Publications by authors named "Sandra Marmiroli"

42 Publications

An Unbiased Approach to Mapping the Signaling Network of the Pseudorabies Virus US3 Protein.

Pathogens 2020 Nov 5;9(11). Epub 2020 Nov 5.

Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.

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.
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http://dx.doi.org/10.3390/pathogens9110916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694389PMC
November 2020

Targeting PI3K/Akt/mTOR in AML: Rationale and Clinical Evidence.

J Clin Med 2020 Sep 11;9(9). Epub 2020 Sep 11.

Haemato-Oncology/Systems Medicine Group, Paul O'Gorman Leukaemia Research Centre, University of Glasgow, Glasgow G12 0ZD, UK.

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.
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http://dx.doi.org/10.3390/jcm9092934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563273PMC
September 2020

Deregulated PTEN/PI3K/AKT/mTOR signaling in prostate cancer: Still a potential druggable target?

Biochim Biophys Acta Mol Cell Res 2020 09 29;1867(9):118731. Epub 2020 Apr 29.

Department of Biomedical, Metabolic, and Neurological Sciences, University of Modena and Reggio Emilia, 41124 Modena, MO, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118731DOI Listing
September 2020

Crosstalks of GSK3 signaling with the mTOR network and effects on targeted therapy of cancer.

Biochim Biophys Acta Mol Cell Res 2020 04 26;1867(4):118635. Epub 2019 Dec 26.

Department of Biomedical and Neuromotor Sciences, University of Bologna, 40126 Bologna, BO, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbamcr.2019.118635DOI Listing
April 2020

Influence of selenium on the emergence of neuro tubule defects in a neuron-like cell line and its implications for amyotrophic lateral sclerosis.

Neurotoxicology 2019 12 1;75:209-220. Epub 2019 Oct 1.

Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, 41125, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.neuro.2019.09.015DOI Listing
December 2019

Development of solvent-casting particulate leaching (SCPL) polymer scaffolds as improved three-dimensional supports to mimic the bone marrow niche.

Mater Sci Eng C Mater Biol Appl 2019 Mar 7;96:153-165. Epub 2018 Nov 7.

Dipartimento di Ingegneria "Enzo Ferrari" (DIEF), Università di Modena e Reggio Emilia, Via P. Vivarelli, 10, 41125 Modena, Italy.

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.
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http://dx.doi.org/10.1016/j.msec.2018.10.086DOI Listing
March 2019

Clusterin enhances AKT2-mediated motility of normal and cancer prostate cells through a PTEN and PHLPP1 circuit.

J Cell Physiol 2019 07 22;234(7):11188-11199. Epub 2018 Nov 22.

Department of Biomedical, Metabolic, and Neural Sciences, Section of Morphology, Signal Transduction Unit, University of Modena and Reggio Emilia, Modena, Italy.

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.
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http://dx.doi.org/10.1002/jcp.27768DOI Listing
July 2019

Nuclear Nox4 interaction with prelamin A is associated with nuclear redox control of stem cell aging.

Aging (Albany NY) 2018 10;10(10):2911-2934

Department of Surgical, Medical, Dental and Morphological Sciences with Interest in Transplant, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, 41124, Italy.

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 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 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.
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http://dx.doi.org/10.18632/aging.101599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224265PMC
October 2018

Dual inhibition of PI3K/mTOR signaling in chemoresistant AML primary cells.

Adv Biol Regul 2018 05 19;68:2-9. Epub 2018 Mar 19.

Cellular Signaling Unit, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena 41125, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jbior.2018.03.001DOI Listing
May 2018

Aberrant Compartment Formation by HSPB2 Mislocalizes Lamin A and Compromises Nuclear Integrity and Function.

Cell Rep 2017 Aug;20(9):2100-2115

Department of Biomedical, Metabolic and Neuronal Sciences, University of Modena and Reggio Emilia, and Center for Neuroscience and Neurotechnology, 41125 Modena, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2017.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583511PMC
August 2017

Cross-talk between the CK2 and AKT signaling pathways in cancer.

Adv Biol Regul 2017 05 28;64:1-8. Epub 2017 Mar 28.

Cell Signaling Unit, Department of Surgery, Medicine, Dentistry and Morphology, University of Modena and Reggio Emilia, 41124 Modena, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jbior.2017.03.002DOI Listing
May 2017

Effects of mutations in Wnt/β-catenin, hedgehog, Notch and PI3K pathways on GSK-3 activity-Diverse effects on cell growth, metabolism and cancer.

Biochim Biophys Acta 2016 12 6;1863(12):2942-2976. Epub 2016 Sep 6.

Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Bologna, Italy.

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.
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http://dx.doi.org/10.1016/j.bbamcr.2016.09.004DOI Listing
December 2016

Critical Roles of EGFR Family Members in Breast Cancer and Breast Cancer Stem Cells: Targets for Therapy.

Curr Pharm Des 2016 ;22(16):2358-88

Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Brody Building 5N98C, Greenville, NC 27858 USA.

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.
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http://dx.doi.org/10.2174/1381612822666160304151011DOI Listing
November 2017

Phosphorylation, Signaling, and Cancer: Targets and Targeting.

Biomed Res Int 2015 21;2015:601543. Epub 2015 Oct 21.

Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/b, 35131 Padova, Italy.

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http://dx.doi.org/10.1155/2015/601543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631856PMC
August 2016

Reversal of the glycolytic phenotype of primary effusion lymphoma cells by combined targeting of cellular metabolism and PI3K/Akt/ mTOR signaling.

Oncotarget 2016 Feb;7(5):5521-37

Department of Surgery, Medicine, Dentistry and Morphology, University of Modena and Reggio Emilia, Modena, Italy.

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.
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http://dx.doi.org/10.18632/oncotarget.6315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868703PMC
February 2016

Inhibition of Ras-mediated signaling pathways in CML stem cells.

Cell Oncol (Dordr) 2015 Dec 12;38(6):407-18. Epub 2015 Oct 12.

Health Research Institute, Research Center of Thalassemia and Hemoglobinopathy, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Background: 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.

Conclusions: A closer investigation of the influence of Ras-mediated signaling pathways on CML progression to blast crisis is warranted to uncover new directions for targeted therapies, particularly in cases that are resistant to current tyrosine kinase inhibitors.
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http://dx.doi.org/10.1007/s13402-015-0248-2DOI Listing
December 2015

Rapamycin treatment of Mandibuloacral dysplasia cells rescues localization of chromatin-associated proteins and cell cycle dynamics.

Aging (Albany NY) 2014 Sep;6(9):755-70

National Research Council of Italy, Institute of Molecular Genetics, IGM-CNR-IOR, Bologna, Italy. Rizzoli Orthopedic Institute, Laboratory of Musculoskeletal Cell Biology, Bologna, Italy.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233654PMC
http://dx.doi.org/10.18632/aging.100680DOI Listing
September 2014

Signaling specificity in the Akt pathway in biology and disease.

Adv Biol Regul 2014 May 19;55:28-38. Epub 2014 Apr 19.

Department of Surgery, Medicine, Dentistry and Morphology, University of Modena and Reggio Emilia, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jbior.2014.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062840PMC
May 2014

Serum Fatty acids and risk of cutaneous melanoma: a population-based case-control study.

Dermatol Res Pract 2013 28;2013:659394. Epub 2013 Jan 28.

Departments of Diagnostic, Clinical and Public Health Medicine, Environmental, Genetic and Nutritional Epidemiology Research Center (CREAGEN), University of Modena and Reggio Emilia, Padiglione De Sanctis, Via Amendola 2, 42122 Reggio Emilia, Italy.

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. The 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-specific direct association with melanoma risk. No other associations emerged for the other saturated, monounsaturated, and polyunsaturated fatty acids, individually or grouped by type. Conclusions. These findings 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.
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http://dx.doi.org/10.1155/2013/659394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3569884PMC
February 2013

The protein kinase Akt/PKB regulates both prelamin A degradation and Lmna gene expression.

FASEB J 2013 Jun 21;27(6):2145-55. Epub 2013 Feb 21.

Cellular Signaling Laboratory, Department of Surgery, Medicine, Dentistry, and Morphology, University of Modena and Reggio Emilia, Modena, Italy.

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 identify those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and the lamin A precursor prelamin A are type V intermediate filament 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 report here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt also regulates Lmna transcription. 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 lamin physiology to therapeutic approaches for lamin A-linked disorders.
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http://dx.doi.org/10.1096/fj.12-218214DOI Listing
June 2013

Monocytosis has adverse prognostic significance and impacts survival in patients with T-cell lymphomas.

Leuk Res 2013 Jun 8;37(6):619-23. Epub 2013 Feb 8.

Department of Diagnostic, Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy.

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<120 g/L, albumin<35 g/L) had a negative influence on outcome, but in multivariate analysis, monocytosis alone had a stronger association with poor OS.
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http://dx.doi.org/10.1016/j.leukres.2013.01.009DOI Listing
June 2013

The need for a reassessment of the safe upper limit of selenium in drinking water.

Sci Total Environ 2013 Jan 7;443:633-42. Epub 2012 Dec 7.

CREAGEN - Environmental, Genetic and Nutritional Epidemiology Research Center, Department of Diagnostic and Clinical Medicine and of Public Health, University of Modena and Reggio Emilia, Modena, Italy.

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 levels 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 from in vitro and 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 concentrations exceeding this level, the public health importance of this issue should not be overlooked, and further epidemiologic research is critically needed in this area.
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http://dx.doi.org/10.1016/j.scitotenv.2012.11.025DOI Listing
January 2013

Melanocytes--a novel tool to study mitochondrial dysfunction in Duchenne muscular dystrophy.

J Cell Physiol 2013 Jun;228(6):1323-31

Department of Medical Science, University of Ferrara, Ferrara, Italy.

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.
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http://dx.doi.org/10.1002/jcp.24290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601437PMC
June 2013

Protein kinase B/AKT isoform 2 drives migration of human mesenchymal stem cells.

Int J Oncol 2013 Jan 15;42(1):118-26. Epub 2012 Nov 15.

Department of Biomedical Sciences, Cellular Signalling Laboratory, Bologna, Italy.

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.
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http://dx.doi.org/10.3892/ijo.2012.1700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583637PMC
January 2013

Nuclear phospholipase C β1 signaling, epigenetics and treatments in MDS.

Adv Biol Regul 2013 Jan 21;53(1):2-7. Epub 2012 Sep 21.

Cellular Signalling Laboratory, Department of Human Anatomical Sciences, University of Bologna, Bologna, Italy.

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.
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http://dx.doi.org/10.1016/j.jbior.2012.09.009DOI Listing
January 2013

Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H(2)O(2).

Mol Biol Cell 2011 Aug 7;22(16):2946-56. Epub 2011 Jul 7.

IGM-CNR, Unit of Bologna c/o IOR, 40136 Bologna, Italy.

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.
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http://dx.doi.org/10.1091/mbc.E10-11-0928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154889PMC
August 2011

Reverse-phase protein microarrays (RPPA) as a diagnostic and therapeutic guide in multidrug resistant leukemia.

Int J Oncol 2011 Feb 2;38(2):427-35. Epub 2010 Dec 2.

Department of Anatomy and Histology and CIPro Proteomics Centre, University of Modena and Reggio Emilia, I-41100 Modena, Italy.

Reverse-phase microarray assays using phospho-specific antibodies (RPPA) can directly measure levels of phosphorylated protein isoforms. In the current study, lysates from parental and multidrug resistant (MDR) CEM leukemia cells were spotted onto reverse-phase protein microarrays and probed with a panel of phospho-antibodies to ERK, PCK and Akt pathways. In particular, the Akt pathway is considered to play significant roles in leukemia and Akt inhibitor therapy has been proposed as a potential tool in the treatment of this disease. The RPPA data prompted us to investigate deeper this pathway. Here, we found that whereas total Akt1 protein level is higher in parental CEM cells, the activated isoform content, 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 up-regulated in MDR-CEM cells. Further exploration of inhibitory therapy in this system was evaluated. The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumor cells. Herein, we describe that in MDR-CEM cells TRAIL responsiveness correlates with a reduced expression of endogenous Akt1, suggesting that the MDR phenotype associated to P-gp sensitizes cells to TRAIL therapy.
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http://dx.doi.org/10.3892/ijo.2010.850DOI Listing
February 2011

Dasatinib plus Nutlin-3 shows synergistic antileukemic activity in both p53 wild-type and p53 mutated B chronic lymphocytic leukemias by inhibiting the Akt pathway.

Clin Cancer Res 2011 Feb 24;17(4):762-70. Epub 2010 Nov 24.

Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy.

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.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-2572DOI Listing
February 2011

Laminopathies and A-type lamin-associated signalling pathways.

Adv Enzyme Regul 2010 14;50(1):248-61. Epub 2009 Nov 14.

Laboratory of Cell Biology, IOR, Bologna, Italy.

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http://dx.doi.org/10.1016/j.advenzreg.2009.10.019DOI Listing
August 2010

A-type lamins and signaling: the PI 3-kinase/Akt pathway moves forward.

J Cell Physiol 2009 Sep;220(3):553-61

Department of Anatomy and Histology and CIPro Proteomics Centre, University of Modena and Reggio Emilia, Via Del Pozzo 71, I-41100 Modena, I.G.M.-CNR, Unit of Bologna, c/o IOR, via di Barbiano, Bologna I-40136, Italy.

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.
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http://dx.doi.org/10.1002/jcp.21807DOI Listing
September 2009