energy balance modulates mouse skin tumor promotion ......described (5, 21, 24). groups were...

Research Article

Energy Balance Modulates Mouse Skin Tumor Promotionthrough Altered IGF-1R and EGFR Crosstalk

Tricia Moore1, Linda Beltran1, Steve Carbajal1, Stephen D. Hursting2, and John DiGiovanni1,2

AbstractObesity, an established risk factor for epithelial cancers, remains prevalent in the United States andmany

other countries. In contrast to positive energy balance states (overweight, obesity), calorie restriction (CR)

has been shown to act as a universal inhibitor of tumorigenesis inmultiple animalmodels of human cancer.

Unfortunately, themechanismsunderlying the enhancing effects of obesity or the inhibitory effects ofCRon

cancer etiology remain elusive. Here, we evaluated the impact of dietary energy balance manipulation on

epithelial carcinogenesis and identified several potential mechanisms that may account for the differential

effects of obesity andCRon cancer.Obesity enhanced tumor promotion during epithelial carcinogenesis, in

part, due to altered insulin-like growth factor-1 receptor (IGF-1R)/EGF receptor (EGFR) crosstalk and

downstream signaling to effectors such as Akt/mTOR. Obesity-induced changes in cellular signaling

subsequently led to altered levels of cell-cycle proteins that favored enhanced epidermal proliferation

during tumor promotion. In contrast, CR reduced susceptibility to tumor promotion, attenuated IGF-1R/

EGFR crosstalk and downstream signaling, and altered levels of cell-cycle proteins that favored reduced

epidermal proliferation during tumor promotion. Collectively, these findings suggest potential targets for

the prevention of epithelial cancers, as well as for reversal of obesity-mediated cancer development and

progression. Cancer Prev Res; 5(10); 1236–46. �2012 AACR.

IntroductionEnergy balance refers to the relationship between caloric

consumption and energy expenditure (1). Epidemiologicand animal studies have established a direct correlationbetween positive energy balance states (e.g., overweight,obesity) and risk of developing multiple cancers (2–5). Incontrast, negative energy balance states, such as calorierestriction (CR), have been shown to consistently inhibittumorigenesis in animal models regardless of mode oftumor induction (1, 6–8). Previous studies have evaluatedthe impact of fat consumption and/or CR on 2-stage skincarcinogenesis (8–10). This model of chemically inducedepithelial carcinogenesis enables mechanistic evaluation ofdietary manipulation during all stages of tumor develop-ment, including tumor initiation, promotion, and progres-sion (11). Boutwell (8) found that CR inhibited skin tumordevelopment using the 2-stage carcinogenesis protocol, andBirt and colleagues (9, 10) showed that 40%CRconsistently

inhibited tumor promotion by 12-O-tetradecanoylphor-bol-13-acetate (TPA), with little or no effects on eithertumor initiation or tumor progression. Although thesefindings indicated a role for energy balance in the modu-lation of 2-stage skin carcinogenesis, especially duringtumor promotion, the effect of diet-induced obesity (DIO)per se has not been adequately studied in this model.

Several mechanisms have been proposed to explain theinhibitory effects of CR on tumor development (12, 13)including elevated serum corticosterone (6, 14) and altera-tions in cellular signaling. TPA-induced epidermal AP-1activation and extracellular signal–regulated kinase (ERK)phosphorylation was reduced in mice subject to 40%CR (15–17). Xie and colleagues (18) reported a reductionin TPA-mediated activation of epidermal phosphoinositide3-kinase (PI3K) and Ras signaling following 20% CR.Recently, we reported that both CR and DIO modulatedsteady-state growth factor signaling pathways in mouseepidermis, liver, and prostate (19). Additional studiessuggested that these effects may be mediated by diet-induced changes in circulating insulin-like growth factor-1 (IGF-1) levels and altered IGF-1 receptor (IGF-1R)signaling (19, 20).

Diets of varying caloric density were used to inducechanges in body mass and body fat content to determinethe impact of both positive and negative energy balance onthe promotion stage of multistage epithelial carcinogenesisin mouse skin. We provide novel evidence showing thatdietary energy balance affects susceptibility to epithelialcarcinogenesis, in part, through diet-induced changes in

Authors' Affiliations: 1Division of Pharmacology and Toxicology, Collegeof Pharmacy, and 2Department of Nutritional Sciences, College of NaturalSciences, The University of Texas at Austin, Austin, Texas

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: John DiGiovanni, Dell Pediatric Research Insti-tute, The University of Texas at Austin, 1400 Barbara Jordan Blvd. Austin,TX 78723. Phone: 512-495-4726; Fax: 512-495-4945; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-12-0234

�2012 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 5(10) October 20121236

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IGF-1R and EGF receptor (EGFR) crosstalk and downstreamsignaling during tumor promotion.

Materials and MethodsAnimalsICR female mice (3–4 weeks of age, Harlan Teklad) were

group housed; however, mice maintained on a 30% CRregimen were separated for 1 hour for feeding. Mice wereweighed before randomization and then every 2 weeks forthe duration of the experiments.

DietsFour diets ranging in caloric density were used (Research

Diets) and have been previously described: 30% CR diet,15% CR diet, 10 kcal% fat (ad libitum), and 60 Kcal% fat (adlibitum; ref. 1). CR was achieved by administering a dailyaliquot equivalent to 70% or 85% of the daily energy con-sumedby the 10 kcal% fat group, as previously detailed (19).

Serum analysis and body fat compositionBlood was collected immediately following CO2 asphyx-

iation, serum was prepared, and serum IGF-1, insulin,leptin, and adiponectin levels were determined as previ-ously described (19, 21). Corticosterone levels were deter-mined by ELISA (Kamiya Biomedical Co.). Percentage ofbody fat was determined as previously described (22).

Two-stage skin carcinogenesisICR female mice were placed on the 10 kcal% fat control

diet at 7 weeks of age and initiated with 25 nmol of 7,12-dimethylbenz[a]anthracene (DMBA; Eastman Kodak Co.).Fourweeks following initiation,micewere randomized andplaced on the 4 diets (n ¼ 30 per group). Four weeks later,mice received twiceweekly topical treatments of 3.4 nmol ofTPA (Alexis Biochemicals) for 50 weeks. Tumor incidence(percentage of mice with papillomas) and tumor multiplic-ity (average number of tumors permouse) were determinedweekly until multiplicity reached a plateau (29 weeks).Carcinoma incidence and average carcinomas per mousewere determined weekly from initial detection until 50weeks of tumor promotion. Squamous cell carcinomas(SCC) were confirmed by histopathology.

Epidermal hyperproliferationFemale ICRmice were maintained on the diets described

above for 15 weeks, after which they were treated twiceweekly for 2 weeks with either acetone vehicle or 3.4 nmolof TPA (n ¼ 3 per group). Mice were injected with bromo-deoxyuridine (BrdUrd; Sigma Aldrich; 100 mg/g bodyweight) 30 minutes before sacrifice. Whole skin sectionswere excised, processed, and evaluated for epidermal thick-ness and labeling index as previously described (20).

Epidermal protein lysatesFor short-term in vivo experiments, ICR mice were main-

tained on the diets for 15 weeks, after which they received asingle application of either acetone (vehicle) or 3.4 nmol of

TPA. Mice were killed either 6 (acetone, TPA) or 18 hours(TPA) after treatment, epidermis was scraped and proteinlysates were prepared as previously described (20).

Cell cultureC50 cells are a nontumorigenic keratinocyte cell line

derived from spontaneously immortalized normal mousekeratinocytes. The cells were obtained from Dr. SusanFischer (UTMDACC, Houston, TX) and were cultured aspreviously described (23) with no further characterizations.When cells reached approximately 80% confluency, theywere serum and growth factor starved for 24 hours. Plateswere then stimulated with either 25 ng/mL recombinanthuman IGF-1 (rhIGF-1; Sigma Aldrich) or 10 ng/mL EGF(BD Biosciences) and harvested at multiple time points forprotein or RNA isolation.

Immunoprecipitation and Western blot analysisImmunoprecipitation and Western blot analyses were

conducted using lysates prepared from either epidermis orcultured keratinocytes. For co-immunoprecipitation experi-ments, lysates were precipitated with IGF-1R (Cell Signal-ing) or EGFR (Millipore) antibodies using the DynabeadProtein G IP Kit (Invitrogen). Western blot analyses wereconducted as previously described (20).

Real-time quantitative reverse transcriptase PCRFor in vivo experiments, ICRmice weremaintained on the

diets for 15 weeks, after which they received a single appli-cation of either acetone vehicle or 3.4 nmol of TPA.Mice were killed either 6 (acetone, TPA) or 18 hours (TPA)after treatment and epidermiswas scraped for RNA isolation(n ¼ 3 per group). For in vitro experiments, C50 cells werestimulatedwith either IGF-1 or EGF, harvested for RNA, andRNAwas isolated using theQiagen RNeasy ProtectMini Kit.RNA quality was assessed using the Agilent 2100 Bioana-lyzer (Agilent Technologies, Inc.), and quantitative PCR(qPCR) was carried out, as previously described, usingassays on demand specific to TGF-a,HB-EGF, amphiregulin,and EGFR (21). RNA was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All measurementswere duplicated.

ResultsDietary energy balance effects on skin tumorpromotion

A 2-stage skin carcinogenesis experiment was conductedusing 4 experimental diet groups to generate lean (30%CR),normal (15% CR), overweight (10 kcal% fat), and obese(DIO, 60 kcal% fat) body phenotypes, as previouslydescribed (5, 21, 24). Groups were initiated with DMBA,maintained on 10 kcal% diet for 4 weeks, randomized intothe experimental groups and promoted twice weekly with3.4 nmol of TPA for 50 weeks. Tumor incidence andmultiplicity (average number of papillomas per mouse)were calculated weekly until the latter reached a plateauat 29 weeks. CR (15% and 30%) significantly inhibited

Energy Balance Modulates IGF-1R/EGFR Crosstalk

www.aacrjournals.org Cancer Prev Res; 5(10) October 2012 1237




papillomamultiplicity when compared with both the over-weight control andDIOgroups (P<0.05;Mann–WhitneyUtest); however, no significant differences were observedbetween the overweight control and DIO groups (Fig. 1A;Supplementary Table S1). Dietary energy balance manipu-lation had no significant effect on papilloma incidenceunder these experimental conditions (Fig. 1B; Supplemen-tary Table S1).

Treatments continued for an additional 21 weeks toevaluate the impact of dietary energy balance on tumorprogression. As shown in Fig. 1C and D, the number andincidence of SCCs was significantly reduced in the 30% CR

group (P < 0.05,Mann–WhitneyU test and P < 0.05, c2 test,respectively), compared with the 15% CR, overweight con-trol and theDIOgroups; however, dietarymanipulation didnot affect the overall rate of malignant conversion (see alsoSupplementary Table S1). A second 2-stage skin carcino-genesis experiment was carried out that yielded nearlyidentical findings (data not shown).

Effect of long-term dietary manipulation on bodyprofiles during two-stage skin carcinogenesis

Body mass, percentage of body fat, and circulatoryprotein levels (i.e., IGF-1, insulin, leptin, adiponectin)

Figure 1. Effect of dietary energybalance on 2-stage skincarcinogenesis and TPA-inducedepidermal proliferation. Two-stageskin carcinogenesis wasconducted using lean (30% CR),normal (15% CR), overweightcontrol (10 kcal% fat), and obese(60 kcal% fat) mice (n ¼ 30 pergroup). A, tumor multiplicity; CR(30%, 15%) significantly reducedtumor multiplicity (P < 0.05, Mann–Whitney U test). B, tumorincidence. C, average carcinomasper mouse; 30% CR significantlyreduced, whereas DIO (60 kcal%)significantly increased the averagenumber of carcinomas per mouse(P < 0.05,Mann–WhitneyU test). D,carcinoma incidence. CR (30%,15%) significantly reducedcarcinoma incidence (P < 0.05, c2

test). E and F, ICR female micewere maintained on the four dietsfor 15 weeks and were treated witheither acetone (white bars) or 3.4nmol TPA (black bars), twiceweekly for 2 weeks (n ¼ 3 pergroup). E, representative BrdUrd-stained skin sections. F, epidermalthickness (top) and percent BrdUrdincorporation (bottom).�, statistically different from allsimilarly treated groups; a,significantly different from similarlylabeled values (P < 0.05, Mann–Whitney U test).

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were evaluated at the conclusion of the 2-stage skin carci-nogenesis study (Supplementary Table S2.I). Body massand percentage of body fat were significantly differentamong all groups (P < 0.05, Mann–Whitney U test). CR(both 30% and 15%) significantly reduced levels of circu-lating IGF-1, insulin, and leptin. In contrast, CR increasedlevels of adiponectin, as compared with both the over-weight control and DIO groups; however, only the 30%CR group yielded a statistically significant difference (P <0.05,Mann–WhitneyU test). Despite significant differencesin bothbodymass andpercentage of body fat, no significantdifferences in the levels of circulatory proteins wereobserved between the overweight control and DIO groupsat the end of the 50-week experimental period.

Dietary energy balance modulates TPA-inducedepidermal hyperproliferationTo evaluate potential mechanisms whereby dietary ener-

gy balancemodulates 2-stage skin carcinogenesis, addition-al mice were maintained on the 4 diets for 15 weeks, thusgenerating lean, normal, overweight, and obese body phe-notypes. Body mass, percentage of body fat, and levels ofcirculatory proteins were then determined at this time point(Supplementary Table S2.II). Body mass and percentage ofbody fat correlated directlywith caloric consumption, yield-ing significant differences in both parameters among allgroups (P< 0.05,Mann–WhitneyU test). Similarly, levels ofIGF-1, insulin, leptin, and adiponectin were significantlydifferent across the groups, with the greatest differencesoccurring between the caloric extremes (P < 0.05, Mann–Whitney U test). Specifically, CR reduced whereas DIOincreased levels of circulating IGF-1, insulin, and leptin,findings consistent with our previous studies (19). Levels ofadiponectin inversely correlated with caloric consumption,with significant differences occurring between each CRgroup and the overweight control and DIO groups (P <0.05, Mann–Whitney U test). In addition, 30% CR signif-icantly increased circulating corticosterone levels relative toboth the overweight control and DIO groups, however, noother statistically significant differences in corticosteronelevels were observed.Given the effect of dietary energy balance on tumor

promotion, we evaluated diet-induced changes in the epi-dermal proliferative response following TPA treatment.Female ICR mice maintained on the 4 diets for 15 weekswere treated twice weekly for 2 weeks with either acetone or3.4 nmol of TPA. Groups of mice were killed 48 hoursfollowing the final treatment and whole skin sections wereremoved and processed. Epidermal hyperplasia (epidermalthickness) and labeling index [LI; BrdUrd incorporation]were determined as previously described (20). Figure 1Eshows representative BrdUrd-stained skin sections, corre-sponding to both acetone and TPA-treated skins excisedfrom lean, normal, overweight, and obese mice and quan-titative analyses are shown in Fig. 1F. In the absence of TPAtreatment, 30% CR significantly reduced both epidermalthickness and LI, when compared with all other groups;however, 15% CR only significantly reduced both para-

meters when compared with the DIO group (P < 0.05,Mann–Whitney U test). No differences in either epidermalthickness or LI were observed between the overweightcontrol and DIO groups in the absence of TPA treatment.In contrast, both epidermal thickness and LI were signifi-cantly different among all dietary groups following TPAtreatment, with the greatest differences again occurringbetween the 30% CR and DIO groups (P < 0.05, Mann–Whitney U test).

Effect of dietary manipulation on growth factorsignaling and cell-cycle regulatory proteins duringtumor promotion

Diet-induced changes in growth factor signaling path-ways were also assessed. ICRmiceweremaintained on the 4diets for 15weeks, treatedwith a single application of eitheracetone or 3.4 nmol of TPA, and killed 6 hours later.Epidermal lysates were prepared for Western blot analysesas previously described (19, 20). As shown in Fig. 2A,phosphorylation of the EGFR, IGF-1R, and downstreamsignaling targets (i.e., Akt, mTOR, S6 ribosomal, GSK-3b,Foxo3a, Stat3) correlated directly with caloric density in theabsence of TPA treatment, corroborating our previous find-ings indicating that dietary energy balance modulatedsteady-state growth factor signaling (19). Diet-inducedchanges in EGFR/IGF-1R activation and downstream sig-naling were further enhanced following TPA treatment,with significant differences in phosphorylation occurringbetween the 30%CR andDIOgroups (Fig. 2A). Specifically,30% CR significantly reduced activation and/or phosphor-ylation of the EGFR, IGF-1R, Akt, mTOR, S6 ribosomal,GSK-3b, Foxo3a, and Stat3, as compared with the DIOgroup (P < 0.05, Student t test; Fig. 2A).

We also evaluated diet-induced changes in cell-cycleregulatory proteins. Epidermal protein lysates were pre-pared as above with the addition of an 18-hour time point.Initially, we analyzed changes in positive cell-cycle regula-tory protein levels. CR reduced epidermal levels of cyclinD1, cyclin E, cyclin A, and c-myc compared with DIO;however, these differences were not statistically significant(Fig. 2B). TPA treatment led to increased levels of cyclin D1(18hours), cyclin E (6, 18hours), cyclin A (6, 18 hours) andc-myc (6, 18 hours; ref. 25; see Fig. 2B). The TPA-mediatedincreases in epidermal cyclin D1, cyclin E, cyclin A, and c-myc were significantly attenuated by 30% CR, comparedwith DIO, at 18 hours (P < 0.05, Student t test). Diet-induced changes in cyclin A and c-myc levels followingTPA treatment were also significant at the 6-hour time point(P < 0.05, Student t test).

Additional analyses were conducted to determine theimpact of dietary energy balance on levels of negativecell-cycle regulatory proteins. Levels of both p27 and p21were significantly higher in 30% CRmice (vehicle control),as compared with DIO mice (Fig. 2C). Following TPAtreatment (6 hours), both p27 and p21 levels were signif-icantly higher in mice maintained on the 30% CR diet, ascompared with mice maintained on the DIO diet (P < 0.05,Student t test). A similar trend was observed at 18 hours,






although these differences were not significantly different.Preliminary experiments (data not shown) showedincreased nuclear localization of both p27 and p21 in theepidermis of CR mice, as compared with mice maintainedon the DIO regimen, suggesting that dietary energy balancemay also modulate these negative cell-cycle regulatoryproteins through additional mechanisms.

Activation of the IGF-1R stimulates IGF-1R/EGFRcrosstalk in cultured mouse keratinocytes

Both our current and previous data suggested a role foraltered IGF-1 levels in modulating the effect of dietaryenergy balance on epithelial carcinogenesis. A reduction incirculating IGF-1, either due to genetic (LID mouse model)or dietary manipulation (CR) attenuated both IGF-1R andEGFR signaling (refs. 19, 20; Fig. 2), indicating a potential

for crosstalk between these 2 receptors. C50 cells were usedto examine the role of IGF-1 inmodulating both IGF-1R andEGFR signaling and crosstalk, as well as erbB2 activation.Cultured cells were stimulated with IGF-1 (25 ng/mL),harvested at multiple time points (0–120 minutes), andanalyzed for IGF-1R activation (measured by IRS-1 phos-phorylation) and for EGFR and erbB2 activation (measuredby phosphorylation) using Western blot analysis. IGF-1treatment significantly induced activation of all 3 cell sur-face receptors (Fig. 3). EGFR phosphorylation was rapid,with maximal activation occurring within 5 minutes andphosphorylation was maintained above the basal level upto 120 minutes. EGFR phosphorylation correlated directlywith IGF-1–induced activation of the IGF-1R. Rapid phos-phorylation of erbB2 also paralleled both IGF-1R and EGFRactivation; however, erbB2 activation was maintained at a

Figure 2. Effect of dietarymanipulation on growth factorsignaling and cell-cycle regulatoryproteins in epidermis followingtreatment with TPA. ICR femalemice maintained on the 4 diets for15weekswere treatedwith a singleapplication of acetone (Ace) or 3.4nmol TPA and killed 6 hours(growth factor signaling, cell cycle)or 18 hours (cell cycle) aftertreatment (n¼6). Epidermal lysateswere pooled for Western blotanalyses. A, growth factorsignaling changes in epidermis 6hours after TPA treatment (left) anddensitometry graph representingmean � SEM of 3 independentexperiments (right; 30% CRacetone, light gray bars; 60 kcal%acetone, dark gray bars; 30% CRTPA, white bars; 60 Kcal% TPA,black bars). Western blot data werenormalized to both actin and totalprotein. a–d, significantly differentfrom similarly labeled values(P < 0.05, Student t test). Changesin positive (B) and negative (C) cell-cycle regulatory proteins at 6 and18 hours following TPA treatment.Densitometry quantitation graphs(shown to the right of the Westernblot analyses) in B and C representmean � SEM of 3 independentexperiments (30% CR, white bars;60 Kcal%, black bars) in whichWestern blot data were normalizedto actin. �, significantly differentfrom the corresponding time point(P < 0.05, Student t test).

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consistent level across all time points examined. Stimula-tion of C50 cells with EGF (10 ng/mL) over the same timecourse led to rapid activation of both the EGFR and erbB2;however, there was no significant effect on IGF-1R activa-tion (Fig. 3).Previous studies have identified several potential

mechanisms for IGF-1R and EGFR crosstalk (26–30). Theability of IGF-1 to induce heterodimerization betweenthe IGF-1R and the EGFR was evaluated in co-immunopre-cipitation experiments carried out using lysates preparedfrom C50 cells treated with either IGF-1 or EGF. As shownin Fig. 4A, IGF-1 treatment induced a statistically significantincrease in IGF-1R/EGFR association from 5 to 60 minutes.In contrast, EGFhadnoobservable effect. Additional experi-ments were carried out to determine the impact of IGF-1stimulation on levels of mRNA for the EGFR and EGFRligands. qPCR was carried out using RNA extractedfromC50 cells (stimulatedwith IGF-1 or EGF and harvestedat 0–120 minutes) to determine the relative expression ofEGFR, TGF-a, HB-EGF, and amphiregulin. Both IGF-1 andEGF stimulation significantly increased expression of HB-EGF and amphiregulin, with a greater induction occurringwith EGF treatment (Fig. 4B). These relative increases inmRNA expression, however, were observed only at later

time points (� 60 minutes). No increases in TGF-a andEGFR mRNA expression were observed following stimula-tion of C50 cells with either IGF-1 or EGF (Fig. 4B).

Dietary energy balance alters IGF-1R/EGFR crosstalkin vivo

Finally, we evaluated the impact of dietary energy balancemanipulation on IGF-1R/EGFR crosstalk during tumorpromotion in mouse epidermis in vivo. Female ICR micewere maintained on the previously described 30% CR andDIO diets for 15 weeks. These lean and obese mice werethen treatedwith a single application of acetone (vehicle) or3.4 nmol of TPA and killed 6 or 18 hours later. Epidermallysates and epidermal RNA were prepared for Western blotand qPCR analyses, respectively. Co-immunoprecipitationexperiments were carried out to evaluate diet-inducedchanges in IGF-1R/EGFR heterodimerization. As shownin Fig. 5A, there was a greater degree of association betweenthe IGF-1R and EGFR in obese mice relative to CR mice inthe absence of TPA treatment. Furthermore, TPA (6 hours)induced a significant increase in IGF-1R and EGFR associ-ation in both the CR and obese mice although the relativeincrease in heterodimerization was significantly greater inobese mice (P < 0.05, Student t test).

Figure 3. Effect of IGF-1 and EGF on receptor activation in keratinocytes. C50 cells were serum and growth factor starved for 24 hours, stimulated with eitherIGF-1 (25 ng/mL) or EGF (10 ng/mL) and harvested at multiple time points (0–120 minutes). A, Western blot analyses evaluating EGFR, erbB2, andIGF-1R activation in cell lysates. B, Quantitation. Data shown represents mean � SEM of 3 independent experiments (IRS-1, white bars; EGFR,black bars; erbB2, gray bars). IGF-1 stimulation significantly increased activation of all targets at all time points, whereas EGF stimulation significantlyincreased EGFR and erbB2 activation at all time points (P < 0.05, Student t test).






Dietary manipulation did not modulate mRNA levels ofEGFR and EGFR ligands in the absence of TPA treatment;however, DIO significantly increased levels of mRNA forEGFR ligands compared with 30% CR, following treatmentwith TPA (Fig. 5B). Specifically, DIO significantly increasedmRNA levels of TGF-a (18 hours), HB-EGF (6 hours), andamphiregulin (6 hours) following TPA treatment. EGFRmRNA levels were not affected by diet or treatment withTPA. Collectively, the in vitro and in vivo results suggestthat dietary energy balance modulates IGF-1R/EGFR cross-talk, at least in part, due to diet-induced changes in mRNAlevels of EFGR ligands and changes in IGF-1R and EGFRheterodimerization.

DiscussionIn this study, potential mechanism(s) underlying the

effects of dietary energy balancemanipulationon skin tumorformation during the promotion phase of 2-stage skin car-cinogenesis were examined. Consistent with previously pub-lisheddata, both15%and30%CRsignificantly inhibited the

promotion of papillomas by TPA compared with both theoverweight control and DIO groups but had no significanteffect on tumor progression (8–10). Notably, no significantdifference in tumor response was seen between the DIO andoverweight control groups, although the DIO regimen sig-nificantly increased both body mass and percentage of bodyfat. Previous mammary carcinogenesis studies also reportedthat despite significant differences in body mass, no signif-icant increase in tumor incidence occurred when dietary fatconsumptionexceeded20Kcal% to30Kcal%(31–33). Thesedata support the hypothesis that in some model systems,there is an upper limit to the effects of a positive energybalance state above which further increases in total caloriesdo not further increase tumor response.

Genetic reduction of circulating IGF-1 levels (i.e., LIDmouse model) attenuated TPA-induced epidermal hyper-proliferation,which correlatedwith reduced responsivenessto skin tumor promotion by TPA (20). In comparison to theoverweight control andDIOgroups, both the 30%and15%CR groups had significantly reduced epidermal thicknessand LI in the absence of TPA. Following TPA treatment, we

Figure 4. Effect of IGF-1 and EGFon IGF-1R/EGFR association andEGFR ligand mRNA levels. C50cells were cultured andharvested as described, andlysate was prepared for co-immunoprecipitation experimentsandmRNA expression analyses. A,effect of IGF-1 and EGF stimulationon IGF-1R/EGFR association.Right, relative quantitation of mean� SEM of 3 independentexperiments (IGF-1, white bars;EGF, black bars). EGFR and IGF-1R were normalized to IgG andthen to each other. �, significantincrease in IGF-1R/EGFRassociation, relative to the0 time point (P < 0.05, Student ttest). B, effect of IGF-1 (white bars)andEGF (black bars) stimulation onEGFR and EGFR ligand (TGF-a,HB-EGF, amphiregulin, and EGFR)mRNA expression by qPCRanalysis. Data shown representsmean � SEM of 3 independentexperiments. �, significant increasein EGFR ligand mRNA expressionfollowing EGF stimulation;��, significant increase in EGFRligand mRNA following both IGF-1and EGF stimulation (P < 0.05,Student t test).

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observed a significantly progressive effect of dietary energybalance across all the diet groups on the epidermal prolif-erative response, with 30% CR exhibiting the lowestresponse and DIO exhibiting the highest. Several otherstudies have shown that energy balance impacts cellularproliferation in both normal and tumorigenic tissue (e.g.,mammary, colon, liver, epidermis, and bladder; refs. 34–38). Collectively, our results suggest that manipulatingdietary energy balance impacts TPA-mediated epidermalhyperproliferation, thus modulating epithelial carcinogen-esis in mouse skin.Previous studies have indicated an important role for IGF-

1R and EGFR activation in TPA-induced epidermal hyper-proliferation and tumor promotion in mouse skin(reviewed in ref. 39). Inhibition of TPA-induced EGFRactivation in vivo, using either RG13022 (tyrosine kinaseinhibitor) orGW2974 (dual specific erbB2/EGFR inhibitor)significantly reduced TPA-induced epidermal hyperproli-feration aswell as skin tumorpromotion (GW2974; refs. 40,41). In addition, epidermal IGF-1 overexpression increasedepidermal proliferation, both in the presence and absenceof TPA, and increased sensitivity to 2-stage skin carcino-genesis (42, 43). Furthermore, dietary energy balanceinduced changes in steady-state activation of these criticalgrowth factor receptors in epidermis and other epithelialtissues of FVB/N and C57BL/6 mice (19). As shown

in Fig. 2A, 30% CR reduced activation and/or phosphory-lation of the IGF-1R, EGFR, and downstream signalingfollowing treatment with TPA compared with the DIOgroup. Xie and colleagues reported a similar reduction inTPA-mediated epidermal Akt activation in CR mice (18).

The impact of dietary manipulation on levels of positiveand negative cell-cycle regulatory proteins was also evalu-ated. Notably, in mice treated with TPA, the levels ofpositive cell-cycle regulatory proteins (cyclin D1, cyclin E,cyclin A, c-myc) correlated directly with caloric consump-tion, whereas the levels of negative cell-cycle regulatoryproteins (i.e., p21, p27) inversely correlated with caloricconsumption. As reported earlier, elevated IGF-1R activa-tion or increasedAkt activity, respectively, leads to increasedlevels of positive cell-cycle regulatory proteins (cyclins D, A,E, c-myc) during tumor promotion, providing amechanismfor the upregulation of cellular proliferation observed inmouse epidermis after treatment with TPA (25, 44). Togeth-er, these observations suggest that dietary energy balance–induced alterations in epidermal proliferation seen duringtumor promotion result from altered upstream growthfactor signaling which modulates levels of both positiveand negative cell-cycle regulatory proteins.

Using C50 cells, we established that IGF-1 treatmentspecifically induced activation of not only the IGF-1R butalso the EGFR and erbB2. Similar to previous studies (29,

Figure 5. Effect of dietarymanipulation on IGF-1R/EGFRheterodimerization and EGFR andEGFR ligand mRNA expression. ICRfemale mice were maintained on a30% CR (white bars) and60 Kcal% fat (black bars) diet andtreated with TPA as described in Fig.2 (n ¼ 6). Epidermal lysates werepooled and prepared forco-immunoprecipitation (IP)experiments and qPCR analysis.A, co-immunoprecipitation withEGFR and subsequent Western blot(WB) analyses for EGFR and IGF-1R(left); representative densitometrygraph (right). EGFR and IGF-1R werenormalized to IgG and then to eachother. Data shown represent mean�SEM of 3 independent experiments.a–c, values with the same letteringindicate statistically significantdifferences (P < 0.05, Student t test).B, qPCR analysis of EGFR and theEGFR ligands TGF-a,HB-EGF, amphiregulin, and EGFR.Data represent mean � SEM.�, significantly different from 30%CRgroup at the corresponding timepoint (P < 0.05, Mann–WhitneyU test).






30), EGF stimulation did not activate the IGF-1R, suggestingthat IGF-1R/EGFR crosstalk may be unidirectional. Thus,IGF-1 modulated activation of the EGFR in cultured kera-tinocytes through multiple mechanisms, including induc-tion of IGF-1R/EGFR heterodimerization and heightenedexpression of EGFR ligand mRNA. Both mechanisms likelycontribute to the sustained activation of the EGFR observedat later time points; however, they do not fully explain therapid induction of EGFR and erbB2 phosphorylationobserved following IGF-1 stimulation. EGFR phosphoryla-tion may also occur as a result of IGF-1–mediated ectodo-main shedding of preformed, membrane-bound EGFRligands (29, 30, 45), whichmay explain the early activationof the EGFRby IGF-1 observed in our studies. The activation(phosphorylation) of erbB2 seen in IGF-1–treated cells islikely due to EGFR:erbB2 heterodimerization, althoughwe cannot completely rule out the possibility that othermechanisms may be involved, including heterodimeriza-tion between IGF-1R and erbB2 (46, 47). Finally, weconducted in vivo experiments to determine whetherdietary energy balance modulates IGF-1R/EGFR crosstalk.CR (30%) not only reduced IGF-1R/EGFR heterodimeriza-tion in epidermis following TPA treatment but also signif-icantly reduced mRNA expression of EGFR ligands, ascompared with DIO. To our knowledge, this is the firstreport showing that dietary energy balance impacts IGF-1Rand EGFR crosstalk. These diet-related changes in receptorcrosstalk account, at least in part, for the observedreduction (CR) or increase (DIO) in epidermal signalingthat controls the proliferative response to TPAduring tumorpromotion.

In the current study, the 2-stage skin carcinogenesismodel was used primarily as a general model of epithelialcarcinogenesis to evaluate mechanisms associated withdietary energy balance manipulation. Nevertheless, theobserved changes may also be relevant to UVB-mediatedskin carcinogenesis. In this regard, diet-induced obesity hasbeen shown to increase UVB-mediated inflammation aswell as inflammation and cell survival signaling (48). Aktactivation (phosphorylation) was found to be elevated inthe epidermis of obesemice exposed toUVB comparedwithmice on a control diet in this study. In a model of leptindeficiency-induced obesity (i.e., ob/ob mice), similarincreases in UVB-induced inflammation signaling (NF-kB,COX-2, TNFa) and survival signaling (Akt activation) wereobserved compared with wild-type mice (49). Elevatedlevels of proliferating cell nuclear antigen (PCNA) andcyclin D1 were also observed in epidermis of the obesemice following exposure toUVB. Thus, a number of changessimilar to those seen in our current study have beenobserved in skin/epidermis of obese mice (either diet-induced or genetically induced obesity) following exposureto UVB. In a study by Hopper and colleagues (50), CR wasreported to inhibitUVB-inducedupregulationof AP-1:DNAbinding and UVB-induced alteration in AP-1 constituentprotein levels suggesting that CR inhibited events associatedwith UVB-mediated skin tumor promotion. Only limitedinformation is available on the potential link between

dietary energy balance and non-melanoma skin cancer(NMSC) in humans (reviewed in ref. 51); however, strongerevidence points to a relationship between obesity andmelanoma skin cancer (reviewed in ref. 51). Further studiesevaluating the role of dietary energy balance in bothNMSCsand melanoma skin cancer seem warranted on the basis ofthe data presented in our current studies as well as thosepublished on UV effects in mouse models of obesity asnoted above.

In conclusion, we have shown that CR reduced, whereasDIO increased, signaling through the IGF-1R and EGFRfollowing TPA treatment. Both in vitro and in vivo studies(20) suggest that levels of circulating IGF-1, which aremodulated by dietary energy balance, regulate activationof the IGF-1R which in turn regulates crosstalk with theEGFR (and erbB2). These diet-induced changes in IGF-1Rand EGFR activation subsequently altered downstreamsignaling to Akt andmTOR (and other signaling pathways),thus modulating levels and activity of cell-cycle regulatoryproteins. These changes in cell-cycle regulatory proteinscorrelated directly with diet-induced changes in TPA-induced epidermal proliferation. Supplementary FigureS1 summarizes the effects of dietary energy balance onsignaling pathways and cell-cycle proteins observed in thepresent study in relation to altered epidermal proliferationand tumor promotion. Taken together, these effects ofdietary energy balance on IGF-1R/EGFR signaling and cross-talk, cell-cycle regulation, and epidermal proliferation pro-vide a plausible mechanism for the inhibitory effects ofnegative energy balance and the enhancing effects of pos-itive energy balance on susceptibility to skin tumor promo-tion during 2-stage skin carcinogenesis. These studies pro-vide new information about potential molecular targets forprevention of epithelial cancers and for reversing the effectsof obesity on cancer development.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Moore, S.D. Hursting, J. DiGiovanniDevelopment of methodology: T. Moore, J. DiGiovanniAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): T. Moore, L. Beltran, S. Carbajal, J. DiGiovanniAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): T. Moore, S.D. Hursting, J. DiGiovanniWriting, review, and/or revision of themanuscript: T. Moore, L. Beltran,S. Carbajal, S.D. Hursting, J. DiGiovanniAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T. Moore, J. DiGiovanniStudy supervision: J. DiGiovanni

Grant SupportThis work was supported by NIH Grant CA129409 and NIEHS Center

Grant P30ES007784. T. Moore was supported by NIEHS training grantES007247.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 30, 2012; revised July 9, 2012; accepted July 24, 2012;published OnlineFirst August 15, 2012.

Moore et al.





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2012;5:1236-1246. Published OnlineFirst August 15, 2012.Cancer Prev Res Tricia Moore, Linda Beltran, Steve Carbajal, et al. Altered IGF-1R and EGFR CrosstalkEnergy Balance Modulates Mouse Skin Tumor Promotion through

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energy balance modulates mouse skin tumor promotion ......described (5, 21, 24). groups were...

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