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Absence of the Birt-Hogg-Dubé gene product is associated with

Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Dr. Andy Tee Cardiff University (Medical Genetics) United Kingdom. Cowden’s Syndrome. Neurofibromatosis. Peutz-Jeghers Syndrome.

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Absence of the Birt-Hogg-Dubé gene product is associated with

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  1. Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility Dr. Andy Tee Cardiff University (Medical Genetics) United Kingdom

  2. Cowden’s Syndrome Neurofibromatosis Peutz-Jeghers Syndrome Tuberous Sclerosis Complex Birt-Hogg-Dubé Rapamycin Regulation of mTOR by Tumour Suppressors PTEN PIP2 PIP3 PDK1 PI3K SoS Akt NF1 Ras AMPK LKB1 Raf TSC1/TSC2 MEK ERK FLCN Rheb RSK ? mTOR Complex 1 (mTORC1) mTOR Raptor Lst8 Cell Growth

  3. Birt Hogg Dubé syndrome renal cancer & cysts Lung cysts Hair follicle tumours / Fibrofolliculoma • Clear cell carcinoma • (Inactivation of VHL) • Type 1 Papillary RCC • (Activation of c-MET • Chromophobe RCC • Oncocytoma • Oncocytic hybrid • Chromophobe • + Oncocytoma Tuberous Sclerosis Complex (TSC) Lung cysts renal angiomyolipomas & cysts facial angiofibroma

  4. TSC2 null cells have high Hypoxia Inducible Factor (HIF) activity mTORC1 Tumour HIF * oxygen • HIF induces expression of >170 genes • Cell survival • Energy metabolism • Angiogenesis • Cell proliferation Kayleigh Dodd

  5. < mTOR> < AMPK> Mitochondrial Biogenesis Energy Metabolism HIF1 Molecularly Targeted Therapy < Birt-Hogg-Dubé > Phenotype Drug Interventions Gene(s) ? < BHD > ? Functions Pathways

  6. UOK257-2 UOK257 -BHD 66kDa - + BHD UOK257 cells have elevated HIF-mediated gene expression VEGF-A VEGF mRNA levels * - - - - + + + + BHD - - - - Rap + + + + oxygen 21% 1% * * Cells provided by Dr. Laura S. Schmidt CCND1 * CCND1 mRNA levels - - - - + + + + BHD - - - - Rap + + + + 21% 1% oxygen 1% oxygen 21% - - + + BHD -BHD -VEGF -CCND1 Rachael Preston - actin

  7. Knockdown of BHD in ACHN cells increases HIF activity 1% 21% oxygen - - + + Scrambled shRNA ACHN cells - - + + BHD shRNA BHD HIF1 HIF2 actin Cells provided by Dr. Arnim Pause HIF activity VEGF-A * * VEGF-A mRNA levels HIF activity Luminescence - - - - + + + + Scrambled shRNA - - + + - - - - Scrambled shRNA + + + + BHD shRNA ACHN cells ACHN cells - - + + BHD shRNA - - - - Rap + + + + 1% oxygen 21% oxygen 21% 1% Note: We also see similar observations in BHD -/- MEFS

  8. HIF gene expression is upregulated in a BHD tumour Chromophobe carcinoma Unaffected Tissue A B Glomerulus Increased cell survival -BNIP3 loop of Henle C D Increased glucose uptake glycolysis Glomerulus -GLUT1 L Gijezen and T Brinkhuizen from Prof. M. van Steensel lab

  9. FLCN ? O2 Inherited disease and HIF : Renal cancer Birt-Hogg-Dubé Von Hippel Lindau Tuberous Sclerosis Complex VHL Protein degradation TSC1/TSC2 OH- Hereditary leiomyomatosis and renal cell cancer HIF1 Fumarate Hydratase mTOR Proline Hydroxylase ? HIF1 Raptor Lst8 Mutations in Fumarate Hydratase Lead to increase cellular fumarate Fumarate Ca2+ PKD Polycystic Kidney Disease Primary Cilia sense oxygen and flow rates (kidneys are highly metabolic and act as oxygen sensors)

  10. Metabolic Profile suggests that the BHD null (UOK257) cells prefer anaerobic respiration – ‘Warburg Effect’ A Pyruvate Kinase C B Glucose Hexokinase ( ) B Hexokinase * Pyruvate anaerobic ( ) D ( ) C Lactate Kinase respiration Dehydrogenase Fatty Acids Pyruvate L - Lactate Mitochondria Fold activation Acyl CoA Pyruvate aerobic Pyruvate respiration Dehydrogenase Dehydrogenase ( ) E Kinase HOAD Acetyl CoA ( ) F Citrate ( ) G Malate Krebs Synthase BHD- BHD- BHD+ BHD+ Dehydrogenase HEK293 HEK293 Cycle Lactate Dehydrogenase 3-hydroxyacyl-CoA dehydrogenase Malate Dehydrogenase D E F G Citrate Synthase * * Fold activation BHD- BHD- BHD- BHD- BHD+ BHD+ BHD+ BHD+ HEK293 HEK293 HEK293 HEK293

  11. BHD null cells retain and consume lactic acid through enhanced MCT1 expression NS L-Lactate secretion (mM) - + BHD Glut1 Glucose Glucose - + BHD BHD * L-lactate Lactate Dehydrogenase (LDH) Oxygen Consumption (% Change) MCT1 Pyruvate L-lactate Fatty Acids Acyl CoA 10 15 20 5 MCT1 Lactate (mM) HOAD mitochondrial LDH Acetyl CoA - - + Rapamycin + Krebs Cycle BHD MCT1 Mitochondria MCT4

  12. BHD patient tumour show high levels of LDH and MCT1 expression Chromophobe carcinoma Unaffected Tissue A B Higher glycolysis LDH-A C D Increased Lactic acid uptake MCT-1 L Gijezen and T Brinkhuizen from Prof. M. van Steensel lab

  13. Tumour enviroment in the context of HIF dysfunction Lactic acid (Oxygen dependent) Glucose feeds the hypoxic core

  14. 2-deoxyglucose oxamate BHD null cells are sensitive to LDH inhibition by oxamate as well as 2-deoxyglucose Glut1 Glucose Glucose L-lactate Lactate Dehydrogenase (LDH) MCT1 Pyruvate L-lactate Fatty Acids Acyl CoA MCT1 HOAD mitochondrial LDH Acetyl CoA Krebs Cycle Mitochondria

  15. BHDnull cells are energy stressed, and undergo apoptosis when treated with oxamate or 2-deoxyglucose BHD+(UOK257-2) Cell Death + - BHD BHD BHD- (UOK257) Dr. Elaine Dunlop

  16. Mitochondrial DNA - - - - + + + + BHD - - - - + + + + Rap 21% 1% Do BHD-null cells have high levels of cellular mitochondria? • Similarities between TSC and BHD clinically • It is known that loss of TSC1 or TSC2 upregulates mitochondrial biogenesis • Tumours from BHD patients were observed to have high levels of mitochondria * C N M Fraction C:cytosol N:Nuclear M:Mitochondrial BHD LDH-A (cytosol) Lamin A/C (nuclear) HK2 cells (100% confluent) - - + + BHD oxygen Work carried out by Rachael Preston

  17. Mitochondria biogenesis is upregulated in (BHD null) UOK257 cells PGC1 expression PGC1 mRNA ATP5G1 mRNA * * * PGC1 mRNA levels ATP5G1 mRNA levels Pgc1αgene expression - - - - + + + + BHD BHD - - - - + + + + Rap Rap - - + + BHD oxygen 21% 1% Work carried out by Rachael Preston

  18. BHD null cells are energy stressed 1% 21% oxygen - - + + BHD -AMPK -AMPK(P) -ACC(P) -ACC UOK257 cells Work carried out by Rachael Preston

  19. Mitochondrial membrane potential is compromised in BHD null cells JC1 Aggregate = respiring mitos JC1 Monomer Merge BHD - BHD + UOK257 cells Work carried out by Steve Land

  20. Mitochondrial membrane potential is compromised in BHD null cells Mitochondrial membrane potential is compromised in BHD null cells * BHD null cells contain foci of JC1 monomer. Feature absent from BHD plus cells Work carried out by Steve Land

  21. Higher levels of Reactive Oxygen Species in BHD null cells H2O2increase SOD2 levels increase - - + + Rap - - + + BHD H2O2 (M) BHD SOD2 b-actin - + BHD UOK257 cells Work carried out by Elaine Dunlop Note: We also see similar observations in other BHD cell models

  22. Anti-oxidants inhibit HIF activity in BHD null cells HIF activity ** NS * *** *** *** *** - - - + - + - Flag-BHD - - NAC (mM) 0.1 1 5 10 10 BHD -/- MEFs N-acetyl-L-cysteine (NAC) Work carried out by Elaine Dunlop

  23. Cancer Progression in Birt-Hogg-Dubé Mitochondria biogenesis / Function -> Aging? Cancer? Birt-Hogg-Dubé FLCN Reactive Oxygen Species (ROS) HIF1 DNA-damage Hypoxia -> Cell survival/ Metabolism/Angiogenesis

  24. Translating Basic Research Basic Research Pre-clinical Studies Clinical Trials Rapamycin is cool!

  25. < mTOR> < AMPK> Mitochondrial Biogenesis Energy Metabolism HIF1 Molecularly Targeted Therapy < Birt-Hogg-Dubé > Phenotype Drug Interventions Gene(s) < BHD > Functions Pathways

  26. Similarly to TSC-tumours: high mTORC1 activity and low insulin signalling in BHD tumour Chromophobe carcinoma Unaffected Tissue A B Akt P-S6(S235/236) mTOR C D Raptor Lst8 P-AKT(S473) S6K1 L Gijezen and T Brinkhuizen from Prof. M. van Steensel lab

  27. Cowden’s Syndrome Neurofibromatosis Peutz-Jeghers Syndrome Tuberous Sclerosis Complex Birt-Hogg-Dubé Rapamycin Regulation of mTOR by Tumour Suppressors PTEN PIP2 PIP3 PDK1 PI3K SoS Akt NF1 Ras AMPK LKB1 Raf TSC1/TSC2 MEK ERK FLCN Rheb RSK ? mTOR Complex 1 (mTORC1) mTOR Raptor Lst8 Cell Growth

  28. Andy’s Lab Dr Elaine Dunlop (AICR fellow) Kayleigh Dodd (AICR Ph.D student) David Hunt (AICR Ph.D student) Lyndsey Seymour (MRC/TS Association Ph.D student) Rachael Preston (MRC/Myrovlytis Trust Ph.D student) Rienk Doetjes (Tenovus Ph.D student) Tijs Claessens (Ph.D student) Dr. Mark Davies (Medical Genetics, Cardiff) Collaborators Prof. Maurice van Steensel (University Hospital Maastricht) Dr Arnim Pause (University Hospital Maastricht) Dr. Keith Baar (University of California ) Dr. Steven Land (Ninewells, Dundee University)

  29. Myrovlytis Trust • Medical research charity, founded 2007 • Promotes research into rare genetic disorders • Focussed initially on BHD syndrome: • Research grants worth ~£4m GBP to date • Annual BHD Symposia • Now considering broadening its support to related conditions, while maintaining support for BHD www.MyrovlytisTrust.org www.BHDSyndrome.org

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