Caffeine and Caffeic Acid Inhibit Growth and ... · caffeine (0–5 mmol/L) or caffeic acid (0–50 mmol/L) with or without tamoxifen (1 mmol/L) for 48 hours in SFM. Cell prolif-eration

Cancer Therapy: Clinical

Caffeine and Caffeic Acid Inhibit Growth andModify Estrogen Receptor and Insulin-like GrowthFactor I Receptor Levels inHumanBreastCancerAnn H. Rosendahl1, Claire M. Perks2, Li Zeng2, Andrea Markkula1, Maria Simonsson1,Carsten Rose3, Christian Ingvar4, Jeff M.P. Holly2, and Helena Jernstr€om1

Abstract

Purpose: Epidemiologic studies indicate that dietary factors,such as coffee, may influence breast cancer and modulate hor-mone receptor status. The purpose of this translational study wasto investigate how coffee may affect breast cancer growth inrelation to estrogen receptor-a (ER) status.

Experimental Design: The influence of coffee consumption onpatient and tumor characteristics and disease-free survival wasassessed in a population-based cohort of 1,090 patients withinvasive primary breast cancer in Sweden. Cellular andmoleculareffects by the coffee constituents caffeine and caffeic acid wereevaluated in ERþ (MCF-7) and ER� (MDA-MB-231) breast cancercells.

Results: Moderate (2–4 cups/day) to high (�5 cups/day)coffee intake was associated with smaller invasive primary tumors(Ptrend ¼ 0.013) and lower proportion of ERþ tumors (Ptrend ¼

0.018), compared with patients with low consumption (�1 cup/day). Moderate to high consumption was associated with lowerrisk for breast cancer events in tamoxifen-treated patients with ERþ

tumors (adjusted HR, 0.51; 95% confidence interval, 0.26–0.97).Caffeine and caffeic acid suppressed the growth of ERþ (P� 0.01)and ER� (P � 0.03) cells. Caffeine significantly reduced ER andcyclin D1 abundance in ERþ cells. Caffeine also reduced theinsulin-like growth factor-I receptor (IGFIR) and pAkt levels inbothERþ andER� cells. Together, these effects resulted in impairedcell-cycle progression and enhanced cell death.

Conclusions: The clinical and experimental findings demon-strate various anticancer properties of caffeine and caffeic acidagainst both ERþ and ER� breast cancer that may sensitize tumorcells to tamoxifen and reduce breast cancer growth. Clin Cancer Res;21(8); 1877–87. �2015 AACR.

IntroductionBreast cancer is the major cancer affecting women with a

lifetime risk of more than 10% in the general population inWestern countries. The prevalence is, however, much lower inAsia and many economically developing countries (1). However,Asian immigrants to the United States acquire the prevalence ofthat country within one generation, indicating a strong effect ofenvironmental exposures. Although the majority of primarybreast cancer is estrogen-driven and often responsive to endocrinetreatment, many patients progress to estrogen-insensitive diseasewith poor prognosis (2, 3). A critical determinant of such pro-

gression is the loss of the estrogen receptor-a (ER), which isassociated with a more aggressive tumor phenotype and the lossof sensitivity to endocrine therapies such as tamoxifen (3). ER canstimulate cellular proliferation via progesterone receptor (PgR)induction and transcriptional upregulation of cyclin D1, leadingto enhanced cell-cycle progression, whereas ER loss has beenassociated with enhanced motility and metastatic abilities ofbreast cancer cells. Although loss of ER activity and expressionhas critical consequences for breast cancers, it generally occurswithout evidence of ER gene mutation or deletion. Alterations inER levels can be regulated at transcriptional or posttranscriptionalstages, or through epigenetic mechanisms (4). Given that theprevalence of breast cancer varies between different geographiclocations, potential modifiable environmental, dietary, and life-style determinants may also influence the ER status.

The insulin-like growth factor (IGF) family has been identifiedas an additional driver of breast cancer. The mitogenic andantiapoptotic signals are mediated via the IGF type I receptor(IGFIR) and its downstream targets MAPK–ERK or PI3K–Akt. TheER and IGFIR signaling systems are tightly linked and subject tofeedback cross-talk. For example, estrogen and IGFs coregulateseveral genes that affect breast cancer outcome (5). In addition,hyperactive IGFIR–PI3K–Akt signaling has been implicated tocontribute in the acquisition of resistance to endocrine therapyduring breast cancer progression (6). Estrogen increases IGFIRmRNA and antiestrogens, like tamoxifen, can reduce serum IGFIlevels and IGF-mediated growth (7, 8). Conversely, blocking IGFactions can inhibit estrogen-mediated growth (9). Similar to ER

1LundUniversityandSka�neUniversityHospital,DepartmentofClinical

Sciences, Lund, Division of Oncology and Pathology, Lund, Sweden.2University of Bristol, School of Clinical Sciences, IGFs and MetabolicEndocrinology Group, Southmead Hospital, Bristol, United Kingdom.3Lund University, CREATE Health and Department of Immunotech-nology, Medicon Village, Lund, Sweden. 4Lund University and Ska

�ne

University Hospital, Department of Clinical Sciences, Lund, Division ofSurgery, Lund, Sweden.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthor:AnnH.Rosendahl, LundUniversity andSka�neUniversity

Hospital, Barngatan 2B, SE-221 85 Lund, Sweden. Phone: 46-46-17-7567; Fax:46-46-14-7327; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-1748

�2015 American Association for Cancer Research.

ClinicalCancerResearch

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Published OnlineFirst February 17, 2015; DOI: 10.1158/1078-0432.CCR-14-1748

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activity, various anthropometric, lifestyle, and dietary factors caninfluence the IGF systemwith impact on cancer development andprogression.

In recent years, mechanisms involved in mediating cancerchemopreventive effects of naturally derived plant polyphenolsfrom green tea and coffee have been widely studied in severalneoplasias, including breast and prostate cancer (10–13). How-ever, there have been few translational studies on anticancereffects of coffee in relation to ER status in breast cancer. Prospec-tive and retrospective epidemiologic studies show an associationbetween coffee consumption and a significantly reduced risk,delayed onset, and reduced growth of breast cancer (14, 15).Coffee beans are one of the richest dietary sources of the anti-oxidants caffeine and the plant phenolic compound caffeic acid,both of which may be mechanistically involved in the cancer-suppressive properties of coffee. Various dietary and lifestylefactors are also known to influence the sensitivity to cancertreatment (10, 16). In line with this, our group recently showedin a subset of the present study cohort, that moderate to highcoffee consumption was associated with improved disease-freesurvival among ERþ tamoxifen-treated women (17).

The aimof the present translational studywas to investigate theeffect of coffee consumption on tumor characteristics and disease-free survival in an extended cohort ofwomenwith breast cancer inSweden, and to establish underlying molecular mechanisms bywhich coffee constituents may affect breast cancer cell growth inrelation to ER status. Understanding how exposure to compo-nents of our daily diet can modify tumor growth or molecularadaptations that occur within a tumor could lead to better patientinformation for managing disease.

Materials and MethodsReagents

All chemicals and reagents were purchased from Sigma-Aldrichunless stated otherwise. Cell culture media, penicillin–strepto-mycin, and FBS were purchased from Invitrogen. For immuno-blotting, the anti-ERa antibody (1D5)was obtained fromAbcam;

anti-IGFIRb (C-20) was purchased from Santa Cruz Biotechnol-ogy; anti–phospho-AktSer473 (#4060), anti-Akt (#9272), anti–Bcl-2 (#2870), anti–Bcl-xL (#2764), anti-cleaved caspase-7 (#9491),anti-cleaved PARP (#9541), anti-cyclin D1 (#2926), and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #3683)were purchased from Cell Signaling Technology Inc.

Study populationThe present study is based on an extended cohort of 1,090

women, ages 24 to 99 years, diagnosed with primary invasivebreast cancer between October 2002 and December 2012 at theSka

�ne University Hospital (Lund, Sweden), without preoperative

treatment and with no other cancer history within the past 10years. This is an ongoing cohort (BC Blood study) and is nowupdated to include almost twice as many women as recentlyreported by our group (17). The inclusion and exclusion criteriawere compliant to the REporting recommendations for tumorMARKer prognostic study (REMARK) guidelines (18). During thepreoperative visit, body measurements of weight, height, waistand hip circumference, and breast volume were collected (aspreviously described; refs. 19, 20). Extensive questionnaire datawith information on reproductive factors, the use of exogenoushormones or other medications, lifestyle factors, including smok-ing, alcohol, and coffee consumption, etc., were obtained fromthe cohort members at the preoperative visit. Information ontumor size, lymph node status, histologic grade, proliferativeindex (Ki67), and hormone receptor status (ER and PgR) wereobtained through pathology reports and medical records. ERastatus was routinely determined until December 2009, usingantibody clone 1D5 (DAKO), and from 2010 using clone SP1(Ventana Medical Systems), as previously described (17, 21).Follow-up information of breast cancer recurrence or death wasobtained from patient charts, pathology reports, the RegionalTumor Registry or the Population Registry. Written informedconsents were obtained from all participating women and thestudy was approved by the local ethics committee at LundUniversity (LU 75-02, LU 37-08, LU 658-09, LU 58-12, LU379-12, and LU 227-13).

Cell cultureThe human breast cancer cell lines MCF-7, T47D (both ERa-

positive) and MDA-MB-231 (ERa�) were purchased from andvalidated by ATCC-LGC Standards and maintained in DMEMsupplemented with 10% FBS and antibiotics (100 U/mL penicil-lin and 100 mg/mL streptomycin) in a humidified 5% CO2

atmosphere at 37�C. Tamoxifen-resistant (Tam-R) MCF-7 cellswere kindly supplied by Julia Gee (Cardiff University, UK) andwere derived and maintained as outlined previously (22). For invitro experiments, cells were seeded in growth media for 24 hoursfollowed by transfer to phenol red- and serum-free media [SFM;DMEM:Ham's nutrient mix F-12 supplemented with BSA (0.2mg/mL), sodium bicarbonate (1.2 mg/mL), transferrin (0.01mg/mL), L-glutamine (2 mmol/L), and antibiotics as above] anddosed as specified below.

Cell proliferation assayMCF-7, MDA-MB-231, T47D, and Tam-R cells were exposed to

caffeine (0–5 mmol/L) or caffeic acid (0–50 mmol/L) with orwithout tamoxifen (1 mmol/L) for 48 hours in SFM. Cell prolif-eration was determined following [3H]thymidine incorporationas a measure of DNA synthesis for a period of 4 hours before the

Translational Relevance

Despite improved diagnostic and treatment modalities,breast cancer remains the leading cause of cancer-related deathamong women worldwide. An increased understanding of theclinical and mechanistic effects of modifiable components ofour daily diet on breast cancer characteristics and prognosiscould lead to better patient information formanaging disease.This study reveals that increasing coffee consumption isassociated with significantly smaller invasive breast tumorsizes, a lower proportion of ERþ tumors, and improved dis-ease-free survival among tamoxifen-treated women with ERþ

breast cancer. Mechanistically, this study shows that predom-inantly caffeine, but also caffeic acid, mimics the actions ofantiestrogens andmodifiesmajor growth regulatory pathwaysER/cyclin D1 and IGFIR/pAkt, resulting in impaired cell-cycleprogression and reduced cellular proliferation. Together, thesetranslational findings provide a more comprehensive under-standing of the clinical andmechanistic effects of these dietaryfactors that can modify breast cancer progression.

Rosendahl et al.

Clin Cancer Res; 21(8) April 15, 2015 Clinical Cancer Research1878




end of the treatment protocol, as described in ref. 23. Total cellnumber and viability was assessed by trypan blue exclusion usinga hemocytometer.

Cell-cycle analysisCells were dosedwith caffeine (0–5mmol/L) or caffeic acid (0–

50 mmol/L) in SFM for 48 hours. Floating and adherent cells werecollected,fixed in 70%cold ethanol, and incubatedwithPI/RNasebuffer (BD Biosciences). Approximately 2 � 104 events (cells)were collected for each sample using a FACSCalibur (BD Bios-ciences) dual laser, flow cytometer with CellQuest Pro Software(BD Biosciences). Cell-cycle distribution was determined usingFlowJo Software (TreeStar Inc.).

Western immunoblottingCells were exposed to caffeine (0–5mmol/L) or caffeic acid (0–

50 mmol/L) in SFM for 72 hours after which cell lysates wereprepared and protein concentration determined, as describedpreviously (24). Protein samples (50–60 mg/lane) were resolvedby SDSPAGE and transferred to nitrocellulose membranes. Themembranes were blocked with 5% (w/v) milk in TBS Tween-20(TBST) and probed with antibodies to Bcl-2, Bcl-xL, cleavedcaspase-7, cleaved PARP, ERa, IGFIRb, pAkt, Akt, cyclin D1, orGAPDH as a loading control. Protein abundance was detectedwith horseradish peroxidase–specific secondary antibodies andvisualized by SuperSignal West Extended Duration Substrate(Thermo Fisher Scientific) using the Alpha Innotech FluorChemFC2 imaging system and AlphaView version 3.0.3.0 software(ProteinSimple) or ImageJ software (NIH).

Statistical analysisClinical data. Baseline characteristics of patients with low (�1cup/day),moderate (2–4 cups/day), or high (�5 cups/day) coffeeconsumption were compared as specified by using the Jonc-kheere–Terpstra test for continuous variables (not normally dis-tributed) and linear-by-linear association test for categorical vari-ables. In survival analyses, patients were followed from inclusionuntil first breast cancer recurrence, last follow-up, or death,whichever came first, before January 1, 2013. Of 1,090 women,8 were excluded because of generalized disease within 3 monthsof diagnosis, leaving 1,082 women included in the survivalanalyses. Disease-free survival was estimated using the Kaplan–Meier method. The log-rank test was used for univariable survivalanalysis of the risk of early breast cancer recurrence in relation tocoffee consumption. The influence of coffee consumption onprognosis was also analyzed using multivariable Cox regressionto calculate HR with 95% confidence intervals (CI), adjusted forpotential confounding factors; age (<60 years), histologic grade 3(yes/no), invasive tumor size (>20 mm, or muscular or skininvolvement) and any axillary lymph node involvement (yes/no). Because few patients had an invasive tumor size�51mm, ormuscular or skin involvement, these patients were combinedwiththe patients with invasive tumor sizes between 21 and 50 mm inthemultivariable analyses. All statistical analyses were performedusing SPSS Statistics 19.0 software (IBM). A P value of <0.05 wasconsidered statistically significant. Because this was an explorato-ry study, nominal P values are presented without adjustment formultiple testing.

Experimental data.Aminimumof three independent repeats wereperformed for each experiment. Proliferation data are expressed as

means � SE of triplicate wells. Densitometry measurements ofWestern blot data are expressed as means � SE of at least threeindividual repeats. Statistical analyses were performed using theunpaired Student t test with Excel 2007 software. A P value of<0.05 was considered statistically significant.

ResultsAssociations between tumor size, ER status, and coffeeconsumption among women with breast cancer

To evaluate the impact of coffee consumption on patient andtumor characteristics among women with breast cancer, coffeeconsumption was categorized into low (�1 cup/day), moderate(2–4 cups/day), or high (�5 cups/day), whereas no furtherdefinition of a cup was specified. Patient and tumor character-istics according to coffee consumption for the 1,090 patientsincluded in the study are shown in Table 1. Women that wereyounger at their first full-term pregnancy showed a trend ofdrinking moderate to high amounts of coffee daily. In addition,the proportion of current smokers was higher with increasingcoffee consumption, whereas the proportion of alcohol abstai-ners was higher with decreasing coffee consumption. Interest-ingly, moderate to high coffee consumption was associatedwith significantly smaller invasive tumor sizes (Ptrend ¼ 0.013),whereas concomitantly lower proportion of ERþ tumors andconversely higher proportion of ER� tumors (Ptrend ¼ 0.018).After stratification according to ER status (Table 2), increasingcoffee consumption remained significantly associated withsmaller invasive tumor sizes among women with ERþ tumors(Ptrend ¼ 0.009), whereas no significant association was foundamong ER� tumors (Ptrend ¼ 0.512). However, a nonsignificanttrend toward lower tumor proliferation measured by the Ki67index (Ptrend ¼ 0.092) was observed with increasing coffeeintake among women with ER� tumors. These results suggestthat coffee may have growth-suppressing properties on breastcancer cells and that ERþ tumors may be more sensitive to thecellular effects by coffee.

Effects of caffeine and caffeic acid on ERþ and ER� breast cancercell growth and viability

Given the clinical observations of smaller breast tumor sizesand lower frequency of ERþ tumors with increasing coffee con-sumption, we next examined how two major coffee constituentsmay affect breast cancer growth in relation to ER status at thecellular level. Increasing doses of caffeine significantly suppressedthe proliferation and total cell number of both ERþ MCF-7 andER� MDA-MB-231 (Fig. 1A) breast cancer cells, with a maximuminhibition at 5 mmol/L, compared with control (P < 0.01). At 1mmol/L caffeine, the proliferation was reduced by 80% inMCF-7cells (P < 0.01) and by 40% in MDA-MB-231 cells (P ¼ 0.054).Similarly, caffeic acid reduced bothMCF-7 andMDA-MB-231 cellgrowth, although to a lesser extent than caffeine (Fig. 1A). Aconcomitant increase in cell death was observed for MCF-7, butnot for MDA-MB-231 (Fig. 1A). The response to caffeine andcaffeic acid were identical in the Tam-R cells as with the parentalMCF-7 cells (Fig. 1B). The caffeine-induced cell death in MCF-7was associated with reduced prosurvival Bcl-xL levels whereasincreased active caspase-7 and cleaved PARP, a signature event ofapoptosis (Fig. 1C and Supplementary Fig. S1). These resultsdemonstrate that caffeine, but also caffeic acid, suppress theproliferation of human breast cancer cells, and suggest that

Impact of Coffee on Breast Cancer

www.aacrjournals.org Clin Cancer Res; 21(8) April 15, 2015 1879




ERþ tumors may be more sensitive to the growth inhibitingproperties of the coffee constituents.

Coffee constituents impair the cell-cycle progression in breastcancer cells

Having demonstrated that caffeine and caffeic acid suppressedthe proliferation and viability of breast cancer cells, we furtherassessed their effect on cell-cycle distribution. Increasing doses ofcaffeine resulted in impairedG1 to S transition inboth ERþMCF-7(P� 0.003; Fig. 2A andC) and ER�MDA-MB-231 (P� 0.001; Fig.2B and D) cells, with an accumulation of cells in G0–G1, asubsequent reductionof cells in S andG2–Mphases and enhanced

cell death. Although caffeic acid reduced MCF-7 and MDA-MB-231 cell densities (Fig. 2A and B), only minor modulations of thecell-cycle distribution by caffeic acid were observed in the ERþ

(Fig. 2A and C) as well as ER� cells (Fig. 2B and D).

Caffeine and caffeic acid alter hormone receptor levels anddownstream effectors in breast cancer cells

The role of coffee constituents in modulating molecularmechanisms with impact on breast cancer cell growth in relationto ER status was further investigated. Exposure to caffeine signif-icantly reduced the ER abundance in ERþ MCF-7 cells. In fact, 5mmol/L caffeine almost completely abolished the ER levels

Table 1. Patient and tumor characteristics of the 1,090 patients in relation to daily coffee consumption (cups/day)

All Low (0–1) Moderate (2–4) High (5þ)(n ¼ 1,090) (n ¼ 202) (n ¼ 668) (n ¼ 220) Ptrend

Patient characteristics, median (IQR)Age at diagnosis, y 61.2 (52.1–68.1) 59.4 (48.6–67.8) 62.7 (55.0–69.3) 58.6 (49.5–65.6) >0.3a

Weight (kg) 69.0 (62.0–78.0) 68.0 (60.0–74.6) 70.0 (62.0–79.0) 69.2 (61.0–79.0) >0.3a

Missing 36 8 18 10Height (m) 1.65 (1.62–1.70) 1.65 (1.61–1.69) 1.65 (1.62–1.70) 1.67 (1.63–1.70) >0.3a

Missing 36 8 18 10BMI (kg/m2) 25.1 (22.5–28.4) 24.5 (21.9–27.7) 25.2 (22.8–28.6) 25.0 (22.6–27.9) >0.3a

Missing 38 8 20 10Waist-to-hip ratio 0.86 (0.81–0.91) 0.86 (0.81–0.90) 0.86 (0.81–0.90) 0.87 (0.82–0.91) >0.3a

Missing 53 13 23 17Total breast volume (mL) 1,000 (650–1,500) 925 (550–1,463) 1,000 (700–1,600) 950 (600–1,400) >0.3a

Missing 178 36 101 41Age at first full-term pregnancy (y) 25.0 (22.0–28.0) 26.0 (23.0–29.0) 24.0 (21.0–28.0) 24.0 (21.0–28.0) 0.009a

Missing 140 33 85 22Current smoker, n (%) 219 (20.1%) 18 (8.9%) 119 (17.8%) 82 (37.3%) <0.001b

Missing 4 1 3 0Alcohol abstainer, n (%) 116 (10.6%) 33 (16.3%) 70 (10.5%) 13 (5.9%) 0.001b

Missing 3 0 2 1Tumor characteristics, n (%)Invasive tumor size (pT)1 785 (72.0%) 134 (66.3%) 485 (72.6%) 166 (75.5%) 0.013b

2 287 (26.3%) 60 (29.7%) 175 (26.2%) 52 (23.6%)3 16 (1.5%) 8 (4.0%) 6 (0.9%) 2 (0.9%)4 2 (0.2%) 0 2 (0.3%) 0

Node status0 668 (61.4%) 116 (57.7%) 419 (62.8%) 133 (60.5%) >0.3b

1–3 326 (30.0%) 65 (32.3%) 192 (28.8%) 69 (31.4%)4þ 94 (8.6%) 20 (10.0%) 56 (8.4%) 18 (8.2%)Missing (n) 2 1 1 0

Histologic grade1 266 (24.4%) 43 (21.3%) 167 (25.0%) 56 (25.5%) >0.3b

2 540 (49.6%) 110 (54.5%) 324 (48.6%) 106 (48.2%)3 283 (26.0%) 49 (24.3%) 176 (26.4%) 58 (26.4%)Missing (n) 1 0 1 0

Ki67Negative/low (�20%) 300 (63.0%) 63 (63.0%) 173 (62.0%) 64 (66.0%) >0.3b

High (>20%) 176 (37.0%) 37 (37.0%) 106 (38.0%) 33 (34.0%)Missing (n) 614 102 389 123

Hormone receptor statusERþ 956 (87.9%) 185 (91.6%) 586 (88.1%) 185 (84.1%) 0.018b

ER� 131 (12.1%) 17 (8.4%) 79 (11.9%) 35 (15.9%)Missing (n) 3 0 3 0PgRþ 774 (71.2%) 139 (68.8%) 483 (72.6%) 152 (69.1%) >0.3b

PgR� 313 (28.8%) 63 (31.2%) 182 (27.4%) 68 (30.9%)Missing (n) 3 0 3 0ERþPgRþ 767 (70.6%) 138 (68.3%) 479 (72%) 150 (68.2%) >0.3b

ERþPgR� 189 (17.4%) 47 (23.3%) 107 (16.1%) 35 (15.9%) 0.052b

ER�PgR� 124 (11.4%) 16 (7.9%) 75 (11.3%) 33 (15.0%) 0.022b

ER�PgRþ 7 (0.6%) 1 (0.5%) 4 (0.6%) 2 (0.9%) >0.3b

Missing (n) 3 0 3 0aThe Jonckheere–Terpstra test.bThe linear-by-linear association test.

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(Fig. 3A). Although an approximately 50% reduction of MCF-7proliferation was obtained by caffeic acid treatment, the effect onER abundance was less pronounced (Fig. 3A). The ER suppressionwas associated with a concomitant cyclin D1 decrease, and nodetectable levels remained after 5 mmol/L caffeine exposure (Fig.3A). In contrast, caffeine exposure resulted in reexpression of ERand cyclin D1 in ER� MDA-MB-231 cells (Fig. 3B). However,caffeine did not under these conditions reactivate ER proteinexpression to the endogenous ER levels seen in untreated MCF-7 cells.

Caffeine exposurewas further associatedwith a 70%and a 25%reduction in IGFIR abundance in MCF-7 (Fig. 3A) and MDA-MB-231 cells (Fig. 3B), respectively, when compared with control.Increasing doses of caffeine resulted in a concomitant dose-dependent and complete inhibition of AktSer473 phosphorylationin both MCF-7 and MDA-MB-231 cells (Fig. 3). In line with thelower magnitude in growth inhibition by caffeic acid, a 30%reduction in IGFIR levels was observed for MCF-7, but not forMDA-MB-231 cells, although a �50% reduction in Akt phos-phorylation was found for both cell types following caffeic acidexposure. These results demonstrate that caffeine and caffeic acidsignificantly alter ER and cyclinD1 levels, and suppress the IGFIR–pAkt signaling pathway in both ERþ and ER� breast cancer cells.

Coffee consumption and reduced risk of early breast cancerrecurrence

Finally, impact by coffee consumption on breast cancer prog-nosis was evaluated. Patients were followed for up to 9 years(median 3.0 years, IQR 1.0–5.1). During the study period, 100 of1,082 patients were diagnosed with breast cancer recurrence, ofthem 65 patients had distant metastases. In total 76 deathsoccurred, of which 45 had reported breast cancer event beforedeath. Coffee consumption alone was not associated with diseaseprognosis overall or when stratifying according to ER status (alladjusted Ps � 0.22). However, the disease-free survival wassignificantly improved among tamoxifen-treated women withERþ tumors drinking 2 or more cups of coffee per day (log-rankP¼0.013; Fig. 4). Among thesewomen therewas a49%decreased

hazard of early breast cancer recurrence when drinking moderateto high amounts of coffee compared with low coffee consump-tion, adjusted HR 0.51 (95% CI, 0.26–0.97; P ¼ 0.040). Incontrast, therewas no significant difference indisease-free survivalin relation to coffee consumption among women with ERþ

tumors treated with aromatase inhibitors, chemotherapy, orradiotherapy (all adjusted Ps � 0.26). In vitro evaluation of theeffects on cell proliferation of the combined exposure of tamox-ifen and caffeine or caffeic acid showed that the response to thecoffee constituentsweremaintained andadditive to the inhibitoryeffects of tamoxifen in both ERþ MCF-7 and T47D breast cancercells (data not shown).

DiscussionAgrowingbodyof literature indicates thatmodifiable dietary or

lifestyle factors, such as coffee, may have a protective role againstthe risk or progression of breast cancer (14, 15, 17). In the presenttranslational study, we demonstrate an association betweenincreasing coffee consumption and significantly smaller invasivetumor sizes, whereas concomitantly lower proportion of ERþ

tumors among women with breast cancer. Moreover, in thepresent study, mechanistic in vitro data show that caffeine andcaffeic acid attenuated the growth of predominantly ERþ, but alsoER�, humanbreast cancer cells bymodulating ER and IGFIR levelswith impact on downstream effectors and cell-cycle progression.Finally, the present study demonstrate that tamoxifen-treatedwomen with ERþ tumors drinking 2 or more cups of coffee perday show a significantly reduced risk of early breast cancerrecurrence, compared with women with low daily coffeeconsumption.

Several epidemiologic reports suggest that coffee consumptiondecreases breast cancer risk (14, 25, 26), whereas other studiesreport no association between coffee intake and breast cancer risk(27). However, relatively few studies have examined the associ-ation with risk or progression by ER status. Sweden is among thetop coffee consuming countries worldwide, and less than 1% ofthe coffee consumed is decaffeinated (28). Coffee contains a

Table 2. Selected tumor characteristics of 1,087a patients stratified by ER status in relation to daily coffee consumption (cups/day)

All Low (0–1) Moderate (2–4) High (5þ)(n ¼ 1,087) (n ¼ 202) (n ¼ 665) (n ¼ 220) Ptrend

b

ERþ tumors (n ¼ 956)Invasive tumor size (pT)1 704 (73.6%) 124 (67.0%) 436 (74.4%) 144 (77.8%) 0.0092 236 (24.7%) 54 (29.2%) 143 (24.4%) 39 (21.1%)3 14 (1.5%) 7 (3.8%) 5 (0.8%) 2 (1.1%)4 2 (0.2%) 0 2 (0.3%) 0

Ki67Negative/low (�20%) 292 (68.7%) 62 (67.4%) 171 (68.1%) 59 (72.0%) 0.527High (>20%) 133 (31.3%) 30 (32.6%) 80 (31.9%) 23 (28.0%)Missing (n) 531 93 335 103

ER� tumors (n ¼ 131)Invasive tumor size (pT)1 79 (60.3%) 10 (58.8%) 47 (59.5%) 22 (62.9%) 0.5122 50 (38.2%) 6 (35.3%) 31 (39.2%) 13 (37.1%)3 2 (1.5%) 1 (5.9%) 1 (1.3%) 04 0 0 0 0

Ki67Negative/low (�20%) 8 (15.7%) 1 (12.5%) 2 (7.1%) 5 (33.3%) 0.092High (>20%) 43 (84.3%) 7 (87.5%) 26 (92.9%) 10 (66.7%)Missing (n) 80 9 51 20

aInformation on ER status was missing for 3 patients.bThe linear-by-linear association test.






mixture of bioactive components, and there are several plausiblebiologic pathways whereby coffeemight alter breast cancer risk orprogression. Caffeine and the polyphenol constituents have beensuggested to contribute to its anticancer activities. However,various coffee constituents may differentially affect ERþ andER� breast cancer subtypes. The present findings demonstratesmaller invasive tumor sizes, whereas concomitant lower propor-tion of ERþ tumors and consequently higher proportion of ER�

tumors with increasing coffee consumption. These findings fromalmost twice as many women, confirm the previous report from a

subset of thepresent cohort (17).When stratifying according toERstatus, the association between increasing coffee consumptionand smaller invasive tumor sizewas only observed in ERþ tumors,suggesting that coffee suppresses breast tumor growth, and thatERþ tumors may be more sensitive to these effects. A previousreport demonstrated a protective effect of caffeine intake on bothERþ and ER� breast cancer risk in the Nurses' Health Study,although only significant for the ERþ group (25). Although atthe same time, no association with caffeine intake and overallbreast cancer risk in the Women's Health Study was reported, the

Figure 1.Caffeine or caffeic acid reduces human breast cancer cell growth in vitro. ERþ (MCF-7), ER� (MDA-MB-231), and Tam-R breast cancer cells were treated withthe indicated concentrations of caffeine (Caff) or caffeic acid (CaA) in SFM for 48 (A andB) or 72 hours (C). Proliferation (A and B)was assessed using [3H]thymidineincorporation as a measurement of DNA synthesis. Total cell number and cell death were assessed by cell counting (A). The graphs represent the mean� SEof three (A) or five (B) determinations; � , P <0.05; �� , P <0.01; �� , P <0.001, the Student t test. Total protein (C)was extracted and analyzed by immunoblottingfor the expression of Bcl-2, Bcl-xL, cleaved caspase-7, and cleaved PARP. GAPDH is shown as loading control. One representative image from at least threeindependent experiments is shown.

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proportion of ERþ tumors was decreased and the proportion ER�

tumors increased (29). The shifted ER proportions are in line withboth the clinical and experimental data in the present study.

Several studies have described caffeine and caffeic acid asanticarcinogenic agents in various cell culture and in vivo models(30, 31). For instance, in hepatocellular carcinoma and epidermal

JB6 cells, caffeine attenuated cell growth via induction of G0–G1

cell-cycle arrest (32, 33). Furthermore, caffeine-mediated inhibi-tion of metastatic potential have been demonstrated for melano-ma cells (34). Caffeine has also been shown to inhibit skin cancerin mice by enhancing UVB-induced apoptosis (35). The experi-mental doses used in the present study are equivalent to previous

Figure 2.Cell-cycle distribution after exposure to caffeine or caffeic acid. Representative images showing the morphology and cell-cycle distribution of MCF-7 (A) andMDA-MB-231 cells (B) after 48-hour exposure to the indicated concentrations of caffeine (Caff) or caffeic acid (CaA). Floating and adherent cells were collectedand subjected to cell-cycle analysis using flow cytometry. Graph shows cell-cycle distribution of MCF-7 (C) and MDA-MB-231 cells (D) with the percentage ofcells in sub-G1 (apoptotic), G0–G1, S, and G2–M phases, as the mean of at least three individual repeats.






Rosendahl et al.





in vitro studies (30–33), although the higher doses are above thephysiologic achievable systemic doses. Translating experimentalin vitro doses to human in vivo settings frequently requires signif-icant dose adjustments due to pharmacokinetic and pharmaco-dynamic parameters along with differences in acute in vitroexposure versus cumulative effects following long-term exposure.Our present results demonstrate that predominantly caffeine, andto a lesser extent caffeic acid, inhibited the proliferation of ERþ

MCF-7 and ER� MDA-MB-231 human breast cancer cells. In linewith our clinical observations, the ERþ breast cancer cellsappeared more sensitive to the growth inhibitory effects by thecoffee constituents compared with ER� cells. Interestingly, thegrowth inhibitory property by caffeine on ERþ MCF-7 cells wasassociated with a significant reduction in ER abundance. A mod-erate effect was also found for caffeic acid. Downregulation of ERfunction and expression with subsequent growth inhibition haspreviously been demonstrated for green tea polyphenolic cate-chenins (EGCC) and the structurally related plant flavonoidwogonin (12, 13). In a similar manner to these dietary constitu-ents, caffeinemimicked the actions of the antiestrogen fulvestrant(ICI 182,780), which inhibits ER-dependent functions anddecreases ER expression, resulting in suppression of tumor cellgrowth (36). Similarly, endoxifen, the bioactive metabolite of

tamoxifen, inhibits estrogen-induced breast cancer growth bycompetitively binding ER, leading to inhibition of ER transcrip-tional activity and targeting ER to proteasomal degradation (37).In addition, a recent study described that a derivative of caffeicacid (CAPE) specifically bound and downregulated ER expression(38).

Caffeine affected ER levels differently in ERþ versus ER�

breast cancer cell lines. Surprisingly, the caffeine and caffeicacid–induced growth inhibition of ER� MDA-MB-231 cells wasassociated with a reexpression of ER protein abundance. MDA-MB-231 cells have densely methylated ER CpG islands andepigenetic repression via promoter methylation has been sug-gested to be the predominant mechanism for ER downregula-tion in ER� tumors (39). Caffeic acid has been reported toinfluence epigenetic repression and to reactivate methylation-silenced genes (40). However, the modulation of ER levels inthe present study appeared to involve other mechanisms asinitial investigation of the methylation of the ER promoterusing Combined Bisulphite Restriction Assay revealed no effectof the coffee constituents (data not shown). Further studies areneeded to identify whether other epigenetic changes such ashistone acetylation played a role or whether nonepigeneticmechanisms were involved in the caffeine and caffeic acidinduce reexpression of ER in ER� cells.

ER directly activates the cyclin D1 promoter, and has beendemonstrated to be capable of stimulating cell-cycle progressionin the absence of peptide growth factors (41). In addition, cyclinD1 can stimulate ER transcriptional activity in the absence ofestrogen (42). The caffeine-mediated ER downregulation in ERþ

cells was associated with a concomitant cyclin D1 reduction,impaired G1 to S cell-cycle transition, reduced growth, and induc-tion of apoptosis. This is potentially important as cyclin D1 geneamplification and protein overexpression is associated withimpaired tamoxifen response (43), and if coffee constituentsmodify cyclin D1 levels this may potentially augment tamoxifentreatment response. However, cyclin D1 appears to have contrast-ing effects in ERþ and ER� cells. Although overexpression of cyclinD1maypromote treatment resistance and ametastatic phenotypein ERþ cells, it appears to decrease migration and mammosphereformation in ER� breast cancer cells (43–45). Such contrastingeffects may explain the divergent regulation of ER and cyclin D1by caffeine in ERþ versus ER� cells, both resulting in growthinhibition.

In addition to modulating ER and cyclin D1 levels, caffeinetreatment dose-dependently downregulated the growth and anti-apoptotic IGFIR levels with subsequent impaired AktSer473 phos-phorylation in both MCF-7 and MDA-MB-231 cells, which maypartially contribute to the reduced cell proliferation observed.This is to our knowledge, the first study to show a direct effect ofcaffeine on IGFIR regulation. Previously, inhibition of IGFIRsignaling by green tea polyphenols as measured by reduced pAktand pErk1/2, has been shown to reduce prostate cancer progres-sion and invasion in the TRAMPmouse model (11). The IGF and

Figure 4.Impact by coffee consumption on disease-free survival among tamoxifen-treated women with ERþ breast cancer. Kaplan–Meier estimates of disease-free survival in relation to coffee consumption for 506 tamoxifen-treatedwomen with ERþ breast cancer are shown (log-rank 1 df; P ¼ 0.013). Theadjusted HRwas 0.51 (95%CI, 0.24–0.97; P¼0.040). The number of patientsat each follow-up is indicated. Because this is an ongoing study, the number ofpatients decreases with each follow-up.

Figure 3.Altered hormone receptor levels and downstream effectors by coffee constituents. ERþ MCF-7 (A) and ER� MDA-MB-231 cells (B) were treated with theindicated concentrations of caffeine (Caff) or caffeic acid (CaA) in SFM for 72 hours. Total protein was extracted and analyzed by immunoblotting for the expressionof ER, cyclin D1, IGFIR, pAkt, and Akt. GAPDH is shown as loading control. Relative protein abundance was quantified by densitometry analysis andcorrected forGAPDH (ER, cyclinD1, and IGFIR) or total Akt (pAkt). Thegraphs showmodulationof relative protein abundance versus untreated control and representthe mean � SE of at least three independent experiments; � , P < 0.05; �� , P < 0.01; �� , P < 0.001, the Student t test.






estrogen growth regulatory pathways are tightly linked and com-pensatory mechanisms by the IGFIR have been suggested tomediate cell proliferation in tamoxifen-resistant tumors (46).Therapies targeting IGFIR may, thus, be of clinical benefit fortreatment of such cancers. The association between increasingcoffee consumption and reduced risk of early breast cancerrecurrence among tamoxifen-treated women with ERþ tumors inthe present study may mechanistically be linked to the altered ERand impaired IGFIR signaling by enhanced exposure to the coffeeconstituents. This could sensitize the tumor cells to further inhib-itory effects by tamoxifen with improved clinical response rate.Thesefindings are consistent with and support the previous reportshowing an association between increasing coffee consumptionand significantly increased disease-free survival among tamoxi-fen-treated women with ERþ tumors, in a subset of the presentcohort (17). The present cell work demonstrates that caffeine andcaffeic acidhave inhibitory effects that are at least additivewith theeffects of tamoxifen. It appears that the inhibitory effects ofcaffeine and caffeic acid on ERþ cells are maintained and notdiminished when the ER is either inactive or effectively blocked.Furthermore, it is possible that coffee consumption could alter thepharmacokinetics of tamoxifen via, for example, CYP1A2 orCYP2C8 (17), but that would require examination in an appro-priate in vivo model.

In summary, this study shows inhibitory effects by caffeineand caffeic acid on breast cancer cell growth. These results are inline with previous epidemiologic reports demonstrating pro-tective roles of coffee constituents and the risk or progression ofbreast cancer. The present findings enhance the general under-standing of how modifiable factors present in our daily diet,such as coffee constituents, may alter ER status, IGFIR levels,and contribute to reduced growth of ERþ and ER� breast cancercells.

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

Authors' ContributionsConception and design: A.H. Rosendahl, C.M. Perks, C. Ingvar, H. Jernstr€omDevelopment of methodology: A.H. Rosendahl, C.M. Perks, L. Zeng, J.M.P.Holly, H. Jernstr€omAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.H. Rosendahl, L. Zeng, A. Markkula, M. Simonsson,C. Ingvar, J.M.P. HollyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.H. Rosendahl, L. Zeng, C. Rose, C. Ingvar, J.M.P.Holly, H. Jernstr€omWriting, review, and/or revision of the manuscript: A.H. Rosendahl, C.M.Perks, A.Markkula,M. Simonsson, C. Rose, C. Ingvar, J.M.P.Holly, H. Jernstr€omAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.H. Rosendahl, A. Markkula, M. Simonsson,C. Ingvar, J.M.P. Holly, H. Jernstr€omStudy supervision: A.H. Rosendahl, C. Ingvar, H. Jernstr€omOther (PI of the BC Blood study): H. Jernstr€om

AcknowledgmentsThe authors thank the research nurses Maj-Britt Hedenblad, Karin Henriks-

son, Anette M€oller, Monika Meszaros, Anette Ahlin Gullers, Linda Ågren, andJessica Åkesson. The authors also thank Erika Ba

�geman,MariaHenningson, and

Maria Hjertberg for data entry.

Grant SupportThis study was supported by grants from the Swedish Cancer Society CAN

2011/497, the Swedish Research Council K2012-54X-22027-01-3 (PI,H. Jernstr€om), the Faculty of Medicine at Lund University, the Unit of NursingResearch at Malm€o University, the Faculty of Health and Society at Malm€oUniversity, the South Swedish Health Care Region (Region Ska

�ne ALF), the

Lund Hospital Fund, the Swedish Breast Cancer Group (BRO), the Mrs. BertaKamprad Foundation, the Crafoord Foundation, the Gunnar Nilsson Founda-tion, King Gustaf V's Jubilee Foundation, the Maja and Hjalmar Leander Fund,and the Royal Physiographic Society in Lund.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 8, 2014; revised December 9, 2014; accepted February 2, 2015;published OnlineFirst February 17, 2015.

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2015;21:1877-1887. Published OnlineFirst February 17, 2015.Clin Cancer Res Ann H. Rosendahl, Claire M. Perks, Li Zeng, et al. Human Breast CancerReceptor and Insulin-like Growth Factor I Receptor Levels in Caffeine and Caffeic Acid Inhibit Growth and Modify Estrogen

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Caffeine and Caffeic Acid Inhibit Growth and ... · caffeine (0–5 mmol/L) or caffeic acid (0–50 mmol/L) with or without tamoxifen (1 mmol/L) for 48 hours in SFM. Cell prolif-eration