PRECLINCAL INSIGHTS INTO THEPRECLINCAL INSIGHTS INTO THERELATIONSHIP OBESITY AND BREASTRELATIONSHIP OBESITY AND BREASTCANCERCANCERMargot 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)
OBESITY & BREAST CANCEROBESITY & BREAST CANCER• There are many epidemiological studies supporting a role forThere are many epidemiological studies supporting a role forobesity in breast cancer development and progression butobesity in breast cancer development and progression butstudies 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 inobese women converts androgens to estrogen because ofobese women converts androgens to estrogen because ofincreased activity of aromatase and then elevated estrogenincreased activity of aromatase and then elevated estrogenpromotes tumor development.promotes tumor development.• Elevated insulin levels associated withElevated insulin levels associated with increased bodyincreased body weightweightand 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, proteinsmade in adipose tissue, opened up new possibilities for amade in adipose tissue, opened up new possibilities for adirect connection to fat tissue.direct connection to fat tissue.
PRESENTATIONPRESENTATION• Background.• Studies from my laboratory usingpreclinical animal models.• Highlight other studies.• Summarize and suggest considerationsfor future investigations.
PRECLINICAL ANIMAL MODELS OFPRECLINICAL ANIMAL MODELS OFOBESITY USED IN TUMORIGENESISOBESITY USED IN TUMORIGENESISSTUDIESSTUDIES• Genetic obesity such asLepob, Leprdb, and AvymiceorZucker/Corpulent rats.• Diet-induced obesity(DIO) primarily be feedinghigh fat diets.• Brain damage of theVMH.• Ovariectomy (OVX) aloneor in combination withDIO.
PRECLINICAL ANIMAL MODELS OFPRECLINICAL ANIMAL MODELS OFMAMMARY TUMORIGENESISMAMMARY TUMORIGENESIS• Spontaneous MT developmentin rats & mice.• Carcinogen-induced mostly inrats.• Transgenic animals mostlymice.• Transplanted human tumor tissueor cell lines (xenograft) usingimmunocompromised mice.• Allografts, i.e., malignant cell linesfrom specific mouse strains soimmunocompetent syngenic micecan be used.• Last two better models ofprogression than tumorigenesis.
EARLY ANIMAL STUDIESEARLY ANIMAL STUDIES• Although limited in scope several earlyAlthough limited in scope several earlyrodent studies supported a role for bodyrodent studies supported a role for bodyweight (heavier mice or genetic obesityweight (heavier mice or genetic obesityprimarily) in spontaneous and carcinogen-primarily) in spontaneous and carcinogen-induced MT development but there was littleinduced MT development but there was littleeffort to determine mechanisms.effort to determine mechanisms.• Over the past 15 years expansion of this areaOver the past 15 years expansion of this areaof research combining models of mammaryof research combining models of mammarytumorigenesis with obesity models.tumorigenesis with obesity models.
AN OBESE MAMMARY TUMORAN OBESE MAMMARY TUMORMODEL in MPC LABMODEL in MPC LAB• With the increasingly evidence that obesity affectedWith the increasingly evidence that obesity affectedpostmenopausal breast cancer development wepostmenopausal breast cancer development wewanted 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 (overexpresshuman 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 ageallowing for the detection of increases/decreases inallowing for the detection of increases/decreases inincidence 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 obesemice to eliminate confounding factors of differencesin diet composition.
TGF-TGF-αα && LepLep ((LeprLepr) MICE) MICETGF-α male mice nonTransgenicfemale miceLep and Lepr mice are genetically obese when they inherit anobesity gene from both of their parents.Mice heterozygous for the obese gene were used for breeding.Following birth mice were genotyped for their obesity andtransgene status (one copy for MT development).Lean and obese mice were followed until 104 weeks of age toassess age of tumor appearance and numbers of mice withtumors.
MAMMARY TUMOR INCIDENCE INMAMMARY TUMOR INCIDENCE IN LepLepANDAND LeprLepr MICEMICELEAN LepobLepobOBESE*LeprdbLeprdbOBESE+TOTAL #MICE74 59 42# MICE WITHMTs44 0 0# MICEWITHOUT MTs30 59 42PERCENTAGEOF MICE withMTs59 0 0*Cleary et al BCRT 77:205,2003 and +Cleary et al Exp.Biol.Med. 229:182,2004.
EXPLANATIONS FOR THESEEXPLANATIONS FOR THESERESULTS?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 fattissue.tissue.• LeprLepr mice are hyperleptinemic but have defective leptinmice are hyperleptinemic but have defective leptinreceptors 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 inthe 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 beexpressed in many tissues throughout the body and also inexpressed in many tissues throughout the body and also incancer 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 leptinwas shown to be a growth factor for human breast cancer cellwas shown to be a growth factor for human breast cancer celllines provided strong support for leptin as a growth factor forlines provided strong support for leptin as a growth factor formammary tumorigenesis.mammary tumorigenesis.
OTHER STUDIES USING GENETICALLYOTHER STUDIES USING GENETICALLYOBESE RODENTS AND MAMMARYOBESE RODENTS AND MAMMARYTUMORIGENESISTUMORIGENESIS• Zucker/Corpulent rats with Lepr defects have been usedto study carcinogen-induced induced MTs. MNU treatedrats no MTs but with DMBA yes/no.• LeprdbLeprdbmice crossed with MMTV-PyMT model (veryfast growing tumors) also had reduced tumor growth (Parket al Am.J.Pathol.177:3133,2010).• A recent study compared effects of tumor growth ofMMTV-Wnt-1 cells implanted into Lepoband Leprdbmice toWT mice. Tumor volume was decreased in Lepobleptindeficient mice but was accelerated in Leprdbmice (leptin levels 24.4 ng/ml)compared to WT mice(leptin 0.9 ng.ml)(Zheng, et al End-Rel.Cancer18:491,2011)
MOST DIET-INDUCED OBESITY STUDIES COMPARE ANIMALSMOST DIET-INDUCED OBESITY STUDIES COMPARE ANIMALSFED LOW-FAT vs HIGH-FAT DIETS WITH VERY DIFFERENTFED LOW-FAT vs HIGH-FAT DIETS WITH VERY DIFFERENTNUTRIENT COMPOSITIONSNUTRIENT COMPOSITIONS
OBESITY-PRONE vs OBESITY-OBESITY-PRONE vs OBESITY-RESISTANTRESISTANT MICEMICE• When this approach is used for some strains of rats and mice some will gainweight and become obese = OBESITY-PRONE, while others stay in thesame body weight range of low-fat diet animals = OBESITY-RESISTANT.• This approach allows for comparison of animals all fed the same diet butwith different body weights or to compare those with the same body weightconsuming different diets, i.e., high-fat versus low fat.• We also included the middle group which we termed OVERWEIGHTalthough many researchers in the obesity field do not use this group.Equal body weight oflow fat fed mice.OBESITY-PRONEOBESITY-PRONE OBESITY-RESISTANTOBESITY-RESISTANT
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-fatdiet 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 MTsdetected at this point) were divided intodetected at this point) were divided intoObesity-Prone, Overweight andObesity-Prone, Overweight andObesity-Resistant.Obesity-Resistant.• Followed for MT development until 85Followed for MT development until 85weeks of age.weeks of age.
FINAL BODY WEIGHTS &FINAL BODY WEIGHTS &TUMOR LATENCYTUMOR LATENCYFINAL BODY WEIGHTS OF MICE IN DIET-INDUCED OBESITY EXPERIMENTLOW-FAT OBESITY-RESISTANT OVERWEIGHT OBESITY-PRONE01020304050ANOVA p<0.0001, different superscripts indicate significantlydifferent. n=17/group except LOW-FAT n=25abccGRAMSMAMMARY TUMOR LATENCY FOR TGF-α MICE IN DIET-INDUCEDOBESITY STUDYLOW-FAT OBESITY-RESISTANT OVERWEIGHT OBESITY-PRONE01020304050607080aANOVA p<0.0001, different superscripts indicate significantlydifferent, n=13-18/group.a,bbcWEEKSOFAGESERUM LEPTIN LEVELS OF TGF-α MICE IN DIET-INDUCED OBESITYEXPERIMENT 2LOW-FAT OBESITY-RESISTANT OVERWEIGHT OBESITY-PRONE01020 ANOVA p<0.0001-Different superscriptsindicate significant differences.ab*,#b*,#b** Students 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=13ng/mlMammary tumor latency inverselyrelated to body weight.Serum leptin levels positivelycorrelated with body weight.Aggressive tumors only found inobesity-prone mice.Cleary et al Int.J.Obesity 2004 and Dogan etal Breast Cancer Res 2007
OTHER DIET-INDUCED OBESITYOTHER DIET-INDUCED OBESITYSTUDIES- MICESTUDIES- MICE• MMTV-neu mice that develop ER- MTs were subjected to theMMTV-neu mice that develop ER- MTs were subjected to thesame dietary protocol but there was little effect of diet-inducedsame dietary protocol but there was little effect of diet-inducedobesity 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 bodyweight status. Although time to developing first MT was notweight status. Although time to developing first MT was notdifferent, second tumors developed sooner in the high fat dietdifferent, second tumors developed sooner in the high fat dietmice and average # MT/mouse was increasedmice and average # MT/mouse was increased (Khalid et al BCRT(Khalid et al BCRT122:647,2010122:647,2010).).• To directly compare ER+ to ER- MT developmentTo directly compare ER+ to ER- MT developmentimmunocompromised 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-7or MDA-MB-231 cells, but neither cell line was very responsiveor MDA-MB-231 cells, but neither cell line was very responsivein this approachin this approach (Ray et al Cancer Letters 253:291 (2007)(Ray et al Cancer Letters 253:291 (2007) ..
OBESITY-PRONE VS OBESITY-RESISTANTOBESITY-PRONE VS OBESITY-RESISTANTRATS AND MAMMARY TUMOR PROGRESSIONRATS AND MAMMARY TUMOR PROGRESSION• Wistar rats were fed 46% fat diet and injectedwith MNU at ~7 weeks of age.• At 19 weeks of age a high fat diet rats dividedinto Lean, Mid-weight and Obese.• OVX at ~24 weeks of age @ which time tumorincidence and burden were similar in the threegroups.• With OVX some tumors regressed, but half asmany in Obese rats regressed and many morenew ones appeared compared to Lean rats(MacLean et al Obesity 18:696, 2010).
SECOND STUDY OBESITY-SECOND STUDY OBESITY-PRONE RATSPRONE RATS• High fat diet fed rats were further divided by energyintake minus energy expenditure to identify High Energyvs Low Energy groups.• Obesity increased tumor PR expression (before OVX)-samples obtained by FNA.• At study termination MT PR expression in Obese-HighEnergy Excess group still higher than in Lean-HighEnergy Excess group.• Also at termination MTs from Obese-High EnergyExcess rats had twice the glucose uptake as the ObeseLow Energy and the comparable lean groups.(Giles et al Cancer Res. 72:6490, 2012)
WILL INTERVENTIONS AFFECT THEWILL INTERVENTIONS AFFECT THEDEVELOPMENT AND/OR PROGRESSION OFDEVELOPMENT AND/OR PROGRESSION OFMAMMARY TUMORS IN OBESE ANIMALS?MAMMARY TUMORS IN OBESE ANIMALS?• It is well-documented that calorie restriction prevents and/or delaysmammary tumor development in normal weight rodents.• We and others have reported that leptin and IGF-I are consistentlylowered by calorie restriction again in normal weight rodents inconjunction with the prevention of MTs.• Other interventions have also been implemented in normal weightrodents, i.e., various chemopreventive agents, fatty acids andpharmaceutical agents to prevent MT development.• However, few studies have been done with obese animals.• Since preventative approaches in humans would presumably betargeted to at risk populations such as obese women these types ofstudies should be a high priority in particular those affecting bodyweight such as weight loss.→
EFFECT OF CALORIE RESTRICTION IN OBESEEFFECT OF CALORIE RESTRICTION IN OBESERODENTS ON MAMMARY TUMORIGENESIS-RODENTS ON MAMMARY TUMORIGENESIS-EARLY INTERVENTIONSEARLY INTERVENTIONS• There are several older publishedstudies.• C3H mice which develop spontaneousMTs were made obese with GTG at 2months of age and when clearly obeseat 3 months of age were calorierestricted to reduce their body weightsto the level of control mice or allowedto continue to overeat. Average age ofMT onset was 219 days for Obesemice, 267 days for Control/Lean miceand 365 days of Previously Obesemice (Waxler JNCI 14:1253, 1954).• Genetically obese LA/N (corpulentrats) were given DMBA at 65 days ofage and one week later one half wererestricted by 40%. Rats were followedfor a total of 16 wks (Klurfled et alPSEBM 196: 381,1991).Obese ObeseRest.LeanBODYWEIGHT(grams)~550 ~350 ~200MTIncidence100% 27% 21%MT # perrat4.1 1.5 1.0MT weightper rat22.6 9.6 1.7
MORE RECENT INTERVENTIONSMORE RECENT INTERVENTIONSIN OBESE MODELSIN OBESE MODELS• OVX + DIO (60% fat diet) mice for 17 weeks and then wereswitched to low fat diet and rapidly lost weight over three weeksreaching body weight of mice always fed the low fat diet at whichtime Wnt-1 tumor cells were injected. Tumors that formed over thenext 15 days were twice as big in the formerly obese mice as thosein the control mice. Data interpretation is somewhat difficult as therewas no obese control group so it is possible that the tumor sizewould have been reduced compared to them (DeAngelMol.Carcinogenesis 2012).• OVX+DIO mouse model after 10 weeks were calorie restricted by30% and followed for another 7 or 14 weeks. Not a MT study butassessment of factors associated with inflammation weredetermined in mammary tissue and many were reversed by calorierestriction such as NF-κB. TNF-α, IL-1β,COX-2, aromatase and PRexpression and also detection of crown-like-structures (CLS)reduced (Bhardwaj, et al CPR in press, 2013).
CALORIE RESTRICTIONCALORIE RESTRICTIONMIMETICS- METFORMINMIMETICS- METFORMIN• Human observational studies have indicated that diabetic womentreated with metformin were at decreased risk of breast cancer, andif diagnosed with breast cancer women using metformin were moreresponsive to cancer treatments.• In addition, in vitro studies and preclinical rodent studies havefurther indicated that metformin may have anticancer effects.• However the majority of preclinical studies have been done innormal weight animals.• One recent study using Obesity-Prone rats administered MNU toinduce MTs which I described earlier for effects on calorie restrictionalso presented results for short-term metformin (2mg/mL in drinkingwater) starting one wk before OVX and continued for three weeks.
EFFECTS OF METFORMIN ONEFFECTS OF METFORMIN ONMAMMARY TUMORS IN OBESE RATSMAMMARY TUMORS IN OBESE RATSA. Tumor burden aftertreatment withmetformin for 3 wkpost-OVX.B. Tumor receptor statusafter 1 wk ofmetformin.(*p<0.05)From Giles et al CancerRes.72:6490, 2012)DID not comparemetformin to calorierestriction.
ON GOING STUDY TO COMPARE METFORMINON GOING STUDY TO COMPARE METFORMINDIRECTLY TO CALORIE RESTRICTION ON MTDIRECTLY TO CALORIE RESTRICTION ON MTDEVELOPMENT IN OBESE VS LEAN MICEDEVELOPMENT IN OBESE VS LEAN MICE• MMTV-TGF-α mice consume low or high fat diets until 30 weeks ofage.• 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 givenmetformin at a dose of 250 mg/kg body weight/day.• The mice will then be followed until 90 weeks of age to assess theimpact of the interventions on mammary tumor development.• Serum samples are being taken over the course of the study toassess metformin levels and for measurements of adipokines,insulin and IGF-I.• At termination serum and tissues will be obtained.
BODY WEIGHT OF CALORIEBODY WEIGHT OF CALORIERESTRICTED AND METFORMINRESTRICTED AND METFORMINTREATED MICETREATED MICE15.0020.0025.0030.0035.0040.000 10 20 30 40 50 60 70 80 90Weeks of AgeGramsLF-ALLF MetLF-CCRHF-ALHF-MetHF-CCRInterventions started at 30 weeks of age↑
SUMMARYSUMMARY• Evidence that leptin may be a growth factor for mammary cancer cells.• Recent study reported that prediagnosis leptin and leptin:adiponectin ratiowere elevated prior to postmenopausal breast cancer diagnosis (Ollberding etal CPR 6:188,2013).• Several research groups developing analogues/antagonists of leptin andleptin receptor for therapeutic uses.• Adiponectin another adipokine may also play a role in mammarytumorigenesis.• The ratio of adiponectin to leptin is being considered as a more specificindicator 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 andshould be supported to provide insights into how interventions can beapplied to at risk humans.• NCI does seem to be taking this seriously given that several of theProvactive Questions are targeting obesity.
HOW CAN WE APPLY STUDIES OFHOW CAN WE APPLY STUDIES OFPHYSICAL ACTIVITY TO PRECLINICALPHYSICAL ACTIVITY TO PRECLINICALOBESITY STUDIES?OBESITY STUDIES?
CO-WORKERS and FINANCIALSUPPORTAmitahba RayKatai J. NkhataNancy K. MizunoXin HuMichael E. GrossmannSoner DoganOlga P. RogozinaJill ChavezDavid PotterNita J. MaihleSUPPORTDOD Breast Cancer ProgramEagles Cancer TelethonThe Hormel FoundationThe Breast Cancer ResearchFoundationNational Cancer Institute- NIH