Symposium on “Advances in Endometrial CancerSymposium on Advances in Endometrial Cancer Epidemiology and Biology

March 18th 2014

Shingo Kajimura, Ph.D.University of California San FranciscoUniversity of California, San Francisco UCSF Diabetes Center, Dept. Cell & Tissue Biology

Obesity and Cancer Risk

C l C

Esophageal CancerPancreatic Cancer

Colon Cancer

Obesity (BMI>30)

Renal CancerGastric Cancer

(BMI>30)Breast Cancer (postmenopausal)Gall Bladder Cancer

Liver Cancer Multiple myeloma

Endometrial Cancer

Modified from www.cancer.gov/cancertopics/factsheet/Risk/obesityand from www.nutrino.co

Obesity Results from Disturbed Energy Balance

Weight lossWeight lossWeight gainWeight gain

AdaptiveAdaptiveThermogenesis

ExerciseFood Intake

Cellular functions

ExpenditureExpenditureIntakeIntake

White vs Brown

WAT BAT

• Store energy as triacylglycerol • Dissipate energy through UCP1gy y g y

• Unilocular lipid droplets

• Low number of mitochondria

• Multilocular lipid droplets

• High number of mitochondria with cristae• Low number of mitochondria

• Pro-inflammatory

• High number of mitochondria with cristae

• Anti-Obesity

Active BAT in Adult Humans

mas

sB

AT m

Saito et al. Diabetes 2009

Body Mass Index

Copyright 2011 Joslin Diabetes Center

50g of BAT can burn 4kg of fat in a year.

Goal: Engineering Fat Cell Fate to Fight Obesitto Fight Obesity

Weight lossWeight lossW i ht iW i ht iWeight gainWeight gain

HeatBAT

Food IntakeExercise

C f

BAT

Cellular functions

E ditE ditIntakeIntake

ExpenditureExpendituree.g., Phentermine, Orlistat

Skeletal muscle

Myf5+; MyoD+ MyoD+; Myogenin+

Skeletal muscle

En1+; Myf5+En1+; C/EBPβ+

Myoblast

C/EBPβDEP

OT

DermomyotomalPrecursor

Brown adipocyte(Clasical BAT)

PRDM16+; PGC-1α+; UCP1+

PRDM16

C/EBPβ

BAT

D

Brown preadipocyte

Seale et al., Nature 2008Kajimura et al., Nature 2009

“classical” brown adipocytes

Generation of functional BAT by defined factors

CT PET

eBAT(PRDM16 + C/EBPβ)

Mouse skin fibroblasts expressing

eWAT(PPARγ)

Mouse skin fibroblasts expressing PRDM16 and C/EBPβ

(Green: Bodipy staining, Red: Mitotracker)

Transplantation of engineered BAT

Kajimura at al. Nature, 2009

Transplantation of engineered BAT

“Inducible” form of UCP1 positive cells in WAT (Beige or Brite cells)

α UCP1

“Browning” of White fat

α-UCP1

Murano et al., J Anatomy 2009

Skeletal muscle

Myf5+; MyoD+ MyoD+; Myogenin+

DermomyotomalPrecursor PRDM16+; PGC-1α+; UCP1+

En1+; Myf5+En1+; C/EBPβ+

Myoblast

DEP

OT

C/EBPβ

EHMT1Classical brown adipocyte

Brown preadipocyte

BAT

PRDM16

EHMT1

Pharmacological approaches toBrown preadipocyte

UCP1+Environmental cues

(cold exercise PPARg agonist)

Pharmacological approaches to induce brown/beige development?

Beige / Brite cell

UCP1

DEP

OT

(cold, exercise, PPARg agonist)

EHMT1+

PDGFRa+

WAT

PPARγ+

PRDM16

preadipocyte

preadipocyte

Ohno et al. Cell Met (2012)Ohno et al. Nature (2013)Cohen et al. Cell (2014)

White adipocyte

High-resolution phosphoproteome to identify unique kinases in brown, beige, and whiteq , g ,

(Beige fat)

Beige fat(white fat + PRDM16)Collaboration with Dr. Ishihama lab in Kyoto Uni.

Prediction of kinases from phosphoproteome dataphosphoproteome data

• Matched P-peptides sequences to identify A.A. motifs

otal

)

Matched P peptides sequences to identify A.A. motifs

-pep

tides

/t

Beige fat (white fat + PRDM16)Casein Kinase II substrate motifpSXX(D/E)/pSX(D/E)

chm

ent (

P- pSXX(D/E)/pSX(D/E)

Fold

enr

ic

Time (min)

What is CK2 kinase ?

CK2 active complex

Cell cycleCell survival

CK2α

p(tumorigenesis)

CK2βCK2β Cell differentiation

CK2α2

Circadian rhythmy

CK2 activity in WAT is induced in obesity

Regular diet

*****

(%/)

High fat Diet

CK

2 ac

tivity

(R

elat

ive

C

Subcutaneous Visceral BATSubcutaneous(Inguinal)

WAT

Visceral (Epididymal)

WAT

BAT

15 weeks of HFD in Bl6 mice (60%)

Depletion of CK2α induces browning of white adipocytes

si-Ctrl si-Ck2α

white adipocytesWestern Blot si-Ctrl si Ck2α

β actin

Ucp1**

**t l

UCP1

β-actin

expr

essi

on

control

si-Ck2a**

elat

ive

gene

**

*** e

xpre

ssio

n control

si-Ck2a

Re

**

N.S.

Rela

tive

gene

cAMPDMSO

Chemical inhibition of CK2

CX-4945 • ATP-competitive inhibitor (IC50: 1 nM)CK2 activity • Selective (at least ×500 higher IC50)

• Clinical trial (phase II for multiple myeloma)

(in Inguinal WAT)

• Orally available

utes

CAS-863598

ount

Per

Min

***

ATP-competitive inhibitor (EC50: 100 nM/ IC50: 9 nM)

Co

(EC50: 100 nM/ IC50: 9 nM)Vehicle CX-4945

Chemical inhibition of CK2 induces browning of white fat in vivobrowning of white fat in vivo

Cont

*** ****

on

Vehicle

CX-4945

***

* **

e ex

pres

sio (50mg/kg)

*

elat

ive

gen

CX-4945

Re

UCP1/DAPI

CK2 Substrates in Adipocytes CK2 recombinant

proteinAdipocytesproteome sample

…EESGDEDEEDPDK………IACEEEFSDSDEEGEG…

De-phosphorylation

CTD1HDAC HDAC1438 482

Kinase reaction

p p y

TVEEPSNPEASSSTSVTPDVSDNEPDHYR

Phospho-peptide enrichment

PTEN_C2PTEN350 378

ki d i119 404

GEPNVSYICSR

GSK3α(808 proteins)LC-MS/MS kinase domainGSK3α

Ishihama lab in Kyoto Uni

(808 proteins)

Chemical inhibition of CK2 increases whole-body energy expenditurewhole-body energy expenditure

min

)

Energy expenditure

n)

Locomotor activity

VehicleCX-4945

**

n (m

l/kg/

m

ount

s/m

in

*

sum

ptio

n

ctiv

ity (c

o

O2

cons

Tota

l ac

Day Night Day Night

Chemical inhibition of CK2 reduces body weight gain in micebody weight gain in mice

55

60Fasting plasma Glucose

45

50

eigh

t (g)

g/dl

)

***

40

45

VEBod

y w

e

** Glu

cose

(m

30

35 CX-4945G

301 3 5 7 9 11 13 15 17

Days

Vehicle CX-4945

High-fat diet (15 weeks)

Conclusions

1. Phosphoproteomics identified CK2 as a negative regulator of beige fat development.

2. Genetic and pharmacological inhibition of CK2 induced “browning” of white fat in vivoinduced browning of white fat in vivo.

1. Pharmacological inhibition of CK2 increased gwhole body energy expenditure and decreased body weight gain.

Kajimura Lab Crews

Kosaku Shinoda Golden gate bridge!Si Sonne

Louis SharpDylan Lowe

Golden gate bridge!

Haemin HongYutaka Hasegawa

Svetlana KeylinMiae Kim

CollaboratorsEnrique Saez (The Scripps Institute)Andrea Galmozzi (The Scripps Institute)

Yasushi Ishihama (Kyoto University)

F di

Yasushi Ishihama (Kyoto University)

Allison Xu (UCSF)Christophe Paillart (UCSF)

FundingNIDDK (R00, RO1); DERC Pilot Grant; The Pew Charitable Trusts

CAS-863598

***** Fabp4Elovl3Ucp1

N.S.***

***

CtrlcAMP

mRN

A le

vel

**

Rela

tive

m

DMSO DMSO DMSO

cx-4945

0.1μM~1μM

Fabp4Elovl3Ucp1

CtrlcAMP *

**

A le

vel N.S.

*

**

cAMP

Rela

tive

mRN

A

DMSO DMSO DMSO

R

*** ****

Inguinal White Adipose TissueInguinal White Adipose

Vehicle CX-4945

25kUcp1***

* **

*

Vehicle

CX-4945

β-actin 37k

Cited1 CideaElovl3 AdipoQDio2 Pgc1αUcp1 Otop1

Brown Adipose TissueCK2 activity

(in Inguinal WAT)

Vehicle

CX-4945**

* *

p

Min

utes

*

Cou

nt P

er M

***

Cox7a CideaElovl3 AdipoQDio2 Pgc1αUcp1 Otop1 Vehicle CX-4945

Prediction of kinases from phosphoproteome motif analysisphosphoproteome motif analysis

GSK3

al)

eptid

es/to

tm

ent (

P-pe

ATM

old

enric

hm

0 5 10 20

PKA

F

Time (min)

Time (min)

(A) (A)

(B)

(C)

(B) (C)

( )

(B) (C)

Recruitment of adult human BAT b h i ldby chronic cold

Yoneshiro et al. JCI 2013

Recruitment of adult human BAT by chronic coldby chronic cold

Before After

2h at 19 ˚C per day for 6 weeks Yoneshiro et al. JCI 2013

Gastrocnemius muscleTissue weight

acyl-Coenzyme A dehydrogenase, medium chain (Acadm) mRNA level

Cox1 (mRNA)MCAD/citrate synthasemRNA

Uncoupling protein 1 (UCP1)p g p ( )

LCFA H+ co transporter

Kajimura & Saito, Ann Rev Physiol. 2014

LCFA-H+ co-transporter

Chemical inhibition of CK2 reduces body weight gain in micebody weight gain in mice

50

Body weight (g) Fasting plasma Glucose

ht (g

) *

g/dl

)

***40

Bod

y w

eigh

Glu

cose

(m

30

B G

High-fat diet (15 weeks)

Vehicle CX-4945 Vehicle CX-4945