BHD - Tuberous Sclerosis Alliance

Report
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
Regulation of mTOR by Tumour Suppressors
Cowden’s Syndrome
PIP2
PTEN
PIP3
PDK1
Neurofibromatosis
NF1
PI3K
SoS
Ras
Raf
Tuberous Sclerosis
Complex
MEK
ERK
Peutz-Jeghers
Syndrome
AMPK
LKB1
TSC1/TSC2
FLCN
Rheb
RSK
mTOR
Complex 1
(mTORC1)
Akt
?
Birt-Hogg-Dubé
mTOR
Lst8 Raptor
Cell Growth
Rapamycin
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
Tuberous Sclerosis Complex (TSC)
facial angiofibroma
renal angiomyolipomas & cysts
Lung cysts
mTORC1
TSC2 null cells have high
Hypoxia Inducible Factor
(HIF) activity
Tumour
HIF
*
oxygen
Kayleigh Dodd
HIF induces expression of >170 genes
• Cell survival
• Energy metabolism
• Angiogenesis
• Cell proliferation
Molecularly Targeted Therapy
< Birt-Hogg-Dubé >
Phenotype
Drug
? Interventions
Gene(s) < BHD >
?
Functions
Pathways
< mTOR> < AMPK>
Mitochondrial Biogenesis
HIF1 Energy Metabolism
VEGF
mRNA levels
UOK257 cells have elevated HIF-mediated gene expression
BHD
+
-
Cells provided by Dr. Laura S. Schmidt
*
4
2
0
BHD + + Rap - + oxygen
21%
CCND1
mRNA levels
-BHD
66kDa
VEGF-A
6
2.5
2
1.5
1%
CCND1
*
*
*
1
0.5
0
BHD +
Rap -
+ - - + + - + - + - + - +
oxygen
21%
oxygen
21%
BHD
Rachael Preston
- + + - + - + - +
+ -
1%
1%
+ -
-BHD
-VEGF
-CCND1
- actin
Knockdown of BHD in ACHN cells increases HIF activity
oxygen 21%
Scrambled shRNA + BHD shRNA - +
1%
+ - +
ACHN
cells
BHD
HIF1
HIF2
actin
Cells provided by Dr. Arnim Pause
HIF activity
*
15
10
5
0
Scrambled shRNA + + BHD shRNA - - +
Rap - + oxygen
21%
- + + - + - - + + ACHN
cells
+ - + - +
1%
*
125
Luminescence
20
HIF activity
VEGF-A
mRNA levels
VEGF-A
100
75
50
25
0
Scrambled shRNA + BHD shRNA - +
oxygen 21%
+ - ACHN
- + cells
Note: We also see similar observations in BHD -/- MEFS
1%
HIF gene expression is upregulated in a BHD tumour
Chromophobe
carcinoma
Increased
cell survival
-GLUT1
Glomerulus
loop of Henle
C
Increased
glucose
uptake
glycolysis
B
-BNIP3
A
Unaffected Tissue
D
Glomerulus
L Gijezen and T Brinkhuizen from Prof. M. van Steensel lab
Inherited disease and HIF : Renal cancer
Birt-Hogg-Dubé
Von Hippel Lindau
FLCN
Tuberous Sclerosis
Complex
VHL
Protein
degradation
TSC1/TSC2
OH-
?
HIF1
mTOR
?
Lst8 Raptor
HIF1
Ca2+
Fumarate
Hydratase
Proline
Hydroxylase
O2
Fumarate
PKD
Polycystic Kidney Disease
Hereditary
leiomyomatosis and
renal cell cancer
Mutations in
Fumarate
Hydratase
Lead to increase
cellular fumarate
Primary Cilia
sense oxygen and
flow rates
(kidneys are highly metabolic and act as oxygen sensors)
Metabolic Profile suggests that the BHD null (UOK257)
cells prefer anaerobic respiration – ‘Warburg Effect’
A
Hexokinase (B)
Pyruvate
anaerobic
(D) Lactate
(C)
Kinase
respiration
Dehydrogenase
Fatty Acids
Pyruvate
L-Lactate
(E)
HOAD
D
3
2.5
2
Pyruvate
Dehydrogenase
Kinase
Pyruvate
Dehydrogenase
Acetyl CoA
(G) Malate
Krebs
Dehydrogenase
Fold activation
Fold activation
aerobic
respiration
(F)
2.5
1
*
0.5
1.5
1
0.5
0
Lactate
Dehydrogenase
HOAD
E 3-hydroxyacyl-CoA
5
4
3
dehydrogenase
Citrate Synthase
F Citrate
Synthase
G
1.5
1.5
1
1
0.5
0.5
0
0
*
1.5
1
Pyruvate
Kinase
Pyruvate Kinase
2
0
Citrate
Synthase
C
Cycle
Lactate Dehydrogenase
*
Hexokinase
1.5
Mitochondria
Acyl CoA
HexoKinase
B
Glucose
2
0.5
1
0
0
Malate Dehydrogenase
Malate
Dehydrogenase
Glucose
L-Lactate secretion
(mM)
BHD null cells retain and
consume lactic acid through
enhanced MCT1 expression
NS
1.25
1
0.75
0.5
0.25
0
-
+
BHD
Glucose
Glut1
BHD -
Fatty Acids
Pyruvate
L-lactate
Lactate
Dehydrogenase
(LDH)
MCT1
L-lactate
Acyl CoA
Oxygen Consumption
(% Change)
BHD+
*
5
MCT1
HOAD
mitochondrial
LDH
Acetyl CoA
Krebs
Cycle
Mitochondria
-
+
10
15
Lactate (mM)
-
20
+ Rapamycin
BHD
MCT1
MCT4
BHD patient tumour show high levels of LDH and
MCT1 expression
Chromophobe
carcinoma
Increased
Lactic
acid
uptake
B
C
D
LDH-A
A
MCT-1
Higher
glycolysis
Unaffected Tissue
L Gijezen and T Brinkhuizen from Prof. M. van Steensel lab
Tumour enviroment in the context
of HIF dysfunction
Lactic acid
(Oxygen dependent)
Glucose feeds the hypoxic core
BHD null cells are sensitive to LDH inhibition by oxamate
as well as 2-deoxyglucose
2-deoxyglucose
Glucose
Glut1
Glucose
oxamate
Fatty Acids
Pyruvate
Lactate
Dehydrogenase
(LDH)
L-lactate
MCT1
L-lactate
Acyl CoA
MCT1
HOAD
mitochondrial
LDH
Acetyl CoA
Krebs
Cycle
Mitochondria
BHD null cells are energy stressed, and undergo
apoptosis when treated with oxamate or 2-deoxyglucose
20
untreated
2-DG
Oxamate
2-DG + Oxamate
BHD+
10
0
BHD+
BHD -
Relative ATPLevel
1.25
BHD+
(UOK257-2)
1.00
0.75
0.50
0.25
0.00
Control
2-DG
Oxamate 2-DG+Oxa
BHD-
1.25
Dr. Elaine
Dunlop
Relative ATP Level
Cell Death
30
- (UOK257)
BHDDrug
Treatment
1.00
0.75
0.50
0.25
0.00
Control
2-DG
Oxamate 2-DG+Oxa
Drug Treatment
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
Fraction
C N M
C:cytosol
N:Nuclear
M:Mitochondrial
BHD
LDH-A (cytosol)
Lamin A/C (nuclear)
HK2 cells
(100% confluent)
Mitochondrial DNA
*
100
75
50
25
0
BHD
Rap
oxygen
+
-
+
+
-
21%
+
+
-
+
+
-
+
1%
Work carried out by Rachael Preston
Mitochondria biogenesis is upregulated in (BHD null)
UOK257 cells
PGC1 expression
100
75
50
25
BHD
Rap
75
50
25
0
+
-
+
+
-
+
BHD
Rap
*
100
100
0
120
Pgc1α gene expression
ATP5G1 mRNA
*
ATP5G1
mRNA levels
PGC1
mRNA levels
PGC1 mRNA
*
+
-
+
+
-
+
Work carried out by Rachael Preston
80
60
40
20
0
BHD + oxygen 21%
+ 1%
BHD null cells are energy stressed
BHD
21%
1%
+ -
+ -
oxygen
-AMPK
-AMPK(P)
-ACC(P)
-ACC
UOK257 cells
Work carried out by Rachael Preston
Mitochondrial membrane potential is compromised in
BHD null cells
JC1 Monomer
JC1 Aggregate
= respiring mitos
BHD -
BHD +
UOK257 cells
Work carried out by Steve Land
Merge
Mitochondrial membrane potential is compromised in
BHD null cells
Figure
* 2
Mito - Membrane
Potential (m)
e1
-
2.5
2
1.5
1
0.5
0
BHD
CCCP
+
-
+
+
-
+
BHD null cells contain
foci of JC1 monomer. Feature
absent from BHD plus cells
Work carried out by Steve Land
Higher levels of Reactive Oxygen Species in BHD
null cells
5
H2O2
increase
SOD2 levels
increase
4
H2O2
(M)
Rap
BHD
3
- + - +
+ + - BHD
2
SOD2
1
0
BHD
-actin
+
-
UOK257 cells
Work carried out by Elaine Dunlop
Note: We also see similar observations in other BHD cell models
Anti-oxidants inhibit HIF activity in BHD null cells
HIF activity
Protein adjusted
Relative HIF Activity
1.25
**
NS
1.00
*
*** *** ***
0.75
0.50
***
0.25
0.00
BHD -/- MEFs
10
+
-
+
N
A
C
N
10
5
5
-
N
1
-
FL
C
0.1
-
1
0.
1
-
FL
C
C
NAC (mM)
-
ro
l
on
t
Flag-BHD
10
N-acetyl-L-cysteine (NAC)
Work carried out by Elaine Dunlop
Cancer Progression in Birt-Hogg-Dubé
Birt-Hogg-Dubé
Mitochondria biogenesis
/ Function -> Aging?
Cancer?
FLCN
Reactive Oxygen Species
(ROS)
HIF1
DNA-damage
Hypoxia -> Cell survival/
Metabolism/Angiogenesis
Translating Basic Research
Basic Research
Pre-clinical Studies
Clinical Trials
Rapamycin
is cool!
Molecularly Targeted Therapy
< Birt-Hogg-Dubé >
Phenotype
Drug
Interventions
Gene(s) < BHD >
Functions
Pathways
< mTOR> < AMPK>
Mitochondrial Biogenesis
HIF1 Energy Metabolism
Similarly to TSC-tumours: high mTORC1 activity and
low insulin signalling in BHD tumour
Akt
P-S6(S235/236)
Chromophobe
carcinoma
Lst8 Raptor
S6K1
A
B
C
D
P-AKT(S473)
mTOR
Unaffected Tissue
L Gijezen and T Brinkhuizen from Prof. M. van Steensel lab
Regulation of mTOR by Tumour Suppressors
Cowden’s Syndrome
PIP2
PTEN
PIP3
PDK1
Neurofibromatosis
NF1
PI3K
SoS
Ras
Raf
Tuberous Sclerosis
Complex
MEK
ERK
Peutz-Jeghers
Syndrome
AMPK
LKB1
TSC1/TSC2
FLCN
Rheb
RSK
mTOR
Complex 1
(mTORC1)
Akt
?
Birt-Hogg-Dubé
mTOR
Lst8 Raptor
Cell Growth
Rapamycin
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)
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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