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Sandra MARMIROLI

Professore Ordinario
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze,sede Istituti Anatomici (area Policlinico)

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Pubblicazioni

2024 - The key role of public health in renovating Italian biomedical doctoral programs [Articolo su rivista]
Palandri, Lucia; Urbano, Teresa; Pezzuoli, Carla; Miselli, Francesca; Caraffi, Riccardo; Filippini, Tommaso; Bargellini, Annalisa; Righi, Elena; Mazzi, Davide; Vigezzi, Giacomo Pietro; Odone, Anna; Marmiroli, Sandra; Boriani, Giuseppe; Vinceti, Marco
abstract

Background: A key renovation of doctoral programs is currently ongoing in Italy. Public health and its competencies may play a pivotal role in high-level training to scientific research, including interdisciplinary and methodological abilities. Methods: As a case study, we used the ongoing renovation of the Clinical and Experimental Medicine doctoral program at the University of Modena and Reggio Emilia. We focused on how the program is designed to meet national requirements as well as students' needs, thus improving educational standards for scientific research in the biomedical field, and on the specific contribution of public health and epidemiology in such an effort. Results: The renovation process of doctoral programs in Italy, with specific reference to the biomedical field, focuses on epidemiologic-statistical methodology, ethics, language and communication skills, and open science from an interdisciplinary and international perspective. In the specific context of the doctoral program assessed in the study and from a broader perspective, public health appears to play a key role, taking advantage of most recent methodological advancements, and contributing to the renovation of the learning process and its systematic quality monitoring. Conclusions: From a comparative assessment of this case study and Italian legislation, the key role of public health has emerged in the renovation process of doctoral programs in the biomedical field.

2023 - Improved efficacy of quizartinib in combination therapy with PI3K inhibition in primary FLT3-ITD AML cells [Articolo su rivista]
Darici, Salihanur; Jørgensen, Heather G; Huang, Xu; Serafin, Valentina; Antolini, Ludovica; Barozzi, Patrizia; Luppi, Mario; Forghieri, Fabio; Marmiroli, Sandra; Zavatti, Manuela
abstract

: Acute myeloid leukemia is a heterogeneous hematopoietic malignancy, characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells, with poor outcomes. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) represents the most common genetic alteration in AML, detected in approximately 30% of AML patients, and is associated with high leukemic burden and poor prognosis. Therefore, this kinase has been regarded as an attractive druggable target for the treatment of FLT3-ITD AML, and selective small molecule inhibitors, such as quizartinib, have been identified and trialled. However, clinical outcomes have been disappointing so far due to poor remission rates, also because of acquired resistance. A strategy to overcome resistance is to combine FLT3 inhibitors with other targeted therapies. In this study, we investigated the preclinical efficacy of the combination of quizartinib with the pan PI3K inhibitor BAY-806946 in FLT3-ITD cell lines and primary cells from AML patients. We show here that BAY-806946 enhanced quizartinib cytotoxicity and, most importantly, that this combination increases the ability of quizartinib to kill CD34+ CD38-leukemia stem cells, whilst sparing normal hematopoietic stem cells. Because constitutively active FLT3 receptor tyrosine kinase is known to boost aberrant PI3K signaling, the increased sensitivity of primary cells to the above combination can be the mechanistic results of the disruption of signaling by vertical inhibition.

2022 - Editor's Note: Dasatinib Plus Nutlin-3 Shows Synergistic Antileukemic Activity in Both p53wild-type and p53mutated B Chronic Lymphocytic Leukemias by Inhibiting the Akt Pathway [Articolo su rivista]
Zauli, Giorgio; Voltan, Rebecca; Bosco, Raffaella; Melloni, Elisabetta; Marmiroli, Sandra; Rigolin, Gian Matteo; Cuneo, Antonio; Secchiero, Paola
abstract

2021 - A KDM4A-PAF1-mediated epigenomic network is essential for acute myeloid leukemia cell self-renewal and survival [Articolo su rivista]
Massett, M. E.; Monaghan, L.; Patterson, S.; Mannion, N.; Bunschoten, R. P.; Hoose, A.; Marmiroli, S.; Liskamp, R. M. J.; Jorgensen, H. G.; Vetrie, D.; Michie, A. M.; Huang, X.
abstract

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

2021 - Editorial: Special Issue on Innovative Multi-Disciplinary Approaches for Precision Studies in Leukemia [Recensione in Rivista]
Marmiroli, S.; Huang, X.; Serafin, V.; Michie, A. M.
abstract

By investigating the role of recurrent mutations, epigenetic modifications and aberrant activation of oncogenic signaling (1–4), our understanding of leukemogenesis has advanced to the stage that precision medicine is capable of transforming diagnostic and therapeutic approaches. This Research Topic focuses on the innovative technologies that will aid in the development of a multi-dimensional integrated molecular network, enabling the prediction of therapy responses and creation of novel treatments for those in most clinical need.

2021 - Mechanisms involved in the promoting activity of fibroblasts in HTLV-1-mediated lymphomagenesis: Insights into the plasticity of lymphomatous cells [Articolo su rivista]
Rigotto, G.; Montini, B.; Mattiolo, A.; Lazzari, N.; Piano, M. A.; Remondini, D.; Marmiroli, S.; Bertacchini, J.; Chieco-bianchi, L.; Calabro, M. L.
abstract

Among the mechanisms leading to progression to Adult T-cell Leukaemia/Lymphoma in Human T-cell Leukaemia Virus type 1 (HTLV-1)-infected subjects, the contribution of stromal components remains poorly understood. To dissect the role of fibroblasts in HTLV-1-mediated lymphomagenesis, transcriptome studies, cytofluorimetric and qRT-PCR analyses of surface and intracellular markers linked to plasticity and stemness in coculture, and in vivo experiments were performed. A transcriptomic comparison between a more lymphomagenic (C91/III) and the parental (C91/PL) cell line evidenced hyperactivation of the PI3K/Akt pathway, confirmed by phospho-ELISA and 2-DE and WB analyses. C91/III cells also showed higher expression of mesenchymal and stemness genes. Short-term coculture with human foreskin fibroblasts (HFF) induced these features in C91/PL cells, and significantly increased not only the cancer stem cells (CSCs)-supporting CD10+GPR77+ HFF subpopulation, but also the percentage of ALDH1bright C91/PL cells. A non-cytotoxic acetylsalicylic acid treatment decreased HFF-induced ALDH1bright C91/PL cells, downregulated mesenchymal and stemness genes in cocultured cells, and delayed lymphoma growth in immunosuppressed mice, thus hindering the supportive activity of HFF on CSCs. These data suggest that crosstalk with HFF significantly intensifies the aggressiveness and plasticity of C91/PL cells, leading to the enrichment in lymphoma-initiating cells. Additional research is needed to better characterize these preliminary findings.

2021 - Synergistic cytotoxicity of dual PI3K/mTOR and FLT3 inhibition in FLT3-ITD AML cells [Articolo su rivista]
Darici, S.; Zavatti, M.; Braglia, L.; Accordi, B.; Serafin, V.; Horne, G. A.; Manzoli, L.; Palumbo, C.; Huang, X.; Jorgensen, H. G.; Marmiroli, S.
abstract

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy, characterized by a heterogeneous genetic landscape and complex clonal evolution, with poor outcomes. Mutation at the internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic alterations in AML, associated with high relapse rates and poor survival due to the constitutive activation of the FLT3 receptor tyrosine kinase and its downstream effectors, such as PI3K signaling. Thus, aberrantly activated FLT3-kinase is regarded as an attractive target for therapy for this AML subtype, and a number of small molecule inhibitors of this kinase have been identified, some of which are approved for clinical practice. Nevertheless, acquired resistance to these molecules is often observed, leading to severe clinical outcomes. Therapeutic strategies to tackle resistance include combining FLT3 inhibitors with other antileukemic agents. Here, we report on the preclinical activity of the combination of the FLT3 inhibitor quizartinib with the dual PI3K/mTOR inhibitor PF-04691502 in FLT3-ITD cells. Briefly, we show that the association of these two molecules displays synergistic cytotoxicity in vitro in FLT3-ITD AML cells, triggering 90% cell death at nanomolar concentrations after 48 h.

2020 - An unbiased approach to mapping the signaling network of the pseudorabies virus US3 protein [Articolo su rivista]
Jansens, R. J. J.; Marmiroli, S.; Favoreel, H. W.
abstract

The US3 serine/threonine protein kinase is conserved among the alphaherpesvirus family and represents an important virulence factor. US3 plays a role in viral nuclear egress, induces dramatic alterations of the cytoskeleton, represses apoptosis, enhances gene expression and modulates the immune response. Although several substrates of US3 have been identified, an unbiased screen to identify US3 phosphorylation targets has not yet been described. Here, we perform a shotgun and phosphoproteomics analysis of cells expressing the US3 protein of pseudorabies virus (PRV) to identify US3 phosphorylation targets in an unbiased way. We identified several cellular proteins that are differentially phosphorylated upon US3 expression and validated the phosphorylation of lamin A/C at serine 404, both in US3-transfected and PRV-infected cells. These results provide new insights into the signaling network of the US3 protein kinase and may serve as a basis for future research into the role of the US3 protein in the viral replication cycle.

2020 - Crosstalks of GSK3 signaling with the mTOR network and effects on targeted therapy of cancer [Articolo su rivista]
Evangelisti, C; Chiarini, F; Paganelli, F; Marmiroli, S; Martelli, Am
abstract

Abstract The introduction of therapeutics targeting specific tumor-promoting oncogenic or non-oncogenic signaling pathways has revolutionized cancer treatment. Mechanistic (previously mammalian) target of rapamycin (mTOR), a highly conserved Ser/Thr kinase, is a central hub of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR network, one of the most frequently deregulated signaling pathways in cancer, that makes it an attractive target for therapy. Numerous mTOR inhibitors have progressed to clinical trials and two of them have been officially approved as anticancer therapeutics. However, mTOR-targeting drugs have met with a very limited success in cancer patients. Frequently, the primary impediment to a successful targeted therapy in cancer is drug-resistance, either from the very beginning of the therapy (innate resistance) or after an initial response and upon repeated drug treatment (evasive or acquired resistance). Drug-resistance leads to treatment failure and relapse/progression of the disease. Resistance to mTOR inhibitors depends, among other reasons, on activation/deactivation of several signaling pathways, included those regulated by glycogen synthase kinase-3 (GSK3), a protein that targets a vast number of substrates in its repertoire, thereby orchestrating many processes that include cell proliferation and survival, metabolism, differentiation, and stemness. A detailed knowledge of the rewiring of signaling pathways triggered by exposure to mTOR inhibitors is critical to our understanding of the consequences such perturbations cause in tumors, including the emergence of drug-resistant cells. Here, we provide the reader with an updated overview of intricate circuitries that connect mTOR and GSK3 and we relate them to the efficacy (or lack of efficacy) of mTOR inhibitors in cancer cells.

2020 - Deregulated PTEN/PI3K/AKT/mTOR signaling in prostate cancer: Still a potential druggable target? [Articolo su rivista]
Braglia, L.; Zavatti, M.; Vinceti, M.; Martelli, A. M.; Marmiroli, S.
abstract

Although the prognosis of patients with localized prostate cancer is good after surgery, with a favorable response to androgen deprivation therapy, about one third of them invariably relapse, and progress to castration-resistant prostate cancer. Overall, prostate cancer therapies remain scarcely effective, thus it is mandatory to devise alternative treatments enhancing the efficacy of surgical castration and hormone administration. Dysregulation of the phosphoinositide 3-kinase pathway has attracted growing attention in prostate cancer due to the highly frequent association of epigenetic and post-translational modifications as well as to genetic alterations of both phosphoinositide 3-kinase and PTEN to onset and/or progression of this malignancy, and to resistance to canonical androgen-deprivation therapy. Here we provide a summary of the biological functions of the major players of this cascade and their deregulation in prostate cancer, summarizing the results of preclinical and clinical studies with PI3K signaling inhibitors and the reasons of failure independent from genomic changes.

2020 - Targeting PI3K/Akt/mTOR in AML: Rationale and Clinical Evidence. [Articolo su rivista]
Darici, S; Alkhaldi, H; Horne, G; Jørgensen, Hg; Marmiroli, S; Huang, X.
abstract

Acute myeloid leukemia (AML) is a highly heterogeneous hematopoietic malignancy characterized by excessive proliferation and accumulation of immature myeloid blasts in the bone marrow. AML has a very poor 5-year survival rate of just 16% in the UK; hence, more efficacious, tolerable, and targeted therapy is required. Persistent leukemia stem cell (LSC) populations underlie patient relapse and development of resistance to therapy. Identification of critical oncogenic signaling pathways in AML LSC may provide new avenues for novel therapeutic strategies. The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathway, is often hyperactivated in AML, required to sustain the oncogenic potential of LSCs. Growing evidence suggests that targeting key components of this pathway may represent an effective treatment to kill AML LSCs. Despite this, accruing significant body of scientific knowledge, PI3K/Akt/mTOR inhibitors have not translated into clinical practice. In this article, we review the laboratory-based evidence of the critical role of PI3K/Akt/mTOR pathway in AML, and outcomes from current clinical studies using PI3K/Akt/mTOR inhibitors. Based on these results, we discuss the putative mechanisms of resistance to PI3K/Akt/mTOR inhibition, offering rationale for potential candidate combination therapies incorporating PI3K/Akt/mTOR inhibitors for precision medicine in AML.

2019 - Clusterin enhances AKT2-mediated motility of normal and cancer prostate cells through a PTEN and PHLPP1 circuit [Articolo su rivista]
Bertacchini, Jessika; Mediani, Laura; Beretti, Francesca; Guida, Marianna; Ghalali, Aram; Brugnoli, Federica; Bertagnolo, Valeria; Petricoin, Emanuel; Poti, Francesco; Arioli, Jessica; Anselmi, Laura; Bari, Alessia; Mccubrey, James; Martelli, Alberto M.; Cocco, Lucio; Capitani, Silvano; Marmiroli, Sandra
abstract

Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3′-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.

2019 - Development of solvent-casting particulate leaching (SCPL) polymer scaffolds as improved three-dimensional supports to mimic the bone marrow niche [Articolo su rivista]
Sola, Antonella; Bertacchini, Jessika; D'Avella, Daniele; Anselmi, Laura; Maraldi, Tullia; Marmiroli, Sandra; Messori, Massimo
abstract

The need for new approaches to investigate ex vivo the causes and effects of tumor and to achieve improved cancer treatments and medical therapies is particularly urgent for malignant pathologies such as lymphomas and leukemias, whose tissue initiator cells interact with the stroma creating a three-dimensional (3D) protective environment that conventional mono- and bi-dimensional (2D) models are not able to simulate realistically. The solvent-casting particulate leaching (SCPL) technique, that is already a standard method to produce polymer-based scaffolds for bone tissue repair, is proposed here to fabricate innovative 3D porous structures to mimic the bone marrow niche in vitro. Two different polymers, namely a rigid polymethyl methacrylate (PMMA) and a flexible polyurethane (PU), were evaluated to the purpose, whereas NaCl, in the form of common salt table, resulted to be an efficient porogen. The adoption of an appropriate polymer-to-salt ratio, experimentally defined as 1:4 for both PMMA and PU, gave place to a rich and interconnected porosity, ranging between 82.1 vol% and 91.3 vol%, and the choice of admixing fine-grained or coarse-grained salt powders allowed to control the final pore size. The mechanical properties under compression load were affected both by the polymer matrix and by the scaffold's architecture, with values of the elastic modulus indicatively varying between 29 kPa and 1283 kPa. Preliminary tests performed with human stromal HS-5 cells co-cultured with leukemic cells allowed us to conclude that stromal cells grown associated to the supports keep their well-known protective and pro-survival effect on cancer cells, indicating that these devices can be very useful to mimic the bone marrow microenvironment and therefore to assess the efficacy of novel therapies in pre-clinical studies.

2019 - Influence of selenium on the emergence of neuro tubule defects in a neuron-like cell line and its implications for amyotrophic lateral sclerosis [Articolo su rivista]
Maraldi, T.; Beretti, F.; Anselmi, L.; Franchin, C.; Arrigoni, G.; Braglia, L.; Mandrioli, J.; Vinceti, M.; Marmiroli, S.
abstract

Impairment of the axonal transport system mediated by intracellular microtubules (MTs) is known to be a major drawback in neurodegenerative processes. Due to a growing interest on the neurotoxic effects of selenium in environmental health, our study aimed to assess the relationship between selenium and MTs perturbation, that may favour disease onset over a genetic predisposition to amyotrophic lateral sclerosis. We treated a neuron-like cell line with sodium selenite, sodium selenate and seleno-methionine and observed that the whole cytoskeleton was affected. We then investigated the protein interactome of cells overexpressing αTubulin-4A (TUBA4A) and found that selenium increases the interaction of TUBA4A with DNA- and RNA-binding proteins. TUBA4A ubiquitination and glutathionylation were also observed, possibly due to a selenium-dependent increase of ROS, leading to perturbation and degradation of MTs. Remarkably, the TUBA4A mutants R320C and A383 T, previously described in ALS patients, showed the same post-translational modifications to a similar extent. In conclusion this study gives insights into a specific mechanism characterizing selenium neurotoxicity.

2018 - Dual inhibition of PI3K/mTOR signaling in chemoresistant AMLprimary cells. [Articolo su rivista]
Bertacchini, Jessika; Frasson, Chiara; Chiarini, Francesca; D'Avella, Daniele; Accordi, Benedetta; Anselmi, Laura; Barozzi, Patrizia; Foghieri, Fabio; Luppi, Mario; Martelli, Alberto M.; Basso, Giuseppe; Najmaldin, Saki; Khosravi, Abbas; Rahim, Fakher; Marmiroli, Sandra
abstract

A main cause of treatment failure for AML patients is resistance to chemotherapy. Survival of AML cells may depend on mechanisms that elude conventional drugs action and/or on the presence of leukemia initiating cells at diagnosis, and their persistence after therapy. MDR1 gene is an ATP-dependent drug efflux pump known to be a risk factor for the emergence of resistance, when combined to unstable cytogenetic profile of AML patients. In the present study, we analyzed the sensitivity to conventional chemotherapeutic drugs of 26 samples of primary blasts collected from AML patients at diagnosis. Detection of cell viability and apoptosis allowed to identify two group of samples, one resistant and one sensitive to in vitro treatment. The cells were then analyzed for the presence and the activity of P-glycoprotein. A comparative analysis showed that resistant samples exhibited a high level of MDR1 mRNA as well as of P-glycoprotein content and activity. Moreover, they also displayed high PI3K signaling. Therefore, we checked whether the association with signaling inhibitors might resensitize resistant samples to chemo-drugs. The combination showed a very potent cytotoxic effect, possibly through down modulation of MDR1, which was maintained also when primary blasts were co-cultured with human stromal cells. Remarkably, dual PI3K/mTOR inactivation was cytotoxic also to leukemia initiating cells. All together, our findings indicate that signaling activation profiling associated to gene expression can be very useful to stratify patients and improve therapy.

2018 - Nuclear Nox4 interaction with prelamin A is associated with nuclear redox control of stem cell aging [Articolo su rivista]
Casciaro, Francesca; Beretti, Francesca; Zavatti, Manuela; McCubrey, James A.; Ratti, Stefano; Marmiroli, Sandra; Follo, Matilde Y.; Maraldi, Tullia
abstract

Mesenchymal stem cells have emerged as an important tool that can be used for tissue regeneration thanks to their easy preparation, differentiation potential and immunomodulatory activity. However, an extensive culture of stem cells in vitro prior to clinical use can lead to oxidative stress that can modulate different stem cells properties, such as self-renewal, proliferation, differentiation and senescence. The aim of this study was to investigate the aging process occurring during in vitro expansion of stem cells, obtained from amniotic fluids (AFSC) at similar gestational age. The analysis of 21 AFSC samples allowed to classify them in groups with different levels of stemness properties. In summary, the expression of pluripotency genes and the proliferation rate were inversely correlated with the content of reactive oxygen species (ROS), DNA damage signs and the onset premature aging markers, including accumulation of prelamin A, the lamin A immature form. Interestingly, a specific source of ROS, the NADPH oxidase isoform 4 (Nox4), can localize into PML nuclear bodies (PML-NB), where it associates to prelamin A. Besides, Nox4 post translational modification, involved in PML-NB localization, is linked to its degradation pathway, as it is also for prelamin A, thus possibly modulating the premature aging phenotype occurrence.

2017 - Aberrant Compartment Formation by HSPB2 Mislocalizes Lamin A and Compromises Nuclear Integrity and Function [Articolo su rivista]
Morelli F., F.; Verbeek D., S.; Bertacchini, Jessika; Vinet, Jonathan; Mediani, Laura; Marmiroli, Sandra; Cenacchi, G.; Nasi, Milena; DE BIASI, Sara; Brunsting J., F.; Lammerding, J.; Pegoraro, E.; Angelini, C.; Tupler, Rossella; Alberti, S.; Carra, Serena
abstract

Small heat shock proteins (HSPBs) contain intrinsically disordered regions (IDRs), but the functions of these IDRs are still unknown. Here, we report that, in mammalian cells, HSPB2 phase separates to form nuclear compartments with liquid-like properties. We show that phase separation requires the disordered C-terminal domain of HSPB2. We further demonstrate that, in differentiating myoblasts, nuclear HSPB2 compartments sequester lamin A. Increasing the nuclear concentration of HSPB2 causes the formation of aberrant nuclear compartments that mislocalize lamin A and chromatin, with detrimental consequences for nuclear function and integrity. Importantly, phase separation of HSPB2 is regulated by HSPB3, but this ability is lost in two identified HSPB3 mutants that are associated with myopathy. Our results suggest that HSPB2 phase separation is involved in reorganizing the nucleoplasm during myoblast differentiation. Furthermore, these findings support the idea that aberrant HSPB2 phase separation, due to HSPB3 loss-of-function mutations, contributes to myopathy.

2017 - Clusterin enhances migration and invasion of prostate cancer cells through an isoform-specific Akt2/miR-190/PHLPP1 circuit. [Abstract in Rivista]
Jessika Bertacchini, Marianna Guida; Mediani, Laura; Aram, Ghalali; Poti', Francesco; Arioli, Jessica; Federica, Brugnoli; Valeria, Bertagnolo; Beretti, Francesca; Lucio, Cocco; Capitani, Silvano; Palumbo, Carla; Marmiroli, Sandra
abstract

During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2.

2017 - Cross-talk between the CK2 and AKT signaling pathways in cancer [Articolo su rivista]
Ruzzene, Maria; Bertacchini, Jessika; Toker, Alex; Marmiroli, Sandra
abstract

CK2 and AKT display a high degree of cross-regulation of their respective functions, both directly, through physical interaction and phosphorylation, and indirectly, through an intense cross-talk of key downstream effectors, ultimately leading to sustained AKT activation.Being CK2 and AKT attractive targets for therapeutic intervention, here we would like to emphasize how AKT and CK2 might influence cell fate through their complex isoform-specific and contextual-dependent cross-talk, to the extent that such functional interplay should be considered when devising therapies that target one or both these key signaling kinases.

2017 - Phosphoproteomic analysis reveals hyperactivation of mTOR/STAT3 and LCK/Calcineurin axes in pediatric early T-cell precursor ALL [Articolo su rivista]
Serafin, V.; Lissandron, V.; Buldini, B.; Bresolin, S.; Paganin, M.; Grillo, F.; Andriano, N.; Palmi, C.; Cazzaniga, G.; Marmiroli, Sandra; Conter, V.; Basso, G.; Accordi, B.
abstract

Early T-cell Precursor ALL (ETP-ALL) is a subgroup of acute T leukemias with a peculiar immunophenotypic profile. Patients display a poor early response to conventional induction treatment, but an outcome after intensified therapy not worse than the other T-ALL. ETP-ALL was extensively characterized from a genetic point of view in the paper published by Zhang et al in 2012, but information about proteomic aberrancies is still lacking. In this study we profiled, by Reverse Phase Protein Arrays, the activation and expression of a considerable number of proteins in a significative cohort of 16 pediatric patients affected by ETP-ALL at diagnosis. We compared protein activation in these patients versus a group of 62 other T-ALL pediatric patients. Phosphoprotemic data were also related to mutational status of ETP-ALL patients. Our most interesting findings concern the hyperactivation of the LCK/Calcineurin and mTOR/STAT3 signaling pathways in ETP-ALL patients. LCK, despite of a lower expression, resulted strikingly boosted in these patients with a concomitant overexpression of the downstream target Calcineurin. The LCK/Calcineurin axis, followed by the activation of the NFAT family of transcription factors, was already demonstrated to be critical for T cell leukemogenesis. We also observed a possible feedback loop which could sustain mTOR activation through p70 S6Kinase hyperphosphorylation. mTOR, in turn, phosphorylates and activates STAT3, counteracting cell differentiation. The mTOR/STAT3 axis activity thus could sustain cell proliferation and survival while maintaining the markedly undifferentiated phenotype typical of ETP-ALL. Interestingly, the activation of the mTOR/STAT3 pathways is not influenced by the presence of FLT3 or PTEN mutations. Our paper also reports in ETP-ALL patients the hyperactivation of the RAS/MAPK and the JAK/STAT signaling pathways, that is not dependant from BRAF, NRAS, IL7R, JAK1 or JAK3 somatic mutations. In conclusion, our study is the first one investigating the activation of a such number of proteins in a wide cohort of pediatric ETP-ALL patients. We identified the specific activation of signaling pathways, such as the LCK/Calcineurin and the mTOR/STAT3 ones, important for T cell leukemogenesis and for the support of cancer cell viability and block of differentiation. Our results will help to explain the mechanism responsible of the poor response to conventional treatment of these patients, and might also suggest new druggable targets for personalized and less toxic therapies.

2016 - Critical Roles of EGFR Family Members in Breast Cancer and Breast Cancer Stem Cells: Targets for Therapy [Articolo su rivista]
Steelman, Linda S; Fitzgerald, Timothy; Lertpiriyapong, Kvin; Cocco, Lucio; Follo, Matilde Y; Martelli, Alberto M; Neri, Luca M; Marmiroli, Sandra; Libra, Massimo; Candido, Saverio; Nicoletti, Ferdinando; Scalisi, Aurora; Fenga, Concettina; Drobot, Lyudmyla; Rakus, Dariusz; Gizak, Agnieszka; Laidler, Piotr; Dulinska Litewka, Joanna; Basecke, Joerg; Mijatovic, Sanja; Maksimovic Ivanic, Danijela; Montalto, Giuseppe; Cervello, Melchiorre; Milella, Michelle; Tafuri, Agustino; Demidenko, Zoya; Abrams, Stephen L; Mccubrey, James A.
abstract

The roles of the epidermal growth factor receptor (EGFR) signaling pathway in various cancers including breast, bladder, brain, colorectal, esophageal, gastric, head and neck, hepatocellular, lung, neuroblastoma, ovarian, pancreatic, prostate, renal and other cancers have been keenly investigated since the 1980's. While the receptors and many downstream signaling molecules have been identified and characterized, there is still much to learn about this pathway and how its deregulation can lead to cancer and how it may be differentially regulated in various cell types. Multiple inhibitors to EGFR family members have been developed and many are in clinical use. Current research often focuses on their roles and other associated pathways in cancer stem cells (CSCs), identifying sites where therapeutic resistance may develop and the mechanisms by which microRNAs (miRs) and other RNAs regulate this pathway. This review will focus on recent advances in these fields with a specific focus on breast cancer and breast CSCs. Relatively novel areas of investigation, such as treatments for other diseases (e.g., diabetes, metabolism, and intestinal parasites), have provided new information about therapeutic resistance and CSCs.

2016 - Effects of mutations in Wnt/β-catenin, hedgehog, Notch and PI3K pathways on GSK-3 activity—Diverse effects on cell growth, metabolism and cancer [Articolo su rivista]
Mccubrey, James A; Rakus, Dariusz; Gizak, Agnieszka; Steelman, Linda S; Abrams, Steve L; Lertpiriyapong, Kvin; Fitzgerald, Timothy L; Yang, Li V; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Nicoletti, Ferdinando; Scalisi, Aurora; Torino, Francesco; Fenga, Concettina; Neri, Luca M; Marmiroli, Sandra; Cocco, Lucio; Martelli, Alberto M.
abstract

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that participates in an array of critical cellular processes. GSK-3 was first characterized as an enzyme that phosphorylated and inactivated glycogen synthase. However, subsequent studies have revealed that this moon-lighting protein is involved in numerous signaling pathways that regulate not only metabolism but also have roles in: apoptosis, cell cycle progression, cell renewal, differentiation, embryogenesis, migration, regulation of gene transcription, stem cell biology and survival. In this review, we will discuss the roles that GSK-3 plays in various diseases as well as how this pivotal kinase interacts with multiple signaling pathways such as: PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK, Wnt/beta-catenin, hedgehog, Notch and TP53. Mutations that occur in these and other pathways can alter the effects that natural GSK-3 activity has on regulating these signaling circuits that can lead to cancer as well as other diseases. The novel roles that microRNAs play in regulation of the effects of GSK-3 will also be evaluated. Targeting GSK-3 and these other pathways may improve therapy and overcome therapeutic resistance.

2016 - Reversal of the glycolytic phenotype of primary effusion lymphoma cells by combined targeting of cellular metabolism and PI3K/Akt/ mTOR signaling [Articolo su rivista]
Mediani, Laura; Gibellini, Federica; Bertacchini, Jessika; Frasson, Chiara; Bosco, Raffaella; Accordi, Benedetta; Basso, Giuseppe; Bonora, Massimo; Calabrò, Maria Luisa; Mattiolo, Adriana; Sgarbi, Gianluca; Baracca, Alessandra; Pinton, Paolo; Riva, Giovanni; Rampazzo, Enrico; Petrizza, Luca; Prodi, Luca; Milani, Daniela; Luppi, Mario; Potenza, Leonardo; De Pol, Anto; Cocco, Lucio; Capitani, Silvano; Marmiroli, Sandra
abstract

PEL is a B-cell non-Hodgkin lymphoma, occurring predominantly as a lymphomatous effusion in body cavities, characterized by aggressive clinical course, with no standard therapy. Based on previous reports that PEL cells display a Warburg phenotype, we hypothesized that the highly hypoxic environment in which they grow in vivo makes them more reliant on glycolysis, and more vulnerable to drugs targeting this pathway. We established here that indeed PEL cells in hypoxia are more sensitive to glycolysis inhibition. Furthermore, since PI3K/Akt/mTOR has been proposed as a drug target in PEL, we ascertained that pathway-specific inhibitors, namely the dual PI3K and mTOR inhibitor, PF-04691502, and the Akt inhibitor, Akti 1/2, display improved cytotoxicity to PEL cells in hypoxic conditions. Unexpectedly, we found that these drugs reduce lactate production/extracellular acidification rate, and, in combination with the glycolysis inhibitor 2-deoxyglucose (2-DG), they shift PEL cells metabolism from aerobic glycolysis towards oxidative respiration. Moreover, the associations possess strong synergistic cytotoxicity towards PEL cells, and thus may reduce adverse reaction in vivo, while displaying very low toxicity to normal lymphocytes. Finally, we showed that the association of 2-DG and PF-04691502 maintains its cytotoxic and proapoptotic effect also in PEL cells co-cultured with human primary mesothelial cells, a condition known to mimic the in vivo environment and to exert a protective and pro-survival action. All together, these results provide a compelling rationale for the clinical development of new therapies for the treatment of PEL, based on combined targeting of glycolytic metabolism and constitutively activated signaling pathways.

2015 - AKT1-mediated Lamin A/C degradation is required for nuclear degradation and normal epidermal terminal differentiation [Articolo su rivista]
Naeem, A. S; Zhu, Y; Di, W. L; Marmiroli, Sandra; O'Shaughnessy, R. F. L.
abstract

Nuclear degradation is a key stage in keratinocyte terminal differentiation and the formation of the cornified envelope that comprises the majority of epidermal barrier function. Parakeratosis, the retention of nuclear material in the cornified layer of the epidermis, is a common histological observation in many skin diseases, notably in atopic dermatitis and psoriasis. Keratinocyte nuclear degradation is not well characterised, and it is unclear whether the retained nuclei contribute to the altered epidermal differentiation seen in eczema and psoriasis. Loss of AKT1 function strongly correlated with parakeratosis both in eczema samples and in organotypic culture models. Although levels of DNAses, including DNase1L2, were unchanged, proteomic analysis revealed an increase in Lamin A/C. AKT phosphorylates Lamin A/C, targeting it for degradation. Consistent with this, Lamin A/C degradation was inhibited and Lamin A/C was observed in the cornified layer of AKT1 knockdown organotypic cultures, surrounding retained nuclear material. Using AKT-phosphorylation-dead Lamin A constructs we show that the retention of nuclear material is sufficient to cause profound changes in epidermal terminal differentiation, specifically a reduction in Loricrin, Keratin 1, Keratin 10, and filaggrin expression. We show that preventing nuclear degradation upregulates BMP2 expression and SMAD1 signalling. Consistent with these data, we observe both parakeratosis and evidence of increased SMAD1 signalling in atopic dermatitis. We therefore present a model that, in the absence of AKT1-mediated Lamin A/C degradation, DNA degradation processes, such as those mediated by DNAse 1L2, are prevented, leading to parakeratosis and changes in epidermal differentiation.

2015 - Inhibition of Ras-mediated signaling pathways in CML stem cells [Articolo su rivista]
Bertacchini, Jessika; Ketabchi, Neda; Mediani, Laura; Capitani, Silvano; Marmiroli, Sandra; Saki, Najmaldin
abstract

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the presence of the BCR-ABL1 oncoprotein in cells with a hematopoietic stem cell (HSC) origin. BCR-ABL1 tyrosine kinase activity leads to constitutive activation of Ras, which in turn acts as a branch point to initiate multiple downstream signaling pathways governing proliferation, self-renewal, differentiation and apoptosis. As aberrant regulation of these cellular processes causes transformation and disease progression particularly in advanced stages of CML, investigation of these signaling pathways may uncover new therapeutic targets for the selective eradication of CML stem cells. Transcription factors play a crucial role in unbalancing the Ras signaling network and have recently been investigated as potential modulators in this regard. In this review, we first briefly summarize the Ras-associated molecular pathways that are involved in the regulation of CML stem cell properties. Next we discuss the relevance of Ras-associated transcription factors as nuclear targets in combination treatment strategies for CML.

2015 - Phosphorylation, Signaling, and Cancer: Targets and Targeting [Articolo su rivista]
Marmiroli, Sandra; Fabbro, Doriano; Miyata, Yoshihiko; Pierobon, Mariaelena; Ruzzene, Maria
abstract

After 60 years from the first report of an enzymatic phosphorylation of proteins, protein kinases are well-established key signaling molecules that impact all major biological processes (reviewed in [1, 2]). Protein and lipid kinases fulfill essential roles in many signaling pathways that regulate normal cell functions [1–5]. Deregulation of kinase activities leads to a variety of pathologies ranging from cancer to inflammatory diseases, diabetes, infectious diseases, cardiovascular disorders, and cell growth and survival [1, 2, 5–11]. A much larger proportion of additional kinases are present in parasites and bacterial, fungal, and viral genomes that are susceptible to exploitation as drug targets [12]. Since many human diseases result from overactivation of protein and lipid kinases due to mutations and/or overexpression, this enzyme class represents an important target for the pharmaceutical industry [6]. Approximately one-third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases and to date 33 small molecular weight kinase inhibitors (SMWKIs) and a handful of therapeutic antibodies have been approved for various indications mainly in oncology and many more in various stages of clinical and preclinical development [5]. Kinase inhibitor drugs, which are in clinical trials, target all stages of signal transduction from the receptor protein tyrosine kinases that initiate intracellular signaling, through second-messenger dependent lipid and protein kinases and protein kinases that regulate the cell cycle [10, 13]. While treating chronic phase CML (an almost monogenic disease) with imatinib has been very successful, the treatment of more advanced cancers with kinase inhibitors has proven more difficult due to the heterogeneity of these cancer types as well as due to kinase inhibitor resistance resulting from selection for mutant alleles and/or upregulation of alternative signaling pathways [5, 10].

2014 - Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis [Articolo su rivista]
Bertacchini, Jessika; Guida, M; Accordi, B; Mediani, Laura; Martelli, A. M; Barozzi, Patrizia; Petricoin, E; Liotta, L; Milani, G; Giordan, M; Luppi, Mario; Forghieri, Fabio; DE POL, Anto; Cocco, L; Basso, G; Marmiroli, Sandra
abstract

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

2014 - Rapamycin treatment of Mandibuloacral dysplasia cells rescues localization of chromatin-associated proteins and cell cycle dynamics [Articolo su rivista]
Cenni, Vittoria; Capanni, Cristina; Mattioli, Elisabetta; Columbaro, Marta; Wehnert, Manfred; Ortolani, Michela; Fini, Milena; Novelli, Giuseppe; Bertacchini, Jessika; Maraldi, Nadir M; Marmiroli, Sandra; D'Apice, Maria Rosaria; Prencipe, Sabino; Squarzoni, Stefano; Lattanzi, Giovanna
abstract

Lamin A is a key component of the nuclear lamina produced through post-translational processing of its precursor known as prelamin A.LMNA mutations leading to farnesylated prelamin A accumulation are known to cause lipodystrophy, progeroid and developmental diseases, including Mandibuloacral dysplasia, a mild progeroid syndrome with partial lipodystrophy and altered bone turnover. Thus, degradation of prelamin A is expected to improve the disease phenotype. Here, we show different susceptibilities of prelamin A forms to proteolysis and further demonstrate that treatment with rapamycin efficiently and selectively triggers lysosomal degradation of farnesylated prelamin A, the most toxic processing intermediate. Importantly, rapamycin treatment of Mandibuloacral dysplasia cells, which feature very low levels of the NAD-dependent sirtuin SIRT-1 in the nuclear matrix, restores SIRT-1 localization and distribution of chromatin markers, elicits release of the transcription factor Oct-1 and determines shortening of the prolonged S-phase. These findings indicate the drug as a possible treatment for Mandibuloacral dysplasia.

2014 - Signaling specificity in the Akt pathway in biology and disease [Articolo su rivista]
Toker, Alex; Marmiroli, Sandra
abstract

Akt/PKB is a key master regulator of a wide range of physiological functions including metabolism, proliferation, survival, growth, angiogenesis and migration and invasion. The Akt protein kinase family comprises three highly related isoforms encoded by different genes. The initial observation that the Akt isoforms share upstream activators as well as several downstream effectors, together with the high sequence homology suggested that their functions were mostly redundant. By contrast, an increasing body of evidence has recently uncovered the concept of Akt isoform signaling specificity, supported by distinct phenotypes displayed by animal strains genetically modified for each of the three genes, as well as by the identification of isoform-specific substrates and association with discrete subcellular locations. Given that Akt is regarded as a promising therapeutic target in a number of pathologies, it is essential to dissect the relative contributions of each isoform, as well as the degree of compensation in pathophysiological function. Here we summarize our view of how Akt selectivity is achieved in the context of subcellular localization, isoform-specific substrate phosphorylation and context-dependent functions in normal and pathophysiological settings.

2013 - Monocytosis has adverse prognostic significance and impacts survival in patients with T-cell lymphomas [Articolo su rivista]
Bari, Alessia; Tadmor, T.; Sacchi, Stefano; Marcheselli, L.; Liardo, ELIANA VALENTINA; Pozzi, Samantha; Luminari, Stefano; Baldini, L.; Marmiroli, Sandra; Federico, Massimo; Polliack, A.
abstract

In this retrospective study we evaluated the prognostic impact of peripheral blood monocytosis in patients with T-cell non Hodgkin lymphomas with "aggressive-typically nodal presentation". In this dataset monocytes >0.8×10(9)/L had a strong and statistically significant negative impact on overall survival (OS). In univariate analysis several parameters, including age >60 years, advanced stage, bone marrow involvement, ECOG PS >1, high LDH level, monocytes >0.8×10(9)/L, hemoglobin<120g/L, albumin<35g/L) had a negative influence on outcome, but in multivariate analysis, monocytosis alone had a stronger association with poor OS.

2013 - Nuclear phospholipase C β1 signaling, epigenetics and treatments in MDS. [Articolo su rivista]
Follo, Matilde Y.; Marmiroli, Sandra; Faenza, Irene; Fiume, Roberta; Ramazzotti, Giulia; Martelli, Alberto M.; Gobbi, Pietro; Mccubrey, James A.; Finelli, Carlo; Manzoli, Francesco A.; Cocco, Lucio
abstract

Myelodysplastic syndromes (MDS), clonal hematopoietic stem-cell disorders mainly affecting older adult patients, show ineffective hematopoiesis in one or more of the lineages of the bone marrow. Most MDS are characterized by anemia, and a number of cases progresses to acute myeloid leukemia (AML). Indeed, the molecular mechanisms underlying the MDS evolution to AML are still unclear, even though the nuclear signaling elicited by PI-PLCβ1 has been demonstrated to play an important role in the control of the balance between cell cycle progression and apoptosis in MDS cells. Here we review both the role of epigenetic therapy on PI-PLCβ1 promoter and the changes in PI-PLCβ1 expression in MDS patients treated for anemia.

2013 - Risk Assessment for Metals and PAHs by Mediterranean Seafood [Articolo su rivista]
Chiara, Copat; Gea Oliveri, Conti; Carlo, Signorelli; Marmiroli, Sandra; Salvatore, Sciacca; Vinceti, Marco; Margherita, Ferrante
abstract

Fish is a very important food because of its high nutritional value. Fish consumption is largely recommended in all countries, so quality and safety of seafood are becoming of great concern. Especially in Mediterranean Sea, where many pollutants, as metals and Polycyclic Aromatic Hydrocarbons (PAHs), are often relieved also in high concentrations, seafood safety has to be checked by a methodologically rigorous risk assessment. So we propose in this paper a stages- risk assessment methodology to estimate the seafood potential risk for human health and point-out critical topics in or-der to support fish advisories.

2013 - Serum fatty acids and risk of cutaneous melanoma: a population-based case-control study [Articolo su rivista]
Vinceti, Marco; Malagoli, Carlotta; Iacuzio, Laura; Catherine M., Crespi; Sabina, Sieri; Vittorio, Krogh; Marmiroli, Sandra; Pellacani, Giovanni; Elisabetta, Venturelli
abstract

Background. Some observational studies have suggested that excess dietary intake of polyunsaturated fatty acids such as linoleic acid increases cutaneous melanoma risk. We aimed at examining the association between serum fatty acids and melanoma risk by conducting a population-based case-control study in a northern Italy community. Methods. e percentage composition of 12 fatty acids was determined in 51 newly diagnosed melanoma patients and 51 age- and sex-matched population controls by extracting total lipids from serum samples using thin layer and gas chromatography. Conditional logistic regression was used to estimate the relative risk of melanoma associated with tertiles of percentage composition of each fatty acid as well as groupings including saturated, monounsaturated, and polyunsaturated fatty acids. Results. We found a slightly increased melanoma risk for stearic and arachidic acids proportion, with and without adjustment for potential confounders. For an n-3 polyunsaturated fatty acid, docosapentaenoic acid, we found a male-specic direct association with melanoma risk. o other associations emerged for the other saturated, monounsaturated, and polyunsaturated fatty acids, individually or grouped by type. Conclusions. These fndings do not suggest a major role of fatty acids, including linoleic acid, on risk of cutaneous melanoma, though their evaluation is limited by the small sample size.

2013 - The need for a reassessment of the safe upper limit of selenium in drinking water [Articolo su rivista]
Vinceti, Marco; C. M., Crespi; Bonvicini, Francesca; Malagoli, Carlotta; M., Ferrante; Marmiroli, Sandra; S., Stranges
abstract

Results of recent epidemiologic studies suggest the need to reassess the safe upper limit in drinking water of selenium, a metalloid with both toxicological and nutritional properties. Observational and experimental human studies on health effects of organic selenium compounds consumed through diet or supplements, and of inorganic selenium consumed through drinking water, have shown that human toxicity may occur at much lower concentrations than previously surmised. Evidence indicates that the chemical form of selenium strongly influences its toxicity, and that its biological activity may differ in different species, emphasizing the importance of the few human studies on health effects of the specific selenium compounds found in drinking water. Epidemiologic studies that investigated the effects of selenate, an inorganic selenium species commonly found in drinking water, together with evidence of toxicity of inorganic selenium at low levels in vitro and in animal studies, indicate that health risks may occur at exposures below the current European Union and World Health Organization upper limit and guideline of 10 and 40 µg/l, respectively, and suggest reduction to 1 µg/l in order to adequately protect human health. Although few drinking waters are currently known to have selenium levels exceeding this level, the public health importance of this issue should not be overlooked, and further epidemiologic research is critically needed in this area.

2013 - The protein kinase Akt/PKB regulates both prelamin A degradation and Lmna gene expression [Articolo su rivista]
Bertacchini, Jessika; Beretti, Francesca; Vittoria, Cenni; Guida, Marianna; Federica, Gibellini; Mediani, Laura; Oriano, Marin; Nadir M., Maraldi; DE POL, Anto; Giovanna, Lattanzi; Lucio, Cocco; Marmiroli, Sandra
abstract

The serine/threonine kinase Akt/PKB is a major signaling hub integrating metabolic, survival, growth and cell cycle regulatory signals. The definition of the phospho-motif cipher driving phosphorylation by Akt led to the identification of hundreds of putative substrates, and it is therefore pivotal to name those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and their precursor prelamin A (collectively called A-type lamins) are type V intermediate filaments proteins forming a filamentous meshwork, the lamina, underneath the inner nuclear membrane, for nuclear envelope structures organization and interphase chromatin anchoring. In our previous work we reported that A-type lamins are phosphorylated by Akt at S301 and S404 in physiological conditions, and are therefore bona fide substrates of Akt. We describe here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt regulates also Lmna transcription. All together, our study unveils a previously unknown function of Akt in the control of prelamin A stability and expression. Moreover, given the large number of diseases related to prelamin A, our findings represent a further important step bridging basic A-type lamins physiology to therapeutic approaches for lamin A-linked disorders.

2012 - Environmental risk factors for amyotrophic lateral sclerosis: methodological issues in epidemiologic studies [Articolo su rivista]
Vinceti, Marco; M., Fiore; C., Signorelli; A., Odone; M., Tesauro; M., Consonni; Arcolin, Elisa; Malagoli, Carlotta; Mandrioli, Jessica; Marmiroli, Sandra; Sciacca, S. v.; M., Ferrante
abstract

The exact role of environmental risk factors in the etiology of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) is still unknown. Their hypothetical contribution ranges from a minimal impact to a major role. Among the environmental factors strictu sensu (i.e., not life-style factors) suspected to play a role in ALS etiology, we consider pesticides, the metalloid selenium, some heavy metals, magnetic fields and cyanobacteria. However, the possibility exists that these factors exert their activity only in genetically susceptible persons and only after long-term exposures, thus further hampering epidemiologic studies. The recent availability of powerful tools such as population-based ALS registries for case ascertainment and clustering detection, and of environmental modeling techniques and of geographical information systems, may yield unique opportunities for offering insight into the etiology of the disease.

2012 - Melanocytes – A novel tool to study mitochondrial dysfunction in Duchenne Muscular Dystrophy [Articolo su rivista]
Pellegrini, C.; Zulian, A.; Gualandi, F.; Manzati, E.; Merlini, L.; Michelini, M. E.; Benassi, Luisa; Marmiroli, Sandra; Ferlini, A.; Sabatelli, P.; Bernardi, P.; Maraldi, N. M.
abstract

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal epidermal junction. In addition the full length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA and western blot analysis. Melanocytes of Duchenne Muscular Dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments.

2012 - Nuclear damages and oxidative stress: new perspectives for laminopathies [Articolo su rivista]
G., Lattanzi; Marmiroli, Sandra; A., Facchini; N. M., Maraldi
abstract

Mutations in genes encoding nuclear envelope proteins, particularly LMNA encoding the A-type lamins, cause a broad range of diverse diseases, referred to as laminopathies. The astonishing variety of diseased phenotypes suggests that different mechanisms could be involved in the pathogenesis of laminopathies. In this review we will focus mainly on two of these pathogenic mechanisms: the nuclear damages affecting the chromatin organization, and the oxidative stress causing un-repairable DNA damages. Alteration in the nuclear profile and in chromatin organization, which are particularly impressive in systemic laminopathies whose cells undergo premature senescence, are mainly due to accumulation of unprocessed prelamin A. The toxic effect of these molecular species, which interfere with chromatin-associated proteins, transcription factors, and signaling pathways, could be reduced by drugs which reduce their farnesylation and/or stability. In particular, inhibitors of farnesyl transferase (FTIs), have been proved to be active in rescuing the altered cellular phenotype, and statins, also in association with other drugs, have been included into pilot clinical trials. The identification of a mechanism that accounts for accumulation of un-repairable DNA damage due to reactive oxygen species (ROS) generation in laminopathic cells, similar to that found in other muscular dystrophies (MDs) caused by altered expression of extracellular matrix (ECM) components, suggests that anti-oxidant therapeutic strategies might prove beneficial to laminopathic patients.

2012 - Protein kinase B/AKT isoform 2 drives migration of human mesenchymal stem cells. [Articolo su rivista]
Zrinka, Bulj; Serena, Duchi; Alessandro, Bevilacqua; Alessandro, Gherardi; Barbara, Dozza; Filippo, Piccinini; GIULIA ADALGISA, Mariani; Enrico, Lucarelli; Sandro, Giannini; Davide, Donati; Marmiroli, Sandra
abstract

This study was designed to investigate the migratory behavior of adult human mesenchymal stem cells (MSC) and the underlying mechanism. Cell migration was assessed by transwell, wound healing and time-lapse in vivo motility assays. Pharmacological inhibitors were used to determine the potential mechanism responsible for cell migration and invasion. The tests that were implemented revealed that MSC were fairly migratory. Protein kinase B (AKT) was strongly activated at the basal level. Through our analyses we demonstrated that pharmacological inactivation of AKT2 but not AKT1 significantly decreased cell migration and invasion. Although preliminary, collectively our results indicate that AKT2 activation plays a critical role in enabling MSC migration.

2011 - Ankrd2/ARPP is a novel Akt2 specific substrate andregulates myogenic differentiation upon cellular exposure to H(2)O(2). [Articolo su rivista]
Cenni, Vittoria; Bavelloni, A; Beretti, Francesca; Tagliavini, F; Manzoli, L; Lattanzi, G; Maraldi, Nm; Cocco, L; Marmiroli, Sandra
abstract

Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C(2)C(12) murine muscle cells exploiting protein characterization databases in combination with an anti-phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H(2)O(2) triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C(2)C(12) myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions.

2011 - Autophagic degradation of farnesylated prelamin A as a therapeutic approach to lamin-linked progeria. [Articolo su rivista]
Cenni, V; Capanni, C; Columbaro, M; Ortolani, M; D'Apice, Mr; Novelli, G; Fini, M; Marmiroli, Sandra; Scarano, E; Maraldi, Nm; Squarzoni, S; Prencipe, S; Lattanzi, G.
abstract

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.

2011 - Dasatinib Plus Nutlin-3 Shows Synergistic Antileukemic Activity inBoth p53wild-type and p53mutated B Chronic Lymphocytic Leukemias by Inhibitingthe Akt Pathway [Articolo su rivista]
Zauli, G; Voltan, R; Bosco, Raffaella; Melloni, E; Marmiroli, Sandra; Rigolin, Gm; Cuneo, A; Secchiero, P.
abstract

PURPOSE: To analyze the effect of the combination of Dasatinib, a multikinase inhibitor, plus Nutlin-3, a nongenotoxic activator of the p53 pathway, in primary B chronic lymphocytic leukemia (B-CLL) patient samples and B leukemic cell line models.EXPERIMENTAL DESIGN: The induction of cytotoxicity was evaluated in both primary B-CLL cell samples (n = 20) and in p53(wild-type) (EHEB, JVM-2) and p53(deleted/mutated) (MEC-2, BJAB) B leukemic cell lines. The role of Akt in modulating leukemic cell survival/apoptosis in response to Dasatinib or Dasatinib + Nutlin-3 was documented by functional experiments carried out using specific pharmacological inhibitors and by overexpression of membrane-targeted constitutively active form of Akt.RESULTS: The combination of Dasatinib + Nutlin-3 exhibited a synergistic cytotoxicity in the majority (19 out of 20) of B-CLL samples, including patients carrying 17p- (n = 4), and in both p53(wild-type) and p53(deleted/mutated) B leukemic cell lines. At the molecular level, Dasatinib significantly counteracted the Nutlin-3-mediated induction of the p53 transcriptional targets MDM2 and p21 observed in p53(wild-type) leukemic cells. Conversely, Nutlin-3 did not interfere with the ability of Dasatinib to decrease the phosphorylation levels of ERK1/2, p38/MAPK, and Akt in both p53(wild-type) and p53(deleted/mutated) B leukemic cell lines. A critical role of Akt downregulation in mediating the antileukemic activity of Dasatinib and Dasatinib + Nutlin-3 was demonstrated in experiments carried out by specifically modulating the Akt pathway.CONCLUSIONS: These findings suggest that Dasatinib + Nutlin-3 might represent an innovative therapeutic combination for both p53(wild-type) and p53(deleted/mutated) B-CLL. Clin Cancer Res; 17(4); 1-9. ©2010 AACR.

2011 - Reverse-phase protein microarrays (RPPA) as a diagnosticand therapeutic guide in multidrug resistant leukemia [Articolo su rivista]
Maraldi, Tullia; Bertacchini, Jessika; Benincasa, Marta; M., Guida; DE POL, Anto; L., Liotta LA; E., Petricoin; L., Cocco; Marmiroli, Sandra
abstract

Abstract. Reverse-phase microarray assays using phosphospecificantibodies (RPPA) can directly measure levels ofphosphorylated protein isoforms. In the current study, lysatesfrom parental and multidrug resistant (MDR) CEM leukemiacells were spotted onto reverse-phase protein microarraysand probed with a panel of phospho-antibodies to ERK, PCKand Akt pathways. In particular, the Akt pathway is consideredto play significant roles in leukemia and Akt inhibitor therapyhas been proposed as a potential tool in the treatment of thisdisease. The RPPA data prompted us to investigate deeperthis pathway. Here, we found that whereas total Akt1 proteinlevel is higher in parental CEM cells, the activated isoformcontent, p-Akt1, increases in doxorubicin-selected CEM cells(MDR-CEM). This was backed up by Western blot analysis,confirming that Akt1 activity/phosphorylation may be upregulatedin MDR-CEM cells. Further exploration of inhibitorytherapy in this system was evaluated. The TNF-relatedapoptosis-inducing ligand, TRAIL, has been shown toselectively kill tumor cells. Herein, we describe that in MDRCEMcells TRAIL responsiveness correlates with a reducedexpression of endogenous Akt1, suggesting that the MDRphenotype associated to P-gp sensitizes cells to TRAIL therapy.

2010 - Laminopathies and A-type lamin-associated signalling pathways [Articolo su rivista]
Maraldi, Nm; Lattanzi, G; Cenni, Vittoria; Bavelloni, A; Marmiroli, Sandra; Manzoli, Fa
abstract

no abstract available

2009 - A-type lamins and signaling: the PI 3-kinase/Akt pathway moves forward [Articolo su rivista]
Marmiroli, Sandra; Bertacchini, Jessika; Beretti, Francesca; V., Cenni; M., Guida; DE POL, Anto; N. M., Maraldi; G., Lattanzi
abstract

Lamin A/C is a nuclear lamina constituent mutated in a number of human inherited disorders collectively referred to as laminopathies. The occurrence and significance of lamin A/C interplay with signaling molecules is an old question, suggested by pioneer studies performed in vitro. However, this relevant question has remained substantially unanswered, until data obtained in cellular and organismal models of laminopathies have indicated two main aspects of lamin A function. The first aspect is that lamins establish functional interactions with different protein platforms, the second aspect is that lamin A/C activity and altered function may elicit different effects in different cells and tissue types and even in different districts of the same tissue. Both these observations strongly suggest that signaling mechanisms targeting lamin A/C or its binding partners may regulate such a plastic behavior. A number of very recent data show involvement of kinases, as Akt and Erk, or phosphatases, as PP1 and PP2, in lamin A-linked cellular mechanisms. Moreover, altered activation of signaling in laminopathies and rescue of the pathological phenotype in animal models by inhibitors of signaling pathways, strongly suggest that signaling effectors related to lamin A/C may be implicated in the pathogenesis of laminopathies and may represent targets of therapeutic intervention. In face of such an open perspective of basic and applied research, we review current evidence of lamin A/C interplay with signaling molecules, with particular emphasis on the lamin A-Akt interaction and on the biological significance of their relationship.

2009 - Human MATER localization in specific cell domains of oocytes and follicular cells [Articolo su rivista]
Sena, Paola; Riccio, Massimo; Marzona, Laura; A., Nicoli; T., Marsella; Marmiroli, Sandra; Bertacchini, Jessika; Fano, Rita Adriana; LA SALA, Giovanni Battista; DE POL, Anto
abstract

MATER (Maternal Antigen That Embryos Require) is an oocyte-specific protein dependent on the maternal genome and required for early embryonic development. The gene products expressed in oocytes play important roles in folliculogenesis, fertilization and pre-implantation development. The aim of this study was to characterize the localization and distribution pattern of the human MATER protein during follicular development and after ovulation, to determine its functional role. Immunocytochemistry experiments coupled with confocal and electron microscopy analysis were carried out to determine the ultrastructural localization of MATER in human ovarian tissue and in isolated oocytes, obtained during IVF procedures. Human cumulus cells were cultured, with or without cycloheximide, to confirm endogenous biosynthesis of the protein. Human MATER is detectable at the onset of the follicular maturation process, suggesting this protein has a role at earlier stages in the human compared with other mammalian species. The presence of MATER is specific to the oocyte and follicular cells that, during maturation, are spatially and functionally associated with the oocyte. The nuclear, nucleolar and mitochondrial localization hints at a possible role in RNA processing and the metabolic activity of the cell.

2009 - MATER protein as substrate of PKCepsilon in human cumulus cells. [Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Sena, Paola; Marzona, Laura; A., Nicoli; Marca, A. L.; Marmiroli, Sandra; Bertacchini, Jessika; LA SALA, Giovanni Battista; DE POL, Anto
abstract

High activity of the phosphoinositide 3-kinase/Akt pathway in cumulus cells plays an important role in FSH regulation of cell function and Protein Kinase C epsilon (PKCepsilon) collaborates with these signalling pathways to regulate cell proliferation. Relevant roles in follicular development are played by Maternal Antigen That Embryos Require (MATER) that is a cumulus cell- and oocyte-specific protein dependent on the maternal genome. We recently demonstrated that human MATER localizes at specific domains of oocytes and, for the first time, also in cumulus cells. MATER contains a carboxy-terminal leucine-rich repeat domain involved in protein-protein interactions regulating different cellular functions. Here we investigated the functional role of MATER. Thus, we performed coimmunoprecipitation experiments using HEK293T cells expressing human MATER; a similar approach was then followed in human cumulus/follicular cells. In MATER(+)HEK293T cells, we observed that this protein acts as a phosphorylation substrate of PKCepsilon. Western blot experiments indicate that, unlike oocytes, human cumulus cells express PKCepsilon. Immunoprecipitation and confocal analysis suggest for the first time that MATER protein interacts with this protein kinase in cumulus cells under physiological conditions. Since PKCepsilon is known to collaborate with antiapoptotic signalling pathways, this suggests a novel mechanism for the function of MATER in follicular maturation.

2008 - Lamin A Ser404 Is a Nuclear Target of Akt Phosphorylation in C2C12 Cells [Articolo su rivista]
Vittoria, Cenni; Bertacchini, Jessika; Beretti, Francesca; Giovanna, Lattanzi; Alberto, Bavelloni; Riccio, Massimo; Maria, Ruzzene; Oriano, Marin; Giorgio, Arrigoni; Veena, Parnaik; Manfred, Wehnert; Nadir M., Maraldi; DE POL, Anto; Lucio, Cocco; Marmiroli, Sandra
abstract

Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli. Although both localization and activity of Akt in the nuclear compartment are well-documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404 phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response. Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly, we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of the nuclear lamina.

2008 - The oral protein-kinase C beta inhibitor enzastaurin (LY317615) suppresses signalling through the AKT pathway, inhibits proliferation and induces apoptosis in multiple myeloma cell lines [Articolo su rivista]
Antonino, Neri; Marmiroli, Sandra; Pierfrancesco, Tassone; Luigia, Lombardi; Lucia, Nobili; Donata, Verdelli; Monica, Civallero; Maria, Cosenza; Bertacchini, Jessika; Federico, Massimo; DE POL, Anto; Giorgio Lambertenghi, Deliliers; Sacchi, Stefano
abstract

Deregulation of the protein kinase C (PKC) signalling pathway has been implicated in tumor progression. Here we investigated the PKC inhibitor enzastaurin for its activity against multiple myeloma (MM) cells. Enzastaurin suppresses cell proliferation in a large panel of human myeloma cell lines (HMCLs), with IC50 values ranging from 1.3 to 12.5 mu M and induces apoptosis, which is prevented by the ZVAD-fmk broad caspase inhibitor. These results are consistent with decreased phosphorylation of AKT and GSK3-beta, a downstream target of the AKT pathway and a pharmacodynamic marker for enzastaurin. Furthermore, enzastaurin cytotoxicity is retained when HMCLs were cocultured with multipotent mesenchymal stromal cells. Enzastaurin has additive or synergistic cytotoxic effects with bortezomib or thalidomide. Considering the strong anti-myeloma activity of enzastaurin in vitro and in animal models and its safe toxicity profile, phase II studies in MM patients of enzastaurin alone or in combination with other drugs are warranted.

2007 - Pharmacological inhibition of protein kinase CK2 reverts the multidrug resistance phenotype of a CEM cell line characterized by high CK2 level [Articolo su rivista]
DI MAIRA, G; Brustolon, F; Bertacchini, Jessika; Tosoni, K; Marmiroli, Sandra; Pinna, La; Ruzzene, M.
abstract

Protein kinase CK2 is an ubiquitous and constitutively active kinase, which phosphorylates many cellular proteins and is implicated in the regulation of cell survival, proliferation and transformation. We investigated its possible involvement in the multidrug resistance phenotype (MDR) by analysing its level in two variants of CEM cells, namely S-CEM and R.-CEM, normally sensitive or resistant to chemical apoptosis, respectively. We found that, while the CK2 regulatory subunit beta was equally expressed in the two cell variants, CK2 alpha catalytic subunit was higher in R-CEM and this was accompanied by a higher phosphorylation of endogenous protein substrates. Pharmacological downregulation of CK2 activity by a panel of specific inhibitors, or knockdown of CK2 alpha expression by RNA interference, were able to induce cell death in R-CEM. CK2 inhibitors could promote an increased uptake of chemotherapeutic drugs inside the cells and sensitize them to drug-induced apoptosis in a co-operative manner. CK2 blockade was also effective in inducing cell death of a different MDR line (U2OS). We therefore conclude that inhibition of CK2 can be considered as a promising tool to revert the MDR phenotype.

2006 - Anticancer agents sensitize osteosarcoma cells to TNF-related apoptosis-inducing ligand downmodulating IAP family proteins. [Articolo su rivista]
Mirandola, P; Sponzilli, I; Gobbi, G; Marmiroli, Sandra; Rinaldi, L; Binazzi, R; Piccari, Gg; Ramazzotti, G; Gaboardi, Gc; Cocco, L; Vitale, M.
abstract

Although TNF-related apoptosis-inducing ligand (TRAIL) usually induces cell death in tumor cells, there are some tumor cell types that are resistant to its apoptogenic effects. Some chemotherapeutic drugs, however, can sensitize resistant cancer cells to TRAIL by either upregulating surface TRAIL death receptor expression or by modulating intracellular signalling pathways emanating from TRAIL receptors. U2OS human osteosarcoma cells express TRAIL-R2 but are resistant to TRAIL-induced apoptosis. However, the genotoxic drugs, Doxorubicin and Cisplatin, are able to sensitize U20S cells to TRAIL, without affecting their surface expression of either death or decoy TRAIL receptors. We demonstrate that Doxorubicin and Cisplatin downmodulate X-IAP, while not affecting FLIP levels in U20S cells. Selective downmodulation of X-IAP protein synthesis by specific small interference RNA transfection induced a sensitization of U20S cells to TRAIL comparable to that induced by pharmacological treatment with genotoxic drugs. TRAIL-R2 downmodulation by siRNAs completely abolished the TRAIL-induced apoptosis of genotoxin-treated U20S cells. Our findings demonstrate that Doxorubicin and Cisplatin do not sensitize U20S osteosarcoma cells to TRAIL by surface receptor modulation but rather by the removal of the intracellular signalling inhibition generated by X-IAP, suggesting a foreseeable relevant advantage to the therapy of these tumors by the combined regimen of genotoxin-based chemotherapy and TRAIL.

2006 - The Oral PKC-ß Inhibitor Enzastaurin (LY317615) Suppress Phosphorylation and Induces Apoptosis in Multiple Myeloma Cell Lines by Inhibition of AKT Pathway. [Abstract in Rivista]
Antonino, Neri; Marmiroli, Sandra; Pierfrancesco, Tassone; Luigia, Lombardi; Lucia, Nobili; Donata, Verdelli; Civallero, Monica; Cosenza, Maria; Jessika, Bertacchini; Federico, Massimo; DE POL, Anto; Giorgio Lambertenghi, Deliliers; Sacchi, Stefano
abstract

The PKC pathway has been shown to play a role in the regulation of cell proliferation in several hematologic malignancies. In this study we tested the oral PKC-ß inhibitor, Enzastaurin (LY317615 - Eli Lilly) for its therapeutic efficacy in Multiple Myeloma (MM). We first analyzed PKC-ß I and II expression by Western blot in a panel of 19 human MM cell lines, showing that 9 cell lines express either 1 or both isoforms. We next examined the growth inhibition effect of Enzastaurin in the same panel of MM cell lines using either WST-1 or MTT assay and cell viability assessment by Tripan Blue exclusion. Eighteen cell lines have IC50 value ranging from 1,2 µM to 12,5 µM. To examine molecular mechanisms whereby Enzastaurin induces cytotoxicity, we performed cell cycle profiling using PI and observed a significant increase of the percentage of cells in the sub G0–G1 fraction. To determine whether Enzastaurin-induced cell death is mediated by apoptosis, we studied by ELISA and Western blot caspase 3 and PARP cleavage. We observed induction of caspase 3 and PARP cleavage in a dose and time dependent fashion. Notably, the broad caspase (Z-VAD-FMK) inhibitor reduced Enzastaurin-induced cytotoxicity. We next determined whether Enzastaurin could inhibit AKT phosphorylation in MM cell lines with constitutive phosphorylation of AKT. Enzastaurin decreased AKT phosphorylation in a dose and time dependent fashion. Phosphorylation of GSK3ß, a downstream target protein of AKT, was also markedly inhibited. Phosphorylation of PDK-1, a known upstream activator of AKT, was not affected by Enzastaurin. In conclusion, our results indicate that Enzastaurin-induced cytotoxicity is mediated via activation of caspase. This effect is associated with significant inhibition of AKT activity and its downstream target GSK3 ß. Enzastaurin does not alter the phosphorylation of the upstream AKT activator PDK-1. These data suggest that Enzastaurin inhibit AKT signalling pathway and support its evaluation in a murine model of human MM.

2005 - Lamin A N-terminal phosphorylation is associated with myoblast activation: impairment in Emery-Dreifuss muscular dystrophy [Articolo su rivista]
Cenni, Vittoria; P., Sabatelli; E., Mattioli; Marmiroli, Sandra; C., Capanni; A., Ognibene; S., Squarzoni; Nm, Maraldi; G., Bonne; M., Columbaro; L., Merlini; G., Lattanzi
abstract

Background: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms.Objective: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells.Methods: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery-Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres.Results: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts.Conclusions: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B.

2004 - New roles for lamins, nuclear envelope proteins and actin in the nucleus [Articolo su rivista]
Nm, Maraldi; G., Lattanzi; Marmiroli, Sandra; S., Squarzoni; Fa, Manzoli
abstract

Non Disponibile

2004 - Sensitization of multidrug resistant human ostesarcoma cells to Apo2 Ligand/TRAIL-induced apoptosis by inhibition of the Akt/PKB kinase [Articolo su rivista]
V., Cenni; Nm, Maraldi; A., Ruggeri; P., Secchiero; R., Del Coco; DE POL, Anto; L., Cocco; Marmiroli, Sandra
abstract

Chemotherapeutic agents have been used for the treatment of patients with osteosarcoma (OS). However, inherent or acquired resistance to these agents is a serious problem in the management of OS patients. The emergence of the multidrug resistance (MDR) phenotype in cancer cells is often associated with the overexpression of P-glycoprotein, encoded by the multidrug resistance gene MDR-1. The administration of some of the most common chemotherapeutic agents to these cells becomes ineffective because of their P-gp-driven efflux from the cell. Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that is considered to induce death of cancer cells but not normal cells. Its powerful apoptotic activity is mediated through its cell surface death domain-containing receptors, TRAIL-R1/DR4 and TRAIL-R2/DR5, which in turn spread the signal in the cytosol through the activation of the caspase cascade. The Akt/PKB kinase is an important cell survival protein which is regulated by D3-phosphoinositides. High Akt expression and activity levels are well documented in many types of tumors., which very often show an altered PI3-K/Akt/PTEN pathway. In this study the U2OS human osteosarcoma cell line and its multidrug resistant (MDR) subline that overexpresses MDR-1 gene, MDR-U2OS. have been analyzed for their responsiveness to TRAIL. In conflict with the presence of active DR4 and DR5 receptors in both clones, U2OS cells exhibited only a low responsiveness to TRAIL, while the MDR-U2OS subline did exhibit a marked TRAIL sensitivity. An analysis of the post-receptor events showed that TRAIL responsiveness correlates with a reduced expression of endogenous Akt. In fact, expression in MDR-U2OS cells of a constitutively active Akt strongly decreased their sensitivity to TRAIL. The identification of Akt as a key modulator of TRAIL responsiveness could help to design TRAIL-based combinations for treatment of osteosarcoma. Moreover, the discovery that multidrug resistant osteosarcomas are highly sensitive to TRAIL-induced apoptosis indicates TRAIL as a new candidate for the treatment of multidrug resistant bone malignancies.

2003 - Association of emerin with nuclear and cytoplasmic actin is regulated in differentiating myoblasts [Articolo su rivista]
Lattanzi, G.; Cenni, V.; Marmiroli, Sandra; Capanni, C.; Mattioli, E.; Merlini, L.; Squarzoni, S.; Maraldi, N. M.
abstract

Emerin is a nuclear envelope protein whose biological function remains to be elucidated. Mutations of emerin gene cause the Emery-Dreifuss muscular dystrophy, a neuromuscular disorder also linked to mutations of lamin A/C. In this paper, we analyze the interaction between emerin and actin in differentiating mouse myoblasts. We demonstrate that emerin and lamin A/C are bound to actin at the late stages of myotube differentiation and in mature muscle. The interaction involves both nuclear alpha and beta actins and cytoplasmic actin. A serine-threonine phosphatase activity markedly increases emerin-actin binding even in cycling myoblasts. This effect is also observed with purified nuclear fractions in pull-down assay. On the other hand, active protein phosphatase 1, a serine-threonine phosphatase known to associate with lamin A/C, inhibits emerin-actin interaction in myotube extracts. These data provide evidence of a modulation of emerin-actin interaction in muscle cells, possibly through differentiation-related stimuli.

2003 - At the nucleus of the problem: Nuclear proteins and disease [Articolo su rivista]
Nm, Maraldi; G., Lattanzi; S., Squarzoni; P., Sabatelli; Marmiroli, Sandra; A., Ognibene; Fa, Manzoli
abstract

no abstract available

2003 - Interleukin-1-receptor-associated kinase 2 (IRAK2)-mediated interleukin-1-dependent nuclear factor kappa B transactivation in Saos2 cells requires the Akt/protein kinase B kinase [Articolo su rivista]
V., Cenni; A., Sirri; DE POL, Anto; N. M., Maraldi; Marmiroli, Sandra
abstract

The post-receptor pathway that leads to nuclear factor kappaB (NF-kappaB) activation begins with the assembly of a membrane-proximal complex among the interleukin 1 (IL-1) receptors and the adaptor molecules, myeloid differentiation protein 88 (MyD88), IL-1-receptor-associated kinases (IRAKs) and tumour-necrosis-factor-receptor-associated factor 6. Eventually, phosphorylation of the inhibitor of NF-kappaB (IkappaB) by the IkappaB kinases releases NF-kappaB, which translocates to the nucleus and modulates gene expression. In this paper, we report that IRAK2 and MyD88, but not IRAK1, interact physically with Akt, as demonstrated by coimmunoprecipitation and pull-down experiments. Interestingly, the association of Akt with recombinant IRAK2 is decreased by stimulation with IL-1, and is favoured by pre-treatment with phosphatase. Likewise, Akt association with IRAK2 is increased considerably by overexpression of PTEN (phosphatase and tensin homologue deleted on chromosome 10), while it is completely abrogated by overexpression of phosphoinositide-dependent protein kinase 1. These data indicate that Akt takes part in the formation of the signalling complex that conveys the signal from the IL-1 receptors to NF-kappaB, a step that is much more membrane-proximal than was reported previously. We also demonstrate that Akt activity is necessary for IL-1-dependent NF-kappaB transactivation, since a kinase-defective mutant of Akt impairs IRAK2- and MyD88-dependent, but not IRAK1-dependent, NF-kappaB activity, as monitored by a gene reporter assay. Accordingly, IRAK2 failed to trigger inducible nitric oxide synthase and IL-1beta production in cells expressing dominant-negative Akt. However, NF-kappaB binding to DNA was not affected by inhibition of Akt, indicating that Akt regulates NF-kappaB at a level distinct from the dissociation of p65 from IkappaBalpha and its translocation to the nucleus, possibly involving phosphorylation of the p65 transactivation domain.

2003 - Targeting of the Akt/PKB kinase to the actin skeleton [Articolo su rivista]
V., Cenni; A., Sirri; Riccio, Massimo; G., Lattanzi; S., Santi; DE POL, Anto; Nm, Maraldi; Marmiroli, Sandra
abstract

Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.

2003 - Tumour necrosis factor-related apoptosis-inducing ligand sequentially activates pro-survival and pro-apoptotic pathways in SK-N-MC neuronal cells [Articolo su rivista]
Milani, D; Zauli, G; Rimondi, E; Celeghini, C; Marmiroli, Sandra; Narducci, P; Capitani, S; Secchiero, P.
abstract

The SK-N-MC neuroblastoma cell line, which expresses surface tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors TRAIL-R2 and TRAIL-R4, was used as a model system to examine the effect of TRAIL on key intracellular pathways involved in the control of neuronal cell survival and apoptosis. TRAIL induced distinct short-term (1-60 min) and long-term (3-24 h) effects on the protein kinase B (PKB)/Akt (Akt), extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-kappaB) and caspase pathways. TRAIL rapidly (from 20 min) induced the phosphorylation of Akt and ERK, but not of c-Jun NH2-terminal kinase (JNK). Moreover, TRAIL increased CREB phosphorylation and phospho-CREB DNA binding activity in a phosphatidylinositol 3-kinase (PI 3K)/Akt-dependent manner. At later time points (from 3 to 6 h onwards) TRAIL induced a progressive degradation of inhibitor of kappaB (IkappaB)beta and IkappaBepsilon, but not IkappaBalpha, coupled to the nuclear translocation of NF-kappaB and an increase in its DNA binding activity. In the same time frame, TRAIL started to activate caspase-8 and caspase-3, and to induce apoptosis. Remarkably, caspase-dependent cleavage of NF-kappaB family members as well as of Akt and CREB proteins, but not of ERK, became prominent at 24 h, a time point coincident with the peak of caspase-dependent apoptosis. IF 4.825

2002 - Nuclear PLCβ1 acts as a negative regulator of p45/NF-E2 expression levels in Friend erythroleukemia cells [Articolo su rivista]
Faenza, I.; Matteucci, A.; Bavelloni, A.; Marmiroli, Sandra; Martelli, A. M.; Gilmour, R. S.; Suh, P. G.; Manzoli, L.; Cocco, L.
abstract

t is well established that phospholipase C (PLC) beta(1) plays a role in the nuclear compartment and is involved in the signalling pathway that controls the switching of the erythroleukemia cells programming from an undifferentiated to a differentiated state. Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend cells differentiation. For further characterization, we investigated the localization of PLCbeta(1)a and PLCbeta(1)b in Friend cells by fusing their cDNA to enhanced green fluorescent protein (GFP). To investigate the potential target of nuclear PLCbeta(1) in Friend differentiation, we studied the expression of p45/NF-E2 transcription factor, which is an enhancer binding protein for expression of the beta-globin gene and the expression of GATA proteins that are important for the survival and differentiation of erythroid cells. Our data suggest that the overexpression of PLCbeta(1) (both 1a and 1b) only in the nuclear compartment significantly reduces the expression of p45/NF-E2. The effect observed is attributable to the specific action of nuclear PLCbeta(1) signalling given that GATA-1 and GATA-3 are not affected at all. Here we show the existence of a unique target, i.e. the transcription factor p45/NF-E2, whose expression is specifically inhibited by the nuclear signalling evoked by PLCbeta(1) forms.

2002 - Troglitazione affects survival of human osteosarcoma cells [Articolo su rivista]
Lucarelli, E; Sangiorgi, L; Maini, V; Lattanzi, G; Marmiroli, Sandra; Reggiani, M; Gobbi, A; Scrimieri, F; ZAMBON BERTOIA, A; Picci, P.
abstract

Activation of PPAR gamma, a transcription factor member of the family of peroxisome proliferator-activated receptors, induces apoptosis in several normal and tumor cell lines. In our study, we investigated whether treatment with troglitazone (TRO), a known PPAR gamma agonist, induced apoptosis in the human osteosarcoma (OS) cell lines G292, MG63, SAOS and U2OS that express PPAR gamma. In our experiments, TRO never induced apoptosis of OS cells; on the contrary, TRO increased cell number, based on MTT proliferation assay. Remarkably, the TRO-induced cell number increase depended on a decrease of apoptosis that naturally occurred in the culture and was not due to an increased cell proliferation rate. TRO also prevented staurosporin-induced apoptosis. The TRO-mediated survival effect correlated with the activation of Akt, a well-known mediator of survival stimuli. Our work describes a new function for TRO and indicates that the Akt survival pathway may be a mediator of TRO-induced increase of survival.IF 4.926

2000 - Inhibition of phosphoinositide 3-kinase impairs pre-commitment cell cycle traverse and prevents differentiation in erythroleukaemia cells. [Articolo su rivista]
Bavelloni, A.; Faenza, I.; Aluigi, M.; Ferri, A.; Toker, A.; Maraldi, N. M.; Marmiroli, Sandra
abstract

During the early hours after exposure to differentiation inducing agents, Friend erythroleukaemia cells undergo alterations which commit them to cessation of growth and development of the characteristics of differentiation. Our current experiments have compared the expression and activity of phosphoinositide 3-kinase (PI 3-kinase) in control cells with cells undergoing differentiation which has been induced by dimethyl sulfoxide (DMSO). When the cultures were initiated with stationary phase cells and DMSO was added at the time of seeding, PI 3-kinase activity was stimulated in both treated and control cells during the first 3 h from seeding. This event appears to be a rate limiting step in commitment since pretreatment of cells with 10 microM LY294002 or down-regulation of p85 expression prior to adding DMSO completely prevents commitment to erythropoiesis. Accordingly, PI 3-kinase inhibition during the commitment period prevents DNA-binding of the transcription factor GATA-1, essential for erythroid differentiation. However, once cells are committed to differentiate, PI 3-kinase activity and expression dramatically decreases along with the differentiation programme, to become barely detectable after 96 h. Remarkably, LY294002 treatment leads to accumulation of cell in G1 phase and prevents DMSO-dependent cyclin D3 induction. Based on these data, we suggest that PI 3-kinase is rate limiting for the completion of the first round cycle of cell division required for initiation of erythrocytic differentiation. On the other hand, the late decrease of PI 3-kinase associated with the differentiation process seems to be part of the programmed shut off of genes not needed in mature erythrocytes.

2000 - Unusual laminin alpha2 processing in myoblasts from a patient with a novel variant of congenital muscular dystrophy. [Articolo su rivista]
Lattanzi, G; Muntoni, F; Sabatelli, P; Squarzoni, S; Columbaro, M; Cenni, V; Merlini, ; Maraldi, Nm; Marmiroli, Sandra
abstract

We recently described a novel congenital muscular dystrophy (CMD) syndrome characterized by mental retardation, microcephaly, and partial merosin deficiency on muscle biopsy. Linkage analysis excluded involvement of the known CMD loci. We now report on a study performed on the differentiation of cultured myoblasts from one patient affected by this condition to evaluate the potential to form myotubes and merosin processing in these cells. The differentiation rate was comparable to controls and myotubes were stable in culture. Biochemical analysis showed the expected 80-kDa merosin subunit in myoblasts. However, a shifted 60-kDa protein was detected in myotubes. Matrix-metalloproteinases (MMPs) zymography showed increased gelatinolytic activity, and immunoblotting identified an increased amount of membrane-type 1 matrix-metalloproteinase in pathological myotube preparations. Our results show that these CMD-derived myotubes contain a low molecular weight merosin. They further suggest that an altered regulation of MMPs can be involved in basal lamina damage.

1999 - Phosphatidylinositol 3-kinase translocation to the nucleus is an early event in the interleukin-1 signalling mechanism in human osteosarcoma Saos-2 cells. [Articolo su rivista]
Maraldi, Nm; Marmiroli, Sandra; Rizzoli, R; Mazzotti, G; Manzoli, F. A.
abstract

no abstract available

1999 - Phosphatidylinositol 3-kinase translocation to the nucleus is induced by interleukin 1 and prevented by mutation of interleukin 1 receptor in human osteosarcoma Saos-2 cells [Articolo su rivista]
A., Bavelloni; S., Santi; A., Sirri; Riccio, Massimo; I., Faenza; N., Zini; S., Cecchi; A., Ferri; P., Auron; N. M., Maraldi; Marmiroli, Sandra
abstract

Although interleukin 1 (IL-1) functions have been extensively characterized, the mechanisms by which IL-l signals are transduced from the plasma membrane to the nucleus are less known. Recent evidence indicates that phosphatidylinositol 3-kinase (PI3-kinase) could be activated by a direct association with the activated IL-1 receptor. In this study we analyzed the effects of IL-1 on the intracellular distribution of PI3-kinase in wild-type Saos-2 human osteosarcoma cells, and in cell clones overexpressing type I IL-I receptor (IL-1RI), PI3-kinase intracellular distribution displays two distinct patterns, In quiescent cells, PI3-kinase is distributed through the cytoplasm, although a portion is present in the nucleus; following stimulation with IL-1, PI3-kinase is redistributed, increasing in the nuclear compartment. Both immunoblotting and immunofluorescence data indicate that IL-1 causes a rapid and transient translocation of PIS-kinase from the cytoplasm to the nucleus, This phenomenon is prevented by PI3-kinase inhibitors, suggesting that the maintenance of PI3-kinase activity is essential for IL-l-induced translocation, Indeed, in cell clones stably transfected with Y479F receptor mutant, in which the binding of the enzyme to the activated receptor is blocked, IL-l-induced PI3-kinase translocation to the nucleus is completely prevented, These data suggest that PI3-kinase translocation to the nucleus upon IL-1R activation is an early event in IL-l signaling mechanism, and may be involved in transcriptional activation.

1998 - Phosphatidylinositol 3-kinase is recruited to a specific site in the activated IL-1 receptor I. [Articolo su rivista]
Marmiroli, Sandra; Bavelloni, Faenza; Sirri, Ognibene; Cenni, Tsukada; Koyama, Ruzzene; Ferri, Auron
abstract

Phosphatidylinositol 3-kinase is recruited to a specific site in the activated IL-1 receptor I. IF 3.609

1997 - Inhibition of the expression of ornithine decarboxylase and c-Myc by cell-permeant ceramide in difluoromethylornithine-resistant leukaemia cells. [Articolo su rivista]
F., Flamigni; I., Faenza; Marmiroli, Sandra; I., Stanic; A., Giaccari; C., Muscari; C., Stefanelli; C., Rossoni
abstract

Ceramide has emerged as a novel lipid mediator in cell growth and apoptosis. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, the cell-permeant analogues of ceramide N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide) inhibited the induction of ornithine decarboxylase (ODC) activity with IC50 of 8.3 and 1.5 microM respectively. This effect was strictly related to the ability to inhibit cell growth and [3H]thymidine incorporation. The suppression of cell growth was also associated with apoptosis. The addition of bacterial sphingomyelinase resulted in a significant, but limited, reduction of ODC induction and [3H]thymidine incorporation. Bacterial lipopolysaccharide, which may act as a ceramide analogue, also inhibited the induction of the enzyme. Moreover, C6-ceramide largely prevented the accumulation of ODC mRNA and its precursor, ODC heterogeneous nuclear RNA, that accompanied the induction of ODC activity. A slight increase in ODC turnover was also observed. The DNA-binding activity of some transcription factors known to bind and transactivate the ODC gene was investigated by gel mobility-shift assay under the same experimental conditions. However, only the binding of Myc/Max was negatively affected by the treatment with C6-ceramide. Furthermore, the amount of immunoreactive c-Myc, which increased after stimulation of the cells to growth, was strongly reduced by C6-ceramide. These results suggest that the inhibition of c-Myc and ODC expression may be early events in the response of leukaemia cells to ceramide.IF 5.016

1997 - Nuclear lipid-dependent signal transduction in human osteosarcoma cells. [Articolo su rivista]
N. M., Maraldi; Marmiroli, Sandra; L., Cocco; S., Capitani; O., Bernabei; F. A., Manzoli
abstract

no abstract available

1997 - Phosphatidylinositol 3-kinase is required for the induction of ornithine decarboxylase in leukemia cells stimulated to growth. [Articolo su rivista]
F., Flamigni; Marmiroli, Sandra; C., Capanni; C., Stefanelli; C., Guarnieri; C. M., Caldarera
abstract

The involvement of phosphatidylinositol 3-kinase (PI3K) in the induction of ornithine decarboxylase (ODC) was investigated by using specific PI3K inhibitors. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, treatment with LY294002 inhibited cell growth and provoked a complete block of the induction of ODC activity (IC50 approximately 2 microM) and ODC protein. Some reduction in the accumulation of ODC mRNA was also observed, whereas ODC turnover was not affected significantly. Wortmannin, another specific inhibitor of PI3K, structurally unrelated to LY294002, also inhibited ODC induction with an IC50 of about 10 nM. These results indicate that PI3K activity is required for the induction of ODC, possibly affecting both ODC mRNA level and translation. Since p70 S6 kinase (p70S6K) is considered an important mediator of PI3K action in several experimental systems, the effect of rapamycin, which can lead to selective inhibition of p70S6K, was also investigated. Rapamycin inhibited p70S6K activity and produced ODC inhibiting effects similar to those elicited by LY294002. However, LY294002 and wortmannin at concentrations which inhibited almost completely PI3K activity did not decrease p70S6K activity, suggesting that p70S6K does not mediate the PI3K effects on ODC, but may lie on a separate pathway in this experimental model.

1996 - IL-1 receptor signalling mechanism in SAOS-2 osteosarcoma cells [Articolo su rivista]
Faenza, I.; Bavelloni, A.; Cecchi, S.; Ognibene, A.; Maraldi, N. M.; Marmiroli, S.
abstract

1996 - Transfected Saos-2 cells overexpressing phosphoinositidase C beta1 isoform accumulate it within the nucleus. [Articolo su rivista]
Marmiroli, Sandra; N., Zini; A., Bavelloni; I., Faenza; A., Ognibene; N. M., Maraldi
abstract

The subcellular partitioning of the phosphoinositidase C (PIC) isoforms involved in signal transduction, with the selective localization of the PIC beta(1) isoform in the nucleus, represents a crucial aspect of the complex mechanism of cell response to agonists. In order to further elucidate this phenomenon, we utilized human osteosarcoma Saos-2 cells, transfected with the cDNA for rat PIC beta(1). In the cells overexpressing this isoform, immunocytochemical analyses at the electron microscope level reveal an increased synthesis at the cytoplasm and a significant accumulation within the nucleus of the protein. Interestingly, the sites of intranuclear localization are, as in wild type cells, the interchromatin domains. These results indicate that the transfected cells maintain the capability of accumulating the enzyme within the nucleus and can be considered a model for functional studies on the nuclear signal transduction also in response to specific agonists.

1995 - The intranuclear amount of phospholipase C beta1 decreases following cell differentiation in Friend cells, whereas gamma1 isoform is not affected. [Articolo su rivista]
N., Zini; A., Ognibene; Marmiroli, Sandra; A., Bavelloni; M. C., Maltarello; I., Faenza; A., Valmori; N. M., Maraldi
abstract

The intranuclear amount of phospholipase C beta1 decreases following cell differentiation in Friend cells, whereas gamma1 isoform is not affected. IF 2.244

1995 - Tiazofurin induces a down modulation of ICAM-1 expression on K562 target cells impairing NK adhesion and killing. [Articolo su rivista]
L., Zamai; G., Zauli; A., Bavelloni; Marmiroli, Sandra; A., Cataldi; G., Weber; M., Vitale
abstract

Tiazofurin induces a down modulation of ICAM-1 expression on K562 target cells impairing NK adhesion and killing. IF 2.6

1994 - Immunocytochemical detection of the specific association of different PIC isoforms with cytoskeletal and nuclear matrix compartments in PC12 cells [Articolo su rivista]
Zini, N.; Mazzoni, M.; Neri, L. M.; Bavelloni, A.; Marmiroli, S.; Capitani, S.; Maraldi, N. M.
abstract

The increasing evidence of discrete roles of phosphoinositidase C (PIC) isoforms and the assessment of their localization in the cytoskeleton and in the nucleus support the involvement of particular isotypes of this enzyme in signal transduction at multiple levels. PC12 rat pheochromocytoma is one of the few cell lines expressing three immunologically distinct isoforms of PIC. We have analyzed the subcellular distribution of the PIC β2, γ1 and δ1 isoforms using confocal and electron microscope immunocytochemistry. PIC β1 is mainly found in the nucleus and is associated with interchromatin domains. On the other hand, the PIC γ1 isoform is found in the nucleus and in the cytosol, while PIC δ1 is exclusively cytoplasmic. Immunoblot and immunocytochemical experiments indicate that the various PIC isoforms are differently bound to structural cell compartments, such as cytoskeletal and nuclear matrix elements. In fact, PIC β1 and PIC γ1 isoforms are tightly associated with the nuclear matrix, while only about 50% of PIC γ1 is associated with the cytoskeleton after DNase I and high salt extractions. PIC γ1 is almost completely soluble under these conditions. These results further confirm the complexity of the inositide signal transduction mechanism, which involves several PIC isoforms, specifically localized in different cell compartments and support the existence of a membrane-unrelated inositol lipid-dependent signalling in the nuclear interior.

1994 - Inositol lipid phosphorylation and breakdown in rat liver nuclei is affected by hydrocortisone blood levels [Articolo su rivista]
Santi, P.; Marmiroli, S.; Falcieri, E.; Bertagnolo, V.; Capitani, S.
abstract

The possibility that inositol lipid metabolism is related to nuclear events accompanying steroid hormone action has been investigated by comparing lipid phosphorylation and breakdown in normal rat liver nuclei and in hypo‐ and hypercortisolemic conditions. Lipid phosphorylation in vitro showed the presence of diacylglycerol (DAG)‐, phosphatidylinositol (PI)‐ and phosphatidylinositol‐4‐phosphate (PIP)‐kinase activity, with differences between total tissue homogenates and isolated nuclei, relevant to the treatment in vivo. Administration of hydrocortisone (HC) produced a marked decrease in the phosphorylated nuclear products without influencing the homogenate kinase activity. Under conditions which were optimal for the kinase activities, nuclear PIP‐kinase was strongly increased in presence of a high blood level of HC whereas PI‐kinase activity was reduced. From these observations it appears that the observed differences were due to specific modulation of kinase activities rather than to changes in the availability of substrates. The phosphoinositide‐specific phospholipase C (PLC) activity was also investigated. In the presence of a high HC blood level, the phosphodiesteratic cleavage of PIP strongly increased, while that of phosphatidylinositol bisphosphate (PIP2) was similar in normal and hypercortisolemic conditions. Nuclear phosphoinositide hydrolysis was affected by PLC, β and γ isoforms, which were equally represented in all the conditions investigated, indicating that the observed changes of activity were due to a modulation rather than to a change in the amount of enzyme. These results suggest that inositol lipid metabolism plays a role in the nuclear modifications accompanying steroid hormone induction of transcriptional activity. Copyright © 1994 John Wiley &amp; Sons Ltd.

1994 - Interleukin-1alfa stimulates nuclear phosphoinositidase C activity in human osteosarcoma SaOS-2 cells. [Articolo su rivista]
Marmiroli, Sandra; A., Ognibene; A., Bavelloni; C., Cinti; L., Cocco; N. M., Maraldi
abstract

Interleukin-1alfa stimulates nuclear phosphoinositidase C activity in human osteosarcoma SaOS-2 cells

1993 - Age-related events in human active T limphocytes. Changes in the phosphoinositidase C activity. [Articolo su rivista]
R., DI PIETRO; R. A., Rana; A., Sciscio; Marmiroli, Sandra; A. M., Billi; A., Cataldi; L., Cocco
abstract

Since PHA-stimulated active T lymphocytes from aged humans showed changes in the metabolic pattern of inositol lipids in comparison with young subjects, we studied the possible role of phosphoinositidase C (PIC) in the generation of this phenomenon. The breakdown of exogenous [3H]phosphatidylinositol 4,5-bisphosphate was found to be optimal at neutral pH and Ca++ concentrations close to millimolar levels. Under these conditions PIC activity of resting lymphocytes did not differ in aged and young subjects, while, after short periods of PHA stimulation (up to 4 hr) the substrate hydrolysis was lower and delayed in the elderly group in comparison with that of controls. Our findings support the hypothesis that the age-related default of this enzyme, responsible for the age-related changes in the inositol lipid pathway of this peculiar subpopulation, could be involved, as a primary event, in the mechanisms leading to the reduced proliferative response of aged active T lymphocytes. IF 2.9476

1993 - Phosphoinositidase C isozymes in SaOS-2 cells: immunocytochemical detection in nuclear and cytoplasmic compartments. [Articolo su rivista]
N. M., Maraldi; N., Zini; S., Santi; A., Bavelloni; A., Valmori; Marmiroli, Sandra; A., Ognibene
abstract

SaOS-2 cell line presents osteoblastic characteristics which can be modulated by specific agonists involving also phosphoinositide breakdown. In order to determine whether SaOS-2 cells display a phosphoinositide signalling system not only at the cytosol-cell membrane level but also, as recently reported for other cell lines, at the nuclear level, a study has been performed to evaluate the phosphoinositidase C (PIC) activity and to localize different isoforms of PIC in nuclear and cytoplasmic compartments. By immunochemicals methods, and by confocal and electron microscope immunocytochemistry, both PIC beta 1 and gamma 1 have been detected in the nucleus, while only PIC gamma 1 was found in the cytoplasm. A specific association with the inner nuclear matrix has been demonstrated for PIC beta 1 and gamma 1; this latter resulted, on the other hand, in relationship with cytoskeletal filaments after high salt extraction. These findings suggest that these enzymes are not completely soluble but functionally related with cytoskeletal and nucleoskeletal structures.

1991 - Nuclear protein kinases in rat liver: evidence for increased Histone H1 phosphorylating activity during liver regeneration. [Articolo su rivista]
A. M., Martelli; C., Carini; Marmiroli, Sandra; M., Mazzoni; P. J., Barker; R. S., Gilmour; S., Capitani
abstract

Comparison of protein kinase activity in normal and regenerating rat liver nuclei indicates that exogenous histone H1 is hyperphosphorylated in 22-h regenerating nuclei. The protein kinase involved is not sensitive to protein kinase A inhibitor, is inhibited by staurosporine and by an anti-PKC polyclonal antibody, utilizes only ATP, and also phosphorylates the C-terminal fragment of histone H1. These data suggest that protein kinase C is responsible for the observed effects, in agreement with the presence of this enzyme in normal and regenerating nuclei demonstrated by immunoblotting.IF 3.949

1990 - Effect of ATP depletion and phenanthroline on the spermidine-mediated decay of ornithine decarboxylase in erythroleukemia cells [Articolo su rivista]
Flamigni, F.; Marmiroli, S.; Guarnieri, C.; Caldarera, C. M.
abstract

Addition of spermidine to Friend erythroleukemia cells caused a rapid decay of ornithine decarboxylase (ODC) activity and the accumulation of a ODC-antizyme complex. The induction of antizyme only partially accounted for the decrease of ODC activity by a direct inhibition of the enzyme. However, the antizyme induction was accompanied by a marked reduction of the half-life of ODC. Shift of the cells to an ATP-depleting medium prevented the spermidine-elicited decay of ODC activity as well as the accumulation of ODC-antizyme complex. However, ODC appeared to be stabilized even when ATP depletion was performed 40 min after spermidine addition, in the presence of high levels of antizyme. Similar results were obtained by treating the cells with phenanthroline, a heavy metal chelator and protease inhibitor. These findings indicate that ATP and some metalloprotease(s) may be involved in the degradation pathway of ODC, even in the presence of high levels of polyamines. © 1990.

1990 - Phosphorylation of ornithine decarboxylase in intact erythroleukemia cells [Articolo su rivista]
Flamigni, F.; Marmiroli, S.; Meggio, F.; Guarnieri, C.; Pinna, L. A.
abstract

32P-labeled ornithine decarboxylase was isolated by immunoprecipitation from murine erythroleukemia cells incubated in a medium containing [32P]ortophosphoric acid. Analysis of immunoprecipitate by SDS-polyacrylamide gel electrophoresis and autoradiography revealed a radiolabeled band, which corresponded to the position of mouse ornithine decarboxylase, phosphorylated in vitro by casein kinase-2. A preparation of casein kinase-2 purified from nuclei of erythroleukemia cells could also phosphorylate mouse ornithine decarboxylase. © 1990.

1989 - Effect of sodium arsentie on the induction and turnover of ornithine decarboxylase activity in erythroleukemia cells [Articolo su rivista]
Flamigni, F.; Marmiroli, S.; Caldarera, C. M.; Guarnieri, C.
abstract

Sodium arsenite proved effective in preventing the induction of ornithine decarboxylase (ODC) activity elicited by dilution of Friend erythroleukemia cells in fresh medium. A 50 per cent inhibition was produced at approximately 1 μM arsenite and complete inhibition was obtained at concentrations above 10 μM. However, addition of arsenite 5 h after cell dilution, i.e. when ODC was already induced, appeared to stabilize the enzyme. The half‐life of ODC activity, measured after cycloheximide treatment, increased almost six‐fold after addition of sodium arsenite. Agents known to provoke oxidative alteration of the thiol‐redox status in cells, also caused a similar effect on the induction and stability of ODC. Copyright © 1989 John Wiley &amp; Sons Ltd.

1989 - Involvement of thiol transferase- and thioredoxin-dependent systems in the protection of 'essential' thiol groups of ornithine decarboxylase [Articolo su rivista]
Flamigni, F.; Marmiroli, S.; Caldarera, C. M.; Guarnieri, C.
abstract

Ornithine decarboxylase (ODC), an enzyme with 'essential' thiol group(s), may be inactivated in vitro by removal of thiol reducing agents and re-activated by soluble factors from rat liver in the presence of NADPH or GSH. The NADPH- and GSH-dependent reducing systems were sepated and resolved into three components, called factors A, B1 and B2, by chromatographic techniques. Factor B1 (M(r) 12000) could reactivate ODC in the presence of GSH of co-purified with thiol transferase activity. Factor B2 (M(r) 12000) and factor A (M(r) approx. 110000) were both needed to re-activate ODC in the presence of NADPH, and co-purified with thioredoxin and thioredoxin reductase activity respectively. In an attempt to investigate the physiological role of the 'essential' thiol group(s) of ODC, erythroleukemia cells were incubated with NN-bis-(2-chloroethyl)-N'-nitrosourea, t-butyl hydroperoxide and vinblastine, which are known to increase the cellular GSSG/GSH ratio, azelaic acid, an inhibitor of thioredoxin reductase, and sodium arsenite, a strong inhibitor of the ODC-re-activating factors. All these compounds were able to decrease significantly the ODC activity induced in these cells. These results suggest that the thiol transferase- and thioredoxin-dependent systems may be physiologically relevant in maintaining ODC in the active, reduced, state.

1989 - Stabilization of ornithine decarboxylase in erythroleukemia cells depleted of ATP [Articolo su rivista]
Flamigni, F.; Marmiroli, S.; Guarnieri, C.; Caldarera, C. M.
abstract

Ornithine decarboxylase activity in Friend erythroleukemia cells decayed with a half-life of 50 minutes after addition of cycloheximide and at a faster rate after addition of spermidine. Incubation with a medium containing dinitrophenol and 2-deoxyglucose in place of glucose caused ATP depletion and blocked the turnover of ornithine decarboxylase, even after addition of spermidine. Dinitrophenol in the presence of glucose was able to provoke only a slight increase of the half-life of the enzyme. These results suggest that degradation of ornithine decarboxylase in erythroleukemia cells is ATP-dependent. © 1989.

1988 - Study on the role of endogenous polyamines in glucagon, isoproterenol or serum-mediated induction of tyrosine aminotransferase in cultured heart cells [Articolo su rivista]
Carlo, C.; Tantini, B.; Marmiroli, S.; Pignatti, C.; Caldarera, C. M.
abstract

In confluent and serum-starved embryonic heart cell cultures, the addition of serum (10%), glucagon (GLU, 0.1 μM) or isoproterenol (ISO, 10 μM), causes the onset of ornithine decarboxylase (ODC) activity, with a maximum after 5-6 hr. This is paralleled by polyamine accumulation and by the induction of TAT, which, in the case of GLU and ISO, exhibits maximal activity at 4-3hr respectively, followed by a net decline. Cyclic AMP (cAMP) also accumulates after exposure to GLU or ISO. However, under different conditions of ODC inhibition, serum fails to induce TAT, thus supporting a relevant role of cellular polyamines in serum action. Conversely, cAMP and TAT responses to GLU or ISO are markedly improved under prevention of polyamine accumulation, which also leads to a longer lasting TAT inducibility. The suggestion is made that polyamines are not required in the cAMP-dependent mechanism of TAT induction, but rather in the restoration of the basal activity of the enzyme. © 1988 Academic Press, Inc.

1987 - A different involvement of polyamines in the induction of tyrosine aminotransferase by glucagon, isoproterenol or serum in cultured heart cells [Articolo su rivista]
Clo, C.; Manfroni, S.; Marmiroli, S.
abstract

1987 - Regulation of hormonal sensitivity of cardiac cells in culture by polyamines [Articolo su rivista]
Clo, C.; Pignatti, C.; Tantini, B.; Manfroni, S.; Marmiroli, S.; Caldarera, C. M.
abstract

1985 - Inhibition of rat heart ornithine decarboxylase by basic polypeptides [Articolo su rivista]
Flamigni, F.; Guarnieri, C.; Marmiroli, S.; Caldarera, C. M.
abstract