role of leptin signaling in mammary tumor...

Ruben R. GonzalezRuben R. Gonzalez--Perez, Ph.D.Perez, Ph.D.Assistant ProfessorAssistant Professor

Dept Microbiology, Biochemistry & Immunology,Dept Microbiology, Biochemistry & Immunology,

Morehouse School of MedicineMorehouse School of Medicine

Role of leptin signaling in Role of leptin signaling in mammary tumor progressionmammary tumor progression

Pathological processesPathological processes

Leptin Leptin (1994)(1994)

Functions:Regulator of energy balance/appetite

Angiogenesis ProliferationInflammation

Anti-apoptosis

20082008

reproductive processreproductive processOvulation

Endometrial receptivityEmbryo implantation

CancerCancerendometriosis endometriosis

diabetes diabetes arthritis, etc.arthritis, etc.

Elevated leptin levels are associated with increased Elevated leptin levels are associated with increased incidence, poor outcome and worse prognosis of incidence, poor outcome and worse prognosis of

breast cancerbreast cancer

Leptin receptor (OBLeptin receptor (OB--R)R)

Several isoforms

OB-Rb long isoform /functional

LeptinLeptin

Small protein (16 kDa)

Helical cytokine

Product of the obese gene (ob)

Secreted mainly by adipocytes

JAK2JAK2

JAK2JAK2

p-Stat3

SuperfamilySuperfamily of cytokine of cytokine receptor Ireceptor I

Signaling pathways activated by leptin

Leptin

OB-Rcancer

MAPK

JAK2-STAT3

PI-3K

p38 kinaseJNK

AMPKPKC

VEGFVEGFR2

LIFRLIF

IL-1R tIIL-1β

Bcl-2

Cyclin D1

?

Hypothesis:Hypothesis:

Disruption of leptin signaling will Disruption of leptin signaling will negatively impact mammary tumor negatively impact mammary tumor

growth by decreasing leptingrowth by decreasing leptin--induced expression of proinduced expression of pro--

angiogenic, proliferation and antiangiogenic, proliferation and anti--apoptosis factorsapoptosis factors

Designing Leptin Peptide Receptor Designing Leptin Peptide Receptor Antagonists (LPrA)Antagonists (LPrA)

Leptin Leptin (similar to G(similar to G--CSF)CSF)

GG--CSF RCSF R

(similar to OBsimilar to OB--RR)

GG--CSFCSF

Designing Leptin AntagonistsDesigning Leptin Antagonists

H3H3

7070

9595 LPrALPrA--11

LPrALPrA--22

LPrALPrA--22 LPrALPrA--2 Sc2 Sc

Structural differences between LPrAStructural differences between LPrA--2 2 and LPrAand LPrA--2Sc peptides (control)2Sc peptides (control)

Gonzalez, Gonzalez, UnpublishedUnpublished

The LPrA effects were dose dependent.

The peptides solutions (0.15-0.3 nmoles) were assayed in the CAM assay.

Experiments were repeated 20 times.

In contrast to PBS (A) and LPrA-Sc (B) (arrows), LPrA-1 (C) and LPrA-2 (D) (arrowhead) inhibit the formation of capillaries.

Inhibition of leptin signaling with Inhibition of leptin signaling with LPrAsLPrAs disrupts angiogenesisdisrupts angiogenesis

PBSPBS LPrALPrA--ScSc

LPrALPrA--11 LPrALPrA--22

Chicken Chicken chorioallantoicchorioallantoic membrane membrane (CAM) assay (CAM) assay

A

C

B

D

E F

Negative ControlNegative Control OBOB--RR

Human MCFHuman MCF--77

Human MDAHuman MDA--MB 231MB 231

Mouse 4T1Mouse 4T1

ER+ER+

ER+ER+

ERER--

Mammary cancer cells

express OB-R

LPrA2LPrA2 inhibitsinhibits leptinleptin--inducedinduced adhesionadhesion andandproliferationproliferation ofof breastbreast cancercancer cellscells

Fig 2. Non-pegylated 3H-w-LPA2 pharmacokinetics in female mice

0

500

1000

1500

2000

2500

3000

3500

0.3 0.5 1.0 2.0

time (hs)

mamm

liver

lung

horns+ovaries

CNSAbd Fat

kidney

BloodBlood

Abd fat Uterine horns

Liver

kidney

cpm

Fig 1. PEG20-3H-w-LPA2 pharmacokinetics in female mice

0

200

400

600

800

1000

1200

1.0 2.0 4.0 8.0 12.0 24.0 36.0 hs

mamm

liver

lung

horns+ovaries

CNS

Abd Fat

kidney

Blood

MM

Blood

liver

abd fat

hornskidney

lung

CNS

cpm

Half-life ︽ 1 h

Half-life ︽ 18 h

Intraperitonealinjections

UNCONJUGATED 3H-wLPrA2

PEGYLATED 3H-wLPrA2

323.80.1 x 10-70.000080.0040.004561.792505.00subdermal

63.12.4 x 10-50.0100.0420.093408.7138.3201.25i.v.

67.7 3.9 x10-50.0100.1410.295255.2675.3962.50i.v.

59.6 10-40.0111.5701.471103.23151.8885.00i.v.

19.10.0060.3620.98613.44654.68735.29412.50i.p.

T1/2( h)

CLmg/Kg

Kelmg/L

Cptmg

Cpomg

Vdliters/Kg

Dose(mg/Kg)

Dose(mg)

Via

Pharmacokinetics of PEG-3H-LPrA-2 in female mice

0.1338400.3753.5701.50vaginal 128.82 x 10-70.00540.025

PEG-wLPrA2 treatment does not affect food intake, body weight, glucose, insulin serum levels

or HOMAX-index in mice.

Body WeightBody Weight

0

5

10

15

20

25

30

D0 D3 D19 D25 D48 D52 D61 D64 D66 D77

PEG-LPrA2 Treated Untreated

DayDay

Food IntakeFood Intake

012345678

D0 D3 D19 D25 D48 D52 D61 D64 D66 D77

PEG-LPrA2 Treated Untreated

(g/mouse/day(g/mouse/day)

DayDay

BW (g)BW (g)

0

days

15 21

LPrAsLPrAs100 100 µµll

twice dailytwice daily

2nd row mammary glands2nd row mammary glands

Right nippleRight nipple

♀♀BALB/c BALB/c syngeneicsyngeneic sisterssisters

In vivo studiesIn vivo studiesSyngeneicSyngeneic mouse model of MTmouse model of MT

m4T1 cells

LPrA2 delays the onset and reduces

the growth of mouse 4T1-MT in a

syngeneic model

A

0

50

100

150

b

a

MT

volu

me

(%)

PBS LPrA2 PEG-LPrA2Sc

B

LPrA2Sc

C

0

50

100

150

MT

volu

me

(%)

PBS PEG-Sc PEG-LPrA2NT

b

0

50

100

150

PBS PEG-Sc PEG-LPrA2NT

MT

volu

me

(%)

D

b

Treatment

Chemoprevention

One week

Two weeks

MT

volu

me

(%)

Gonzalez RR, et al., J Biol Chem. 2006, 281(36):26320-8

C(-)

Sc PEG-LPrA2

PEG-LPrA2Sc

b

a

LPrA2 PEG-LPrA20

50

100

150

Tumor bearing

b

VEG

F (%

)

ScPBS

a

LPrA2 PEG-LPrA20

50

100

150

200 C

Sc

NTControl

VEG

F (p

g/m

l)

42 kDa

β-actin195 kDa

VEGFR2

PEG-LPrA2

VEG

F-R

2 (%

)

a

Sc LPrA20

50

100

150

PBS

a

D

B

A

PEGPEG--LPrA2 and LPrA2 and LPrA2 treatments LPrA2 treatments

decrease VEGF and decrease VEGF and VEGFR2 levelsVEGFR2 levels


ControlsControls

MCFMCF--7 tumor Onset7 tumor Onset

MCF-7 inoculation

MT detection

D23D23 D45D0 D7

PEG-wLPrA2

PEG-wLPrA2

MT detection

LPrA chemoprevention significantly delays the onset of MCF-7 MT in SCID mice.

Ovariectomized & E2 supplemented SCID mice

(8-weeks old)

7 days21 50

PEGPEG--wLPrA2 wLPrA2 or Scor Sc



MCFMCF--7 cells7 cells

Treatment of MTTreatment of MT--ER+ in SCID miceER+ in SCID mice

0

♀♀ SCID SCID ovariectomizedovariectomized mice mice E2/cholesterolE2/cholesterol--capsulecapsule

0

25

50

75

100

125

PEG-wLPrA2Controls

*

MT Relative Growth (%)

0

0.5

1

PEG-wLPrA2Controls

*

MT weight (g)

PEG-wLPrA2 reduces growth

of MCF-7 derived tumors

in SCID mice

0

25

50

75

100

125

PEG-wLPrA2Controls

*

VEGF (%)

0

25

50

75

100

125

VEGFR2 (%)

*

212 kDa VEGFR2

PEG-wLPrA2Controls

694 695 696 697 699 700

PEG-wLPrA2 treatment

decreases the levels of

VEGF/VEGFR2 in MCF-7 mammary

tumors

98 kDa IL-1 R tI

PEG-wLPrA2Controls

IL-1 (%)

PEGPEG--wLPrA2 decreases ILwLPrA2 decreases IL--1 and IL1 and IL--1R 1R tItIlevels within MCFlevels within MCF--7 MT7 MT

*p<0.05*p<0.05

0

2 5

5 0

7 5

1 0 0

1 2 5

PEG-wLPrA2Controls

*

PEG-wLPrA2 treatment decreases CD31, VEGFR2, CD68 (TAM), IL-1R tI, VEGF, leptin

and OB-R expression within MCF-7 MT

PECAM (CD31)

VEGFR2 (Flk-1) CD68 LEPTIN OB-R

SC

PEG-LPrA

0 days13 28

PEGPEG--wLPrA2 wLPrA2 or Scor Sc



MDAMDA--MB231cellsMB231cells

♀♀ SCID miceSCID mice

Human MT ERHuman MT ER--: In vivo studies: In vivo studies

-7

0

500In MT

In blood

Control

PEG-LPrA2

VEGF pg/ml/mg prot

Control

PEG-LPrA2

**

PEGPEG--wLPrA2 decreased wLPrA2 decreased VEGF levels in MDAVEGF levels in MDA--MB231 MTMB231 MT

ELISA DETERMINATIONS

050

100150200250300350

VEGF bcl-2

VEGFBcl-2

Control

PEG-LPrA2

Relative expression

Control

PEG-LPrA2

* *

21 kDaVEGF

PEG-wLPrA2Controls

PEG-wLPrA2Controls

26 kDa Bcl-2

PEGPEG--wLPrA2 decreased BclwLPrA2 decreased Bcl--2 levels in 2 levels in MDAMDA--MB231 MTMB231 MT

0

50

100

Control

IL-1R tIIL-1β

Control

*

*

LPrA LPrA

PEGPEG--wLPrA2 decreased ILwLPrA2 decreased IL--1 and IL1 and IL--1R 1R tItIlevels in MDAlevels in MDA--MB231 MTMB231 MT

30 kDa

Basal Lep LPrA20

250

500

SOC

S-3

(%) * DSOCS-3

Leptin (62.5nM)

ERK 1/21/242 kDa

0

1 0 0

2 0 0

3 0 0

ERK

1/2

(%

) * A*

Leptin (62.5nM)

Basal Lep LPrA2 Sc

Leptin (62.5nM)

92 kDa

0

50

100

150

pSTA

T3 (%

) * C

Basal Lep LPrA2 Sc AG490 siRNA

*pSTAT3

Leptin (62.5nM)

0

100

200

pAK

T1 (

%) * B*

pAKT1

Basal Lep LPrA2 Sc

60 kDa

LPrA2 blocks the leptin canonical signaling

pathways and SOCS3 down-regulation in 4T1 cells

Leptin activated the ERK 1/2/MAPK, Leptin activated the ERK 1/2/MAPK, PIPI--3K/AKT1 and JAK2/STAT3 3K/AKT1 and JAK2/STAT3

pathwayspathways

SOCSSOCS--3 was 3 was upregulatedupregulated by leptin and by leptin and inhibited by inhibited by LPrAsLPrAs


0

50

100

150

200

Cyc

linC

yclin

D1

(%)

D1

(%)

**

Basal

siRNA

AG490

Wor

tm

+ Leptin 62.5 nM

PD98

059

Lept

in

Cyclin D1D1

36 kDa36 kDa

C

195 kDa

0

50

100

150

200

250

* *

VEG

FR2

VEG

FR2

(%)

VEGFR2

Basal

siRNA

AG490

Wor

tm

+ Leptin 62.5 nM

PD98

059

Lept

in

0

50

100

150

200

250 *** *

VEG

F (%

)VE

GF

(%)

+Leptin 62.5(nM)

Lept

inBas

al

Ab VEGFR

Wor

tmPD98

059

siRNA

AG490

Signaling intermediates involved in the leptin-induced levels of VEGF/ VEGFR2 and

Cyclin D1 in 4T1 cells

ERK 1/2


STAT3 PI-3K MAPK

STAT3 PI-3K

Luciferase reporter constructs of mouse VEGF promoter and 5'-end deletions

Mouse mammary cancer cells:

4T1

MMT

EM6

Ramanathan et al. Exp Biol Med228:697-705 (2003)

EM6

4T1

MMT

Leptin regulation of VEGF promoter in mouse mammary

cancer cells

Full-length

-HRE -HRE -AP1

-HRE -AP1 -AP2

-NFκΒ

-HRE -AP1 -AP2

-NFκΒ

0

100

200

300

** *

**

* **

0

100

200

*** * *

*

0

100

200

**** * **

B HL L+ H

B HL L+ H

B HL L+ H

B HL L+ H

*** *

B HL L+ H

B HL L+ H

-HRE -AP1 –AP2 -SP1

AP1 + AP2 -

NFκΒ+

4T1

EM6

MMT

HRE + AP2 –

NFκΒ –

SP1+

VEGF

Leptin

HRE + AP2 –NFκΒ+

SP1+

Molecular mechanisms of leptin induction of pro-

angiogenic/pro-inflammatory factors in

breast cancer cells

Leptin OBOB--RR

Breast CC

mTORPI-3K

VEGFVEGF

VEGFR2VEGFR2

MAPK

JAK2

LIFRLIFR

LIFLIF

IL-1β

ILIL--1R1R tI

HREHRE

AP1AP1

NFNFκΒκΒ

SP1SP1

LPrA decreases MT growth by inhibiting leptinLPrA decreases MT growth by inhibiting leptin--induced induced mitogenic, angiogenic and antimitogenic, angiogenic and anti--apoptotic effects.apoptotic effects.

MT ER+ were more sensitive to LPrA effects than MT ERMT ER+ were more sensitive to LPrA effects than MT ER--

LPrALPrA

LeptinLeptinVEGF VEGF

VEGFR2VEGFR2

Cyclin D1Cyclin D1

OBOB--RR

ILIL--1 1 ILIL--1R 1R tItI

CD31CD31BclBcl--22

SUMMARY

This work was supported in part by

CONRAD (CIG-02-87, 06-113 and 07-114)

The Susan G Komen Foundation for the Cure

The Cancer Research and Prevention Foundation

NIH/UAB SPORE Breast Cancer

FINANCIAL SUPPORT

Daniel Okenu, Ph.D.

Salandre Cherfils

The Cardiovascular Research CenterNew Jersey Med School

Bo R. Rueda, Ph.D.

Ramey D. Littell. M.D.

Maureen P. Lynch, Ph.D.

Aaron K. Styer, M.D.

Brian T. Sullivan

Hideo Sakamoto, Ph.D.

Takehiro Serikawa, MD, Ph.D.

Alexander B Olawaiye M.D

MSM Vincent Center for Reproductive Biology (VCRB), Massachusetts General Hospital,

Harvard Medical School

COLLABORATORS

Amber Watters

Yanbo Xu, Ph.D.David Mann, Ph.D.

Samuel J. Leibovich, Ph.D.

Udai Singh, Ph.D.

role of leptin signaling in mammary tumor...

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