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

Birt-Hogg-Dubé

Tuberous SclerosisComplex

Peutz-JeghersSyndrome

Neurofibromatosis

TSC1/TSC2

ERK

MEK

Raf

RasNF1 Akt

AMPK LKB1

Rheb FLCN

PI3K

RSK

SoS

PTENPIP2 PIP3

PDK1

?

Regulation of mTOR by Tumour Suppressors

Raptor

mTOR

Lst8

mTORComplex 1(mTORC1)

Cell Growth

Rapamycin

Tuberous Sclerosis Complex (TSC)facial angiofibroma renal angiomyolipomas & cysts Lung cysts

Birt Hogg Dubé syndromeHair follicle tumours / Fibrofolliculoma

renal cancer & cysts Lung cysts

Clear cell carcinoma(Inactivation of VHL)Type 1 Papillary RCC(Activation of c-METChromophobe RCCOncocytomaOncocytic hybrid

Chromophobe + Oncocytoma

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

*

Molecularly Targeted Therapy

Phenotype

Gene(s)

FunctionsPathways

DrugInterventions

< Birt-Hogg-Dubé >

< BHD >

< mTOR>

Mitochondrial Biogenesis

< AMPK>

HIF1 Energy Metabolism

?

?

UOK257 cells have elevated HIF-mediated gene expression

Rachael Preston

Cells provided by Dr. Laura S. Schmidt

66kDa -BHDU

OK2

57-2

UO

K257

+ -BHD

0

2

4

6

VEG

F m

RN

A le

vels

- - - -+ + + +Rap+ - -+ +BHD - + -

21% 1%

*

0

0.5

1

1.5

2

2.5

- - - -+ + + +Rap+ - -+ +BHD - + -

CC

ND

1 m

RN

A le

vels

*

* *

21% 1%

-VEGF-CCND1

BHD1%21%

-BHD- -+ +

- actin

VEGF-A

CCND1oxygen

oxygen

oxygen

0

25

50

75

100

125

0

5

10

15

20

VE

GF-

A m

RN

A le

vels

- - - -+ + + +Rap

+ - -+ +BHD shRNA

- + -

21% 1%

*

Scrambled shRNA- -+- + - + +

Lum

ines

cenc

e1%21%

+ - +BHD shRNA

-Scrambled shRNA- +-+

*

HIF1BHD

HIF2

1%21%

actin

+ - +BHD shRNA

-Scrambled shRNA- +-+

ACHNcells

ACHNcells

ACHNcells

VEGF-A

HIF

act

ivity

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

oxygen oxygen

HIF gene expression is upregulated in a BHD tumour

A B

C D

-B

NIP

3

-GLU

T1

Chromophobecarcinoma Unaffected Tissue

loop of Henle

Glomerulus

Glomerulus

Increased cell survival

Increasedglucose uptake

glycolysis

L Gijezen and T Brinkhuizen from Prof. M. van Steensel lab


Birt-Hogg-Dubé

Von Hippel Lindau

TSC1/TSC2 OH-

Raptor

mTOR

Lst8

ProlineHydroxylaseHIF1

VHL

HIF1

Protein degradation

Fumarate O2

Mutations in Fumarate Hydratase

Lead to increase cellular fumarate

Fumarate Hydratase

FLCN

? Hereditary leiomyomatosis and

renal cell cancer

?

Polycystic Kidney DiseasePKD

Primary Cilia sense oxygen and

flow rates

Ca2+

Inherited disease and HIF : Renal cancer

(kidneys are highly metabolic and act as oxygen sensors)

A

Pyruvate L-Lactate

Glucose

Acetyl CoA

Acyl CoA

LactateDehydrogenase

anaerobicrespiration

PyruvateKinase

PyruvateDehydrogenase

PyruvateDehydrogenase

Kinase

aerobicrespiration

KrebsCycle

HOAD

Fatty Acids

CitrateSynthase

MalateDehydrogenase

Hexokinase

Mitochondria

(B)

(C) (D)

(E)(F)

(G)

Metabolic Profile suggests that the BHD null (UOK257) cells prefer anaerobic respiration – ‘Warburg Effect’

B

0

0.5

1

1.5

Fold

act

ivat

ion

HexokinasePyruvate Kinase

0

0.5

1

1.5

2

2.5

PyruvateKinase

HE

K29

3

BH

D-

BH

D+

HE

K29

3

BH

D-

BH

D+

C

*

Malate Dehydrogenase

0

0.5

1

1.5

0

1

2

3

4

5

0

0.5

1

1.5

2

2.5

3

Lactate Dehydrogenase

Fold

act

ivat

ion

Citrate Synthase

0

0.5

1

1.5

3-hydroxyacyl-CoA dehydrogenase Citrate Synthase Malate

Dehydrogenase

HE

K29

3

BH

D-

BH

D+

HE

K29

3

BH

D-

BH

D+

HE

K29

3

BH

D-

BH

D+

HE

K29

3

BH

D-

BH

D+

D E F G

**

Acetyl CoA

Pyruvate L-lactate

Glucose

Acyl CoA


(LDH)

HOAD

KrebsCycle

Fatty Acids

MCT1

Mitochondria

mitochondrialLDH

MCT1

L-lactate

Glut1 Glucose

Oxy

gen

Con

sum

ptio

n (%

Cha

nge)

BHD+

Lactate (mM)5 10 15 20

BHD-

*

0

0.25

0.5

0.75

1

1.25

BHD + -L-La

ctat

e se

cret

ion

(mM

)

NS

MCT1

MCT4

Rapamycin++ --BHD

BHD null cells retain and consume lactic acid through enhanced MCT1 expression


LDH

-AB

C D

A

MC

T-1

Higher glycolysis

IncreasedLactic acid

uptake

BHD patient tumour show high levels of LDH and MCT1 expression


Lactic acid(Oxygen dependent)

Glucose feeds the hypoxic core

Tumour enviroment in the context of HIF dysfunction

BHD null cells are sensitive to LDH inhibition by oxamateas well as 2-deoxyglucose

Acetyl CoA

Pyruvate L-lactate

Glucose

Acyl CoA


(LDH)

HOAD

KrebsCycle

Fatty Acids

MCT1

Mitochondria

mitochondrialLDH

MCT1

L-lactate

Glut1 Glucose

oxamate

2-deoxyglucose

BHD null cells are energy stressed, and undergo apoptosis when treated with oxamate or 2-deoxyglucose

BHD+

Control 2-DG Oxamate 2-DG+Oxa0.00

0.25

0.50

0.75

1.00

1.25

Drug Treatment

Rel

ativ

e A

TPLe

vel

BHD-

Control 2-DG Oxamate 2-DG+Oxa0.00

0.25

0.50

0.75

1.00

1.25

Drug Treatment

Rel

ativ

e A

TP L

evel

BHD+ (UOK257-2)

BHD- (UOK257)

0

10

20

30 untreated2-DGOxamate2-DG + Oxamate

Cel

l Dea

th

BHD+ BHD-

Dr. ElaineDunlop

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

0

25

50

75

100

- - - -+ + + +Rap+ - -+ +BHD - + -

21% 1%

Mito

chon

dria

l DN

A

+ -+BHD -

Work carried out by Rachael Preston

Fraction C MN

Lamin A/C (nuclear)

BHDLDH-A (cytosol)

C:cytosolN:NuclearM:Mitochondrial

HK2 cells

(100% confluent)

*

oxygen

Mitochondria biogenesis is upregulated in (BHD null) UOK257 cells


Se-ries1

0

20

40

60

80

100

120

BHD

Pgc

1α g

ene

expr

essi

on

- ++ -

PGC1 expression

*

21% 1%

- -+ +Rap+ -+BHD -

PG

C1

mR

NA

leve

ls

0

25

50

75

100

- -+Rap+ -+BHD

ATP

5G1

mR

NA

leve

ls

0

25

50

75

100

+-

PGC1 mRNA ATP5G1 mRNA* *

oxygen

BHD null cells are energy stressed


-AMPK-AMPK(P)-ACC(P)

BHD

-ACC

1%21%- -+ +

UOK257 cells

oxygen

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



0

25

50

75

100

+BHD

Mito

chon

drial D

NA

-

Figure 1

0

0.5

1

1.5

2

2.5Figure 2

Mito

-

Mem

bran

e Po

tent

ial (

m

)

+BHD -++ -CCCP + - +-

0

25

50

75

100

+BHD

Mito

chon

drial D

NA

-

Figure 1

0

0.5

1

1.5

2

2.5Figure 2

Mito

-

Mem

bran

e Po

tent

ial (

m

)

+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

Work carried out by Elaine Dunlop

++

-+ --+-

BHD

SOD2

-actin

RapBHD

0

1

2

3

4

5

H2O

2 (M

)H2O2

increase SOD2 levels increase

+ -BHD

UOK257 cells

Note: We also see similar observations in other BHD cell models

Anti-oxidants inhibit HIF activity in BHD null cells

Protein adjusted

0.00

0.25

0.50

0.75

1.00

1.25R

elat

ive

HIF

Act

ivity

Flag-BHD -NAC (mM)

+0.1- -

- - +--1 5 10 10

**** *** ***

***

NS **

HIF activity

Work carried out by Elaine Dunlop

BHD -/- MEFs N-acetyl-L-cysteine (NAC)

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

Translating Basic Research

Basic Research Pre-clinical Studies Clinical TrialsRapamycin

is cool!

Molecularly Targeted Therapy

Phenotype

Gene(s)

FunctionsPathways

DrugInterventions

< Birt-Hogg-Dubé >

< BHD >

< mTOR>

Mitochondrial Biogenesis

< AMPK>

HIF1 Energy Metabolism

A B

C D

P-A

KT(

S47

3)P

-S6(

S23

5/23

6)


Raptor

mTOR

Lst8

S6K1

Akt

Similarly to TSC-tumours: high mTORC1 activity and low insulin signalling in BHD tumour


Cowden’s Syndrome

Birt-Hogg-Dubé


Peutz-JeghersSyndrome

Neurofibromatosis

TSC1/TSC2

ERK

MEK

Raf

RasNF1 Akt

AMPK LKB1

Rheb FLCN

PI3K

RSK

SoS

PTENPIP2 PIP3

PDK1

?

Regulation of mTOR by Tumour Suppressors

Raptor

mTOR

Lst8

mTORComplex 1(mTORC1)

Cell Growth

Rapamycin

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)

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

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

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