Publications by authors named "Sonia Missiroli"

36 Publications

Cancer metabolism and mitochondria: Finding novel mechanisms to fight tumours.

EBioMedicine 2020 Sep 17;59:102943. Epub 2020 Aug 17.

Department of Medical Sciences, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy. Electronic address:

Mitochondria are dynamic organelles that have essential metabolic activity and are regarded as signalling hubs with biosynthetic, bioenergetics and signalling functions that orchestrate key biological pathways. However, mitochondria can influence all processes linked to oncogenesis, starting from malignant transformation to metastatic dissemination. In this review, we describe how alterations in the mitochondrial metabolic status contribute to the acquisition of typical malignant traits, discussing the most recent discoveries and the many unanswered questions. We also highlight that expanding our understanding of mitochondrial regulation and function mechanisms in the context of cancer cell metabolism could be an important task in biomedical research, thus offering the possibility of targeting mitochondria for the treatment of cancer.
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http://dx.doi.org/10.1016/j.ebiom.2020.102943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452656PMC
September 2020

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy.

Cell Death Differ 2021 Jan 13;28(1):123-138. Epub 2020 Jul 13.

Basic and Translational Myology laboratory, Université de Paris BFA, UMR 8251, CNRS, F-75013, Paris, France.

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.
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http://dx.doi.org/10.1038/s41418-020-0587-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853070PMC
January 2021

The Role of Mitochondria in Inflammation: From Cancer to Neurodegenerative Disorders.

J Clin Med 2020 Mar 9;9(3). Epub 2020 Mar 9.

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, 44121 Ferrara, Italy.

The main features that are commonly attributed to mitochondria consist of the regulation of cell proliferation, ATP generation, cell death and metabolism. However, recent scientific advances reveal that the intrinsic dynamicity of the mitochondrial compartment also plays a central role in proinflammatory signaling, identifying these organelles as a central platform for the control of innate immunity and the inflammatory response. Thus, mitochondrial dysfunctions have been related to severe chronic inflammatory disorders. Strategies aimed at reestablishing normal mitochondrial physiology could represent both preventive and therapeutic interventions for various pathologies related to exacerbated inflammation. Here, we explore the current understanding of the intricate interplay between mitochondria and the innate immune response in specific inflammatory diseases, such as neurological disorders and cancer.
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http://dx.doi.org/10.3390/jcm9030740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141240PMC
March 2020

The role of mitochondria-associated membranes in cellular homeostasis and diseases.

Int Rev Cell Mol Biol 2020 3;350:119-196. Epub 2019 Dec 3.

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy; Maria Cecilia Hospital, GVM Care & Research, Cotignola (RA), Italy. Electronic address:

Mitochondria and endoplasmic reticulum (ER) are fundamental in the control of cell physiology regulating several signal transduction pathways. They continuously communicate exchanging messages in their contact sites called MAMs (mitochondria-associated membranes). MAMs are specific microdomains acting as a platform for the sorting of vital and dangerous signals. In recent years increasing evidence reported that multiple scaffold proteins and regulatory factors localize to this subcellular fraction suggesting MAMs as hotspot signaling domains. In this review we describe the current knowledge about MAMs' dynamics and processes, which provided new correlations between MAMs' dysfunctions and human diseases. In fact, MAMs machinery is strictly connected with several pathologies, like neurodegeneration, diabetes and mainly cancer. These pathological events are characterized by alterations in the normal communication between ER and mitochondria, leading to deep metabolic defects that contribute to the progression of the diseases.
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http://dx.doi.org/10.1016/bs.ircmb.2019.11.002DOI Listing
December 2020

Glioblastoma: Prognostic Factors and Predictive Response to Radio and Chemotherapy.

Curr Med Chem 2020 ;27(17):2814-2825

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy.

Glioblastoma multiforme (GBM) is characterized by poor prognosis despite an aggressive therapeutic strategy. In recent years, many advances have been achieved in the field of glioblastoma biology. Here we try to summarize the main clinical and biological factors impacting clinical prognostication and therapy of GBM patients. From that standpoint, hopefully, in the near future, personalized therapies will be available.
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http://dx.doi.org/10.2174/0929867327666200131095256DOI Listing
August 2020

Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca levels and tumor growth.

EMBO J 2019 01 30;38(2). Epub 2018 Nov 30.

Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy

Although mitochondria play a multifunctional role in cancer progression and Ca signaling is remodeled in a wide variety of tumors, the underlying mechanisms that link mitochondrial Ca homeostasis with malignant tumor formation and growth remain elusive. Here, we show that phosphorylation at the N-terminal region of the mitochondrial calcium uniporter (MCU) regulatory subunit MICU1 leads to a notable increase in the basal mitochondrial Ca levels. A pool of active Akt in the mitochondria is responsible for MICU1 phosphorylation, and mitochondrion-targeted Akt strongly regulates the mitochondrial Ca content. The Akt-mediated phosphorylation impairs MICU1 processing and stability, culminating in reactive oxygen species (ROS) production and tumor progression. Thus, our data reveal the crucial role of the Akt-MICU1 axis in cancer and underscore the strategic importance of the association between aberrant mitochondrial Ca levels and tumor development.
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http://dx.doi.org/10.15252/embj.201899435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331721PMC
January 2019

A maladaptive ER stress response triggers dysfunction in highly active muscles of mice with SELENON loss.

Redox Biol 2019 01 26;20:354-366. Epub 2018 Oct 26.

Dulbecco Telethon Institute at Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy. Electronic address:

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to human SELENON-related myopathies. SelenoN knockout (KO) mouse limb muscles, however, are protected from the disease, and display no major alterations in muscle histology or contractile properties. Interestingly, we find that the highly active diaphragm muscle shows impaired force production, in line with the human phenotype. In addition, after repeated stimulation with a protocol which induces muscle fatigue, also hind limb muscles show altered relaxation times. Mechanistically, muscle SELENON loss alters activity-dependent calcium handling selectively impinging on the Ca uptake of the sarcoplasmic reticulum and elicits an ER stress response, including the expression of the maladaptive CHOP-induced ERO1. In SELENON-devoid models, ERO1 shifts ER redox to a more oxidised poise, and further affects Ca uptake. Importantly, CHOP ablation in SelenoN KO mice completely prevents diaphragm dysfunction, the prolonged limb muscle relaxation after fatigue, and restores Ca uptake by attenuating the induction of ERO1. These findings suggest that SELENON is part of an ER stress-dependent antioxidant response and that the CHOP/ERO1 branch of the ER stress response is a novel pathogenic mechanism underlying SELENON-related myopathies.
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http://dx.doi.org/10.1016/j.redox.2018.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223234PMC
January 2019

Regulation of PKCβ levels and autophagy by PML is essential for high-glucose-dependent mesenchymal stem cell adipogenesis.

Int J Obes (Lond) 2019 05 6;43(5):963-973. Epub 2018 Aug 6.

Department of Morphology, Surgery and Experimental Medicine, Section of General Pathology and Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy.

Background/objectives: Obesity is a complex disease characterized by the accumulation of excess body fat, which is caused by an increase in adipose cell size and number. The major source of adipocytes comes from mesenchymal stem cells (MSCs), although their roles in obesity remain unclear. An understanding of the mechanisms, regulation, and outcomes of adipogenesis is crucial for the development of new treatments for obesity-related diseases. Recently an unexpected role for the tumor suppressor promyelocytic leukemia protein (PML) in hematopoietic stem cell biology and metabolism regulation has come to light, but its role in MSC biology remains unknown. Here, we investigated the molecular pathway underlying the role of PML in the control of adipogenic MSC differentiation.

Subjects/methods: Muscle-derived stem cells (MDSCs) and adipose-derived stem cells (ADSCs) obtained from mice and voluntary patients (as a source of MSCs) were cultured in the presence of high glucose (HG) concentration, a nutrient stress condition known to promote MSCs differentiation into mature adipocytes and the adipogenic potential of PML was assessed.

Results: PML is essential for a correct HG-dependent adipogenic differentiation, and the enhancement of PML levels is fundamental during adipogenesis. Increased PML expression enables the upregulation of protein kinase Cβ (PKCβ), which, in turn, by controlling autophagy levels permits an increase in peroxisome proliferator-activated receptor γ (PPARγ) that leads the adipogenic differentiation. Therefore, genetic and pharmacological depletion of PML prevents PKCβ expression, and by increasing autophagy levels, impairs the MSCs adipogenic differentiation. Human ADSCs isolated from overweight patients displayed increased PML and PKCβ levels compared to those found in normal weight individuals, indicating that the PML-PKCβ pathway is directly involved in the enhancement of adipogenesis and human metabolism.

Conclusions: The new link found among PML, PKCβ, and autophagy opens new therapeutic avenues for diseases characterized by an imbalance in the MSCs differentiation process, such as metabolic syndromes and cancer.
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http://dx.doi.org/10.1038/s41366-018-0167-1DOI Listing
May 2019

Discovery of Novel 1,3,8-Triazaspiro[4.5]decane Derivatives That Target the c Subunit of F/F-Adenosine Triphosphate (ATP) Synthase for the Treatment of Reperfusion Damage in Myocardial Infarction.

J Med Chem 2018 08 9;61(16):7131-7143. Epub 2018 Aug 9.

Maria Cecilia Hospital , GVM Care & Research , 48033 , Cotignola , Ravenna , Italy.

Recent cardiology research studies have reported the role, function, and structure of the mitochondrial permeability transition pore (mPTP) and have shown that its opening plays a key role in the progression of myocardial cell death secondary to reperfusion. In this manuscript, we validated a new pharmacological approach as an adjunct to reperfusion in myocardial infarction (MI) treatment and describe the discovery, optimization, and structure-activity relationship (SAR) studies of the first small-molecule mPTP opening inhibitors based on a 1,3,8-triazaspiro[4.5]decane scaffold that targets the c subunit of the F/F-ATP synthase complex. We identified three potential compounds with good mPTP inhibitory activity and beneficial effects in a model of MI, including a decreased apoptotic rate in the whole heart and overall improvement of cardiac function upon administration during reperfusion. The selected compounds did not show off-target effects at the cellular and mitochondrial levels. Moreover, the compounds preserved the mitochondrial ATP content despite interacting with the ATP synthase complex.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00278DOI Listing
August 2018

Mitochondria-associated membranes (MAMs) and inflammation.

Cell Death Dis 2018 02 28;9(3):329. Epub 2018 Feb 28.

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy.

The endoplasmic reticulum (ER) and mitochondria are tightly associated with very dynamic platforms termed mitochondria-associated membranes (MAMs). MAMs provide an excellent scaffold for crosstalk between the ER and mitochondria and play a pivotal role in different signaling pathways that allow rapid exchange of biological molecules to maintain cellular health. However, dysfunctions in the ER-mitochondria architecture are associated with pathological conditions and human diseases. Inflammation has emerged as one of the various pathways that MAMs control. Inflammasome components and other inflammatory factors promote the release of pro-inflammatory cytokines that sustain pathological conditions. In this review, we summarize the critical role of MAMs in initiating inflammation in the cellular defense against pathogenic infections and the association of MAMs with inflammation-mediated diseases.
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http://dx.doi.org/10.1038/s41419-017-0027-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832426PMC
February 2018

Calcium Dynamics as a Machine for Decoding Signals.

Trends Cell Biol 2018 04 3;28(4):258-273. Epub 2018 Feb 3.

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy; Cecilia Hospital, GVM Care & Research, E.S: Health Science Foundation, Cotignola, Italy; CNR Institute of Cell Biology and Neurobiology, Monterotondo, Italy. Electronic address:

Calcium (Ca) is considered one of the most-important biological cations, because it is implicated in cell physiopathology and cell fate through a finely tuned signaling system. In support of this notion, Ca is the primary driver of cell proliferation and cell growth; however, it is also intimately linked to cell death. Functional abnormalities or mutations in proteins that mediate Ca homeostasis usually lead to a plethora of diseases and pathogenic states, including cancer, heart failure, diabetes, and neurodegenerative disease. In this review, we examine recent discoveries in the highly localized nature of Ca-dependent signal transduction and its roles in cell fate, inflammasome activation, and synaptic transmission.
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http://dx.doi.org/10.1016/j.tcb.2018.01.002DOI Listing
April 2018

Novel function of the tumor suppressor PML at ER-mitochondria sites in the control of autophagy.

Oncotarget 2017 Oct 4;8(47):81723-81724. Epub 2017 Jul 4.

Carlotta Giorgi: Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies, University of Ferrara, Ferrara, Italy.

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http://dx.doi.org/10.18632/oncotarget.18974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669838PMC
October 2017

Endoplasmic Reticulum-Mitochondria Communication Through Ca Signaling: The Importance of Mitochondria-Associated Membranes (MAMs).

Adv Exp Med Biol 2017 ;997:49-67

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy.

The execution of proper Ca signaling requires close apposition between the endoplasmic reticulum (ER) and mitochondria. Hence, Ca released from the ER is "quasi-synaptically" transferred to mitochondrial matrix, where Ca stimulates mitochondrial ATP synthesis by activating the tricarboxylic acid (TCA) cycle. However, when the Ca transfer is excessive and sustained, mitochondrial Ca overload induces apoptosis by opening the mitochondrial permeability transition pore. A large number of regulatory proteins reside at mitochondria-associated ER membranes (MAMs) to maintain the optimal distance between the organelles and to coordinate the functionality of both ER and mitochondrial Ca transporters or channels. In this chapter, we discuss the different pathways involved in the regulation of ER-mitochondria Ca flux and describe the activities of the various Ca players based on their primary intra-organelle localization.
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http://dx.doi.org/10.1007/978-981-10-4567-7_4DOI Listing
January 2018

Use of luciferase probes to measure ATP in living cells and animals.

Nat Protoc 2017 Aug 6;12(8):1542-1562. Epub 2017 Jul 6.

Section of Pathology, Oncology and Experimental Biology, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy.

ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.
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http://dx.doi.org/10.1038/nprot.2017.052DOI Listing
August 2017

PTEN counteracts FBXL2 to promote IP3R3- and Ca-mediated apoptosis limiting tumour growth.

Nature 2017 06 14;546(7659):554-558. Epub 2017 Jun 14.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA.

In response to environmental cues that promote IP3 (inositol 1,4,5-trisphosphate) generation, IP3 receptors (IP3Rs) located on the endoplasmic reticulum allow the 'quasisynaptical' feeding of calcium to the mitochondria to promote oxidative phosphorylation. However, persistent Ca release results in mitochondrial Ca overload and consequent apoptosis. Among the three mammalian IP3Rs, IP3R3 appears to be the major player in Ca-dependent apoptosis. Here we show that the F-box protein FBXL2 (the receptor subunit of one of 69 human SCF (SKP1, CUL1, F-box protein) ubiquitin ligase complexes) binds IP3R3 and targets it for ubiquitin-, p97- and proteasome-mediated degradation to limit Ca influx into mitochondria. FBXL2-knockdown cells and FBXL2-insensitive IP3R3 mutant knock-in clones display increased cytosolic Ca release from the endoplasmic reticulum and sensitization to Ca-dependent apoptotic stimuli. The phosphatase and tensin homologue (PTEN) gene is frequently mutated or lost in human tumours and syndromes that predispose individuals to cancer. We found that PTEN competes with FBXL2 for IP3R3 binding, and the FBXL2-dependent degradation of IP3R3 is accelerated in Pten mouse embryonic fibroblasts and PTEN-null cancer cells. Reconstitution of PTEN-null cells with either wild-type PTEN or a catalytically dead mutant stabilizes IP3R3 and induces persistent Ca mobilization and apoptosis. IP3R3 and PTEN protein levels directly correlate in human prostate cancer. Both in cell culture and xenograft models, a non-degradable IP3R3 mutant sensitizes tumour cells with low or no PTEN expression to photodynamic therapy, which is based on the ability of photosensitizer drugs to cause Ca-dependent cytotoxicity after irradiation with visible light. Similarly, disruption of FBXL2 localization with GGTi-2418, a geranylgeranyl transferase inhibitor, sensitizes xenotransplanted tumours to photodynamic therapy. In summary, we identify a novel molecular mechanism that limits mitochondrial Ca overload to prevent cell death. Notably, we provide proof-of-principle that inhibiting IP3R3 degradation in PTEN-deregulated cancers represents a valid therapeutic strategy.
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http://dx.doi.org/10.1038/nature22965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627969PMC
June 2017

Mitochondrial and endoplasmic reticulum calcium homeostasis and cell death.

Cell Calcium 2018 01 5;69:62-72. Epub 2017 May 5.

Dept. of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy. Electronic address:

The endoplasmic reticulum (ER) and mitochondria cannot be considered as static structures, as they intimately communicate, forming very dynamic platforms termed mitochondria-associated membranes (MAMs). In particular, the ER transmits proper Ca signals to mitochondria, which decode them into specific inputs to regulate essential functions, including metabolism, energy production and apoptosis. Here, we will describe the different molecular players involved in the transfer of Ca ions from the ER lumen to the mitochondrial matrix and how modifications in both ER-mitochondria contact sites and Ca signaling can alter the cell death execution program.
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http://dx.doi.org/10.1016/j.ceca.2017.05.003DOI Listing
January 2018

Endoplasmic reticulum-mitochondria Ca crosstalk in the control of the tumor cell fate.

Biochim Biophys Acta Mol Cell Res 2017 Jun 4;1864(6):858-864. Epub 2017 Jan 4.

Dept. of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy. Electronic address:

Mitochondria-associated membranes are juxtaposed between the endoplasmic reticulum and mitochondria and have been identified as a critical hub in the regulation of apoptosis and tumor growth. One key function of mitochondria-associated membranes is to provide asylum to a number of proteins with tumor suppressor and oncogenic properties. In this review, we discuss how Ca flux manipulation represents the primary mechanism underlying the action of several oncogenes and tumor-suppressor genes and how these networks might be manipulated to provide novel therapies for cancer. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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http://dx.doi.org/10.1016/j.bbamcr.2016.12.024DOI Listing
June 2017

PML at Mitochondria-Associated Membranes Is Critical for the Repression of Autophagy and Cancer Development.

Cell Rep 2016 08 18;16(9):2415-27. Epub 2016 Aug 18.

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology and LTTA Center, University of Ferrara, Ferrara 44121, Italy. Electronic address:

The precise molecular mechanisms that coordinate apoptosis and autophagy in cancer remain to be determined. Here, we provide evidence that the tumor suppressor promyelocytic leukemia protein (PML) controls autophagosome formation at mitochondria-associated membranes (MAMs) and, thus, autophagy induction. Our in vitro and in vivo results demonstrate how PML functions as a repressor of autophagy. PML loss promotes tumor development, providing a growth advantage to tumor cells that use autophagy as a cell survival strategy during stress conditions. These findings demonstrate that autophagy inhibition could be paired with a chemotherapeutic agent to develop anticancer strategies for tumors that present PML downregulation.
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http://dx.doi.org/10.1016/j.celrep.2016.07.082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011426PMC
August 2016

Methods to Study PTEN in Mitochondria and Endoplasmic Reticulum.

Methods Mol Biol 2016 ;1388:187-212

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology and LTTA center, University of Ferrara, Via Fossato di Mortara 70 c/o CUBO, 44121, Ferrara, Italy.

Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity.Here, we describe different methods that have been used to highlight PTEN subcellular localization.First, a protocol to extract nuclear and cytosolic fractions has been described to assess the "canonical" PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.
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http://dx.doi.org/10.1007/978-1-4939-3299-3_13DOI Listing
December 2016

Alterations in Mitochondrial and Endoplasmic Reticulum Signaling by p53 Mutants.

Front Oncol 2016 25;6:42. Epub 2016 Feb 25.

Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, University of Ferrara , Ferrara , Italy.

The p53 protein is probably the most important tumor suppressor, acting as a nuclear transcription factor primarily through the modulation of cell death. However, currently, it is well accepted that p53 can also exert important transcription-independent pro-cell death actions. Indeed, cytosolic localization of endogenous wild-type or transactivation-deficient p53 is necessary and sufficient for the induction of apoptosis and autophagy. Here, we present the extra-nuclear activities of p53 associated with the mitochondria and the endoplasmic reticulum, highlighting the activities of the p53 mutants on these compartments. These two intracellular organelles play crucial roles in the regulation of cell death, and it is now well established that they also represent sites where p53 can accumulate.
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http://dx.doi.org/10.3389/fonc.2016.00042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766755PMC
March 2016

Defective autophagy is a key feature of cerebral cavernous malformations.

EMBO Mol Med 2015 Nov;7(11):1403-17

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy

Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3-0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.
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http://dx.doi.org/10.15252/emmm.201505316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4644374PMC
November 2015

Mitochondria-Associated Endoplasmic Reticulum Membranes Microenvironment: Targeting Autophagic and Apoptotic Pathways in Cancer Therapy.

Front Oncol 2015 27;5:173. Epub 2015 Jul 27.

Laboratory for Technologies of Advanced Therapies (LTTA), Section of Pathology, Oncology and Experimental Biology, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara , Ferrara , Italy.

Autophagy is a tightly regulated catabolic pathway that terminates in the lysosomal compartment after the formation of a cytoplasmic vacuole that engulfs macromolecules and organelles. Notably, autophagy is associated with several human pathophysiological conditions, playing either a cytoprotective or cytopathic role. Many studies have investigated the role of autophagy in cancer. However, whether autophagy suppresses tumorigenesis or provides cancer cells with a rescue mechanism under unfavorable conditions remains unclear. Mitochondria-associated membranes (MAMs) are juxtaposed between the endoplasmic reticulum and mitochondria and have been identified as critical hubs in the regulation of apoptosis and tumor growth. One key function of MAMs is to provide asylum to a number of proteins with tumor suppressor and oncogenic properties. Accordingly, mechanistic studies during tumor progression suggest a strong involvement of these proteins at various steps of the autophagic process. This paper discusses the present state of our knowledge about the intimate molecular networks between MAMs and autophagy in cancer cells and addresses how these networks might be manipulated to improve anticancer therapeutics.
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http://dx.doi.org/10.3389/fonc.2015.00173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515599PMC
August 2015

The endoplasmic reticulum mitochondrial calcium cross talk is downregulated in malignant pleural mesothelioma cells and plays a critical role in apoptosis inhibition.

Oncotarget 2015 Sep;6(27):23427-44

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy.

The failure of apoptosis may contribute to the formation of cancer and to its resistance to therapy. Malignant pleural mesothelioma (MPM) is an aggressive tumor that responds poorly to standard chemo- and radio-therapies. Several studies have demonstrated that a plethora of oncogenes and tumor suppressors contribute to MPM onset/progression. Importantly, most of these genes are involved in the regulation of calcium (Ca2+)-handling. Cellular Ca2+ signaling is an important regulator of many physiological processes, and it has been widely reported to participate in the regulation of apoptotic cell death in cancer cells and tissues. However, in MPM the role of cellular Ca2+ has been poorly investigated. Therefore, we examined whether Ca2+ is involved in MPM. We found that mesothelioma cell lines and short-term cultures obtained from MPM-affected patients exhibited a critical dysregulation in Ca2+ signaling. We determined that this characteristic was associated with resistance to apoptotic stimuli and that correction of intracellular Ca2+ signaling resulted in the rescue of efficient apoptotic responses. In addition, we discovered that mitochondrial Ca2+-uptake plays a pivotal role as an inducer of apoptosis in MPM. Altogether, these findings suggest the identification of new MPM markers, which in turn could be potential targets for new therapeutic approaches.
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http://dx.doi.org/10.18632/oncotarget.4370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695128PMC
September 2015

p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner.

Proc Natl Acad Sci U S A 2015 Feb 26;112(6):1779-84. Epub 2015 Jan 26.

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara 44121, Italy;

The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca(2+)) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca(2+) homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca(2+)-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca(2+) load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca(2+) overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca(2+) signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca(2+) signal-dependent apoptosis.
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http://dx.doi.org/10.1073/pnas.1410723112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330769PMC
February 2015

Mitochondria-associated membranes: composition, molecular mechanisms, and physiopathological implications.

Antioxid Redox Signal 2015 Apr 13;22(12):995-1019. Epub 2015 Mar 13.

1 Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, University of Ferrara , Ferrara, Italy .

Significance: In all cells, the endoplasmic reticulum (ER) and mitochondria are physically connected to form junctions termed mitochondria-associated membranes (MAMs). This subcellular compartment is under intense investigation because it represents a "hot spot" for the intracellular signaling of important pathways, including the synthesis of cholesterol and phospholipids, calcium homeostasis, and reactive oxygen species (ROS) generation and activity.

Recent Advances: The advanced methods currently used to study this fascinating intracellular microdomain in detail have enabled the identification of the molecular composition of MAMs and their involvement within different physiopathological contexts.

Critical Issues: Here, we review the knowledge regarding (i) MAMs composition in terms of protein composition, (ii) the relationship between MAMs and ROS, (iii) the involvement of MAMs in cell death programs with particular emphasis within the tumor context, (iv) the emerging role of MAMs during inflammation, and (v) the key role of MAMs alterations in selected neurological disorders.

Future Directions: Whether alterations in MAMs represent a response to the disease pathogenesis or directly contribute to the disease has not yet been unequivocally established. In any case, the signaling at the MAMs represents a promising pharmacological target for several important human diseases.
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http://dx.doi.org/10.1089/ars.2014.6223DOI Listing
April 2015

Intravital imaging reveals p53-dependent cancer cell death induced by phototherapy via calcium signaling.

Oncotarget 2015 Jan;6(3):1435-45

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology and LTTA center, University of Ferrara, Ferrara, Italy.

One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca²⁺). In the present study, we established conditions that allow the in vivo detection of Ca²⁺ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca²⁺ concentrations and, consequently, an increase in cell death in a p53-dependent pathway.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359305PMC
http://dx.doi.org/10.18632/oncotarget.2935DOI Listing
January 2015

Silencing of mitochondrial Lon protease deeply impairs mitochondrial proteome and function in colon cancer cells.

FASEB J 2014 Dec 25;28(12):5122-35. Epub 2014 Aug 25.

Department of Surgery, Medicine, Dentistry, and Morphological Sciences, and Dipartimento Sperimentale Interaziendale, Campus San Lazzaro, University of Modena and Reggio Emilia, Reggio Emilia, Italy

Lon is a nuclear-encoded, mitochondrial protease that assists protein folding, degrades oxidized/damaged proteins, and participates in maintaining mtDNA levels. Here we show that Lon is up-regulated in several human cancers and that its silencing in RKO colon cancer cells causes profound alterations of mitochondrial proteome and function, and cell death. We silenced Lon in RKO cells by constitutive or inducible expression of Lon shRNA. Lon-silenced cells displayed altered levels of 39 mitochondrial proteins (26% related to stress response, 14.8% to ribosome assembly, 12.7% to oxidative phosphorylation, 8.5% to Krebs cycle, 6.3% to β-oxidation, and 14.7% to crista integrity, ketone body catabolism, and mtDNA maintenance), low levels of mtDNA transcripts, and reduced levels of oxidative phosphorylation complexes (with >90% reduction of complex I). Oxygen consumption rate decreased 7.5-fold in basal conditions, and ATP synthesis dropped from 0.25 ± 0.04 to 0.03 ± 0.001 nmol/mg proteins, in the presence of 2-deoxy-d-glucose. Hydrogen peroxide and mitochondrial superoxide anion levels increased by 3- and 1.3-fold, respectively. Mitochondria appeared fragmented, heterogeneous in size and shape, with dilated cristae, vacuoles, and electrondense inclusions. The triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid, a Lon inhibitor, partially mimics Lon silencing. In summary, Lon is essential for maintaining mitochondrial shape and function, and for survival of RKO cells.
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http://dx.doi.org/10.1096/fj.14-255869DOI Listing
December 2014

Downregulation of the mitochondrial calcium uniporter by cancer-related miR-25.

Curr Biol 2013 Jan 13;23(1):58-63. Epub 2012 Dec 13.

Section of General Pathology, Department of Morphology, Surgery and Experimental Medicine, Interdisciplinary Center for the Study of Inflammation, Laboratory for Technologies of Advanced Therapies, University of Ferrara, 44121 Ferrara, Italy.

The recently discovered mitochondrial calcium uniporter (MCU) promotes Ca(2+) accumulation into the mitochondrial matrix. We identified in silico miR-25 as a cancer-related MCU-targeting microRNA family and demonstrate that its overexpression in HeLa cells drastically reduces MCU levels and mitochondrial Ca(2+) uptake, while leaving other mitochondrial parameters and cytosolic Ca(2+) signals unaffected. In human colon cancers and cancer-derived cells, miR-25 is overexpressed and MCU accordingly silenced. miR-25-dependent reduction of mitochondrial Ca(2+) uptake correlates with resistance to apoptotic challenges and can be reversed by anti-miR-25 overexpression. Overall, the data demonstrate that microRNA targeting of mitochondrial Ca(2+) signaling favors cancer cell survival, thus providing mechanistic insight into the role of mitochondria in tumorigenesis and identifying a novel therapeutic target in neoplasia.
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http://dx.doi.org/10.1016/j.cub.2012.11.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540261PMC
January 2013

ATP synthesis and storage.

Purinergic Signal 2012 Sep 12;8(3):343-57. Epub 2012 Apr 12.

Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation (ICSI), Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Via Borsari, 46, 44121, Ferrara, Italy.

Since 1929, when it was discovered that ATP is a substrate for muscle contraction, the knowledge about this purine nucleotide has been greatly expanded. Many aspects of cell metabolism revolve around ATP production and consumption. It is important to understand the concepts of glucose and oxygen consumption in aerobic and anaerobic life and to link bioenergetics with the vast amount of reactions occurring within cells. ATP is universally seen as the energy exchange factor that connects anabolism and catabolism but also fuels processes such as motile contraction, phosphorylations, and active transport. It is also a signalling molecule in the purinergic signalling mechanisms. In this review, we will discuss all the main mechanisms of ATP production linked to ADP phosphorylation as well the regulation of these mechanisms during stress conditions and in connection with calcium signalling events. Recent advances regarding ATP storage and its special significance for purinergic signalling will also be reviewed.
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http://dx.doi.org/10.1007/s11302-012-9305-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360099PMC
September 2012

Mitochondrial Ca(2+) and apoptosis.

Cell Calcium 2012 Jul 3;52(1):36-43. Epub 2012 Apr 3.

Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation (ICSI), Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy.

Mitochondria are key decoding stations of the apoptotic process. In support of this view, a large body of experimental evidence has unambiguously revealed that, in addition to the well-established function of producing most of the cellular ATP, mitochondria play a fundamental role in triggering apoptotic cell death. Various apoptotic stimuli cause the release of specific mitochondrial pro-apoptotic factors into the cytosol. The molecular mechanism of this release is still controversial, but there is no doubt that mitochondrial calcium (Ca(2+)) overload is one of the pro-apoptotic ways to induce the swelling of mitochondria, with perturbation or rupture of the outer membrane, and in turn the release of mitochondrial apoptotic factors into the cytosol. Here, we review as different proteins that participate in mitochondrial Ca(2+) homeostasis and in turn modulate the effectiveness of Ca(2+)-dependent apoptotic stimuli. Strikingly, the final outcome at the cellular level is similar, albeit through completely different molecular mechanisms: a reduced mitochondrial Ca(2+) overload upon pro-apoptotic stimuli that dramatically blunts the apoptotic response.
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http://dx.doi.org/10.1016/j.ceca.2012.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396846PMC
July 2012