Publications by authors named "Anna Maria Porcelli"

54 Publications

Plasma-activated Ringer's Lactate Solution Displays a Selective Cytotoxic Effect on Ovarian Cancer Cells.

Cancers (Basel) 2020 Feb 18;12(2). Epub 2020 Feb 18.

Centro di Studio e Ricerca sulle Neoplasie Ginecologiche, Alma Mater Studiorum-University of Bologna, 40138 Bologna, Italy.

Epithelial Ovarian Cancer (EOC) is one of the leading causes of cancer-related deaths among women and is characterized by the diffusion of nodules or plaques from the ovary to the peritoneal surfaces. Conventional therapeutic options cannot eradicate the disease and show low efficacy against resistant tumor subclones. The treatment of liquids via cold atmospheric pressure plasma enables the production of plasma-activated liquids (PALs) containing reactive oxygen and nitrogen species (RONS) with selective anticancer activity. Thus, the delivery of RONS to cancer tissues by intraperitoneal washing with PALs might be an innovative strategy for the treatment of EOC. In this work, plasma-activated Ringer's Lactate solution (PA-RL) was produced by exposing a liquid substrate to a multiwire plasma source. Subsequently, PA-RL dilutions are used for the treatment of EOC, non-cancer and fibroblast cell lines, revealing a selectivity of PA-RL, which induces a significantly higher cytotoxic effect in EOC with respect to non-cancer cells.
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http://dx.doi.org/10.3390/cancers12020476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072540PMC
February 2020

A Humanized Bone Niche Model Reveals Bone Tissue Preservation Upon Targeting Mitochondrial Complex I in Pseudo-Orthotopic Osteosarcoma.

J Clin Med 2019 12 11;8(12). Epub 2019 Dec 11.

Dipartimento di Scienze Mediche e Chirurgiche, Università di Bologna, Via Massarenti 9, 40138 Bologna, Italy.

A cogent issue in cancer research is how to account for the effects of tumor microenvironment (TME) on the response to therapy, warranting the need to adopt adequate in vitro and in vivo models. This is particularly relevant in the development of strategies targeting cancer metabolism, as they will inevitably have systemic effects. For example, inhibition of mitochondrial complex I (CI), despite showing promising results as an anticancer approach, triggers TME-mediated survival mechanisms in subcutaneous osteosarcoma xenografts, a response that may vary according to whether the tumors are induced via subcutaneous injection or by intrabone orthotopic transplantation. Thus, with the aim to characterize the TME of CI-deficient tumors in a model that more faithfully represents osteosarcoma development, we set up a humanized bone niche ectopic graft. A prominent involvement of TME was revealed in CI-deficient tumors, characterized by the abundance of cancer associated fibroblasts, tumor associated macrophages and preservation of osteocytes and osteoblasts in the mineralized bone matrix. The pseudo-orthotopic approach allowed investigation of osteosarcoma progression in a bone-like microenvironment setting, without being invasive as the intrabone cell transplantation. Additionally, establishing osteosarcomas in a humanized bone niche model identified a peculiar association between targeting CI and bone tissue preservation.
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http://dx.doi.org/10.3390/jcm8122184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947153PMC
December 2019

Lithium and Not Acetoacetate Influences the Growth of Cells Treated with Lithium Acetoacetate.

Int J Mol Sci 2019 Jun 25;20(12). Epub 2019 Jun 25.

Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, 5020 Salzburg, Austria.

The ketogenic diet (KD), a high-fat/low-carbohydrate/adequate-protein diet, has been proposed as a treatment for a variety of diseases, including cancer. KD leads to generation of ketone bodies (KBs), predominantly acetoacetate (AcAc) and 3-hydroxy-butyrate, as a result of fatty acid oxidation. Several studies investigated the antiproliferative effects of lithium acetoacetate (LiAcAc) and sodium 3-hydroxybutyrate on cancer cells in vitro. However, a critical point missed in some studies using LiAcAc is that Li ions have pleiotropic effects on cell growth and cell signaling. Thus, we tested whether Li ions per se contribute to the antiproliferative effects of LiAcAc in vitro. Cell proliferation was analyzed on neuroblastoma, renal cell carcinoma, and human embryonic kidney cell lines. Cells were treated for 5 days with 2.5, 5, and 10 mM LiAcAc and with equimolar concentrations of lithium chloride (LiCl) or sodium chloride (NaCl). LiAcAc affected the growth of all cell lines, either negatively or positively. However, the effects of LiAcAc were always similar to those of LiCl. In contrast, NaCl showed no effects, indicating that the Li ion impacts cell proliferation. As Li ions have significant effects on cell growth, it is important for future studies to include sources of Li ions as a control.
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http://dx.doi.org/10.3390/ijms20123104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628210PMC
June 2019

The multifaceted contribution of α-ketoglutarate to tumor progression: An opportunity to exploit?

Semin Cell Dev Biol 2020 02 10;98:26-33. Epub 2019 Jun 10.

Department of Pharmacy and Biotechnology (FaBit), University of Bologna, Via Selmi 3, 40126, Bologna, Italy; Interdepartmental Center of Industrial Research (CIRI) Life Science and Health Technologies, University of Bologna, Via Tolara di Sopra, 41/E, 40064 Ozzano dell'Emilia, Italy. Electronic address:

The thriving field that constitutes cancer metabolism has unveiled some groundbreaking facts over the past two decades, at the heart of which is the TCA cycle and its intermediates. As such and besides its metabolic role, α-ketoglutarate was shown to withstand a wide range of physiological reactions from protection against oxidative stress, collagen and bone maintenance to development and immunity. Most importantly, it constitutes the rate-limiting substrate of 2-oxoglutarate-dependent dioxygenases family enzymes, which are involved in hypoxia sensing and in the shaping of cellular epigenetic landscape, two major drivers of oncogenic transformation. Based on literature reports, we hereby review the benefits of this metabolite as a possible novel adjuvant therapeutic opportunity to target tumor progression. This article is part of the special issue "Mitochondrial metabolic alterations in cancer cells and related therapeutic targets".
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http://dx.doi.org/10.1016/j.semcdb.2019.05.031DOI Listing
February 2020

Potential Prognostic Role of F-FDG PET/CT in Invasive Epithelial Ovarian Cancer Relapse. A Preliminary Study.

Cancers (Basel) 2019 May 23;11(5). Epub 2019 May 23.

Department of Obstetrics and Gynecology, Unit of Oncologic Gynecology, S. Orsola-Malpighi Hospital, University of Bologna, 40138 Bologna, Italy.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with relapse occurring in about 70% of advanced cases with poor prognosis. Fluorine-18-2-fluoro-2-deoxy-d-glucose PET/CT (F-FDGPET/CT) is the most specific radiological imaging used to assess recurrence. Some intensity-based and volume-based PET parameters, maximum standardized uptake values (SUV), metabolic tumor volume (MTV) and total lesion glycolysis (TLG), are indicated to have a correlation with treatment response. The aim of our study is to correlate these parameters with post relapse survival (PRS) and overall survival (OS) in Epithelial Ovarian Cancer (EOC) relapse. The study included 50 patients affected by EOC relapse who underwent F-FDGPET/CT before surgery. All imaging was reviewed and SUV, MTV and TLG were calculated and correlated to PRS and OS. PRS and OS were obtained from the first relapse and from the first diagnosis to the last follow up or death, respectively. SUV, MTV and TLG were tested in a univariate logistic regression analysis, only SUV demonstrated to be significantly associated to PRS and OS ( = 0.005 and = 0.024 respectively). Multivariate analysis confirmed the results. We found a cut-off of SUV of 13 that defined worse or better survival ( = 0.003). In the first relapse of EOC, SUV is correlated to PRS and OS, and when SUV is greater than 13, it is an unfavorable prognostic factor.
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http://dx.doi.org/10.3390/cancers11050713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562912PMC
May 2019

The multifaceted effects of metformin on tumor microenvironment.

Semin Cell Dev Biol 2020 02 22;98:90-97. Epub 2019 May 22.

Dipartimento di Scienze Mediche e Chirurgiche, Università di Bologna, Via Massarenti 9, 40138, Bologna, Italy; Centro di Ricerca Biomedica Applicata (CRBA), Università di Bologna, Via Massarenti 9, 40138, Bologna, Italy. Electronic address:

The efficacy of metformin in treating cancer has been extensively investigated since epidemiologic studies associated this anti-diabetic drug with a lower risk of cancer incidence. Since tumors are complex systems, in which cancer cells coexist and interact with several different types of non-malignant cells, it is not surprising that anti-cancer drugs affect not only cancer cells, but also the abundance and functions of cells of the tumor microenvironment. Recent years have seen a wide collection of reports showing how metformin, as well as other complex I inhibitors, may influence cancer progression by modulating the phenotype of non-transformed cells in a tumor. In this review, we particularly focus on the effect of metformin on angiogenesis, cancer-associated fibroblasts, tumor-associated macrophages and cancer immunosuppression.
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http://dx.doi.org/10.1016/j.semcdb.2019.05.010DOI Listing
February 2020

Inducing cancer indolence by targeting mitochondrial Complex I is potentiated by blocking macrophage-mediated adaptive responses.

Nat Commun 2019 02 22;10(1):903. Epub 2019 Feb 22.

Dipartimento di Scienze Mediche e Chirurgiche, Università di Bologna, Via Massarenti 9, 40138, Bologna, Italy.

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.
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http://dx.doi.org/10.1038/s41467-019-08839-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385215PMC
February 2019

A Nonsense Mitochondrial DNA Mutation Associates with Dysfunction of HIF1 in a Von Hippel-Lindau Renal Oncocytoma.

Oxid Med Cell Longev 2019 9;2019:8069583. Epub 2019 Jan 9.

Department of Medical and Surgical Sciences (DIMEC), Unit of Medical Genetics, University of Bologna Medical School, Bologna 40138, Italy.

The Von Hippel-Lindau (VHL) syndrome has been rarely associated with renal oncocytomas, and tumors usually show HIF1 chronic stabilization. By contrast, oncocytomas mainly associated with respiratory chain (RC) defects due to severe mitochondrial DNA (mtDNA) mutations are incapable of stabilizing HIF1, since oxygen consumption by the RC is dramatically diminished and prolylhydroxylase activity is increased by -ketoglutarate accumulation following Krebs cycle slowdown. Here, we investigate the cooccurrence of a pseudohypoxic condition with oncocytic transformation in a case of VHL-associated renal oncocytoma. While HIF1 was abundant in nuclei concordantly with defects in VHL, negative staining of its targets carbonic anhydrase IX (CAIX) and glucose transporter GLUT1, usually overexpressed in VHL-associated neoplasms, suggested HIF1 to be present in its inactive (hydroxylated) form. MtDNA sequencing and immunohistochemistry analyses revealed a stop-gain mutation and cytochrome c oxidase loss. We suggest that a mitochondrial respiration impairment may lead to hyperhydroxylation of the transcription factor, which we confirmed by specific staining of hydroxylated HIF1. Such inactive form hence accumulated in the VHL-deficient tumor, where it may contribute to the benign nature of the neoplasm. We propose that the protumorigenic role of HIF1 in VHL cancers may be blunted through drugs inhibiting mitochondrial respiratory complexes, such as metformin.
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http://dx.doi.org/10.1155/2019/8069583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343171PMC
March 2019

Mice harbouring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity.

Neurobiol Dis 2019 04 30;124:14-28. Epub 2018 Oct 30.

Department of Neuroscience, University of Torino, Torino, Italy; Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano, Italy.

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.
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http://dx.doi.org/10.1016/j.nbd.2018.10.018DOI Listing
April 2019

Potential for Mitochondrial DNA Sequencing in the Differential Diagnosis of Gynaecological Malignancies.

Int J Mol Sci 2018 Jul 13;19(7). Epub 2018 Jul 13.

Unit of Medical Genetics, Department of Medical and Surgical Sciences (DIMEC), Sant Orsola Hospital, Pav.11, via Massarenti 9, 40138 Bologna, Italy.

In the event of multiple synchronous gynecological lesions, a fundamental piece of information to determine patient management, prognosis, and therapeutic regimen choice is whether the simultaneous malignancies arise independently or as a result of metastatic dissemination. An example of synchronous primary tumors of the female genital tract most frequently described are ovarian and endometrial cancers. Surgical findings and histopathological examination aimed at resolving this conundrum may be aided by molecular analyses, although they are too often inconclusive. High mitochondrial DNA (mtDNA) variability and its propensity to accumulate mutations has been proposed by our group as a tool to define clonality. We showed mtDNA sequencing to be informative in synchronous primary ovarian and endometrial cancer, detecting tumor-specific mutations in both lesions, ruling out independence of the two neoplasms, and indicating clonality. Furthermore, we tested this method in another frequent simultaneously detected gynecological lesion type, borderline ovarian cancer and their peritoneal implants, which may be monoclonal extra-ovarian metastases or polyclonal independent masses. The purpose of this review is to provide an update on the potential use of mtDNA sequencing in distinguishing independent and metastatic lesions in gynecological cancers, and to compare the efficiency of molecular analyses currently in use with this novel method.
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http://dx.doi.org/10.3390/ijms19072048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073261PMC
July 2018

The Paradigm of Respiratory Complex I.

Genes (Basel) 2018 May 7;9(5). Epub 2018 May 7.

Dipartimento di Farmacia e Biotecnologie (FaBit), Univeristà di Bologna, 40126 Bologna, Italy.

Mitochondrial respiratory function is now recognized as a pivotal player in all the aspects of cancer biology, from tumorigenesis to aggressiveness and chemotherapy resistance. Among the enzymes that compose the respiratory chain, by contributing to energy production, redox equilibrium and oxidative stress, complex I assumes a central role. Complex I defects may arise from mutations in mitochondrial or nuclear DNA, in both structural genes or assembly factors, from alteration of the expression levels of its subunits, or from drug exposure. Since cancer cells have a high-energy demand and require macromolecules for proliferation, it is not surprising that severe complex I defects, caused either by mutations or treatment with specific inhibitors, prevent tumor progression, while contributing to resistance to certain chemotherapeutic agents. On the other hand, enhanced oxidative stress due to mild complex I dysfunction drives an opposite phenotype, as it stimulates cancer cell proliferation and invasiveness. We here review the current knowledge on the contribution of respiratory complex I to cancer biology, highlighting the double-edged role of this metabolic enzyme in tumor progression, metastasis formation, and response to chemotherapy.
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http://dx.doi.org/10.3390/genes9050243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5977183PMC
May 2018

Mutant MYO1F alters the mitochondrial network and induces tumor proliferation in thyroid cancer.

Int J Cancer 2018 10 7;143(7):1706-1719. Epub 2018 May 7.

Department of Medical and Surgical Sciences, DIMEC, St. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy.

Familial aggregation is a significant risk factor for the development of thyroid cancer and familial non-medullary thyroid cancer (FNMTC) accounts for 5-7% of all NMTC. Whole exome sequencing analysis in the family affected by FNMTC with oncocytic features where our group previously identified a predisposing locus on chromosome 19p13.2, revealed a novel heterozygous mutation (c.400G > A, NM_012335; p.Gly134Ser) in exon 5 of MYO1F, mapping to the linkage locus. In the thyroid FRTL-5 cell model stably expressing the mutant MYO1F p.Gly134Ser protein, we observed an altered mitochondrial network, with increased mitochondrial mass and a significant increase in both intracellular and extracellular reactive oxygen species, compared to cells expressing the wild-type (wt) protein or carrying the empty vector. The mutation conferred a significant advantage in colony formation, invasion and anchorage-independent growth. These data were corroborated by in vivo studies in zebrafish, since we demonstrated that the mutant MYO1F p.Gly134Ser, when overexpressed, can induce proliferation in whole vertebrate embryos, compared to the wt one. MYO1F screening in additional 192 FNMTC families identified another variant in exon 7, which leads to exon skipping, and is predicted to alter the ATP-binding domain in MYO1F. Our study identified for the first time a role for MYO1F in NMTC.
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http://dx.doi.org/10.1002/ijc.31548DOI Listing
October 2018

Unravelling the Effects of the Mutation m.3571insC/MT-ND1 on Respiratory Complexes Structural Organization.

Int J Mol Sci 2018 Mar 7;19(3). Epub 2018 Mar 7.

Dipartimento di Farmacia e Biotecnologie (FABIT), Università di Bologna, Via Francesco Selmi 3, 40126 Bologna, Italy.

Mammalian respiratory complex I (CI) biogenesis requires both nuclear and mitochondria-encoded proteins and is mostly organized in respiratory supercomplexes. Among the CI proteins encoded by the mitochondrial DNA, NADH-ubiquinone oxidoreductase chain 1 (ND1) is a core subunit, evolutionary conserved from bacteria to mammals. Recently, ND1 has been recognized as a pivotal subunit in maintaining the structural and functional interaction among the hydrophilic and hydrophobic CI arms. A critical role of human ND1 both in CI biogenesis and in the dynamic organization of supercomplexes has been depicted, although the proof of concept is still missing and the critical amount of ND1 protein necessary for a proper assembly of both CI and supercomplexes is not defined. By exploiting a unique model in which human ND1 is allotopically re-expressed in cells lacking the endogenous protein, we demonstrated that the lack of this protein induces a stall in the multi-step process of CI biogenesis, as well as the alteration of supramolecular organization of respiratory complexes. We also defined a mutation threshold for the m.3571insC truncative mutation in mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (), below which CI and its supramolecular organization is recovered, strengthening the notion that a certain amount of human ND1 is required for CI and supercomplexes biogenesis.
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http://dx.doi.org/10.3390/ijms19030764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877625PMC
March 2018

Peculiar combinations of individually non-pathogenic missense mitochondrial DNA variants cause low penetrance Leber's hereditary optic neuropathy.

PLoS Genet 2018 02 14;14(2):e1007210. Epub 2018 Feb 14.

Neurology Unit, IRCCS Institute of Neurological Sciences of Bologna, Bologna, Italy.

We here report on the existence of Leber's hereditary optic neuropathy (LHON) associated with peculiar combinations of individually non-pathogenic missense mitochondrial DNA (mtDNA) variants, affecting the MT-ND4, MT-ND4L and MT-ND6 subunit genes of Complex I. The pathogenic potential of these mtDNA haplotypes is supported by multiple evidences: first, the LHON phenotype is strictly inherited along the maternal line in one very large family; second, the combinations of mtDNA variants are unique to the two maternal lineages that are characterized by recurrence of LHON; third, the Complex I-dependent respiratory and oxidative phosphorylation defect is co-transferred from the proband's fibroblasts into the cybrid cell model. Finally, all but one of these missense mtDNA variants cluster along the same predicted fourth E-channel deputed to proton translocation within the transmembrane domain of Complex I, involving the ND1, ND4L and ND6 subunits. Hence, the definition of the pathogenic role of a specific mtDNA mutation becomes blurrier than ever and only an accurate evaluation of mitogenome sequence variation data from the general population, combined with functional analyses using the cybrid cell model, may lead to final validation. Our study conclusively shows that even in the absence of a clearly established LHON primary mutation, unprecedented combinations of missense mtDNA variants, individually known as polymorphisms, may lead to reduced OXPHOS efficiency sufficient to trigger LHON. In this context, we introduce a new diagnostic perspective that implies the complete sequence analysis of mitogenomes in LHON as mandatory gold standard diagnostic approach.
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http://dx.doi.org/10.1371/journal.pgen.1007210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828459PMC
February 2018

Non-Canonical Mechanisms Regulating Hypoxia-Inducible Factor 1 Alpha in Cancer.

Front Oncol 2017 27;7:286. Epub 2017 Nov 27.

Dipartimento di Scienze Mediche e Chirurgiche, Università di Bologna, Bologna, Italy.

Hypoxia-inducible factor 1 alpha (HIF-1α) orchestrates cellular adaptation to low oxygen and nutrient-deprived environment and drives progression to malignancy in human solid cancers. Its canonical regulation involves prolyl hydroxylases (PHDs), which in normoxia induce degradation, whereas in hypoxia allow stabilization of HIF-1α. However, in certain circumstances, HIF-1α regulation goes beyond the actual external oxygen levels and involves PHD-independent mechanisms. Here, we gather and discuss the evidence on the non-canonical HIF-1α regulation, focusing in particular on the consequences of mitochondrial respiratory complexes damage on stabilization of this pleiotropic transcription factor.
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http://dx.doi.org/10.3389/fonc.2017.00286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711814PMC
November 2017

Mild phenotypes and proper supercomplex assembly in human cells carrying the homoplasmic m.15557G > A mutation in cytochrome b gene.

Hum Mutat 2018 01 12;39(1):92-102. Epub 2017 Oct 12.

Dipartimento di Farmacia e Biotecnologie (FABIT), Università di Bologna, Bologna, Italy.

Respiratory complex III (CIII) is the first enzymatic bottleneck of the mitochondrial respiratory chain both in its native dimeric form and in supercomplexes. The mammalian CIII comprises 11 subunits among which cytochrome b is central in the catalytic core, where oxidation of ubiquinol occurs at the Qo site. The Qo- or PEWY-motif of cytochrome b is the most conserved through species. Importantly, the highly conserved glutamate at position 271 (Glu271) has never been studied in higher eukaryotes so far and its role in the Q-cycle remains debated. Here, we showed that the homoplasmic m.15557G > A/MT-CYB, which causes the p.Glu271Lys amino acid substitution predicted to dramatically affect CIII, induces a mild mitochondrial dysfunction in human transmitochondrial cybrids. Indeed, we found that the severity of such mutation is mitigated by the proper assembly of CIII into supercomplexes, which may favor an optimal substrate channeling and buffer superoxide production in vitro.
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http://dx.doi.org/10.1002/humu.23350DOI Listing
January 2018

Platinum-induced mitochondrial DNA mutations confer lower sensitivity to paclitaxel by impairing tubulin cytoskeletal organization.

Hum Mol Genet 2017 08;26(15):2961-2974

Department of Medical and Surgical Sciences, Unit of Medical Genetics, University Hospital S.Orsola-Malpighi, 40138 Bologna, Italy.

Development of chemoresistance is a cogent clinical issue in oncology, whereby combination of anticancer drugs is usually preferred also to enhance efficacy. Paclitaxel (PTX), combined with carboplatin, represents the standard first-line chemotherapy for different types of cancers. We here depict a double-edge role of mitochondrial DNA (mtDNA) mutations induced in cancer cells after treatment with platinum. MtDNA mutations were positively selected by PTX, and they determined a decrease in the mitochondrial respiratory function, as well as in proliferative and tumorigenic potential, in terms of migratory and invasive capacity. Moreover, cells bearing mtDNA mutations lacked filamentous tubulin, the main target of PTX, and failed to reorient the Golgi body upon appropriate stimuli. We also show that the bioenergetic and cytoskeletal phenotype were transferred along with mtDNA mutations in transmitochondrial hybrids, and that this also conferred PTX resistance to recipient cells. Overall, our data show that platinum-induced deleterious mtDNA mutations confer resistance to PTX, and confirm what we previously reported in an ovarian cancer patient treated with carboplatin and PTX who developed a quiescent yet resistant tumor mass harboring mtDNA mutations.
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http://dx.doi.org/10.1093/hmg/ddx186DOI Listing
August 2017

Mitochondrial DNA sequencing demonstrates clonality of peritoneal implants of borderline ovarian tumors.

Mol Cancer 2017 02 27;16(1):47. Epub 2017 Feb 27.

Department of Medical and Surgical Sciences (DIMEC) - Unit of Medical Genetics, University of Bologna Medical School, Via G. Massarenti 9, 40138, Bologna, Italy.

Borderline ovarian tumors are rare low malignant potential neoplasms characterized by the absence of stromal invasion, whose main prognostic factors are stage and type of peritoneal implants. The latter are defined as invasive when cell proliferation invades the underlying tissue (peritoneal surface, omentum and intestinal wall), or noninvasive. It is still unknown if these implants are metastatic spread from the primary ovarian mass or a neoplastic transformation de novo of the peritoneal surface. Mitochondrial DNA sequencing was performed to assess clonality in eight patients presenting both borderline ovarian tumors and implants. In 37.5% of the cases, the same mitochondrial DNA mutation was present in both borderline ovarian tumors and the peritoneal implant, being this evidence that implants may arise as a consequence of a spread from a single ovarian site.
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http://dx.doi.org/10.1186/s12943-017-0614-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5327524PMC
February 2017

Mitochondrial metabolism and energy sensing in tumor progression.

Biochim Biophys Acta Bioenerg 2017 Aug 14;1858(8):582-590. Epub 2017 Feb 14.

Dipartimento Farmacia e Biotecnologie (FABIT), Università di Bologna, Via Selmi 3, 40126 Bologna, Italy; Centro Interdipartimentale di Ricerca Industriale Scienze della Vita e Tecnologie per la Salute, Università di Bologna, Via Tolara di Sopra, 41/E, 40064 Ozzano dell'Emilia, Italy.

Energy homeostasis is pivotal for cell fate since metabolic regulation, cell proliferation and death are strongly dependent on the balance between catabolic and anabolic pathways. In particular, metabolic and energetic changes have been observed in cancer cells even before the discovery of oncogenes and tumor suppressors, but have been neglected for a long time. Instead, during the past 20years a renaissance of the study of tumor metabolism has led to a revised and more accurate sight of the metabolic landscape of cancer cells. In this scenario, genetic, biochemical and clinical evidences place mitochondria as key actors in cancer metabolic restructuring, not only because there are energy and biosynthetic intermediates manufacturers, but also because occurrence of mutations in metabolic enzymes encoded by both nuclear and mitochondrial DNA has been associated to different types of cancer. Here we provide an overview of the possible mechanisms modulating mitochondrial energy production and homeostasis in the intriguing scenario of neoplastic cells, focusing on the double-edged role of 5'-AMP activated protein kinase in cancer metabolism. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.
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http://dx.doi.org/10.1016/j.bbabio.2017.02.006DOI Listing
August 2017

The α-ketoglutarate dehydrogenase complex in cancer metabolic plasticity.

Cancer Metab 2017 2;5. Epub 2017 Feb 2.

Dipartimento Farmacia e Biotecnologie (FABIT), Università di Bologna, Via Selmi 3, 40126 Bologna, Italy.

Deregulated metabolism is a well-established hallmark of cancer. At the hub of various metabolic pathways deeply integrated within mitochondrial functions, the α-ketoglutarate dehydrogenase complex represents a major modulator of electron transport chain activity and tricarboxylic acid cycle (TCA) flux, and is a pivotal enzyme in the metabolic reprogramming following a cancer cell's change in bioenergetic requirements. By contributing to the control of α-ketoglutarate levels, dynamics, and oxidation state, the α-ketoglutarate dehydrogenase is also essential in modulating the epigenetic landscape of cancer cells. In this review, we will discuss the manifold roles that this TCA enzyme and its substrate play in cancer.
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http://dx.doi.org/10.1186/s40170-017-0165-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289018PMC
February 2017

Molecular and metabolic features of oncocytomas: Seeking the blueprints of indolent cancers.

Biochim Biophys Acta Bioenerg 2017 Aug 20;1858(8):591-601. Epub 2017 Jan 20.

Department of Medical and Surgical Sciences-DIMEC, Unit of Medical Genetics, University Hospital S.Orsola-Malpighi, Bologna, Italy.

Oncocytic tumors are a peculiar subset of human neoplasms in which mitochondria have been proven to have a prominent role. A number of paradoxes render these clinical entities interesting from the translational research point of view. Most oncocytic tumors are generally metabolically constrained due to the impaired respiratory capacity and lack of the ability to respond to hypoxia, yet they maintain features that allow them to strive and persist in an indolent form. Their unique molecular and metabolic characteristics are an object of investigation that may reveal novel ways for therapeutic strategies based on metabolic targeting. With this aim in mind, we here examine the current knowledge on oncocytomas and delve into the molecular causes and consequences that revolve around the oncocytic phenotype, to understand whether we can learn to design therapies from the dissection of benign neoplasms. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.
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http://dx.doi.org/10.1016/j.bbabio.2017.01.009DOI Listing
August 2017

Defective ciliogenesis in thyroid hürthle cell tumors is associated with increased autophagy.

Oncotarget 2016 Nov;7(48):79117-79130

Research Center for Endocrine and Metabolic Diseases, Division of Endocrinology, Department of Internal Medicine, Chungnam National University School of Medicine, Jung-gu, Daejeon 35015, Republic of Korea.

Primary cilia are found in the apical membrane of thyrocytes, where they may play a role in the maintenance of follicular homeostasis. In this study, we examined the distribution of primary cilia in the human thyroid cancer to address the involvement of abnormal ciliogenesis in different thyroid cancers. We examined 92 human thyroid tissues, including nodular hyperplasia, Hashimoto's thyroiditis, follicular tumor, Hürthle cell tumor, and papillary carcinoma to observe the distribution of primary cilia. The distribution and length of primary cilia facing the follicular lumen were uniform across variable-sized follicles in the normal thyroid gland. However, most Hürthle cells found in benign and malignant thyroid diseases were devoid of primary cilia. Conventional variant of papillary carcinoma (PTC) displayed longer primary cilia than those of healthy tissue, whereas both the frequency and length of primary cilia were decreased in oncocytic variant of PTC. In addition, ciliogenesis was markedly defective in primary Hürthle cell tumors, including Hürthle cell adenomas and carcinomas, which showed higher level of autophagosome biogenesis. Remarkably, inhibition of autophagosome formation by Atg5 silencing or treatment with pharmacological inhibitors of autophagosome formation restored ciliogenesis in the Hürthle cell carcinoma cell line XTC.UC1 which exhibits a high basal autophagic flux. Moreover, the inhibition of autophagy promoted the accumulation of two factors critical for ciliogenesis, IFT88 and ARL13B. These results suggest that abnormal ciliogenesis, a common feature of Hürthle cells in diseased thyroid glands, is associated with increased basal autophagy.
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http://dx.doi.org/10.18632/oncotarget.12997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346702PMC
November 2016

A unique combination of rare mitochondrial ribosomal RNA variants affects the kinetics of complex I assembly.

Int J Biochem Cell Biol 2016 06 19;75:117-22. Epub 2016 Apr 19.

Dip. Scienze Mediche e Chirurgiche (DIMEC), U.O. Genetica Medica, Pol. Universitario S. Orsola-Malpighi, Università di Bologna, via Massarenti 9, 40138 Bologna, Italy. Electronic address:

Mitochondrial DNA (mtDNA) mutations in respiratory complexes subunits contribute to a large spectrum of human diseases. Nonetheless, ribosomal RNA variants remain largely under-investigated from a functional point of view. We here report a unique combination of two rare mitochondrial rRNA variants detected by serendipity in a subject with chronic granulomatous disease and never reported to co-occur within the same mitochondrial haplotype. In silico prediction of the mitochondrial ribosomal structure showed a dramatic rearrangement of the rRNA secondary structure. Functional investigation of cybrids carrying this unique haplotype demonstrated that the co-occurrence of the two rRNA variants determines a slow-down of the mitochondrial protein synthesis, especially in cells with an elevated metabolic rate, which impairs the assembly kinetics of Complex I, induces a bioenergetic defect and stimulates reactive oxygen species production. In conclusion, our results point to a sub-pathogenic role for these two rare mitochondrial rRNA variants, when found in the unique combination here reported in a single individual.
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http://dx.doi.org/10.1016/j.biocel.2016.04.007DOI Listing
June 2016

Dysregulation of Parkin-mediated mitophagy in thyroid Hürthle cell tumors.

Carcinogenesis 2015 Nov 8;36(11):1407-18. Epub 2015 Sep 8.

Division of Endocrinology, Department of Internal Medicine, Research Center for Endocrine and Metabolic Diseases, Chungnam National University School of Medicine, 282 Munhwa-ro, Jung-gu, Daejeon 301-721, Republic of Korea,

Abnormal accumulation of defective mitochondria is the hallmark of oncocytes, which are frequently observed in thyroid Hürthle cell lesions. Autophagy is an essential cellular catabolic mechanism for the degradation of dysfunctional organelles and has been implicated in several human diseases. It is yet unknown how autophagic turnover of defective mitochondria in Hürthle cell tumors is regulated. We characterized the expression patterns of molecular markers including Beclin1, LC3, PINK1 and Parkin, which are required for autophagy or mitophagy, in human oncocytic lesions of the thyroid. To undertake mechanistic studies, we investigated autophagy and mitophagy using XTC.UC1 cells, the only in vitro model of Hürthle cell tumors. Beclin1 and LC3 were highly expressed in oncocytes of Hürthle cell tumors. XTC.UC1 showed autophagic responses to starvation and rapamycin treatment, whereas they displayed ineffective activation of mitophagy, which is triggered by the coordinated action of PINK1 and Parkin in response to CCCP. This resulted in a decreased turnover of abnormal mitochondria. The mechanisms underlying defective mitophagy and mitochondrial turnover were investigated by genetic analysis of the PARK2 gene in XTC.UC1 and Hürthle cell tumor tissues. XTC.UC1 and several tumors harbored the V380L mutation, resulting in dysfunctional autoubiquitination and decreased E3 ligase activity. Consistently, oncocytes in Hürthle cell tumors displayed comparable expression of PINK1 but decreased Parkin expression in comparison to normal thyrocytes. The introduction of wild-type Parkin sensitized XTC.UC1 to death induced by CCCP. This study provides a possible etiological basis for oncocytic formation in heterogeneous Hürthle cell tumors through insufficient mitophagy leading to ineffective turnover of aberrant mitochondria caused by dysfunctional Parkin-mediated pathways of mitochondria quality control.
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http://dx.doi.org/10.1093/carcin/bgv122DOI Listing
November 2015

High-resolution genomic profiling of thyroid lesions uncovers preferential copy number gains affecting mitochondrial biogenesis loci in the oncocytic variants.

Am J Cancer Res 2015 15;5(6):1954-71. Epub 2015 May 15.

Medical Genetics Unit, Department of Medical and Surgical Sciences (DIMEC), University of Bologna, S. Orsola-Malpighi Hospital Bologna, Italy.

Oncocytic change is the result of aberrant mitochondrial hyperplasia, which may occur in both neoplastic and non-neoplastic cells and is not infrequent in the thyroid. Despite being a well-characterized histologic phenotype, the molecular causes underlying such a distinctive cellular change are poorly understood. To identify potential genetic causes for the oncocytic phenotype in thyroid, we analyzed copy number alterations in a set of oncocytic (n=21) and non-oncocytic (n=20) thyroid lesions by high-resolution microarray-based comparative genomic hybridization (aCGH). Each group comprised lesions of diverse histologic types, including hyperplastic nodules, adenomas and carcinomas. Unsupervised hierarchical clustering of categorical aCGH data resulted in two distinct branches, one of which was significantly enriched for samples with the oncocytic phenotype, regardless of histologic type. Analysis of aCGH events showed that the oncocytic group harbored a significantly higher number of genes involved in copy number gains, when compared to that of conventional thyroid lesions. Functional annotation demonstrated an enrichment for copy number gains that affect genes encoding activators of mitochondrial biogenesis in oncocytic cases but not in their non-oncocytic counterparts. Taken together, our data suggest that genomic alterations may represent additional/alternative mechanisms underlying the development of the oncocytic phenotype in the thyroid.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529616PMC
August 2015

Syndromic parkinsonism and dementia associated with OPA1 missense mutations.

Ann Neurol 2015 Jul 10;78(1):21-38. Epub 2015 Jun 10.

Mitochondrial Biology Unit, Medical Research Council, Cambridge, United Kingdom.

Objective: Mounting evidence links neurodegenerative disorders such as Parkinson disease and Alzheimer disease with mitochondrial dysfunction, and recent emphasis has focused on mitochondrial dynamics and quality control. Mitochondrial dynamics and mtDNA maintenance is another link recently emerged, implicating mutations in the mitochondrial fusion genes OPA1 and MFN2 in the pathogenesis of multisystem syndromes characterized by neurodegeneration and accumulation of mtDNA multiple deletions in postmitotic tissues. Here, we report 2 Italian families affected by dominant chronic progressive external ophthalmoplegia (CPEO) complicated by parkinsonism and dementia.

Methods: Patients were extensively studied by optical coherence tomography (OCT) to assess retinal nerve fibers, and underwent muscle and brain magnetic resonance spectroscopy (MRS), and muscle biopsy and fibroblasts were analyzed. Candidate genes were sequenced, and mtDNA was analyzed for rearrangements.

Results: Affected individuals displayed a slowly progressive syndrome characterized by CPEO, mitochondrial myopathy, sensorineural deafness, peripheral neuropathy, parkinsonism, and/or cognitive impairment, in most cases without visual complains, but with subclinical loss of retinal nerve fibers at OCT. Muscle biopsies showed cytochrome c oxidase-negative fibers and mtDNA multiple deletions, and MRS displayed defective oxidative metabolism in muscle and brain. We found 2 heterozygous OPA1 missense mutations affecting highly conserved amino acid positions (p.G488R, p.A495V) in the guanosine triphosphatase domain, each segregating with affected individuals. Fibroblast studies showed a reduced amount of OPA1 protein with normal mRNA expression, fragmented mitochondria, impaired bioenergetics, increased autophagy and mitophagy.

Interpretation: The association of CPEO and parkinsonism/dementia with subclinical optic neuropathy widens the phenotypic spectrum of OPA1 mutations, highlighting the association of defective mitochondrial dynamics, mtDNA multiple deletions, and altered mitophagy with parkinsonism.
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http://dx.doi.org/10.1002/ana.24410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5008165PMC
July 2015

Targeting respiratory complex I to prevent the Warburg effect.

Int J Biochem Cell Biol 2015 Jun 7;63:41-5. Epub 2015 Feb 7.

Dipartimento di Farmacia e Biotecnologie (FABIT), Università di Bologna, via Irnerio 42, 40126 Bologna, Italy; Centro Interdipartimentale di Ricerca Industriale Scienze della Vita e Tecnologie per la Salute, Università di Bologna, 40100 Bologna, Italy. Electronic address:

In the last 10 years, studies of energetic metabolism in different tumors clearly indicate that the definition of Warburg effect, i.e. the glycolytic shift cells undergo upon transformation, ought to be revisited considering the metabolic plasticity of cancer cells. In fact, recent findings show that the shift from glycolysis to re-established oxidative metabolism is required for certain steps of tumor progression, suggesting that mitochondrial function and, in particular, respiratory complex I are crucial for metabolic and hypoxic adaptation. Based on these evidences, complex I can be considered a lethality target for potential anticancer strategies. In conclusion, in this mini review we summarize and discuss why it is not paradoxical to develop pharmacological and genome editing approaches to target complex I as novel adjuvant therapies for cancer treatment. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.
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http://dx.doi.org/10.1016/j.biocel.2015.01.017DOI Listing
June 2015

A comprehensive characterization of mitochondrial DNA mutations in glioblastoma multiforme.

Int J Biochem Cell Biol 2015 Jun 7;63:46-54. Epub 2015 Feb 7.

Department of Medical and Surgical Sciences (DIMEC), Medical Genetics Unit, University of Bologna, Bologna, Italy. Electronic address:

Glioblastoma multiforme (GBM) is the most malignant brain cancer in adults, with a poor prognosis, whose molecular stratification still represents a challenge in pathology and clinics. On the other hand, mitochondrial DNA (mtDNA) mutations have been found in most tumors as modifiers of the bioenergetics state, albeit in GBM a characterization of the mtDNA status is lacking to date. Here, a characterization of the burden of mtDNA mutations in GBM samples was performed. First, investigation of tumor-specific vs. non tumor-specific mutations was carried out with the MToolBox bioinformatics pipeline by analyzing 45 matched tumor/blood samples, from whole genome or whole exome sequencing datasets obtained from The Cancer Genome Atlas (TCGA) consortium. Additionally, the entire mtDNA sequence was obtained in a dataset of 104 fresh-frozen GBM samples. Mitochondrial mutations with potential pathogenic interest were prioritized based on heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. A preliminary biochemical analysis of the activity of mitochondrial respiratory complexes was also performed on fresh-frozen GBM samples. Although a high number of mutations was detected, we report that the large majority of them does not pass the prioritization filters. Therefore, a relatively limited burden of pathogenic mutations is indeed carried by GBM, which did not appear to determine a general impairment of the respiratory chain. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.
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http://dx.doi.org/10.1016/j.biocel.2015.01.027DOI Listing
June 2015

Analysis of the mitochondrial proteome of cybrid cells harbouring a truncative mitochondrial DNA mutation in respiratory complex I.

Mol Biosyst 2014 Jun 18;10(6):1313-9. Epub 2014 Mar 18.

CNR - Institute of Biomembranes and Bioenergetics, Bari, Italy.

Transmitochondrial cytoplasmic hybrids (cybrids) are well established model systems to reveal the effects of mitochondrial DNA (mtDNA) mutations on cell metabolism excluding the interferences of a different nuclear background. The m.3571insC mutation in the MTND1 gene of respiratory complex I (CI) is commonly detected in oncocytic tumors, in which it causes a severe CI dysfunction leading to an energetic impairment when present above 83% mutant load. To assess whether the energetic deficit may alter the mitochondrial proteome, OS-78 and OS-93 cybrid cell lines bearing two different degrees of the m.3571insC mutation (78% and 92.8%, respectively) and control cybrids bearing wild-type mtDNA (CC) were analyzed. Two-dimensional electrophoresis and mass spectrometry revealed significant alterations only in cybrids above the threshold (OS-93). All differentially expressed proteins are decreased. In particular, the levels of the pyruvate dehydrogenase E1 chain B subunit (E1β), of lipoamide dehydrogenase (E3), the enzyme component of pyruvate and 2-oxoglutarate dehydrogenase complexes, and of lactate dehydrogenase B (LDHB) were reduced. Moreover, a significant decrease of the pyruvate dehydrogenase complex activity was found when OS-93 cybrid cells were grown in galactose medium, a metabolic condition that forces cells to use respiration. These results demonstrate that the energetic impairment caused by the almost homoplasmic m.3571insC mutation perturbs cellular metabolism leading to a decreased steady state level of components of very important mitochondrial NAD-dependent dehydrogenases.
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http://dx.doi.org/10.1039/c3mb70542kDOI Listing
June 2014

A platform independent RNA-Seq protocol for the detection of transcriptome complexity.

BMC Genomics 2013 Dec 5;14:855. Epub 2013 Dec 5.

Istituto di Tecnologie Biomediche (ITB), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy.

Background: Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform.

Results: We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol.

Conclusion: We tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes.
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http://dx.doi.org/10.1186/1471-2164-14-855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046740PMC
December 2013