NSAID use reduces breast cancer recurrence in overweight and obese women: Role of prostaglandin-aromatase interactions

Abstract

Obesity is associated with a worse breast cancer prognosis and elevated levels of inflammation, including greater cyclooxygenase-2 (COX-2) expression and activity in adipose-infiltrating macrophages. The product of this enzyme, the pro-inflammatory eicosanoid prostaglandin E2 (PGE2), stimulates adipose tissue aromatase expression and subsequent estrogen production, which could promote breast cancer progression. This study demonstrates that daily use of a non-steroidal anti-inflammatory drug (NSAID), which inhibits COX-2 activity, is associated with reduced estrogen receptor alpha (ERα) positive breast cancer recurrence in obese and overweight women. Retrospective review of data from ERα positive patients with an average BMI >30 revealed that NSAID users had a 52% lower recurrence rate and a 28 month delay in time to recurrence. To examine the mechanisms that may be mediating this effect, we conducted in vitro studies that utilized sera from obese and normal weight breast cancer patients. Exposure to obese patient sera stimulated greater macrophage COX-2 expression and PGE2 production. This was correlated with enhanced pre-adipocyte aromatase expression following incubation in conditioned media (CM) collected from the obese patient sera-exposed macrophages, an effect neutralized by COX-2 inhibition with celecoxib. In addition, CM from macrophage/pre-adipocyte co-cultures exposed to obese patient sera stimulated greater breast cancer cell ERα activity, proliferation, and migration compared to normal weight, and these differences were eliminated or reduced by the addition of an aromatase inhibitor during CM generation. Prospective studies designed to examine the clinical benefit of NSAID use in obese breast cancer patients are warranted.

Introduction

Obesity has become a significant global public health problem in the past 30 years (1). More than 35% of Americans have a body mass index (BMI) of ≥30 kg/m² (2), an alarming percentage given obesity’s association with an increased incidence of and mortality from numerous chronic diseases, including breast cancer. Several studies have demonstrated a link between obesity and a worse breast cancer prognosis in both pre- and postmenopausal women, including a prospective study of almost 500,000 women that found a progressive escalation in the risk of breast cancer mortality with each successive increase in BMI category (3). Other studies have established positive correlations between obesity and breast cancer recurrence (4), a shorter disease-free survival and lower rates of overall survival, independent of tumor stage at diagnosis (5–7).

Obesity is hypothesized to negatively impact outcomes in postmenopausal, hormone-responsive breast cancer patients by promoting adipose tissue expression of aromatase, the rate-limiting enzyme in estradiol production. This theory has been supported by the results of clinical studies with anastrazole and letrozole showing a reduction in the efficacy of these aromatase inhibitors with increasing BMI (8–9). Plasma estradiol and estrone sulfate levels in obese patients remain significantly elevated in comparison to non-obese patients following letrozole treatment (10), indicating that this reduced response rate may be related to suboptimal inhibition of obesity-associated aromatase activity. Given that aromatase inhibitors are prescribed at a fixed dose, it is possible that their decreased efficacy in obese women is due to under-dosing. However, two phase III clinical trials of anastrozole found that a ten-fold increase in dose provided no additional overall benefit. It is likely that these trials included a number of obese women, suggesting that greater dosage will not solve the problem of obesity-related aromatase inhibitor resistance (11–12).

Recent findings by Dannenberg et al. confirm the presence of elevated aromatase expression in the breast tissue of overweight and obese women (13–14). Crucially, they have demonstrated high congruence between aromatase levels and local breast tissue inflammation, as measured by the number of crown-like structures (CLS-B). CLS-B are inflammatory foci composed of a necrotic adipocyte surrounded by macrophages, which produce excess amounts of several pro-inflammatory mediators. These include COX-2 derived prostaglandin E2 (PGE2), a potent stimulant of aromatase expression in pre-adipocytes, the predominant site of aromatase expression within adipose tissue (13–15). This obesity-inflammation-aromatase axis may be a significant contributor to the reduced aromatase inhibitor response and increased mortality rate observed in obese postmenopausal patients. Collectively, these studies suggest that obese postmenopausal women with estrogen receptor alpha (ERα) positive breast cancer may benefit from combining hormone therapy with a drug targeting the COX-2 pathway.

In this study, we retrospectively examined the association between non-steroidal anti-inflammatory drug (NSAID) use and recurrence rate in a hormone-responsive breast cancer patient population in order to determine whether this COX-2 pathway-targeting drug group improves prognosis. NSAID use reduced the recurrence rate by approximately 50% and extended the disease-free survival by more than two years in our largely overweight/obese, postmenopausal population of women. Furthermore, to assess whether inflammation-associated pre-adipocyte aromatase expression may be responsible for this effect, we utilized an in vitro model of the obese patient’s tumor microenvironment. Here we demonstrated that obesity-associated circulating factors enhance pre-adipocyte aromatase expression and estradiol production via their stimulation of macrophage COX-2 expression, resulting in greater breast cancer cell ERα activity, proliferation and migration.

Methods

Subjects

Review of medical records dated between 1/01/1987 and 1/12/2011 from the Cancer Therapy and Research Center (CTRC) at the University of Texas Health Science Center at San Antonio (UTHSCSA) and the START Center for Cancer Care clinic in San Antonio, Texas, yielded a patient population of 440 women diagnosed with invasive, ERα positive breast cancer. Exclusion criteria included diagnosis of carcinoma in situ, hormone therapy refusal or non-compliance, unavailable treatment information, discontinuation of adjuvant therapy due to insurance issues or health problems, diagnosis of triple negative breast cancer, breast cancer metastasis at the time of diagnosis, unavailable diagnosis dates, and underweight status. The collection of patient data from these medical records was approved by the Institutional Review Boards (IRB) of UTHSCSA and START and conducted in accordance with the Declaration of Helsinki and good clinical practice. Informed consent was obtained from all patients prior to participation.

Study design and data collection

This exploratory study utilized retrospective data collected from patient charts. Body mass index (BMI) was calculated and patients classified as normal weight (18.5–24.9 kg/m²), overweight (25.0–29.9 kg/m²), or obese (≥30.0 kg/m²). Perimenopausal women were classified as premenopausal because they receive tamoxifen as their first-line hormonal therapy (16–17). Patients were designated as NSAID users if progress notes described daily use of aspirin, ibuprofen, celecoxib, naproxen, meloxican or another COX-2 inhibitor in the list of medications. Information on pre-diagnostic NSAID use was not available, and specific data on post-diagnostic NSAID dosage and length of use was not collected. Use of analgesics that do not target COX-2 as well as prn COX-2 inhibitor use did not qualify for inclusion in the NSAID user group. Recurrence was defined as any local, contralateral, distant tumor or metastasis of the primary breast cancer. Second primaries were not classified as recurrent breast cancer.

Serum samples

Serum was collected from 25 postmenopausal breast cancer patients under an IRB approved biorepository collection protocol at the CTRC of UTHSCSA. The collection and use of these biological samples was conducted in accordance with the Declaration of Helsinki and good clinical practice. Informed consent was obtained prior to participation, and all samples and data were deidentified prior to release to maintain patient confidentiality. Serum was pooled according to patient BMI category (normal weight or obese). Serum from five normal weight and five obese postmenopausal women, who were ineligible control subjects in the Polish Women’s Health Study, was also utilized. The design of this study has been described elsewhere (18). This serum was not pooled.

Cell lines and reagents

The ERα positive MCF-7 and T47D and ERα negative MDA-MB-231 breast cancer cell lines (ATCC) were maintained in IMEM (GIBCO Life Technologies) supplemented with 10% fetal bovine serum (FBS). Pre-adipocytes isolated from women undergoing elective surgical procedures were a generous gift from Dr. Rong Li, UTHSCSA, and have been described previously (19). They were maintained in DMEM/F12 1:1 media (GIBCO Life Technologies) plus 10% FBS. U937 monocytes (ATCC) were cultured in RPMI supplemented with 10% FBS and matured to macrophages at the time of seeding for experimentation by incubating the cells in 10.0 ng/ml phorbol 12-myristate 13-acetate (TPA) for 48 hours. Testosterone, anastrozole, celecoxib, and TPA were purchased from Sigma.

Conditioned media

Macrophage conditioned media (CM) was generated by seeding 4x10⁵ U937 monocytes per well in six-well plates, maturing them to macrophages with TPA, then serum-starving the cells for six hours. The macrophages were then exposed to 2% pooled or individual patient sera in serum-free media (SFM) for one hour, the sera removed and cells washed with phosphate buffered saline (PBS) followed by incubation in SFM for 24 hours. The CM was then collected and stored at -20°C for subsequent in vitro assays. To assess the impact of macrophage COX-2 inhibition, the macrophages were pre-treated with 30 uM celecoxib or vehicle for one hour prior to as well as during sera exposure. Macrophage/pre-adipocyte co-culture CM was generated by first maturing 2x10⁵ U937 monocytes per well into macrophages on top of confluent pre-adipocytes seeded in six-well plates. The co-culture was serum-starved for six hours, exposed to a 2% pooled sera in SFM for one hour, then washed with PBS and incubated for 24 hours in SFM +/- testosterone (100 nM), anastrozole (1 uM), and/or vehicle. The CM was then collected and stored at -20° C for subsequent in vitro assays. This CM was supplemented with 2% charcoal-stripped (CS)-FBS prior to use in all assays in order to provide a baseline level of the nutrients needed for cellular function, absent any hormones that would interfere with our evaluation of the effects of the estradiol produced by pre-adipocytes.

Quantitative RT-PCR

Macrophage PTGS2 (COX-2) mRNA levels were measured following a six-hour serum-starvation period (baseline), then a one-hour exposure to 2% obese or normal weight patient sera in SFM, and after a subsequent 12 and 24 hours in SFM (following the removal of the sera). Pre-adipocyte CYP19A1 (aromatase) mRNA levels were assessed following a 24-hour incubation in macrophage CM. MCF-7 and T47D TFF1 (pS2) and CCND1 (cyclin D1) mRNA levels were measured after a 24-hour incubation in macrophage/pre-adipocyte co-culture CM supplemented with 2% CS-FBS. All cells were serum-starved for six hours prior to exposure to sera or CM. Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with Promega’s ImProm II Reverse Transcription System. The primer sequences are as follows: PTGS2: forward, 5’-CCCTTGGGTGTCAAAGGTAA-3’; reverse, 5’-GCCCTCGCTTATGATCT GTC-3’; CYP19A1: forward, 5’-GCCGAATCGAGAGCTGTAAT-3’; reverse, 5’-GAGAATTCATGCGAGTCTGGA-3’; TFF1: forward, 5’-GGTCGCCTTGGAGCAGA-3’; reverse, 5’-GGGCGAAGATCACCTTGTT-3’; CCND1: forward, 5’-TGGAGGTCTGCGA GGAACAGAA-3’; reverse, 5’-TGCAGGCGGCTCTTTTTCA-3’. The manufacturer’s recommended cycling conditions for the QuantiFast SYBR Green PCR kit (Qiagen) were used. Data shown represents the average of at least three independent experiments, with the exception of pre-adipocyte aromatase expression measured following culture in macrophage CM generated with the serum samples from the Polish Women’s Health Study.

Fatty acid analysis

Serum total fatty acids were extracted and analyzed by gas chromatography as previously described (20).

Prostaglandin E2 and estradiol concentrations

The concentration of prostaglandin E2 in macrophage CM and sera was measured using the Prostaglandin E2 Parameter Assay Kit from R&D Systems (Minneapolis, MN). The estradiol concentration in macrophage/pre-adipocyte co-culture CM and sera was analyzed with the Estradiol EIA Kit from Cayman Chemical (Ann Arbor, MI).

ERE luciferase assay

The ERE luciferase assay was performed as described previously (21), with the MCF-7 and T47D cells exposed for 48 hours to the macrophage/pre-adipocyte co-culture CM supplemented with 2% CS-FBS. Relative ERα activity was calculated by dividing the fluorescence value (standardized to renilla) from cells grown in each experimental condition by that from cells grown in CM generated with normal weight sera exposure followed by incubation in SFM plus testosterone. Data shown represents the average of at least three independent experiments.

Cell proliferation and migration

MCF-7, T47D, and MDA-MB-231 cell proliferation was measured using cell counting by hemocytometer following a 48-hour incubation in macrophage/pre-adipocyte co-culture CM supplemented with 2% CS-FBS. The cells were seeded in 24-well plates at a density of 1x10⁴ cells/well for MCF-7 and MDA-MB-231 cells and 1.5x10⁴ cells/well for T47D cells. Migration of these same cell lines was assessed using the wound healing assay as previously described (22) following a 24-hour incubation in macrophage/pre-adipocyte co-culture CM supplemented with 2% CS-FBS. For both experiments, the cells were serum-starved for six hours prior to treatment. Data shown represents the average of at least three independent experiments.

Statistical analyses

Patient data:

The data was examined for normality. Duplicates were also analyzed to guarantee that one patient was not recorded twice in the event of referrals or transfer of care to the other clinic. Pearson’s chi-squared tests were used to analyze categorical variables of NSAID users and nonusers. Student’s t-test was used to examine mean differences in numerical variables. Time to recurrence was assessed by Wilcox non-parametric test. Age at diagnosis and tumor stage were included as a priori confounding factors. Logistic regression was used to predict recurrence. A p value of <0.05 was considered significant. Statistical analyses were performed in R Foundation for Statistical Computing (version 2.10.1, Vienna, Austria, 2009).

In vitro data:

Differences between cells exposed to two different experimental conditions were measured using Student’s t test. One-way ANOVA was used to analyze COX-2 expression. For experiments involving two independent variables, two-way ANOVA was used. A p value of <0.05 was considered significant.

Results

Patient characteristics

The patient population consisted of 440 women with invasive, ERα positive breast cancer. NSAID users did not significantly differ from nonusers by BMI category, tumor stage, hormone receptor status, tumor type, race or type of surgery. Not surprisingly, NSAID users were more likely to be older, postmenopausal, and diabetic. The majority of patients in our population were overweight/obese (BMI≥25 kg/m²), such that the average BMI for both users and nonusers was >30 kg/m² (Table 1).

Table 1.

Descriptive characteristics of breast cancer patients (n=440)

Characteristics	NSAID nonusers (n=281)	NSAID users (n=159)	p-value
Age at diagnosis, years (meanαSD)	55.5 ± 10.3	60.7 ± 10.7	< 0.001
BMI, kg/m2 (mean ± SD)	30.7 ± 6.21	31.9 ± 6.70	0.072
Time to recurrence, months (median)a	50.6	78.5	0.464
	[-----------------------------N(%---)--------------------]		χ2, p-value for trend
Recurrence
Yes	34(12.1)	10(6.3)	0.050
No	247(87.9)	149(93.7)
BMI category
Normal	50(17.8)	25(15.7)	0.132
Overweight	94(33.4)	41((25.8)
Obese	137(48.8)	93(58.5)
Menopausal status
Premenopausal	110(39.2)	32(20.1)	<0.001
Postmenopausal	171(60.8)	127(79.9)
Race
Hispanic	134(47.7)	74(46.5)	0.135
White	111(39.5)	57(35.8)
African-American	5(1.8)	10(6.3)
Other	4(1.4)	1(0.7)
Missing/Unavailable	27(9.6)	17(10.7)
Tumor stage
I	101(35.9)	60(37.8)	0.389
II	107(38.1)	64(40.2)
III	56(19.9)	23(14.5)
Missing/Unavailable	17(6.1)	12(7.5)
Hormone receptor status
ER+/PR+	246(87.6)	133(83.6)	0.255
ER+/PR−	35(12.4)	26(16.4)
Histological type
Ductal	218(77.6)	121(76.1)	0.156
Lobular	33(11.7)	21(13.2)
Mucinous	1(0.4)	2(1.2)
Missing/Unavailable	29(10.3)	15(9.5)
Type of surgery
Lumpectomy	128(45.5)	72(45.3)	0.405
Mastectomy	122(43.4)	69(43.4)
Bilateral mastectomy	26(9.3)	12(7.5)
Other excisionb	5(1.8)	6(3.8)
Adjuvant hormonal therapy
Aromatase inhibitor	171(60.9)	125(78.6)	<0.001
Tamoxifen	110(39.1)	34(21.4)
Type of NSAIDc
Aspirin	-	129(81.1)
Other	-	30(18.9)
Diabetes status
Diabetic	56(19.9)	57(35.9)	<0.001
Not diabetic	225(80.1)	102(64.1)
Diabetes drug
Metformin	37(66.1)	33(57.9)	0.036
Other	19(33.9)	24(42.1)
Omega-3 fatty acid use
Yes	32(11.4)	40(25.1)	<0.001
No	249(88.6)	119(74.9)
Statin use
Yes	65(23.1)	67(42.1)	<0.001
No	216(76.9)	92(57.9)

Abbreviations: NSAIDs, non-steroidal anti-inflammatory drugs; SD, standard deviation; BMI, body mass index; ER, estrogen receptor; PR, progesterone receptor.

^aWilcox non-parametric test used to analyze time to recurrence analysis in 44 patients who had a recurrence.

^bOther excision includes segmentectomy and quadrantectomy.

^cCalculations pertinent to the 159 patients classified as NSAID users.

NSAID use is associated with a reduced risk of recurrence and longer disease-free survival

The recurrence rate in NSAID users was 52% lower than non-NSAID users (OR, 0.48; 95% CI, 0.22–0.98) (Table 2). After controlling for patient use of statin drugs and omega 3 fatty acids, which also have anti-inflammatory effects, NSAID users’ recurrence rate was still approximately half the rate of non-users (OR 0.52; 95% CI, 0.23 – 1.08). NSAID users remained disease-free for an average of 78.5 months, while non-users averaged 50.6 months, a difference of more than two years (Table 1). Overweight/obese patients had a 1.86-fold higher risk of recurrence versus normal weight when controlling for NSAID use and type of hormone therapy (OR, 1.86; 95% CI, 0.76–5.62) (Table 2). Unfortunately, the small number of normal-weight patients, combined with their low rate of recurrence, precluded our ability to examine whether NSAID use was more effective in preventing recurrence in patients with an elevated BMI. However, despite the small sample size, we found that NSAID use was associated with a substantial reduction in recurrence rate in this predominantly overweight/obese postmenopausal patient population. Larger studies have observed more modest effects (23–25).

Table 2.

Logistic regression model to predict breast cancer recurrence

Predictors	OR	95%CI	p-value
NSAID use
Users	0.48	0.22–0.98	0.05
Nonusers	reference
BMI category
Normal	reference
Overweight + Obese	1.86	0.76–5.62	0.11

Abbreviations: NSAIDs, non-steroidal anti-inflammatory drugs; OR, odds ratio; CI, confidence interval; BMI, body mass index

Obesity stimulates pre-adipocyte aromatase expression and estradiol production via elevated macrophage COX-2 expression

We next sought to determine whether the NSAID-associated reduction in recurrence rate observed in the overweight/obese patient population may be due to the effect of these drugs on local aromatase expression. To this end, we utilized an in vitro model in which cultured cells were exposed to pooled sera samples from obese (OB) or normal weight (N) postmenopausal breast cancer patients in order to mimic the tumor microenvironment of obese versus normal weight women. The characteristics of the serum donors, including serum concentrations of various cytokines, adipokines, and growth factors, have been previously described (21). Following a one hour exposure to OB sera, COX-2 expression in cultured macrophages was a modest 24% higher than cells exposed to N sera (Figure 1a). However, exposure to OB sera for one hour increased macrophage PGE2 production five-fold versus N sera in the 24 hours following sera removal (Figure 1b). Consequently, we assessed whether macrophage COX-2 expression may continue to increase during that 24-hour period following sera removal, finding that mRNA levels were 68% and 92% greater in OB versus N sera-exposed cells at 12 and 24 hours, respectively. (Figure 1c). Pre-adipocytes cultured in conditioned media (CM) from macrophages exposed to OB versus N sera had 52% greater aromatase expression (Figure 1d). Treatment of the macrophages with celecoxib during sera exposure neutralized the difference in pre-adipocyte aromatase expression, suggesting that OB sera-induced macrophage PGE2 production may be responsible for this effect. Pre-adipocyte aromatase expression was also measured following growth in CM from macrophages exposed to serum from ten individual OB or N postmenopausal women, allowing us to examine whether similar results are obtained using non-pooled serum with the data averaged by BMI category. Serum from ineligible control subjects in the Polish Women’s Health Study, a breast cancer case-control study that has been described previously (18), was used, enabling us to additionally determine whether our results are unique to sera obtained from a specific patient population. The average pre-adipocyte aromatase expression was over two-fold greater following culture in macrophage CM generated with OB versus N subjects’ sera (Figure 1e), supporting the findings obtained using pooled sera from the CTRC patients. Finally, the OB sera-induced increase in aromatase expression was correlated with a 16-fold amplification in pre-adipocyte estradiol production in the presence of exogenous testosterone, the substrate for aromatase (Figure 1f). The addition of the aromatase inhibitor anastrozole to the macrophage/pre-adipocyte co-culture following sera exposure completely nullified the difference between OB and N, demonstrating that the increase in estradiol production was solely due to the OB sera’s effect on aromatase expression.

Figure 1. Obesity stimulates macrophage COX-2 expression, resulting in elevated pre-adipocyte aromatase expression and estradiol production.

(a) COX-2 expression in U937 cells matured to macrophages following one hour exposure to obese (OB) versus normal weight (N) patient sera. (b) PGE2 concentration in conditioned media (CM) from macrophages following exposure to OB or N patient sera. (c) COX-2 expression in U937 cells (matured to macrophages) after a six-hour serum-starvation period (Baseline), 12 hours after removal of the sera (OB and N, 12hr), and 24 hours after removal of the sera (OB and N, 24hr). (d) Aromatase expression in pre-adipocytes incubated for 24 hours in macrophage CM generated following patient sera exposure (OB-CM and N-CM) with vehicle or celecoxib treatment. (e) The impact of macrophage CM on pre-adipocyte aromatase expression when individual patient serum samples were utilized for CM generation, with the results averaged according to patient BMI category. (f) Estradiol concentration in CM from a macrophage/pre-adipocyte co-cultures exposed to patient sera (OB-CM and N-CM), then incubated in serum-free media with vehicle, testosterone, and/or anastrozole. Data shown represents the average of at least three independent experiments, with the exception of (e). *, p<0.05; **, p<0.01 in comparison to N or N-CM; different letters indicate significant differences, p<0.05.

Obese patient serum contains higher arachidonic acid and saturated fatty acid levels

Given our finding that exposure to the OB versus N patient sera promotes greater macrophage COX-2 expression and PGE2 production, we next explored two possible mechanisms mediating this effect by profiling the fatty acid content of the sera. The concentration of arachidonic acid, the omega-6 fatty acid substrate utilized by COX-2 for PGE2 production, was significantly higher (p<0.001) in the OB patient sera. The palmitate level as well as the total level of saturated fatty acids, which can stimulate macrophage COX-2 expression (15), were also higher (p<0.001) in the OB patient sera (Table 3). We then measured PGE2 and estradiol levels in the patient sera, as systemic levels of these factors may also impact tumor progression, but there were no significant differences between OB and N (Table 3).

Table 3.

Serum fatty acid, prostaglandin E2 and estradiol concentrations

	Obese	Normal Weight
Fatty acids (nmol/ml)
Arachidonic acid	420.6 (1.30)*	391.3 (2.06)
Palmitate	2205 (0.993)*	1545 (6.95)
Total SFA	3413 (8.29)*	2617 (13.2)
Prostaglandin E2 (pg/ml)	705.8 (151.8)	711.4 (142.3)
Estradiol (pg/ml)	9.39 (1.09)	9.75 (1.73)

^*p<1x10^-3 in comparison to normal weight. Standard error of the mean shown in parentheses. Abbreviations: SFA, saturated fatty acids.

Obesity-associated pre-adipocyte aromatase expression promotes greater breast cancer cell ERα activity

After establishing that obesity-associated circulating factors stimulate pre-adipocyte aromatase expression via induction of macrophage COX-2 expression, we sought to determine whether the resulting elevation in estradiol promotes enhanced breast cancer cell ERα activity. ERα positive breast cancer cells cultured in CM from OB versus N sera-exposed macrophage/pre-adipocyte co-cultures exhibited greater ERα activity, as measured by ERE luciferase assay, with 29% and 42% differences in MCF-7 and T47D cells, respectively (Figures 2a and b). Co-culture treatment with anastrozole after sera exposure significantly decreased the OB-induced ERα activity in both cell lines, eliminating the difference between OB versus N CM-induced breast cancer cell ERα activity. OB and N co-culture CM produced without testosterone stimulated MCF-7 and T47D cell ERα activity that was statistically equivalent to that induced by CM from co-cultures treated with testosterone and anastrozole. Similar results were obtained when the CM was generated with anastrozole treatment but no testosterone. This shows that the presence of testosterone during co-culture CM generation is required for the OB-induced elevation in breast cancer cell ERα activity, further demonstrating that this effect is due to pre-adipocyte aromatase activity. We utilized pS2 and cyclin D1 expression as additional measures of ERα activity, obtaining analogous results. Following growth in OB versus N CM, pS2 expression was 49% higher in MCF-7 cells and 63% greater in T47D cells (Figures 2c and d) while cyclin D1 expression was elevated by 70% in MCF-7 cells and 78% in T47D cells (Figures 2e and f). Co-culture treatment with anastrozole neutralized these differences in pS2 and cyclin D1 expression in both cell lines and, as seen with the ERE luciferase assays, CM generated without testosterone stimulated equivalent expression. Together, these results demonstrate that the obesity-associated, COX-2-induced increase in pre-adipocyte aromatase expression and estradiol production can enhance breast cancer cell ERα activity.

Figure 2. Obesity-associated pre-adipocyte aromatase expression promotes greater breast cancer cell ERα activity.

MCF-7 and T47D breast cancer cell ERα activity after culture in macrophage/pre-adipocyte co-culture CM, as measured by ERE luciferase reporter (a, b) and qPCR analysis of pS2 (c, d) and cyclin D1 (e, f) expression. Experimental conditions include CM from co-cultures exposed to obese or normal weight patient sera (OB-CM and N-CM) followed by incubation in serum-free media with testosterone plus vehicle, testosterone plus anastrozole, vehicle alone, or anastrozole alone. Data shown represents the average of at least three independent experiments. Different letters indicate significant differences, p<0.05.

Breast cancer cell proliferation and migration are induced by obesity-associated pre-adipocyte aromatase expression

In order to determine whether the obesity-associated, COX-2-induced elevation in pre-adipocyte aromatase expression and breast cancer cell ERα activity could result in enhanced disease progression, we assessed the impact of the macrophage/pre-adipocyte co-culture CM on breast cancer cell proliferation and migration. Culturing MCF-7 and T47D cells in OB versus N CM generated with exogenous testosterone increased proliferation by 59% and 55%, respectively (Figures 3a and b). Treatment of the co-culture with anastrozole during CM generation eliminated the difference between OB and N, demonstrating that the OB CM’s elevated estradiol concentration is responsible for its effect on cell proliferation. This conclusion is further supported by the lack of any difference in proliferation levels in MDA-MB-231 cells, an ERα negative breast cancer cell line, following culture in the four CM conditions (Figure 3c). A similar trend was seen when we examined the impact of the co-culture CM on ERα positive breast cancer cell migration. OB CM stimulated 57% more MCF-7 and 46% greater T47D cell migration in comparison to N CM (Figure 4a and b). This effect was neutralized in the T47D cells by treatment of the co-culture with anastrozole during CM production. However, while aromatase inhibition significantly decreased OB-induced MCF-7 cell migration, these cells still migrated farther than those cultured in N CM with anastrozole. Intriguingly, the migration of MDA-MB-231 cells was also significantly enhanced by culture in OB versus N CM (Figure 4c). Consistent with this cell line’s ERα negative status, anastrozole treatment during CM generation did not affect the CM’s impact on migration. Overall, these findings strongly suggest that obesity-associated circulating factors may promote ERα positive breast cancer progression via stimulation of macrophage COX-2 expression and the subsequent increase in pre-adipocyte aromatase expression.

Figure 3. ERα positive breast cancer cell proliferation is induced by obesity-associated pre-adipocyte aromatase expression.

MCF-7 (a), T47D (b), and MDA-MB-231 (c) breast cancer cell proliferation in response to culture in macrophage/pre-adipocyte co-culture CM. Experimental conditions include CM from co-cultures exposed to obese or normal weight patient sera (OB-CM and N-CM) followed by incubation in serum-free media with testosterone plus vehicle or testosterone plus anastrozole. Data shown represents the average of at least three independent experiments. Different letters indicate significant differences, p<0.05.

Figure 4. Obesity-associated pre-adipocyte aromatase expression promotes breast cancer cell migration.

MCF-7 (a), T47D (b), and MDA-MB-231 (c) breast cancer cell migration during incubation in macrophage/pre-adipocyte co-culture CM. Experimental conditions include CM from co-cultures exposed to obese or normal weight patient sera (OB-CM and N-CM) followed by culture in serum-free media with testosterone plus vehicle (OB+T-CM, N+T-CM) or testosterone plus anastrozole (OB+T+AI-CM, N+T+AI-CM). Data shown represents the average of at least three independent experiments. Different letters indicate significant differences, p<0.05.

Discussion

Suboptimal pharmacologic aromatase inhibition in postmenopausal patients with ERα positive breast cancer has detrimental consequences to clinical outcome. COX-2 is a critical node for the convergence of various upstream pathways of inflammation, including IL-1, IL-6, and TNFα signaling (26–27), and it appears to be a key mediator of biologic processes affecting treatment failure, such as PGE2 synthesis and the resulting aromatase expression and estrogen production. Our study is the first to specifically examine the molecular mechanisms that may mediate the impact of daily NSAID use on recurrence rate and time to disease progression in patients with invasive breast cancer receiving adjuvant endocrine therapy. We have demonstrated that the NSAID users in this patient population had a 52% lower recurrence rate. A similar trend was seen after controlling for patient age and tumor stage at diagnosis, though the strength of the association was reduced (data not shown). This is not surprising given that late-stage tumors, as well as the aggressive tumors that disproportionately develop in younger women, seem less likely to significantly benefit from the modest effects of this drug group. NSAID users also remained disease-free for more than two years longer than non-users, a difference that was not statistically significant but may be a clinically relevant variance in outcome.

Similar results have been obtained in some larger prospective studies examining NSAID use after breast cancer diagnosis. Holmes et al. (23) showed that aspirin use 6–7 days/week was associated with a significant reduction in the risk of recurrence (RR, 0.57; 95% CI, 0.39–0.82). Utilizing the Nurses’ Health Study’s substantial pool of subjects, the authors found no change in these results after stratifying by BMI, menopausal status, and ER status. In an analysis of postmenopausal breast cancer patient outcomes, Blair and colleagues (25) observed that any amount of regular NSAID use was correlated with a lower risk of breast cancer death (HR, 0.64; 95% CI, 0.39–1.05). Adjustment for ERα status, but not BMI category, increased the HR and reduced the statistical significance of this association. In contrast with these studies, the recurrence rate in a population of pre and postmenopausal patients was not decreased by aspirin use ≥3 days/week (RR, 1.09; 95% CI, 0.74–1.61), but was affected by use of ibuprofen (RR, 0.56; 95% CI, 0.32–0.98) (24). Controlling for BMI, menopausal status, and ERα did not alter these results.

The variability in design and patient population among these studies makes any comparison with our results difficult. Cumulatively, they appear to indicate that NSAID use may be an effective addition to adjuvant breast cancer treatment, regardless of BMI, ERα, or menopausal status. However, the association between NSAID use and a 50% lower disease recurrence in our study, despite a relatively small patient population, led us to hypothesize that the overwhelming prevalence of overweight/obesity among this population may have increased the NSAID benefit. Our patient population was also largely postmenopausal and included only hormone responsive patients. Because obesity and overweight status are associated with higher PGE2 levels and aromatase expression in female breast tissue (13–14), it seems likely that the efficacy of COX-2 inhibitors in a postmenopausal, ERα positive patient population would increase with greater adiposity. Perhaps the lack of variation in effect among BMI categories in previous studies is due to their failure to stratify the data by BMI, ERα, and menopausal status simultaneously. This question could potentially be addressed by previous trials assessing the clinical benefit of a celecoxib/aromatase inhibitor combination treatment for postmenopausal, hormone-responsive breast cancer. Most of these studies showed a modest benefit with at least three months of combination treatment, including trends towards more clinical complete response, longer duration of clinical benefit, and greater progression-free survival (28–30). Unfortunately, none of these trials analyzed the treatment benefit by BMI category, so it is impossible to determine from this data whether overweight/obese women in this patient population are more likely to benefit from COX-2 inhibition. To our knowledge, no one has examined the efficacy of a COX-2 inhibitor/aromatase inhibitor combination in animal models of obesity and mammary carcinogenesis either.

Given that obesity has been associated with a worse breast cancer prognosis (3–7) as well as a reduced response to aromatase inhibitor therapy among postmenopausal, ERα positive patients (8–9), determination of whether this specific population will benefit from COX-2 inhibition is an important question. Dannenberg et al. have demonstrated an obesity-associated, COX-2-induced elevation in pre-adipocyte aromatase expression using both pre-clinical models and patient breast tissue (13–15). Our aim was to confirm this phenomenon, using an in vitro model of the obese patient’s breast tumor microenvironment, as well as determine whether this increased pre-adipocyte aromatase expression can promote greater epithelial cell ERα activity and subsequent proliferation and migration, two in vitro measures of cancer progression. We show that exposure to sera from obese postmenopausal women stimulates significantly greater macrophage COX-2 expression and a five-fold increase in PGE2 production. Saturated fatty acids can promote COX-2 expression and PGE2 production in cultured macrophages (15, 31), and the increased lipolysis that accompanies obesity results in a higher concentration of circulating free fatty acids, particularly palmitate (32–34). We confirmed that the obese patient sera contains significantly higher levels of palmitate, total saturated fatty acids and arachidonic acid, the omega-6 fatty acid substrate utilized by COX-2 to produce PGE2. Consequently, this difference in macrophage COX-2 expression and PGE2 production may be due to elevated levels of these free fatty acids in the obese patient sera. There is also some evidence that the inflammatory cytokines IL-6 and TNFα, found in higher concentrations in our obese patient serum samples (21), can induce COX-2 expression (27, 35–36). This suggests that more than one mechanism may be responsible for the obesity-associated upregulation of macrophage COX-2 expression and PGE2 production seen in this study.

Given our in vitro results, it may seem surprising that we found no difference between the OB and N patient sera in PGE2 and estradiol concentrations. However, PGE2 has a short half-life, so urinary concentrations of the stable end metabolite of PGE2 catabolism (PGE-M) are typically used to assess systemic PGE2 levels (37). In fact, while an elevated BMI has been associated with increased PGE-M levels (38), we are not aware of any studies demonstrating higher circulating PGE2 levels with obesity. In contrast, the lack of difference in serum estradiol levels by BMI does not agree with the literature, as researchers have generally found that obese postmenopausal women have higher estradiol levels (39–41). However, of the 25 women that provided the sera for this study, nine obese patients and one normal weight patient were receiving an aromatase inhibitor (21), which may have masked any difference in estradiol levels between the groups.

Taken together, our in vitro data provides compelling evidence that an obesity-associated increase in macrophage COX-2 expression promotes greater pre-adipocyte aromatase expression and estradiol production, resulting in an elevation in breast cancer cell ERα activity, proliferation, and migration. Neutralization of the obesity-induced elevation in ERα activity and cell proliferation by the addition of aromatase inhibitor treatment validates our hypothesis that these effects are due to pre-adipocyte aromatase expression. However, while incubation of the ERα negative MDA-MB-231 cells in OB versus N macrophage/pre-adipocyte co-culture CM did not differentially affect cell proliferation, it did stimulate significantly greater cell migration. In addition, OB CM continued to promote more extensive MCF-7 cell migration in comparison to N CM with aromatase inhibition during CM generation, though anastrozole did significantly decrease the OB CM-induced migration. These results indicate that, while locally produced estradiol clearly plays a role in mediating obesity-associated breast cancer cell migration, one or more additional signaling molecules produced by macrophages and/or pre-adipocytes is involved in this effect. Macrophage-derived PGE2 is one possibility, as it is known to promote breast cancer cell migration (42–43). Several studies have concluded that it also stimulates breast cancer cell proliferation based on the ability of COX-2 inhibitors to hinder proliferation (44–45). However, we saw no differential proliferation in the MDA-MB-231 cells following incubation in OB versus N CM, and Robertson et al (46) demonstrated that treatment of MDA-MB-231 cells with exogenous PGE2 does not increase proliferation. The role of additional locally produced signaling molecules in the link between obesity-associated inflammation and breast cancer progression is an area that deserves further exploration in future studies.

This study focused on exploring how COX-2 inhibition via NSAID use could reduce breast cancer recurrence in obese and overweight women. However, a large majority of the NSAID users in our study were taking aspirin, which preferentially inhibits the COX-1 enzyme over COX-2 (47). While most researchers consider suppression of COX-2 activity to be the predominant mechanism by which aspirin reduces cancer risk and progression, a few have examined the role of COX-1 in tumorigenesis. Hwang et al (48) found that a majority of breast tumor samples overexpress COX-1, while Jeong (49) demonstrated that a selective COX-1 inhibitor reduces MCF-7 cell growth. Others have shown that the combined inhibition of COX-1 and COX-2 produces a significantly greater suppression of breast cancer cell growth than either alone (50). Obesity has not been linked to increased COX-1 expression or activity, though, so we did not pursue an examination of this enzyme as part of our study.

Our current investigation strongly suggests that local estradiol production, induced by macrophage COX-2 activity, may be a key mediator in the link between obesity and postmenopausal, hormone-responsive breast cancer progression. This conclusion is supported by the clinical observation of a 52% lower recurrence rate and 28-month extension in time to recurrence with NSAID use in a largely overweight/obese and postmenopausal patient population with ERα positive disease. Collectively, our results provide a strong rationale for further studies regarding the clinical benefit of aromatase inhibitor/COX-2 inhibitor combination treatment for obese, postmenopausal breast cancer patients.