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TRANSMIT: Translational Significance of Mitochondrial DNA Mutations in Tumors Giuseppe Gasparre, PhD University of Bologna Mediterranean School of Oncology.

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Presentation on theme: "TRANSMIT: Translational Significance of Mitochondrial DNA Mutations in Tumors Giuseppe Gasparre, PhD University of Bologna Mediterranean School of Oncology."— Presentation transcript:

1 TRANSMIT: Translational Significance of Mitochondrial DNA Mutations in Tumors Giuseppe Gasparre, PhD University of Bologna Mediterranean School of Oncology Rome – 28 January 2011 TRANSMIT

2 LAYOUT 1.AIMS of TRANSMIT 2.BACKGROUND Mitochondrial genetics, oncocytoma and tumor progression 3.STATE OF ART Objective I Impact of mtDNA mutations on tumor growth Objective IV Develop anti-cancer therapeutic strategies Objective II Develop diagnostic and prognostic tools Definition of oncojanus Biobank assembly Correlative studies Clinical follow-up Allotopic expression Target chaperones Perspectives/Knowledge advances TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 Objective III Uncover the determinants of the homoplasmic shift Genetic predisposition Role of inflammation Dissection of molecular pathways

3 1.AIMS OF TRANSMIT Understanding the functional and clinical implications of the occurrence of mtDNA pathological mutations in cancer cells in vitro and in vivo. TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 Set the ground for gene therapy-based and pharmacological anti- cancer therapeutic strategies targeting the mitochondrial respiratory chain.

4 Human Mitochondrial Genome 16,569 bp D-LOOP (control region) 37 genes (complex subunits, rRNA & tRNA) Oncogene Aug 7;25(34): BACKGROUND TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011

5 Threshold effect Am. J. Hum. Genet. 51: , 1992 CARRIERS AFFECTED 2.BACKGROUND TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 HomoplasmyHeteroplasmyHomoplasmy Oncocytic tumors: mitochondrial hyperplasia mostly benign

6 2.BACKGROUND HIF1  is the main player in the metabolic adaptation of cancer cells to the adverse tumor microenvironment. TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 ND6 HIF1  mut wt Complex I disassembly in vivo correlates with lack of HIF1  stabilization

7 2.BACKGROUND TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 NAD+ Lack of respiratory complex I induces PSEUDONORMOXIA HIF1  chronically destabilized –> block of tumor growth

8 3.STATE OF THE ART Cancer Res Why are mtDNA mutations selected in favour in cancer cells if, once homoplasmic, they abolish both respiration and HIF1  -mediated glycolysis? A PARADOXICAL KEY TO EXPLANATION TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 Porcelli et al., HMG 2010

9 OBJECTIVES OF TRANSMIT I.Demonstrating the impact of homo/heteroplasmy of mtDNA mutations on the tumorigenic potential of cancer cells. II.Testing and implementing IHC screening tools to aid diagnosis/prognosis through prediction of mtDNA mutations occurrence on the basis of respiratory complexes staining patterns. III.Dissecting the molecular mechanisms regulating the homoplasmic shift observed in oncocytic tumors. IV.Develop anti-cancer therapeutic strategies targeting specific mitochondrial functions. TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011

10 Objective I - How mtDNA mutations affect tumor progression TRANSMIT In agreement with the profound metabolic impairment it causes, homoplasmy of mtDNA mutations disassembling respiratory complexes hampers tumorigenic potential of cancer cells. Mediterranean School of Oncology Rome – 28 January 2011 Define a novel class of cancer-related genes that behave neither as oncogenes nor as oncosuppressors. They hamper tumor growth in a mutation threshold-regulated fashion, below which they are either neutral or advantageous to cancer cells. The oncojanus

11 OBJECTIVES OF TRANSMIT TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 I.Demonstrating the impact of homo/heteroplasmy of mtDNA mutations on the tumorigenic potential of cancer cells. II.Testing and implementing IHC screening tools to aid diagnosis/prognosis through prediction of mtDNA mutations occurrence on the basis of respiratory complexes staining patterns. III.Dissecting the molecular mechanisms regulating the homoplasmic shift observed in oncocytic tumors. IV.Develop anti-cancer therapeutic strategies targeting specific mitochondrial functions.

12 Objective II - From IHC patterns to mtDNA mutations - and back Is it possible to predict occurrence of mtDNA mutations from respiratory complexes staining patterns? How overlooked is oncocytic transformation in tumor diagnostics? Can outcome be predicted by combining IHC and genetic analysis of mtDNA? TRANSMIT Tumor biopsies collection Biobank creation mtDNA sequencing Tissue microarray Optimize RC antibodies STRATEGIES Correlate with HIF1  staining Correlate with Ki67 proliferating index Patient follow up Mediterranean School of Oncology Rome – 28 January 2011

13 Endometrial carcinoma type I has rarely been observed to present oncocytic features. IHC combined with genetic analysis helps revealing oncocytic transformation. PRELIMINARY RESULTS ND6 G76STOP Other tumors usually not diagnosed as oncocytic display markers of oncocytic transformation, both in terms of genetic lesions and of respiratory complexes disassembly. IMPLICATIONS Homoplasmic mtDNA mutations will become of high prognostic value implying benignity of the neoplastic lesion. TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 Figures from: Guerra et al., submitted

14 OBJECTIVES OF TRANSMIT TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 I.Demonstrating the impact of homo/heteroplasmy of mtDNA mutations on the tumorigenic potential of cancer cells. II.Testing and implementing IHC screening tools to aid diagnosis/prognosis through prediction of mtDNA mutations occurrence on the basis of respiratory complexes staining patterns. III.Dissecting the molecular mechanisms regulating the homoplasmic shift observed in oncocytic tumors. IV.Develop anti-cancer therapeutic strategies targeting specific mitochondrial functions.

15 Objective III - The homoplasmic shift as a regulated mechanism Is there a genetic predisposition to accumulation of mtDNA mutations? Is the homoplasmic shift another route to cell senescence? Which cellular pathways contribute or determine the homoplasmic shift in cancer? STRATEGY 1 Optimize sensitive methods for low heteroplasmy detection DHPLC F-PCRRT and HRMCloning Collect oncocytic tumor sample… …and blood 1.Detect mutation in mtDNA in tumor 2.Detect low heteroplasmy in blood 3.Reconstruct pedigree, analyze relatives 4.Monitor and follow-up patient TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011

16 STRATEGY 2 Test the role of inflammatory mediators on mitochondrial proliferation Many oncocytic tumors overexpress IL6 The lymphocytic infiltrate in many oncocytic tumors may play a pivotal role in mediating mitochondrial proliferation IL6 is the main regulator of OIS (Oncogene Induced Senescence) which parallels the benign condition of oncocytomas From: Kuilman et al rIL6 1.Check mtDNA copy number increase 2.Measure mitochondrial mass 3.Measure expression changes of biogenesis regulators 4.Detect shift in the mutation load 5.Monitor senescence induction TRANSMIT STRATEGY 2 Test the role of inflammatory mediators on mitochondrial proliferation Mediterranean School of Oncology Rome – 28 January 2011 Bioplex analysis In order not to overlook other cytokines…

17 STRATEGY 3 Pick determinants of the homoplasmic shift from global transcriptomics mtDNA turnover is regulated by diverse stimuli and through several molecular pathways. The tumor microenvironment in vivo must be taken into account when trying to decipher molecular mechanisms! Apply massive pyrosequencing for comparative transcriptomics Data analysis Explant xenografts Measure heteroplasmy Observe shift -Reconstruct differentially activated pathways -Choose candidates Functional validation TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011

18 OBJECTIVES OF TRANSMIT TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 I.Demonstrating the impact of homo/heteroplasmy of mtDNA mutations on the tumorigenic potential of cancer cells. II.Testing and implementing IHC screening tools to aid diagnosis/prognosis through prediction of mtDNA mutations occurrence on the basis of respiratory complexes staining patterns. III.Dissecting the molecular mechanisms regulating the homoplasmic shift observed in oncocytic tumors. IV.Develop anti-cancer therapeutic strategies targeting specific mitochondrial functions.

19 Substitute mutated protein Objective IV – Develop anti-cancer approaches STRATEGY 1 Allotopic expression of dominant negative respiratory subunits Ongoing… Recover tumorigenic potential Re-code mito-genes for cytosolic translation Clone an MTS signal Clone a suitable 5’UTR Clone a suitable 3’UTR for mitochondrial anchorage Tag the allotopic protein Verify assembly within complex Test several dominant negative subunits on the tumorigenic potential in vitro and in vivo. TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 Objective IV – Develop anti-cancer approaches STRATEGY 1 Allotopic expression of dominant negative respiratory subunits Objective IV – Develop anti-cancer approaches STRATEGY 1

20 Target respiratory complexes chaperones – protein translocators (in collaboration with the Neijmegen institute and SME Khondrion B.V. – The Netherlands) Objective IV – Develop anti-cancer approaches STRATEGY 2 TRANSMIT Mediterranean School of Oncology Rome – 28 January 2011 High-throughput compounds screening on cell models Test the effects on mitochondrial morphology and physiology Further develop in mice xenografts-derived cultures and in vivo promising compounds Spinning disk confocal microscopy

21 Perspectives – Knowledge advances TRANSMIT TRANSMIT will reveal the diagnostic/prognostic value of mtDNA mutations in human cancers. TRANSMIT will decipher the molecular mechanisms underlying the homoplasmic shift of mtDNA mutations leading to novel therapeutic targets.

22 Collaborations Università di Bologna Dip. Biologia Ev. Sp. Dip. Scienze Neurologiche Dip. Anatomia Patologica Ospedale Bellaria Università di Bari Dip. Biochimica e Biol. Mol. “E.Quagliariello” Torino Ospedale Regina Margherita Ospedale Evangelico Valdese Az.Osp. CTO M.Adelaide Ospedale San Luigi Dr. N.Gilbert – Cancer Research Centre – Edinburgh, UK Dr. R.Chetty – General Hospital – Toronto, CA Dr. T.Meitinger - Institute of Human Genetics - Neuherberg, DEU Dr. H.Simonnet – CNRS Universitè de Lyon – Lyon, FR Dr. P.Willems – KHONDRION – Amsterdam, NL Dr. P.Chinnery – University of Newcastle upon Tyne – Newcastle, UK Dr. L.Nijtmans – Nijmegen Institute for mitochondrial disorders – Nijmegen, NL CNR – Bari, Milano Milano Ist. C.Besta TRANSMIT

23 Acknowledgements U.O. Medical Genetics - UniBO Giovanni Romeo Anna Bartoletti-Stella Elena Bonora Claudia Calabrese Flora Guerra Ivana Kurelac Martin Lang Elisa Mariani Laura Pradella Roberta Zuntini Dept. of Biology - UniBO Anna Maria Porcelli Michela Rugolo Anna Ghelli Mariantonietta Capristo Dept. of Neurological Sciences - UniBO Valerio Carelli Luisa Iommarini Dept. of Histopathology - UniBO Claudio Ceccarelli Nunzio Salfi Giovanni Tallini Dept. of Biochemistry - UniBA Marcella Attimonelli Universitè de Lyon Hélène Simonnet Dept. of Haematology and Oncological Sciences - UniBO Pier Luigi Lollini Giordano Nicoletti Patrizia Nanni Carla De Giovanni Dept. of Experimental Pathology- UniBO Christine Margaret Betts

24 BACKGROUND Oncocytic tumors display: -Intense haematoxylin/eosin staining -Mitochondrial hyperplasia -Swollen cells with round central nuclei -Occasionally, an inflammatory infiltrate Genetic markers of oncocytic neoplasia are mtDNA mutations disassembling respiratory Complex I Nuclear hit (Pre)neoplasia Mitochondrial hit Oncocytic neoplasia Non-oncocytic neoplasia From: Gasparre et al. – MCE, 2010 Does the shift to homoplasmy frequently observed implicate a selective advantage of mtDNA mutations? TRANSMIT

25 BACKGROUND Homoplasmy of mtDNA complex I mutations implicates disassembly of the complex and respiratory derangement in vitro and in vivo. ND6 NDUFB6 ATP5B Complex I 10952insC ND4 wild type ND4 Alteration of mitochondrial metabolism favour tumor progression through HIF1a stabilization. TRANSMIT

26 BACKGROUND HIF1a mediates the metabolic adaptation of cancer cells and progression to malignancy by inducing the Warburg effect, i.e. the shift towards a glycolitic metabolism. However, oncocytic tumors seem unable to stabilize HIF1a when complex I is disassembled. * * Homoplasmic ND1 mutation Heteroplasmic ND1 mutation thyroid sarcoma Why are mtDNA mutations selected in favour in cancer cells if, once homoplasmic, they abolish both respiration and HIF1a-mediated glycolysis? A PARADOX YET TO BE EXPLAINED TRANSMIT ND6HIF1a mut wt

27 MtDNA mutations are secondary hit to tumorigenesis as they appear after transformation. The multinodular case


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