Presentation is loading. Please wait.

Presentation is loading. Please wait.

Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility.

Similar presentations


Presentation on theme: "Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility."— Presentation transcript:

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 Birt-Hogg-Dubé Tuberous Sclerosis Complex Peutz-Jeghers Syndrome Neurofibromatosis TSC1/TSC2 ERK MEK Raf Ras NF1 Akt AMPK LKB1 Rheb FLCN PI3K RSK SoS PTEN PIP 2 PIP 3 PDK1 ? Regulation of mTOR by Tumour Suppressors Raptor mTOR Lst8 mTOR Complex 1 (mTORC1) Cell Growth Rapamycin

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

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

5 Molecularly Targeted Therapy Phenotype Gene(s) Functions Pathways Drug Interventions Mitochondrial Biogenesis HIF1 Energy Metabolism ? ?

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

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

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

9 Tuberous Sclerosis Complex Birt-Hogg-Dubé Von Hippel Lindau TSC1/TSC2 OH- Raptor mTOR Lst8 Proline Hydroxylase HIF1  VHL HIF1  Protein degradation Fumarate O2O2 Mutations in Fumarate Hydratase Lead to increase cellular fumarate Fumarate Hydratase FLCN ? Hereditary leiomyomatosis and renal cell cancer ? Polycystic Kidney Disease PKD Primary Cilia sense oxygen and flow rates Ca 2+ Inherited disease and HIF : Renal cancer (kidneys are highly metabolic and act as oxygen sensors)

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

11 Acetyl CoA Pyruvate L-lactate Glucose Acyl CoA Lactate Dehydrogenase (LDH) HOAD Krebs Cycle Fatty Acids MCT1 Mitochondria mitochondrial LDH MCT1 L-lactate Glut1 Glucose Oxygen Consumption (% Change) BHD + Lactate (mM) 5 1015 20 BHD - * + - L-Lactate secretion (mM) NS MCT1 MCT4 Rapamycin + + -- BHD BHD null cells retain and consume lactic acid through enhanced MCT1 expression

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

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

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

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

16 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 ---- ++++ Rap + -- ++ BHD - + - 21%1% Mitochondrial DNA + - + BHD - Work carried out by Rachael Preston Fraction CMN Lamin A/C (nuclear) BHD LDH-A (cytosol) C:cytosol N:Nuclear M:Mitochondrial HK2 cells (100% confluent) * oxygen

17 Mitochondria biogenesis is upregulated in (BHD null) UOK257 cells Work carried out by Rachael Preston BHD Pgc1α gene expression - + + - PGC1  expression * 21%1% -- ++ Rap + - + BHD - PGC1  mRNA levels -- + Rap + - + BHD ATP5G1 mRNA levels + - PGC1  mRNA ATP5G1 mRNA * * oxygen

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

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

20 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 Work carried out by Elaine Dunlop + + - + - - + - BHD SOD2  -actin Rap BHD H 2 O 2 (  M) H 2 O 2 increase SOD2 levels increase + - BHD UOK257 cells Note: We also see similar observations in other BHD cell models

22 Anti-oxidants inhibit HIF activity in BHD null cells Flag-BHD - NAC (mM) + 0.1 -- - - + - - 1510 * *** NS ** HIF activity Work carried out by Elaine Dunlop BHD -/- MEFs N-acetyl-L-cysteine (NAC)

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

24 Translating Basic Research Basic ResearchPre-clinical StudiesClinical Trials Rapamycin is cool!

25 Molecularly Targeted Therapy Phenotype Gene(s) Functions Pathways Drug Interventions Mitochondrial Biogenesis HIF1 Energy Metabolism

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

27 Cowden’s Syndrome Birt-Hogg-Dubé Tuberous Sclerosis Complex Peutz-Jeghers Syndrome Neurofibromatosis TSC1/TSC2 ERK MEK Raf Ras NF1 Akt AMPK LKB1 Rheb FLCN PI3K RSK SoS PTEN PIP 2 PIP 3 PDK1 ? Regulation of mTOR by Tumour Suppressors Raptor mTOR Lst8 mTOR Complex 1 (mTORC1) Cell Growth Rapamycin

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

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


Download ppt "Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility."

Similar presentations


Ads by Google