role of leptin signaling in mammary tumor...
TRANSCRIPT
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
Gonzalez RR, et al., J Biol Chem. 2006, 281(36):26320-8
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
2nd row mammary glands2nd row mammary glands
Right nippleRight nipple
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
2nd row mammary glands2nd row mammary glands
Right nippleRight nipple
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
Gonzalez RR, et al., J Biol Chem. 2006, 281(36):26320-8
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
Gonzalez RR, et al., J Biol Chem. 2006, 281(36):26320-8
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.