TRANSMIT: Translational Significance of Mitochondrial DNA Mutations in Tumors

Giuseppe Gasparre, PhD

University of Bologna

Mediterranean School of Oncology

Rome – 28 January 2011

TRANSMIT

LAYOUT

1.AIMS of TRANSMIT

2.BACKGROUNDMitochondrial genetics, oncocytoma

and tumor progression3.STATE OF ART

Objective IImpact of mtDNA

mutations on tumor growth

Objective IVDevelop anti-cancertherapeutic strategies

Objective IIDevelop diagnostic and

prognostic tools

Definition ofoncojanus

Biobank assembly

Correlative studies

Clinical follow-up

Allotopic expression

Target chaperones

Perspectives/Knowledge advances

TRANSMITMediterranean School of Oncology

Rome – 28 January 2011

Objective IIIUncover the determinantsof the homoplasmic shift

Genetic predisposition

Role of inflammation

Dissection of molecular pathways

1.AIMS OF TRANSMIT

Understanding the functional and clinical implications of the Understanding the functional and clinical implications of the occurrence of mtDNA pathological mutations in cancer cells occurrence of mtDNA pathological mutations in cancer cells

in vitroin vitro and and in vivoin vivo..

TRANSMITMediterranean School of Oncology

Rome – 28 January 2011

Set the ground for gene therapy-based and pharmacological anti-Set the ground for gene therapy-based and pharmacological anti-cancer therapeutic strategies targeting the mitochondrial cancer therapeutic strategies targeting the mitochondrial

respiratory chain.respiratory chain.

Human Mitochondrial GenomeHuman Mitochondrial Genome

16,569 bp

D-LOOP (control region)

37 genes (complex subunits, rRNA & tRNA)

Oncogene. 2006 Aug 7;25(34):4663-74.

2.BACKGROUNDTRANSMITMediterranean School of Oncology

Rome – 28 January 2011

Threshold effect

Am. J. Hum. Genet. 51:1201-1212, 1992

CARRIERSAFFECTED

2.BACKGROUNDTRANSMITMediterranean School of Oncology

Rome – 28 January 2011

Homoplasmy Heteroplasmy Homoplasmy

Oncocytic tumors:•mitochondrial hyperplasia•mostly benign

2.BACKGROUND

HIF1 is the main player in the metabolic adaptation of cancer cells to the adverse tumor microenvironment.

TRANSMITMediterranean School of Oncology

Rome – 28 January 2011

ND6 HIF1

mut

wt

Complex I disassembly in vivo correlates with lack of HIF1 stabilization

2.BACKGROUNDTRANSMITMediterranean School of Oncology

Rome – 28 January 2011

NAD+

Lack of respiratory complex I induces PSEUDONORMOXIAHIF1 chronically destabilized –> block of tumor growth

3.STATE OF THE ARTCancer 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 EXPLANATIONA PARADOXICAL KEY TO EXPLANATION

TRANSMITMediterranean School of Oncology

Rome – 28 January 2011

Porcelli et al., HMG 2010

OBJECTIVES OF TRANSMIT

I. Demonstrating the impact of homo/heteroplasmy of mtDNA mutations on the tumorigenic potential of cancer cells.

II.II. Testing and implementing IHC screening tools to aid Testing and implementing IHC screening tools to aid diagnosis/prognosis through prediction of mtDNA diagnosis/prognosis through prediction of mtDNA mutations occurrence on the basis of respiratory mutations occurrence on the basis of respiratory complexes staining patterns.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.

TRANSMITTRANSMITMediterranean School of Oncology

Rome – 28 January 2011

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

OBJECTIVES OF TRANSMITTRANSMITMediterranean 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.II. Testing and implementing IHC screening tools to aid Testing and implementing IHC screening tools to aid diagnosis/prognosis through prediction of mtDNA diagnosis/prognosis through prediction of mtDNA mutations occurrence on the basis of respiratory mutations occurrence on the basis of respiratory complexes staining patterns.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.

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 collectionBiobank creation

mtDNA sequencing

Tissue microarrayOptimize RC antibodies

STRATEGIESSTRATEGIES

Correlate with HIF1 staining

Correlate with Ki67 proliferating index

Patient follow up

Mediterranean School of Oncology

Rome – 28 January 2011

Endometrial carcinoma type I has rarely been observed to present oncocytic features.IHC combined with genetic analysis helps revealing oncocytic transformation.

PRELIMINARY RESULTSPRELIMINARY 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.

IMPLICATIONSIMPLICATIONS

Homoplasmic mtDNA mutations will become of high prognostic value

implying benignity of the neoplastic lesion.

TRANSMITMediterranean School of Oncology

Rome – 28 January 2011

Figures from: Guerra et al., submitted

OBJECTIVES OF TRANSMIT

TRANSMITMediterranean 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.II. Testing and implementing IHC screening tools to aid Testing and implementing IHC screening tools to aid diagnosis/prognosis through prediction of mtDNA diagnosis/prognosis through prediction of mtDNA mutations occurrence on the basis of respiratory mutations occurrence on the basis of respiratory complexes staining patterns.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.

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 1STRATEGY 1 Optimize sensitive methods for low heteroplasmy detection

DHPLC F-PCR RT and HRM Cloning

Collect oncocytic tumor sample…

…and blood

1. Detect mutation in mtDNA in tumor2. Detect low heteroplasmy in blood3. Reconstruct pedigree, analyze relatives4. Monitor and follow-up patient

TRANSMITMediterranean School of Oncology

Rome – 28 January 2011

STRATEGY 2STRATEGY 2 Test the role of inflammatory mediators on mitochondrial proliferation

Many oncocytic tumors overexpress IL6The lymphocytic infiltrate in many oncocytic tumors may play a

pivotal role in mediating mitochondrial proliferationIL6 is the main regulator of OIS (Oncogene Induced Senescence)

which parallels the benign condition of oncocytomas

From: Kuilman et al. - 2008rIL6

1. Check mtDNA copy number increase2. Measure mitochondrial mass3. Measure expression changes of biogenesis

regulators 4. Detect shift in the mutation load5. Monitor senescence induction

TRANSMIT

STRATEGY 2STRATEGY 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…

STRATEGY 3STRATEGY 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 heteroplasmyObserve shift

-Reconstruct differentially activated pathways-Choose candidates

Functional validation

TRANSMITMediterranean School of Oncology

Rome – 28 January 2011

OBJECTIVES OF TRANSMIT

TRANSMITMediterranean 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.II. Testing and implementing IHC screening tools to aid Testing and implementing IHC screening tools to aid diagnosis/prognosis through prediction of mtDNA diagnosis/prognosis through prediction of mtDNA mutations occurrence on the basis of respiratory mutations occurrence on the basis of respiratory complexes staining patterns.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.

Substitute mutated protein

Objective IV – Develop anti-cancer approaches

STRATEGY 1STRATEGY 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.

TRANSMITMediterranean School of Oncology

Rome – 28 January 2011

Objective IV – Develop anti-cancer approaches

STRATEGY 1STRATEGY 1 Allotopic expression of dominant negative respiratory subunits

Objective IV – Develop anti-cancer approaches

STRATEGY 1STRATEGY 1

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 2STRATEGY 2

TRANSMITMediterranean 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

Perspectives – Knowledge advancesTRANSMIT

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.

Collaborations

Università di BolognaDip. Biologia Ev. Sp.Dip. Scienze NeurologicheDip. Anatomia PatologicaOspedale Bellaria

Università di BariDip. Biochimica e Biol. Mol. “E.Quagliariello”

TorinoOspedale Regina MargheritaOspedale Evangelico ValdeseAz.Osp. CTO M.AdelaideOspedale 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

Acknowledgements

U.O. Medical Genetics - UniBOGiovanni RomeoAnna Bartoletti-StellaElena BonoraClaudia CalabreseFlora GuerraIvana KurelacMartin Lang Elisa MarianiLaura PradellaRoberta Zuntini

Dept. of Biology - UniBOAnna Maria PorcelliMichela Rugolo Anna GhelliMariantonietta Capristo

Dept. of Neurological Sciences - UniBOValerio CarelliLuisa Iommarini

Dept. of Histopathology - UniBOClaudio CeccarelliNunzio SalfiGiovanni Tallini

Dept. of Biochemistry - UniBAMarcella Attimonelli

Universitè de LyonHélène Simonnet

Dept. of Haematology and Oncological Sciences - UniBOPier Luigi LolliniGiordano NicolettiPatrizia Nanni Carla De Giovanni

Dept. of Experimental Pathology- UniBOChristine Margaret Betts

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 Genetic markers of oncocytic neoplasia are mtDNA mutations disassembling respiratory Complex Irespiratory Complex I

Nuclear hit

(Pre)neoplasia

Mitochondrial hit

Oncocytic neoplasia

Non-oncocytic neoplasia

12

3

From: Gasparre et al. – MCE, 2010

Does the shift to homoplasmy frequently observed implicate a selective advantage of mtDNA mutations?

TRANSMIT

BACKGROUNDHomoplasmy 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

BACKGROUNDHIF1a 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.

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XTC1 ZTC1 CC HXTC1 HZTC1

Homoplasmic ND1 mutation

Heteroplasmic ND1 mutation

thyroidsarcoma

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 EXPLAINEDA PARADOX YET TO BE EXPLAINED

TRANSMIT

ND6 HIF1a

mut

wt

MtDNA mutations are secondary hit to tumorigenesis as they appear after transformation.

The multinodular caseThe multinodular case