1. PRECLINCAL INSIGHTS INTO THEPRECLINCAL INSIGHTS INTO THE
RELATIONSHIP OBESITY AND BREASTRELATIONSHIP OBESITY AND BREAST
CANCERCANCER
Margot P. ClearyMargot P. Cleary
(no conflicts of interest & will discuss off label use of metformin)(no conflicts of interest & will discuss off label use of metformin)

2. OBESITY & BREAST CANCEROBESITY & BREAST CANCER
• There are many epidemiological studies supporting a role forThere are many epidemiological studies supporting a role for
obesity in breast cancer development and progression butobesity in breast cancer development and progression but
studies that focus on mechanisms of action are limited.studies that focus on mechanisms of action are limited.
• What is it about obesity that stimulates breast cancer ?What is it about obesity that stimulates breast cancer ?
• The most widely accepted theory is thatThe most widely accepted theory is that increasedincreased fat tissue infat tissue in
obese women converts androgens to estrogen because ofobese women converts androgens to estrogen because of
increased activity of aromatase and then elevated estrogenincreased activity of aromatase and then elevated estrogen
promotes tumor development.promotes tumor development.
• Elevated insulin levels associated withElevated insulin levels associated with increased bodyincreased body weightweight
and body fat also has been considered to play a role.and body fat also has been considered to play a role.
• IGF-I also has been considered to be a factor.IGF-I also has been considered to be a factor.
• In the mid 1990’s the identification ofIn the mid 1990’s the identification of adipokinesadipokines, proteins, proteins
made in adipose tissue, opened up new possibilities for amade in adipose tissue, opened up new possibilities for a
direct connection to fat tissue.direct connection to fat tissue.

3. PRESENTATIONPRESENTATION
• Background.
• Studies from my laboratory using
preclinical animal models.
• Highlight other studies.
• Summarize and suggest considerations
for future investigations.

4. PRECLINICAL ANIMAL MODELS OFPRECLINICAL ANIMAL MODELS OF
OBESITY USED IN TUMORIGENESISOBESITY USED IN TUMORIGENESIS
STUDIESSTUDIES
• Genetic obesity such as
Lepob
, Leprdb
, and Avy
mice
orZucker/Corpulent rats.
• Diet-induced obesity
(DIO) primarily be feeding
high fat diets.
• Brain damage of the
VMH.
• Ovariectomy (OVX) alone
or in combination with
DIO.

5. PRECLINICAL ANIMAL MODELS OFPRECLINICAL ANIMAL MODELS OF
MAMMARY TUMORIGENESISMAMMARY TUMORIGENESIS
• Spontaneous MT development
in rats & mice.
• Carcinogen-induced mostly in
rats.
• Transgenic animals mostly
mice.
• Transplanted human tumor tissue
or cell lines (xenograft) using
immunocompromised mice.
• Allografts, i.e., malignant cell lines
from specific mouse strains so
immunocompetent syngenic mice
can be used.
• Last two better models of
progression than tumorigenesis.

6. EARLY ANIMAL STUDIESEARLY ANIMAL STUDIES
• Although limited in scope several earlyAlthough limited in scope several early
rodent studies supported a role for bodyrodent studies supported a role for body
weight (heavier mice or genetic obesityweight (heavier mice or genetic obesity
primarily) in spontaneous and carcinogen-primarily) in spontaneous and carcinogen-
induced MT development but there was littleinduced MT development but there was little
effort to determine mechanisms.effort to determine mechanisms.
• Over the past 15 years expansion of this areaOver the past 15 years expansion of this area
of research combining models of mammaryof research combining models of mammary
tumorigenesis with obesity models.tumorigenesis with obesity models.

7. AN OBESE MAMMARY TUMORAN OBESE MAMMARY TUMOR
MODEL in MPC LABMODEL in MPC LAB
• With the increasingly evidence that obesity affectedWith the increasingly evidence that obesity affected
postmenopausal breast cancer development wepostmenopausal breast cancer development we
wanted to focus on MTs developing later in life.wanted to focus on MTs developing later in life.
• We identified MMTV-TGF-We identified MMTV-TGF-αα mice (overexpressmice (overexpress
human TGF-human TGF-αα)) which were reported to have an ~30%which were reported to have an ~30%
incidence rate of MTs at 15-16 months of ageincidence rate of MTs at 15-16 months of age
allowing for the detection of increases/decreases inallowing for the detection of increases/decreases in
incidence and/or latencyincidence and/or latency (Halter et al Am.J.Pathol.:140:1131,1991).(Halter et al Am.J.Pathol.:140:1131,1991).
• For obesity we decided to use genetically obese
mice to eliminate confounding factors of differences
in diet composition.

8. TGF-TGF-αα && LepLep ((LeprLepr) MICE) MICE
TGF-α male mice nonTransgenic
female mice
Lep and Lepr mice are genetically obese when they inherit an
obesity gene from both of their parents.
Mice heterozygous for the obese gene were used for breeding.
Following birth mice were genotyped for their obesity and
transgene status (one copy for MT development).
Lean and obese mice were followed until 104 weeks of age to
assess age of tumor appearance and numbers of mice with
tumors.

9. MAMMARY TUMOR INCIDENCE INMAMMARY TUMOR INCIDENCE IN LepLep
ANDAND LeprLepr MICEMICE
LEAN Lepob
Lepob
OBESE*
Leprdb
Leprdb
OBESE+
TOTAL #
MICE
74 59 42
# MICE WITH
MTs
44 0 0
# MICE
WITHOUT MTs
30 59 42
PERCENTAGE
OF MICE with
MTs
59 0 0
*Cleary et al BCRT 77:205,2003 and +
Cleary et al Exp.Biol.Med. 229:182,2004.

10. EXPLANATIONS FOR THESEEXPLANATIONS FOR THESE
RESULTS?RESULTS?
• Why were these mice obese?Why were these mice obese?
• LepLep mice have a deficiency of leptin, a protein made in fatmice have a deficiency of leptin, a protein made in fat
tissue.tissue.
• LeprLepr mice are hyperleptinemic but have defective leptinmice are hyperleptinemic but have defective leptin
receptors preventing leptin action at the cell level.receptors preventing leptin action at the cell level.
• Receptors for leptin were first identified in the brain resulting inReceptors for leptin were first identified in the brain resulting in
the conclusion that leptin was a signal of body fat stores.the conclusion that leptin was a signal of body fat stores.
• However, with time leptin receptors were found to beHowever, with time leptin receptors were found to be
expressed in many tissues throughout the body and also inexpressed in many tissues throughout the body and also in
cancer tissues and cancer cell lines.cancer tissues and cancer cell lines.
• These findings in conjunction withThese findings in conjunction with in vitroin vitro studies where leptinstudies where leptin
was shown to be a growth factor for human breast cancer cellwas shown to be a growth factor for human breast cancer cell
lines provided strong support for leptin as a growth factor forlines provided strong support for leptin as a growth factor for
mammary tumorigenesis.mammary tumorigenesis.

11. OTHER STUDIES USING GENETICALLYOTHER STUDIES USING GENETICALLY
OBESE RODENTS AND MAMMARYOBESE RODENTS AND MAMMARY
TUMORIGENESISTUMORIGENESIS
• Zucker/Corpulent rats with Lepr defects have been used
to study carcinogen-induced induced MTs. MNU treated
rats no MTs but with DMBA yes/no.
• Leprdb
Leprdb
mice crossed with MMTV-PyMT model (very
fast growing tumors) also had reduced tumor growth (Park
et al Am.J.Pathol.177:3133,2010).
• A recent study compared effects of tumor growth of
MMTV-Wnt-1 cells implanted into Lepob
and Leprdb
mice to
WT mice. Tumor volume was decreased in Lepob
leptin
deficient mice but was accelerated in Leprdb
mice (leptin levels 24.4 ng/ml)
compared to WT mice
(leptin 0.9 ng.ml)
(Zheng, et al End-Rel.Cancer
18:491,2011)

12. MOST DIET-INDUCED OBESITY STUDIES COMPARE ANIMALSMOST DIET-INDUCED OBESITY STUDIES COMPARE ANIMALS
FED LOW-FAT vs HIGH-FAT DIETS WITH VERY DIFFERENTFED LOW-FAT vs HIGH-FAT DIETS WITH VERY DIFFERENT
NUTRIENT COMPOSITIONSNUTRIENT COMPOSITIONS

13. OBESITY-PRONE vs OBESITY-OBESITY-PRONE vs OBESITY-
RESISTANTRESISTANT MICEMICE
• When this approach is used for some strains of rats and mice some will gain
weight and become obese = OBESITY-PRONE, while others stay in the
same body weight range of low-fat diet animals = OBESITY-RESISTANT.
• This approach allows for comparison of animals all fed the same diet but
with different body weights or to compare those with the same body weight
consuming different diets, i.e., high-fat versus low fat.
• We also included the middle group which we termed OVERWEIGHT
although many researchers in the obesity field do not use this group.
Equal body weight of
low fat fed mice.
OBESITY-PRONEOBESITY-PRONE OBESITY-RESISTANTOBESITY-RESISTANT

14. OBESITY-PRONE vs OBESITY-OBESITY-PRONE vs OBESITY-
RESISTANT ANIMALSRESISTANT ANIMALS
• TGF-TGF-αα mice (n=51) were fed a high-fatmice (n=51) were fed a high-fat
diet from 10 weeks of age.diet from 10 weeks of age.
• At 34 weeks of age mice (no MTsAt 34 weeks of age mice (no MTs
detected at this point) were divided intodetected at this point) were divided into
Obesity-Prone, Overweight andObesity-Prone, Overweight and
Obesity-Resistant.Obesity-Resistant.
• Followed for MT development until 85Followed for MT development until 85
weeks of age.weeks of age.

15. FINAL BODY WEIGHTS &FINAL BODY WEIGHTS &
TUMOR LATENCYTUMOR LATENCY
FINAL BODY WEIGHTS OF MICE IN DIET-INDUCED OBESITY EXPERIMENT
LOW-FAT OBESITY-RESISTANT OVERWEIGHT OBESITY-PRONE
0
10
20
30
40
50
ANOVA p<0.0001, different superscripts indicate significantly
different. n=17/group except LOW-FAT n=25
a
b
c
c
GRAMS
MAMMARY TUMOR LATENCY FOR TGF-α MICE IN DIET-INDUCED
OBESITY STUDY
LOW-FAT OBESITY-RESISTANT OVERWEIGHT OBESITY-PRONE
0
10
20
30
40
50
60
70
80
a
ANOVA p<0.0001, different superscripts indicate significantly
different, n=13-18/group.
a,b
b
c
WEEKSOFAGE
SERUM LEPTIN LEVELS OF TGF-α MICE IN DIET-INDUCED OBESITY
EXPERIMENT 2
LOW-FAT OBESITY-RESISTANT OVERWEIGHT OBESITY-PRONE
0
10
20 ANOVA p<0.0001-Different superscripts
indicate significant differences.
a
b*,#
b*,#
b*
* Student's t test LOW-FATsignificantly different from OBESITY-RESISTANT (p<0.03) &
OVERWEIGHT(p<0.0001). #OBESITY-RESISTANT and OVERWEIGHT different at p<0.09.
n=16 n=10 n=12 n=13
ng/ml
Mammary tumor latency inversely
related to body weight.
Serum leptin levels positively
correlated with body weight.
Aggressive tumors only found in
obesity-prone mice.
Cleary et al Int.J.Obesity 2004 and Dogan et
al Breast Cancer Res 2007

16. OTHER DIET-INDUCED OBESITYOTHER DIET-INDUCED OBESITY
STUDIES- MICESTUDIES- MICE
• MMTV-neu mice that develop ER- MTs were subjected to theMMTV-neu mice that develop ER- MTs were subjected to the
same dietary protocol but there was little effect of diet-inducedsame dietary protocol but there was little effect of diet-induced
obesity on tumor developmentobesity on tumor development (Cleary et al Nutr.Cancer.50:174,2004).(Cleary et al Nutr.Cancer.50:174,2004).
• MT development in MMTV-neu mice fed a high fat diet (45%)MT development in MMTV-neu mice fed a high fat diet (45%)
has also been reported although mice not separated by bodyhas also been reported although mice not separated by body
weight status. Although time to developing first MT was notweight status. Although time to developing first MT was not
different, second tumors developed sooner in the high fat dietdifferent, second tumors developed sooner in the high fat diet
mice and average # MT/mouse was increasedmice and average # MT/mouse was increased (Khalid et al BCRT(Khalid et al BCRT
122:647,2010122:647,2010).).
• To directly compare ER+ to ER- MT developmentTo directly compare ER+ to ER- MT development
immunocompromised C57BL6 mice were used and Obesity-immunocompromised C57BL6 mice were used and Obesity-
Prone and Obesity-Resistant mice inoculated with either MCF-7Prone and Obesity-Resistant mice inoculated with either MCF-7
or MDA-MB-231 cells, but neither cell line was very responsiveor MDA-MB-231 cells, but neither cell line was very responsive
in this approachin this approach (Ray et al Cancer Letters 253:291 (2007)(Ray et al Cancer Letters 253:291 (2007) ..

17. OBESITY-PRONE VS OBESITY-RESISTANTOBESITY-PRONE VS OBESITY-RESISTANT
RATS AND MAMMARY TUMOR PROGRESSIONRATS AND MAMMARY TUMOR PROGRESSION
• Wistar rats were fed 46% fat diet and injected
with MNU at ~7 weeks of age.
• At 19 weeks of age a high fat diet rats divided
into Lean, Mid-weight and Obese.
• OVX at ~24 weeks of age @ which time tumor
incidence and burden were similar in the three
groups.
• With OVX some tumors regressed, but half as
many in Obese rats regressed and many more
new ones appeared compared to Lean rats
(MacLean et al Obesity 18:696, 2010).

18. SECOND STUDY OBESITY-SECOND STUDY OBESITY-
PRONE RATSPRONE RATS
• High fat diet fed rats were further divided by energy
intake minus energy expenditure to identify High Energy
vs Low Energy groups.
• Obesity increased tumor PR expression (before OVX)-
samples obtained by FNA.
• At study termination MT PR expression in Obese-High
Energy Excess group still higher than in Lean-High
Energy Excess group.
• Also at termination MTs from Obese-High Energy
Excess rats had twice the glucose uptake as the Obese
Low Energy and the comparable lean groups.
(Giles et al Cancer Res. 72:6490, 2012)

19. WILL INTERVENTIONS AFFECT THEWILL INTERVENTIONS AFFECT THE
DEVELOPMENT AND/OR PROGRESSION OFDEVELOPMENT AND/OR PROGRESSION OF
MAMMARY TUMORS IN OBESE ANIMALS?MAMMARY TUMORS IN OBESE ANIMALS?
• It is well-documented that calorie restriction prevents and/or delays
mammary tumor development in normal weight rodents.
• We and others have reported that leptin and IGF-I are consistently
lowered by calorie restriction again in normal weight rodents in
conjunction with the prevention of MTs.
• Other interventions have also been implemented in normal weight
rodents, i.e., various chemopreventive agents, fatty acids and
pharmaceutical agents to prevent MT development.
• However, few studies have been done with obese animals.
• Since preventative approaches in humans would presumably be
targeted to at risk populations such as obese women these types of
studies should be a high priority in particular those affecting body
weight such as weight loss.
→

20. EFFECT OF CALORIE RESTRICTION IN OBESEEFFECT OF CALORIE RESTRICTION IN OBESE
RODENTS ON MAMMARY TUMORIGENESIS-RODENTS ON MAMMARY TUMORIGENESIS-
EARLY INTERVENTIONSEARLY INTERVENTIONS
• There are several older published
studies.
• C3H mice which develop spontaneous
MTs were made obese with GTG at 2
months of age and when clearly obese
at 3 months of age were calorie
restricted to reduce their body weights
to the level of control mice or allowed
to continue to overeat. Average age of
MT onset was 219 days for Obese
mice, 267 days for Control/Lean mice
and 365 days of Previously Obese
mice (Waxler JNCI 14:1253, 1954).
• Genetically obese LA/N (corpulent
rats) were given DMBA at 65 days of
age and one week later one half were
restricted by 40%. Rats were followed
for a total of 16 wks (Klurfled et al
PSEBM 196: 381,1991).
Obese Obese
Rest.
Lean
BODY
WEIGHT
(grams)
~550 ~350 ~200
MT
Incidence
100% 27% 21%
MT # per
rat
4.1 1.5 1.0
MT weight
per rat
22.6 9.6 1.7

21. MORE RECENT INTERVENTIONSMORE RECENT INTERVENTIONS
IN OBESE MODELSIN OBESE MODELS
• OVX + DIO (60% fat diet) mice for 17 weeks and then were
switched to low fat diet and rapidly lost weight over three weeks
reaching body weight of mice always fed the low fat diet at which
time Wnt-1 tumor cells were injected. Tumors that formed over the
next 15 days were twice as big in the formerly obese mice as those
in the control mice. Data interpretation is somewhat difficult as there
was no obese control group so it is possible that the tumor size
would have been reduced compared to them (DeAngel
Mol.Carcinogenesis 2012).
• OVX+DIO mouse model after 10 weeks were calorie restricted by
30% and followed for another 7 or 14 weeks. Not a MT study but
assessment of factors associated with inflammation were
determined in mammary tissue and many were reversed by calorie
restriction such as NF-κB. TNF-α, IL-1β,COX-2, aromatase and PR
expression and also detection of crown-like-structures (CLS)
reduced (Bhardwaj, et al CPR in press, 2013).

22. CALORIE RESTRICTIONCALORIE RESTRICTION
MIMETICS- METFORMINMIMETICS- METFORMIN
• Human observational studies have indicated that diabetic women
treated with metformin were at decreased risk of breast cancer, and
if diagnosed with breast cancer women using metformin were more
responsive to cancer treatments.
• In addition, in vitro studies and preclinical rodent studies have
further indicated that metformin may have anticancer effects.
• However the majority of preclinical studies have been done in
normal weight animals.
• One recent study using Obesity-Prone rats administered MNU to
induce MTs which I described earlier for effects on calorie restriction
also presented results for short-term metformin (2mg/mL in drinking
water) starting one wk before OVX and continued for three weeks.

23. EFFECTS OF METFORMIN ONEFFECTS OF METFORMIN ON
MAMMARY TUMORS IN OBESE RATSMAMMARY TUMORS IN OBESE RATS
A. Tumor burden after
treatment with
metformin for 3 wk
post-OVX.
B. Tumor receptor status
after 1 wk of
metformin.(*p<0.05)
From Giles et al Cancer
Res.72:6490, 2012)
DID not compare
metformin to calorie
restriction.

24. ON GOING STUDY TO COMPARE METFORMINON GOING STUDY TO COMPARE METFORMIN
DIRECTLY TO CALORIE RESTRICTION ON MTDIRECTLY TO CALORIE RESTRICTION ON MT
DEVELOPMENT IN OBESE VS LEAN MICEDEVELOPMENT IN OBESE VS LEAN MICE
• MMTV-TGF-α mice consume low or high fat diets until 30 weeks of
age.
• High-fat diet mice will be divided into body weight categories, i.e.,
Obesity-Prone, Overweight and Obesity-Resistant.
• Within each body weight category mice will be allowed to eat freely
(control, ad libitum-fed), be calorie restricted by 25% or be given
metformin at a dose of 250 mg/kg body weight/day.
• The mice will then be followed until 90 weeks of age to assess the
impact of the interventions on mammary tumor development.
• Serum samples are being taken over the course of the study to
assess metformin levels and for measurements of adipokines,
insulin and IGF-I.
• At termination serum and tissues will be obtained.

25. BODY WEIGHT OF CALORIEBODY WEIGHT OF CALORIE
RESTRICTED AND METFORMINRESTRICTED AND METFORMIN
TREATED MICETREATED MICE
15.00
20.00
25.00
30.00
35.00
40.00
0 10 20 30 40 50 60 70 80 90
Weeks of Age
Grams
LF-AL
LF Met
LF-CCR
HF-AL
HF-Met
HF-CCR
Interventions started at 30 weeks of age
↑

26. SUMMARYSUMMARY
• Evidence that leptin may be a growth factor for mammary cancer cells.
• Recent study reported that prediagnosis leptin and leptin:adiponectin ratio
were elevated prior to postmenopausal breast cancer diagnosis (Ollberding et
al CPR 6:188,2013).
• Several research groups developing analogues/antagonists of leptin and
leptin receptor for therapeutic uses.
• Adiponectin another adipokine may also play a role in mammary
tumorigenesis.
• The ratio of adiponectin to leptin is being considered as a more specific
indicator of how body weight and interventions affect tumor growth.
• Interactions of insulin and/or IGF-I with adipokines should be investigated.
• Increasingly studies using obesity models are being undertaken and
should be supported to provide insights into how interventions can be
applied to at risk humans.
• NCI does seem to be taking this seriously given that several of the
Provactive Questions are targeting obesity.

27. HOW CAN WE APPLY STUDIES OFHOW CAN WE APPLY STUDIES OF
PHYSICAL ACTIVITY TO PRECLINICALPHYSICAL ACTIVITY TO PRECLINICAL
OBESITY STUDIES?OBESITY STUDIES?

28. CO-WORKERS and FINANCIAL
SUPPORT
Amitahba Ray
Katai J. Nkhata
Nancy K. Mizuno
Xin Hu
Michael E. Grossmann
Soner Dogan
Olga P. Rogozina
Jill Chavez
David Potter
Nita J. Maihle
SUPPORT
DOD Breast Cancer Program
Eagles Cancer Telethon
The Hormel Foundation
The Breast Cancer Research
Foundation
National Cancer Institute- NIH