Nuova ricerca


Personale tecnico amministrativo
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

Home |


2021 - Hsp90-mediated regulation of DYRK3 couples stress granule disassembly and growth via mTORC1 signaling [Articolo su rivista]
Mediani, L.; Antoniani, F.; Galli, V.; Vinet, J.; Carra, A. D.; Bigi, I.; Tripathy, V.; Tiago, T.; Cimino, M.; Leo, G.; Amen, T.; Kaganovich, D.; Cereda, C.; Pansarasa, O.; Mandrioli, J.; Tripathi, P.; Troost, D.; Aronica, E.; Buchner, J.; Goswami, A.; Sterneckert, J.; Alberti, S.; Carra, S.

Stress granules (SGs) are dynamic condensates associated with protein misfolding diseases. They sequester stalled mRNAs and signaling factors, such as the mTORC1 subunit raptor, suggesting that SGs coordinate cell growth during and after stress. However, the molecular mechanisms linking SG dynamics and signaling remain undefined. We report that the chaperone Hsp90 is required for SG dissolution. Hsp90 binds and stabilizes the dual-specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3) in the cytosol. Upon Hsp90 inhibition, DYRK3 dissociates from Hsp90 and becomes inactive. Inactive DYRK3 is subjected to two different fates: it either partitions into SGs, where it is protected from irreversible aggregation, or it is degraded. In the presence of Hsp90, DYRK3 is active and promotes SG disassembly, restoring mTORC1 signaling and translation. Thus, Hsp90 links stress adaptation and cell growth by regulating the activity of a key kinase involved in condensate disassembly and translation restoration.

2021 - Protein products of nonstop mRNA disrupt nucleolar homeostasis [Articolo su rivista]
Davis, Z. H.; Mediani, L.; Antoniani, F.; Vinet, J.; Li, S.; Alberti, S.; Lu, B.; Holehouse, A. S.; Carra, S.; Brandman, O.

Stalled mRNA translation results in the production of incompletely synthesized proteins that are targeted for degradation by ribosome-associated quality control (RQC). Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the RQC protein LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tails of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress in cell culture and intact flies. Our work elucidates how stalled translation may affect distal cellular processes and may inform studies on the pathology of diseases caused by failures in RQC and characterized by nucleolar stress.

2021 - Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor [Articolo su rivista]
Tiago, T.; Hummel, B.; Morelli, F. F.; Basile, V.; Vinet, J.; Galli, V.; Mediani, L.; Antoniani, F.; Pomella, S.; Cassandri, M.; Garone, M. G.; Silvestri, B.; Cimino, M.; Cenacchi, G.; Costa, R.; Mouly, V.; Poser, I.; Yeger-Lotem, E.; Rosa, A.; Alberti, S.; Rota, R.; Ben-Zvi, A.; Sawarkar, R.; Carra, S.

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.

2020 - ALS and FTD: Where RNA metabolism meets protein quality control [Articolo su rivista]
Mandrioli, J.; Mediani, L.; Alberti, S.; Carra, S.

Recent genetic and biochemical evidence has improved our understanding of the pathomechanisms that lead to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating neurodegenerative diseases with overlapping symptoms and causes. Impaired RNA metabolism, enhanced aggregation of protein-RNA complexes, aberrant formation of ribonucleoprotein (RNP) granules and dysfunctional protein clearance via autophagy are emerging as crucial events in ALS/FTD pathogenesis. Importantly, these processes interact at the molecular level, converging on a common pathogenic cascade. In this review, we summarize key principles underlying ALS and FTD, and we discuss how mutations in genes involved in RNA metabolism, protein quality control and protein degradation meet mechanistically to impair the functionality and dynamics of RNP granules, and how this leads to cellular toxicity and death. Finally, we describe recent advances in understanding signaling pathways that become dysfunctional in ALS/FTD, partly due to altered RNP granule dynamics, but also with stress granule-independent mechanisms and, thus could be promising targets for future therapeutic intervention.

2020 - BAG3 and BAG6 differentially affect the dynamics of stress granules by targeting distinct subsets of defective polypeptides released from ribosomes [Articolo su rivista]
Mediani, L.; Galli, V.; Carra, A. D.; Bigi, I.; Vinet, J.; Ganassi, M.; Antoniani, F.; Tiago, T.; Cimino, M.; Mateju, D.; Cereda, C.; Pansarasa, O.; Alberti, S.; Mandrioli, J.; Carra, S.

Stress granules (SGs) are dynamic ribonucleoprotein granules induced by environmental stresses. They play an important role in the stress response by integrating mRNA stability, translation, and signaling pathways. Recent work has connected SG dysfunction to neurodegenerative diseases. In these diseases, SG dynamics are impaired because of mutations in SG proteins or protein quality control factors. Impaired SG dynamics and delayed SG dissolution have also been observed for SGs that accumulate misfolding-prone defective ribosomal products (DRiPs). DRiP accumulation inside SGs is controlled by a surveillance system referred to as granulostasis and encompasses the molecular chaperones VCP and the HSPB8-BAG3-HSP70 complex. BAG3 is a member of the BAG family of proteins, which includes five additional members. One of these proteins, BAG6, is functionally related to BAG3 and able to assist degradation of DRiPs. However, whether BAG6 is involved in granulostasis is unknown. We report that BAG6 is not recruited into SGs induced by different types of stress, nor does it affect SG dynamics. BAG6 also does not replace BAG3’s function in SG granulostasis. We show that BAG3 and BAG6 target different subsets of DRiPs, and BAG3 binding to DRiPs is mediated by HSPB8 and HSP70. Our data support the idea that SGs are sensitive to BAG3-HSP70-bound DRiPs but not to BAG6-bound DRiPs. Additionally, only BAG3 is strongly upregulated in the stress recovery phase, when SGs dissolve. These data exclude a role for BAG6 in granulostasis and point to a more specialized function in the clearance of a specific subset of DRiPs.

2020 - BAG3 Pro209 mutants associated with myopathy and neuropathy relocate chaperones of the CASA-complex to aggresomes [Articolo su rivista]
Adriaenssens, E.; Tedesco, B.; Mediani, L.; Asselbergh, B.; Crippa, V.; Antoniani, F.; Carra, S.; Poletti, A.; Timmerman, V.

Three missense mutations targeting the same proline 209 (Pro209) codon in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been reported to cause distal myopathy, dilated cardiomyopathy or Charcot-Marie-Tooth type 2 neuropathy. Yet, it is unclear whether distinct molecular mechanisms underlie the variable clinical spectrum of the rare patients carrying these three heterozygous Pro209 mutations in BAG3. Here, we studied all three variants and compared them to the BAG3_Glu455Lys mutant, which causes dilated cardiomyopathy. We found that all BAG3_Pro209 mutants have acquired a toxic gain-of-function, which causes these variants to accumulate in the form of insoluble HDAC6- and vimentin-positive aggresomes. The aggresomes formed by mutant BAG3 led to a relocation of other chaperones such as HSPB8 and Hsp70, which, together with BAG3, promote the so-called chaperone-assisted selective autophagy (CASA). As a consequence of their increased aggregation-proneness, mutant BAG3 trapped ubiquitinylated client proteins at the aggresome, preventing their efficient clearance. Combined, these data show that all BAG3_Pro209 mutants, irrespective of their different clinical phenotypes, are characterized by a gain-of-function that contributes to the gradual loss of protein homeostasis.

2020 - Studying heat shock proteins through single-molecule mechanical manipulation [Articolo su rivista]
Choudhary, D.; Mediani, L.; Carra, S.; Cecconi, C.

Imbalances of cellular proteostasis are linked to ageing and human diseases, including neurodegenerative and neuromuscular diseases. Heat shock proteins (HSPs) and small heat shock proteins (sHSPs) together form a crucial core of the molecular chaperone family that plays a vital role in maintaining cellular proteostasis by shielding client proteins against aggregation and misfolding. sHSPs are thought to act as the first line of defence against protein unfolding/misfolding and have been suggested to act as “sponges” that rapidly sequester these aberrant species for further processing, refolding, or degradation, with the assistance of the HSP70 chaperone system. Understanding how these chaperones work at the molecular level will offer unprecedented insights for their manipulation as therapeutic avenues for the treatment of ageing and human disease. The evolution in single-molecule force spectroscopy techniques, such as optical tweezers (OT) and atomic force microscopy (AFM), over the last few decades have made it possible to explore at the single-molecule level the structural dynamics of HSPs and sHSPs and to examine the key molecular mechanisms underlying their chaperone activities. In this paper, we describe the working principles of OT and AFM and the experimental strategies used to employ these techniques to study molecular chaperones. We then describe the results of some of the most relevant single-molecule manipulation studies on HSPs and sHSPs and discuss how these findings suggest a more complex physiological role for these chaperones than previously assumed.

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

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 - Defective ribosomal products challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin [Articolo su rivista]
Mediani, L.; Guillen-Boixet, J.; Vinet, J.; Franzmann, T. M.; Bigi, I.; Mateju, D.; Carra, A. D.; Morelli, F. F.; Pinheiro Tiago, Tatiana Sofia; Poser, I.; Alberti, S.; Carra, S.

Nuclear protein aggregation has been linked to genome instability and disease. The main source of aggregation-prone proteins in cells is defective ribosomal products (DRiPs), which are generated by translating ribosomes in the cytoplasm. Here, we report that DRiPs rapidly diffuse into the nucleus and accumulate in nucleoli and PML bodies, two membraneless organelles formed by liquid–liquid phase separation. We show that nucleoli and PML bodies act as dynamic overflow compartments that recruit protein quality control factors and store DRiPs for later clearance. Whereas nucleoli serve as constitutive overflow compartments, PML bodies are stress-inducible overflow compartments for DRiPs. If DRiPs are not properly cleared by chaperones and proteasomes due to proteostasis impairment, nucleoli undergo amyloidogenesis and PML bodies solidify. Solid PML bodies immobilize 20S proteasomes and limit the recycling of free ubiquitin. Ubiquitin depletion, in turn, compromises the formation of DNA repair compartments at fragile chromosomal sites, ultimately threatening cell survival.

2019 - Nucleoli and Promyelocytic Leukemia Protein (PML) bodies are phase separated nuclear protein quality control compartments for misfolded proteins [Articolo su rivista]
Mediani, L.; Guillen-Boixet, J.; Alberti, S.; Carra, S.

We uncovered a role for nucleoli and PML-bodies as phase-separated protein quality control organelles that compartmentalize protein quality control factors and misfolded proteins for their efficient clearance. Failure to dispose misfolded proteins converts nucleoli and PML-bodies into a solid state that immobilizes ubiquitin, limiting its recycling for genome integrity maintenance.

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

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 - An interaction study in mammalian cells demonstrates weak binding of HSPB2 to BAG3, which is regulated by HSPB3 and abrogated by HSPB8 [Articolo su rivista]
Morelli, FEDERICA FRANCESCA; Mediani, Laura; Heldens, Lonneke; Bertacchini, Jessika; Bigi, Ilaria; Carrà, Arianna Dorotea; Vinet, Jonathan; Carra, Serena

The ten mammalian small heat shock proteins (sHSPs/HSPBs) show a different expression profile, although the majority of them are abundant in skeletal and cardiac muscles. HSPBs form hetero-oligomers and homo-oligomers by interacting together and complexes containing, e.g., HSPB2/HSPB3 or HSPB1/HSPB5 have been documented in mammalian cells and muscles. Moreover, HSPB8 associates with the Hsc70/Hsp70 co-chaperone BAG3, in mammalian, skeletal, and cardiac muscle cells. Interaction of HSPB8 with BAG3 regulates its stability and function. Weak association of HSPB5 and HSPB6 with BAG3 has been also reported upon overexpression in cells, supporting the idea that BAG3 might indirectly modulate the function of several HSPBs. However, it is yet unknown whether other HSPBs highly expressed in muscles such as HSPB2 and HSPB3 also bind to BAG3. Here, we report that in mammalian cells, upon overexpression, HSPB2 binds to BAG3 with an affinity weaker than HSPB8. HSPB2 competes with HSPB8 for binding to BAG3. In contrast, HSPB3 negatively regulates HSPB2 association with BAG3. In human myoblasts that express HSPB2, HSPB3, HSPB8, and BAG3, the latter interacts selectively with HSPB8. Combining these data, it supports the interpretation that HSPB8-BAG3 is the preferred interaction.

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

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 - Granulostasis: Protein Quality Control of RNP Granules [Articolo su rivista]
Alberti, Simon; Mateju, Daniel; Mediani, Laura; Carra, Serena

Ribonucleoprotein (RNP) granules transport, store, or degrade messenger RNAs, thereby indirectly regulating protein synthesis. Normally, RNP granules are highly dynamic compartments. However, because of aging or severe environmental stress, RNP granules, in particular stress granules (SGs), convert into solid, aggregate-like inclusions. There is increasing evidence that such RNA-protein inclusions are associated with several age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), fronto-temporal dementia (FTD) and Alzheimer's disease (AD). Thus, understanding what triggers the conversion of RNP granules into aggregates and identifying the cellular players that control RNP granules will be critical to develop treatments for these diseases. In this review article, we discuss recent insight into RNP and SG formation. More specifically, we examine the evidence for liquid-liquid phase separation (LLPS) as an organizing principle of RNP granules and the role of aggregation-prone RNA-binding proteins (RBPs) in this process. We further discuss recent findings that liquid-like SGs can sequester misfolded proteins, which promote an aberrant conversion of liquid SGs into solid aggregates. Importantly, very recent studies show that a specific protein quality control (PQC) process prevents the accumulation of misfolding-prone proteins in SGs and, by doing so, maintains the dynamic state of SGs. This quality control process has been referred to as granulostasis and it relies on the specific action of the HSPB8-BAG3-HSP70 complex. Additional players such as p97/valosin containing protein (VCP) and other molecular chaperones (e.g., HSPB1) participate, directly or indirectly, in granulostasis, and ensure the timely elimination of defective ribosomal products and other misfolded proteins from SGs. Finally, we discuss recent findings that, in the stress recovery phase, SGs are preferentially disassembled with the assistance of chaperones, and we discuss evidence for a back-up system that targets aberrant SGs to the aggresome for autophagy-mediated clearance. Altogether the findings discussed here provide evidence for an intricate network of interactions between RNP granules and various components of the PQC machinery. Molecular chaperones in particular are emerging as key players that control the composition and dynamics of RNP granules, which may be important to protect against age-related diseases.

2016 - A Surveillance Function of the HSPB8-BAG3-HSP70 Chaperone Complex Ensures Stress Granule Integrity and Dynamism [Articolo su rivista]
Ganassi, Massimo; Mateju, Daniel; Bigi, Ilaria; Mediani, Laura; Poser, Ina; Lee, Hyun O.; Seguin, SAMUEL JOSEPH ANDRE'; Morelli, FEDERICA FRANCESCA; Vinet, Jonathan; Leo, Giuseppina; Pansarasa, Orietta; Cereda, Cristina; Poletti, Angelo; Alberti, Simon; Carra, Serena

Stress granules (SGs) are ribonucleoprotein complexes induced by stress. They sequester mRNAs and disassemble when the stress subsides, allowing translation restoration. In amyotrophic lateral sclerosis (ALS), aberrant SGs cannot disassemble and therefore accumulate and are degraded by autophagy. However, the molecular events causing aberrant SG formation and the molecular players regulating this transition are largely unknown. We report that defective ribosomal products (DRiPs) accumulate in SGs and promote a transition into an aberrant state that renders SGs resistant to RNase. We show that only a minor fraction of aberrant SGs is targeted by autophagy, whereas the majority disassembles in a process that requires assistance by the HSPB8-BAG3-HSP70 chaperone complex. We further demonstrate that HSPB8-BAG3-HSP70 ensures the functionality of SGs and restores proteostasis by targeting DRiPs for degradation. We propose a system of chaperone-mediated SG surveillance, or granulostasis, which regulates SG composition and dynamics and thus may play an important role in ALS.

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

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 - 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

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 - Targeting PI3K/AKT/mTOR network for treatment of leukemia [Articolo su rivista]
Bertacchini, Jessika; Heidari, Nazanin; Mediani, Laura; Capitani, Silvano; Shahjahani, Mohammad; Ahmadzadeh, Ahmad; Saki, Najmaldin

Objective: Increased activity of PI3K/AKT/mTOR pathway has been observed in a huge number of malignancies. This pathway can function as a prosurvival factor in leukemia stem cells and early committed leukemic precursors and its inhibition is regarded as a therapeutic approach. Accordingly, the aim of this review is to evaluate the PI3K/Akt/mTOR inhibitors used in leukemia models. Discussion: Inhibition of the PI3K/AKT/mTOR pathway has been reported to have beneficial therapeutic effects in leukemias, both in vitro in leukemia cell lines and in vivo in animal models. Overall, the use of dual PI3K/mTOR inhibitor, dual Akt/RTK inhibitor, Akt inhibitor, selective inhibitor of PI3K, mTOR inhibitor and dual PI3K/PDK1 inhibitor in CML, AML, APL, CLL, B-ALL and T-ALL has a better therapeutic effect than conventional treatments. Conclusions: Targeting the PI3K/Akt/mTOR pathway may have pro-apoptotic and antiproliferative effects on hematological malignancies. Furthermore, modulation of miRNA can be used as a novel therapeutic approach to regulate the PI3K/Akt/mTOR pathway. However, both aspects require further clinical studies.

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

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.

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

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.

2007 - Diagnosis of invasive aspergillosis by tracking Aspergillus-specific T cells in hematologic patients with pulmonary infiltrates [Articolo su rivista]
Potenza, Leonardo; Barozzi, Patrizia; Vallerini, Daniela; Bosco, R; Quadrelli, Chiara; Morselli, M; Forghieri, Fabio; Volzone, F; Codeluppi, M; Rossi, G; Tazzioli, Giovanni; Venturelli, C; Torelli, Giuseppe; Luppi, Mario; Mediani, Laura