsive to ATRA induced granulocytic differentiation. To assess whether this resistance to ATRA is related to an impaired function of the Retinoic Acid Receptor alpha (RAR alpha), we performed an analysis of RAR alpha expression and transactivation activity, in several myeloid leukemic cell lines, representative of different types of spontaneous acute myeloid leukemias. Our results indicate that a functionally active RAR alpha nuclear receptor is expressed in all the analyzed cell lines, regardless of their differentiation capacity following exposure to ATRA. The observation that ATRA treatment is able to induce the expression of retinoic acid target genes, in late- but not in early-myetoblastic leukemic cells, raises the possibility that the differentiation block of these cells is achieved through a chromatin mediated mechanism. Acetylation is apparently not involved in this process, since the histone deacetylase inhibitor trichostatin A, is not able to restore the differentiation capacity of early leukemic myeloblasts. Further investigation is needed to clarify whether myeloid transcription factors, distinct to RAR alpha, play a role in the resistance of these cells to ATRA treatment.

2001 - Efficient in vitro and in vivo gene regulation of a retrovirally delivered pro-apoptotic factor under the control of the Drosophila HSP70 promoter [Articolo su rivista]
Romano, G; Reiss, K; Tu, X; Peruzzi, F; Belletti, B; WANG J., Y; ZANOCCO MARANI, Tommaso; Baserga, R.
abstract

We have developed a self-inactivating retroviral vector system with an internal, inducible Drosophila HSP70 promoter. This vector system delivers the desired transgene into cells rapidly and efficiently. It generates mixed populations of transduced cells where the transgene is inducible, and does not require the isolation of specific clones. Since the transgene is not expressed (or poorly expressed) at the restrictive condition (34 degrees C), mixed populations can be selected in which tumor suppressors or other inhibitory genes can be strongly induced upon changing the conditions (39 degrees C or the plant amino acid L-canavanine). This retroviral vector should be very useful for the expression of sequences that are poorly tolerated by cells, and is also active in animals.

2000 - Insulin receptor substrate-1, p70(S6K), and cell size in transformation and differentiation of hemopoietic cells [Articolo su rivista]
Valentinis, B; Navarro, M; ZANOCCO MARANI, T; Edmonds, P; Mccormick, J; Morrione, A; Sacchi, A; Romano, G; Reiss, K; AND BASERGA, R
abstract

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70(S6K), which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70(S6K) and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70(S6K) seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70(S6K) in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70(S6K) activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.

1999 - Biological Activities and Signaling Pathways of the Granulin/Epithelin precursor. [Articolo su rivista]
ZANOCCO MARANI, Tommaso; T, Bateman; A, Romano; G, Valentinis; B, ; Zhi, Heng; He, ; And, Baserga; R., BIOLOGICAL ACTIVITIES AND SIGNALING PATHWAYS OF THE GRANULINEPITHELIN PRECURSOR
abstract

Growth-regulated cells, such as 3T3 mouse embryo fibroblasts (MEFs), require more than one growth factor for growth, usually the insulin-like growth factor I (IGF-I) in combination with either platelet-derived growth factor or epidermal growth factor. Singly, these growth factors cannot sustain the growth of 3T3 cells. However, if the IGF-I receptor (IGF-IR) is even modestly overexpressed, then IGF-I, by itself, stimulates the growth of MEFs in monolayer and makes them capable of forming colonies in soft agar. The granulin/epithelin precursor (GEP) has been identified as the only growth factor, thus far, that can stimulate by itself the growth of R- cells, a 3T3-like cell line in which the genes for the IGF-IR have been deleted. We have expressed GEP in R- cells and show that these cells can now grow in serum-free medium. GEP, however, cannot replace other functions of the IGF-IR, such as protection from apoptosis (anoikis) or transforming activity (colony formation in soft agar). GEP activates, in R- cells, the two signaling pathways that are known to be sufficient for IGF-I-mediated mitogenesis in cells overexpressing the IGF-IR, the mitogen-activated protein kinase and the phosphatidylinositol 3-kinase pathways. This may explain why GEP, by itself, can replace the IGF-IR for growth in monolayer cultures. It also confirms that, for transformation, other pathways must be activated besides the two pathways that are sufficient for mitogenesis.

1999 - Dissociation Between Resistance to Apoptosis and the Transformed Phenotype in IGF-1 Receptor Signaling. [Articolo su rivista]
Romano, G; Prisco, M; ZANOCCO MARANI, Tommaso; T, Peruzzi; F, Valentinis; B, ; And, Baserga
abstract

Programmed Cell Death (PCD) is known to play an important role in both the development and the growth rate of human tumors. It has in fact been suggested that suppression of the apoptotic pathway is a requirement for the establishment of the transformed phenotype. In order to elucidate the relationship between resistance to apoptosis and transformation, we have asked in this investigation whether or not the two processes can be directly correlated. For this purpose, we have used mouse embryo fibroblasts (MEF) expressing either the wild-type or several mutants of the type 1 insulin-like growth factor receptor (IGF-IR). The wild-type IGF-IR has both transforming and anti-apoptotic activities, and we have asked whether these two activities can be or not separated in mutant receptors. Using this well-defined system, our results show that certain mutants of the IGF-IR that have strong anti-apoptotic and mitogenic activities, are incapable of transforming MEF (colony formation in soft agar). We have, instead, a good correlation between mitogenic and anti-apoptotic activities, suggesting the possibility that the two processes may share similar signaling pathways from the IGF-IR. On the other hand, our results indicate that transformation requires an additional signal, above and beyond the mitogenic and survival signals. Our conclusion is that, at least in this system, the establishment of the malignant phenotype and resistance to apoptosis can be dissociated, implying the possibility of separate targeting.

1998 - The Granulin/Epithelin Precursor Abrogates the Requirement for the Insulin-like Growth Factor 1 Receptor for Growth in Vitro [Articolo su rivista]
Xu, Sq; Tang, D; Chamberlain, S; Pronk, G; Masiarz, F; Kaur, S; Prisco, M; ZANOCCO MARANI, T; AND BASERGA, R
abstract

3T3 cells null for the type 1 insulin-like growth factor receptor are refractory to stimulation by a variety of purified growth factors that are known to be required for the stimulation of other 3T3 cells. However, these cells, known as R- cells, grow in serum-supplemented medium and also in media conditioned by certain cell lines. We report here the purification of a growth factor that stimulates DNA synthesis (and growth) of R- cells. The growth factor, purified to homogeneity by SDS-polyacrylamide gel electrophoresis, was identified as the granulin/epithelin precursor by an accurate determination of the masses of endoproteinase Lys-C peptides using matrix-assisted laser desorption ionization mass spectrometry, followed by a data base search. The granulin/epithelin precursor is a little known growth factor, secreted by a variety of epithelial and hemopoietic cells. It is at present the only purified growth factor that can stimulate the growth of mouse embryo fibroblasts null for the type 1 insulin-like growth factor receptor.