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G D Foglesong et al. Lifestyle improvements inhibit breast cancer

483–495

-19-0075

RESEARCH

Enriched environment inhibits breast cancer progression in obese models with intact leptin signaling

Grant D Foglesong1,2, Nicholas J Queen1,2, Wei Huang1,2, Kyle J Widstrom1,2 and Lei Cao1,2

1Department of Cancer Biology and Genetics, The Ohio State University, Columbus, Ohio, USA2The Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA

Correspondence should be addressed to L Cao: Lei.Cao@osumc.edu

Abstract

Obesity is becoming a global epidemic and is a risk factor for breast cancer. Environmental enrichment (EE), a model recapitulating an active lifestyle, leads to leanness, resistance to diet-induced obesity (DIO) and cancer. One mechanism is the activation of the hypothalamic–sympathoneural–adipocyte (HSA) axis. This results in the release of norepinephrine onto adipose tissue inducing a drop of leptin. This study aimed to test the effects of EE on breast cancer onset and progression while considering the effect of leptin by utilizing the transgenic MMTV-PyMT model as well as several models of varied leptin signaling. EE was highly effective at reducing weight gain, regardless of the presence of leptin. However, the effects of EE on tumor progression were dependent on leptin signaling. EE decreased leptin and reduced mammary tumor growth rate in MMTV-PyMT spontaneous and DIO transplantation models; in contrast, the absence of leptin in ob/ob mice resulted in increased tumor growth likely due to elevated norepinephrine levels. Our results suggest that the microenvironment is critical in breast tumorigenesis and that the drop in leptin is an important peripheral mediator of the EE anti-breast cancer effects, offsetting the potential pro-tumorigenic effects of norepinephrine responding to a complex environment.

Introduction

In Western women, breast cancer is the most common malignant disease and is one of the leading causes of cancer-related deaths (Weigelt et  al. 2005, Hanahan & Weinberg 2011), with age and body mass significantly contributing to disease progression. Overweight and obesity have become a global epidemic affecting one third of the world’s population (GBD 2015 Obesity Collaborators et  al. 2017). Obesity is among the most common etiological factors for chronic diseases (Faris & Attlee 2015). Obesity further increases the risk and morbidity of many cancer types including breast cancer

and has contributed to over 120,000 cancer-related deaths worldwide, increasing the risk of dying from cancer by 40–80% (Zheng et al. 2011, Ogden et al. 2014, Gallagher & Leroith 2015). The mechanisms for the obesity–cancer association are multifactorial and include insulin resistance, increased growth factors and anabolic hormones, adipokines, oxidative stress, inflammation, increased bioavailable sex hormones, deterioration in immune surveillance and altered cellular energetic (Longo & Fontana 2010, Ashrafian et al. 2011, Olson et al. 2017). Importantly, progression to obesity can be reversed

Endocrine-Related Cancer (2019) 26, 483–495

5

Key Words

f breast cancer

f obesity

f environmental enrichment

f adipose tissue

f leptin

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484G D Foglesong et al. Lifestyle improvements inhibit breast cancer

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or slowed with lifestyle changes such as weight loss and healthy diet (Uzunlulu et al. 2016), thus also having the potential to mitigate cancer progression.

We have used a mouse model of environmental enrichment (EE) to study an active lifestyle influence on health and diseases (Cao et al. 2004, 2010, 2011, Cao & During 2012, During et  al. 2015, Foglesong et  al. 2016, Xiao et al. 2016, 2019, Mcmurphy et al. 2018). EE refers to a housing with increased space, physical activity and social interactions that facilitate enhanced sensory, cognitive, motor and social stimulation (Nithianantharajah & Hannan 2006). Our work has revealed a novel anti-cancer and anti-obesity phenotype of EE and we have coined a term ‘the hypothalamic–sympathoneural–adipocyte (HSA) axis’ to describe a specific brain–fat axis linking EE to anti-cancer and anti-obesity phenotypes. The key upstream mediator in the brain is brain-derived neurotrophic factor (BDNF) in the hypothalamus. EE induces BDNF expression, thereby elevating the sympathetic tone preferentially to the fat. The HSA axis activation results in adipose tissue remodeling, including induction of beige cells and the suppression of leptin, leading to anti-obesity and anti-cancer phenotype (Cao et al. 2009, 2010, 2011). The EE-induced anti-cancer and anti-obesity phenotypes could not be accounted for by physical activity alone but could be largely reproduced by overexpression of BDNF in the hypothalamus (Cao et al. 2010, 2011). Other labs have adopted EE model and demonstrated that EE inhibits pancreatic cancer (Li et al. 2015), glioma (Garofalo et  al. 2015) and colon cancer (Bice et al. 2017) involving additional mechanisms.

Leptin is a major adipokine whose level is proportional to BMI and plays a critical role in the regulation of energy balance. Leptin has been shown to have mitogenic, proangiogenic and antiapoptotic effects through interactions with its receptor (ob-R) that is overexpressed in many cancers (Hu et al. 2002, Argolo et al. 2016). Our studies using melanoma model have identified leptin as a key peripheral mediator of EE-induced cancer inhibition (Cao et  al. 2010). Given leptin’s oncogenic effects on breast cancer (Lipsey et  al. 2016), we hypothesized that EE inhibits breast cancer growth and requires leptin signaling. Of note, our previous studies have shown that genetically activating the HSA axis by hypothalamic gene transfer of BDNF was effective at reducing EO771 breast cancer growth in middle aged obese mice; however, mechanistic studies to implicate leptin were limited (Liu et al. 2014). Additionally, the amount of fat in the breast is generally proportional to the total fat mass, but these fat depots retain residual independence (Renehan et al. 2008).

The effect of EE and tumorigenesis in this particular depot has not been investigated.

Here, we utilized the commonly used MMTV-PyMT transgenic mouse model to test the effects of EE on spontaneous breast cancer latency and growth (Guy et al. 1992, Lin et al. 2003, Juncker-Jensen et al. 2009). Moreover, we carried this transgene on the C57BL/6 background, because they are more prone to diet-induced obesity (DIO) when fed a high-fat diet (HFD) and have longer tumor latency when compared to FVB/N-Tg(MMTV-PyMT) model, thus providing an excellent model to study the development of this disease in the context of EE. It has been reported that FVB/N-Tg(MMTV-PyMT) mice chronically fed a HFD experienced significantly increased mammary tumor growth, without effect on latency or metastasis compared to mice fed a normal chow diet (NCD) (Cowen et  al. 2015), presumably due to elevated leptin levels. To further investigate to role of leptin in EE, we orthotopically transplanted PyMT-derived primary breast cancer cells into mouse models of obesity with varied leptin signaling. These included (1) WT mice of DIO with elevated leptin levels reflective of the calorie-dense diets and sedentary lifestyle of Western civilizations (Fukumara et al. 2016) and (2) ob/ob mice which are leptin deficient, extremely obese and diabetic.

Methods

Mice

Transgenic MMTV-PyMT mice were previously generated via exogenous DNA microinjection of mouse embryos (Guy et  al. 1992). Females hemizygous for the PyMT gene are unable to lactate, thus, this line was carried on male C57BL/6-Tg (MMTV-PyMT) mice (provided by Dr.  M.  Ostrowski of The Ohio State University) and crossed with WT C57BL/6 females. Pups were weaned at three weeks of age, genotyped (IMR0015-F: CAAATGTTGCTTGTCTGGTG; IMR0016-R: GTCAGT CGAGTGCACAGTTT; IMR0384: GGAAGCAAGTACT TCACAAGGG; IMR0385: GGAAAGTCACTAGGAG CCAGGG) and randomly assigned a housing condition – standard environment (SE) or EE. Six-week-old C57BL/6 mice were purchased from Charles River; six- week-old ob/ob (B6.Cg-Lepob/J) mice were purchased from The Jackson Laboratory. Following a 3-day acclimation upon arrival, mice were randomly assigned to their prospective housing scheme. All mice were housed in temperature- (22–23°C) and humidity-controlled rooms with a 12 h light-12 h darkness cycle and maintained on either NCD

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(7912 rodent chow, Teklad) or HFD (60% kcal from lard; D12492, Research Diets Inc.), where indicated, with food and water ad libitum. All use of animals was approved by and conducted in accordance with the Ohio State University Institutional Animal Care and Use Committees.

EE protocol

Female mice were housed in large cages (63 × 49 × 44 cm, five mice per cage) supplemented with running wheels, tunnels, igloos, huts, retreats, wood toys, a maze and nesting material in addition to chow and water ad libitum (Slater & Cao 2015). Control mice were housed under standard laboratory cages of 30.5 × 17 × 15 cm (five mice per cage).

Body weight/adiposity

Body weights were recorded weekly for the duration of each experiment. A final account of adiposity was determined upon killing by weighing multiple fat depots and normalizing to the body weight of each individual. Food consumption was also recorded at the same time points of body weight as the total food consumption of each cage.

Glucose tolerance test

Mice were intraperitoneally injected with glucose solution (1 mg glucose per g body weight) after an overnight fast. Blood was drawn from the tail at various time points and the blood glucose concentrations were measured with a portable glucose meter (ReliOn Ultima).

Tumor inoculation

MMTV-PyMT mice develop breast tumors spontaneously, and therefore, do not require inoculation. For the transplantation models, primary tumors were harvested from naïve MMTV-PyMT mice and breast cancer cells were dissociated in collagenase and the heterogeneous cell population was collected (the same donor tumor preparation for both DIO and ob/ob mice experiments). WT/DIO and ob/ob mice were placed in their respective housing for specified time followed by orthotopic inoculation of 50,000 PyMT-derived breast cancer cells in 100 μL serum-free media to the fourth right mammary fat pad.

Tumor measurements

Latency was determined via biweekly palpation that started around 12  weeks of age in the MMTV-PyMT

experiment and at 1  week post breast cancer cell inoculation in the transplantation experiments. Once formed, the tumors were measured with calipers and tumor volume was calculated according to the formula V = π/6 × length × width2 (Tomayko & Reynolds 1989). Our IACUC approved euthanasia criteria state that mice are to be euthanized when any one tumor exceeds 2.5 cm3, when the overall tumor burden exceeds 4.5 cm3 or at any other signs of morbidity. Euthanasia times were as follows: MMTV-PyMT at 20  weeks of age; DIO 7  weeks post inoculation; ob/ob 8  weeks post inoculation (this timeline was determined in previous pilot studies; data not shown). Final tumor volumes and weights were determined upon dissection.

Serum harvest and biomarker measurement

Blood was collected following decapitation. Serum was prepared by allowing the blood to clot for 30 min on ice followed by centrifugation. Serum was at least diluted 1:5 in serum assay diluent and assayed using the following DuoSet ELISA Development System (R&D Systems): mouse Leptin, Adiponectin/Acrp30, IGF-1. Insulin was measured using Mercodia ultrasensitive mouse insulin ELISA (ALPCO Diagnostic). Glucose was measured using QuantiChrom Glucose Assay (BioAssay Systems). Total cholesterol was measured using Cholesterol E test kit (Wako Diagnostics). Triglyceride was measured using L-Type test (Wako Diagnostics). Corticosterone was measured using an ELISA kit (Enzo Life Sciences).

Tissue harvest and biomarker measurement

Upon euthanasia tumor and adipose tissues were dissected, weighed and flash frozen for further analysis. For biomarker analysis, tissues were homogenized via sonication in cold RIPA buffer supplemented with protease and phosphatase inhibitors (Pierce) and were centrifuged at 9300 g for 10 min at 4°C. The protein content of the supernatant was quantified by BCA kit (Pierce). The same ELISA kits above were used to measure tissue lysate concentrations.

Quantitative RT-PCR

Hypothalamic dissection was described previously (Liu et  al. 2014). Total RNA was isolated using RNeasy Lipid Kit plus RNase-free DNase treatment (Qiagen). First-strand cDNA was generated using TaqMan Reverse Transcription Reagent (Applied Biosystems). Real-time PCR

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486G D Foglesong et al. Lifestyle improvements inhibit breast cancer

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was carried out using StepOnePlus System (Applied Biosystems) with the Power SYBR Green PCR Master Mix (Applied Biosystems). Primer sequences are available upon request. Data were calibrated to endogenous control β-actin or Hprt1, and the relative gene expression was quantified using the 2−ΔΔCT method (Livak & Schmittgen 2001).

Catecholamine analysis

A Bi-CAT EIA kit (Alpco) was used to determine epinephrine and norepinephrine levels in the serum, tumor and fat per kit instructions. To obtain enough sample volume for the extraction assay, 100 μL of serum from three mice were pooled for a total of 300 μL serum, and at least 50 mg of tissue was homogenized via sonication with 200 μL of 20 mM Tris–HCl, 1 mM EDTA in dH2O. Lysates were then centrifuged at 9300 g for 10 min at 4°C. The protein content of supernatant was quantified by BCA kit (Pierce) to normalize the amount of catecholamine to the protein concentration of the tissue lysate.

Western blotting

Tumors were homogenized in ice-cold Pierce RIPA buffer containing 1× Roche PhosSTOP and Calbiochem protease inhibitor cocktail III, and then spun at 15,700 g for 15 min. Tissue lysates were separated by gradient gel (4–20%, Mini-PROTEAN TGX, Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad). Blots were incubated overnight at 4°C with the following primary antibodies, β-actin (Cell Signaling #3700, 1:1000), P-AKT (S473) (Cell Signaling #9271, 1:1000), total AKT (Cell Signaling #9272, 1:1000), P-p44/42MAPK (T202/Y204) (Cell Signaling #9101, 1:1000), total p44/42MAPK (Cell Signaling #9102, 1:1000), Cyclin D1 (Cell Signaling #2922, 1:1000), P-Stat3 (Cell Signaling #9145, 1:500) and VEGF (Abcam #ab46154, 1:800). Blots were rinsed and incubated with HRP-conjugate secondary antibody (Bio-Rad). Chemiluminescence signal was detected and visualized by LI-COR Odyssey Fc imaging system (LI-COR Biotechnology). Quantification analysis was carried out with image studio software version 5.2 (LI-COR Biotechnology).

Tumor histology

Tumor samples were fixed in 10% formalin. The Comparative Pathology & Mouse Phenotyping Core of OSU provided paraffin section processing, H&E staining and pathological evaluation by a certified pathologist.

Statistical analysis

Data are expressed as mean ± s.e.m. We used JMP software to analyze the following: unpaired, two-tailed Student’s t test for comparison between two groups; log-rank test for Kaplan–Meier analysis of comparison between two groups for the measurement of ‘time to an event’. Differences were considered statistically significant at P < 0.05.

Results

EE delayed breast cancer onset in MMTV-PyMT mice on HFD

Female C57Bl/6-MMTV-PyMT mice were used to investigate the effect of EE on genetically susceptible mouse model of breast cancer. First, to determine the response of MMTV-PyMT mice to EE, a separate cohort of mice, fed a NCD were placed in either SE or EE. Consistent with previous findings (Cowen et  al. 2015), they showed no overt difference in weight gain, but did respond in accordance with our previous studies via a significant drop in serum leptin levels following 8 weeks of EE (Fig. 1A and B). We then sought to investigate the impact of HFD and obesity on breast tumorigenesis by feeding the MMTV-PyMT mice a 60% HFD immediately after weaning at an age of 3–4 weeks. These mice were also randomly placed into either SE or EE at this time. PyMT mice were housed in these conditions for a total of 16  weeks until they reached an age of 20  weeks, which allowed for significant tumor growth, but no other health complications as determined by previous studies (data not shown). EE slightly attenuated weight gain over the course of the study (Fig.  1C), consistent with previous findings that show PyMT mice, regardless of genetic background, are somewhat resistant to excessive weight gain. Nevertheless, mice housed in EE showed a significant decrease in leptin 4 weeks post EE initiation (Fig. 1D). We performed qRT-PCR analysis of the hypothalamus of these mice, which revealed a significant upregulation of Mc4r (encoding melanocortin 4 receptor) and downregulation of Il-1b (encoding interleukin-1b) (Fig. 1E). Kaplan–Meier analysis revealed that breast tumor latency, determined via palpation, was significantly increased (P = 0.01) in response to EE with a median difference of 12 days and mean difference of 15.17 days (Fig. 1F). Tumor progression was also slowed although did not reach significance by the completion of the study (Fig. 1G).

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EE attenuated DIO and decreased engrafted PyMT breast tumor growth

To further explore the effects of EE on DIO and breast tumor progression, 6-week-old C57B/L6 wild-type (WT) female mice were randomly assigned to live in SE or EE and fed with HFD (60% calories from fat). EE robustly attenuated HFD-induced weight gain and reduced adiposity (Fig.  2A and C) while did not change the food consumption relative to body weight (Fig. 2B). After 10-week exposure to EE, serum biomarkers were determined. Insulin-like growth factor 1 (IGF-1), glucose or corticosterone was not significantly different between the two groups (Fig.  2D). Circulating leptin level was significantly reduced in EE mice (Fig. 2D), while adiponectin level was increased (Fig. 2D). Triglyceride and cholesterol levels were significantly decreased in EE mice (Fig. 2D) suggesting an attenuation of DIO-associated dyslipidemia. After the metabolic improvements following EE were observed, PyMT-derived primary breast tumor cells were isolated from a naïve MMTV-PyMT female mouse and then orthotopically transplanted into the right 4th mammary gland after 15 weeks EE.

EE effectively inhibited tumor growth (Fig.  2E). Leptin, IGF-1 and adiponectin were evaluated in the tumor, tumor-adjacent mammary white adipose tissue (T-mWAT), tumor-naïve mWAT and gonadal WAT (gWAT). EE significantly decreased leptin levels in all three WAT

depots while having no effect on leptin levels in the tumor itself (Fig.  2F). The same tissue analysis was also completed for IGF-1 and adiponectin, which showed no overt changes (Fig. 2G and H).

qRT-PCR analysis was performed to profile gene expression in various tissues. In the hypothalamus, Bdnf, Ntrk2 (encoding BDNF receptor TrkB), Mc4r, Agrp (encoding agouti-related peptide) and Pomc (encoding pro-opiomelanocortin), all involved in the regulation of energy balance, were significantly increased in EE mice (Fig.  3A). DIO is associated with hypothalamic inflammation and therefore inflammatory markers were examined. Il6 (encoding interleukins -6) and Il1b expression were significantly reduced in mice housing in EE (Fig.  3A). Next we investigated 3 β-adrenergic receptor genes (Adrb1–3), leptin (Lep) and its receptor, as well as genes associated with inflammation in tumor, T-mWAT and tumor-free mWAT. qRT-PCR revealed only modest variation in the tumor samples, none reaching significance (Fig.  3B). More robust effects on gene expression were observed in tumor-adjacent fat (T-mWAT) and contralateral tumor-free mWAT. Leptin receptor gene expression, both the long form (Obrb) and short form (Obra), was significantly increased in T-mWAT of EE mice (Fig.  3C). Leptin receptor genes, in addition to β-adrenergic receptor genes, were upregulated in tumor-free mWAT (Fig. 3D). The mRNA expression levels of some

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Figure 1EE delayed breast cancer onset in MMTV-PyMT transgenic mice. (A) Weight gain of MMTV-PyMT mice fed a NCD. (B) Leptin levels in the serum of MMTV-PyMT mice fed a NCD 8 weeks post EE. (C) Weight gain of MMTV-PyMT mice fed a HFD. (D) Leptin levels in the serum of MMTV-PyMT mice on HFD 4 weeks post EE. (E) Gene expression profile of the hypothalamus of MMTV-PyMT mice on HFD, n = 5 SE, 6 EE. (F) Kaplan-Meier analysis of latency of tumor occurrence in MMTV-PyMT mice on HFD. (G) Tumor volume in MMTV-PyMT mice on HFD. Data are mean ± s.e.m., n = 12 per group unless specified otherwise, *P < 0.05.

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inflammatory markers were decreased in these adipose depots; specifically, there was a significant downregulation of plasminogen activator inhibitor-1 (Pai-1) and MCP-1 (encoded by Ccl2) in mWAT, and a trend toward decreased

expression in T-mWAT (P = 0.059 and 0.086, respectively) (Fig. 3C and D).

Leptin signaling has been implicated in breast cancer growth and progression (Ando et  al. 2014).

Figure 2EE attenuated DIO and mitigated breast cancer progression in wild type mice transplanted with PyMT-derived breast cancer cells. (A) Weight gain. (B) Food intake relative to body weight monitored for 19 weeks. (C) Adiposity at euthanasia. (D) Serum biomarkers at euthanasia. (E) Tumor volume at euthanasia in DIO mice. (F, G and H) Tissue levels of leptin, IGF-1, and adiponectin. Data are mean ± s.e.m., n = 10 per group, *P < 0.05, **P < 0.01.

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Figure 3Gene expression profiles of the DIO mice inoculated with PyMT-derived breast cancer cells. (A) Hypothalamus, n = 5 per group. (B) Tumor. (C) Tumor-associated mammary fat (T-mWAT). (D) Tumor-naïve mammary fat (mWAT). Data are mean ± s.e.m., n = 6 per group, *P < 0.05.

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Leptin binding to its receptor induces activation of multiple intracellular signaling including Janus kinase signal transducer and activator of transcription 3 (JAK2-STAT3), phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) and mitogen-activated protein kinase (MAPK) pathways involved in various cellular activities (Ahima & Osei 2004). Western blotting was performed to examine the signaling pathways in tumor samples from EE and SE mice (Fig. 4). It is reported that leptin induces Cyclin D1 in breast cancer cells in vitro (Saxena et al. 2007) and Cyclin D1 expression is regulated by active leptin signaling in the MMTV-Wnt-1 transgenic mouse model of breast cancer (Zheng et al. 2012). The Cyclin D1 level in tumors from EE mice was significantly lower than that from SE mice suggesting slower proliferation. Leptin is reported to have proangiogenic property and contribute to tumor angiogenesis partially through induction of vascular endothelial growth factor (VEGF) (Rene Gonzalez et al. 2009, Newman & Gonzalez-Perez 2014). VEGF level was significantly reduced in EE tumors. One of the key downstream signaling pathways of leptin is STAT3. A trend of reduction of phospho-STAT3 was found in EE tumors but not significant. ERK1/2 signaling was reduced in the tumors of mice living in EE, while AKT signaling was not changed (Fig. 4).

Tumor histology and pathological examination was determined by core facility (three tumors per group, three sections per tumor). Fragmentation and necrosis were found in the tumor sections but not qualitatively distinguishable between the two groups (representative images Fig. 5A). Tumors from SE group had a mean of 13 mitoses per 400× field, while EE group had a mean of 12, but not significantly different.

EE inhibited obesity and improved metabolism, but increased PyMT tumor growth in leptin-deficient ob/ob mice

To explore the effects of leptin deficiency on EE and mammary tumorigenesis, 6-week-old female ob/ob mice were randomly placed in either SE or EE and fed with NCD. EE significantly attenuated weight gain just 2 weeks post EE initiation, which lasted the remainder of the experiment (Fig.  6A). The food consumption relative to body weight was higher in EE mice (Fig. 6B) indicating the decrease of weight not due to suppression of food intake. EE significantly improved glucose tolerance (Fig. 6C). EE caused a significant reduction in mWAT (Fig. 6D). Since ob/ob mice do not express leptin protein, it was omitted from the serum and tissue profile analysis. EE significantly decreased circulating insulin levels, but had little effect on the other metabolic markers assayed (Fig. 6E). Final tumor volume revealed that tumor growth was significantly increased in ob/ob mice housed in EE (Fig. 6F), a finding opposite to that in WT DIO model. EE had little effect on tissue IGF-1 or adiponectin (Fig. 6G and H).

qRT-PCR analysis showed a significant reduction in Agrp and Ccl2, as well as an increase in Pomc in the hypothalamus (Fig.  7A), a pattern distinct to DIO mice (Fig.  7A). In the tumor, EE significantly upregulated proangiogenic Vegf and downregulated Il-6 and showed a trend (P = 0.053) of reduction in the suppressor of cytokine signaling 3 (Socs3) (Fig.  7B). Adrb3 was upregulated in both T-mWAT and mWAT, whereas adiponectin (Adipoq) was increased in iWAT and Il-1b was increased in T-mWAT (Fig. 7C and D).

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Figure 4Western blotting of tumors from DIO mice. (A) Western blotting. (B) Quantification of (A). Data are mean ± s.e.m., n = 5 per group, *P < 0.05, **P < 0.01.

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A pathologist examined tumor histology (three tumors per group, three sections per tumor). The tumors from EE mice appeared to have larger areas of coagulative to liquefactive necrosis that spans multiple lobular structures and includes the intervening stromal

septa. The tumors in EE mice also appeared fragmented, with individualized neoplastic cells present in clear spaces in areas where the tumor has broken apart (representative images Fig.  5B). Tumors from SE group had a mean of 9.3 mitoses per 400× field, while

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Figure 6EE decreased weight gain and improved metabolism, but increased PyMT-derived breast cancer growth in the ob/ob mice fed a NCD. (A) Weight gain; syringe indicates when tumor cells were inoculated. (B) Food intake relative to body weight. (C) Glucose tolerance test. (D) Tissue weights at euthanasia. (E) Serum biomarkers at euthanasia. (F) Tumor volume at euthanasia. (G and H) Tissue levels of IGF-1 and adiponectin. Data are mean ± s.e.m., n = 9 per group, *P < 0.05.

Figure 5Histology of PyMT mammary tumors. (A) Representative H&E images of DIO mice implanted with PyMT tumor cells. (B) Representative H&E images of ob/ob mice implanted with PyMT tumor cells. Scale bar: 100 µm.

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EE group had a mean of 11, but did not reach significance.

To explain the EE-induced anti-cancer effect in leptin-proficient DIO mice and pro-cancer effect in leptin-deficient ob/ob mice, two catecholamines were measured due to their known roles in response to stress and EE: epinephrine (E) and norepinephrine (NE), in the tumor, T-mWAT, mWAT and the serum. There was no difference in E in either group and also no difference in NE in the DIO group (Fig. 8A, B and C). In contrast, NE was significantly elevated in the mWAT and serum of ob/ob mice following EE (Fig. 8D).

Discussion

With the epidemic of excessive weight, there are increasing numbers of cancer patients that are also overweight or obese, which worsens prognosis and limits treatment options. Some data suggest that weight loss can reduce the risk of breast cancer for both pre- and postmenopausal women (Moley & Colditz 2016). One study in particular discovered that a one unit reduction in BMI in the overweight and obese population improved overall health and decreased the risk of colon and breast cancer (Verhaeghe et al. 2016). However, concrete evidence as to whether sustained weight loss reduces breast cancer incidence is limited due to the low long-term success rates associated with weight loss interventions (Moley & Colditz 2016). It is also clear that all aspects of metabolic syndrome negatively affect cancer progression, but it is unclear whether these affects are additive or synergistic (Uzunlulu et  al. 2016). The local as well as systemic improvements in metabolism and overall health attributed to our model of EE could provide potential therapeutic targets or approaches that lack such limitations. Herein, we further described the mechanism of EE and subsequent HSA axis activation with regards to the unique microenvironment of breast cancer.

Our preliminary studies revealed that the MMTV-PyMT mice were somewhat resistant to weight modulation, consistent with previous findings (Cowen et  al. 2015), but did respond via a significant drop in leptin (Fig. 1A, B, C and D). The occurrence of palpable mammary tumor was delayed in EE mice compared to SE (Fig. 1F and G). The MMTV-PyMT gene may induce tumor formation in any or all of the ten mammary

Figure 7Gene expression profiles of the ob/ob mice inoculated with PyMT-derived breast cancer cells. (A) Hypothalamus. n = 6 per group. (B) Tumor. (C) Tumor-associated mammary adipose tissue (T-mWAT). (D) Tumor-naïve mammary adipose tissue (mWAT). Data are mean ± s.e.m., n = 9 per group, *P < 0.05.

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Figure 8Catecholamine analysis: (A and B) Epinephrine; (C and D) norepinephrine, in tumor, tumor-associated mammary adipose tissue (T-mWAT), tumor-naïve mammary adipose tissue (mWAT), and serum of wild type DIO mice and ob/ob mice on NCD. Data are mean ± s.e.m., n = 9 per group, *P < 0.05.

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glands present in a mouse which progress very quickly once formed, providing a reasonable explanation for the lack of significant difference and wide variability in additive tumor volume at study termination. Thus, PyMT mice represented an interesting model to study cancer prevention, alluding to the substantial effects of EE and its significant inhibition on cancer onset in a highly aggressive form of breast cancer. To investigate the effects of EE on tumor growth, we shifted our focus to orthotopic transplantation models where we also aimed to examine the role of leptin in tumorigenesis. Previous studies have investigated the effect of impaired leptin signaling in the MMTV-PyMT × ob/ob mouse, but results were confounded due to the impaired mammary gland development associated with this cross (Park et al. 2010). Therefore, orthotopic transplantation of PyMT-derived primary breast cancer cells proved to be a better model to investigate the effect of leptin signaling on tumorigenesis following EE.

Our data showed that both DIO and ob/ob mice responded to EE via improved metabolism and attenuation of weight gain. However, intact leptin signaling, specifically its drop following EE, was necessary to impede mammary tumor growth (Figs 2 and 6). We observed a significant increase in tumor latency in the MMTV-PyMT model of spontaneous breast cancer and a substantial attenuation of tumor growth in WT DIO mice following EE, associated with a substantial drop in leptin. However, in the absence of leptin (ob/ob mice), PyMT breast cancer progression was amplified in EE, relative to SE (Fig. 7F). This finding was unexpected and might seem paradoxical, but the central driver of EE’s anti-cancer effect is the activation of the HSA axis (Cao et  al. 2010). Our previous research indicates that HSA axis activation preferentially elevates SNS tone and NE release onto adipose tissue, which is essential to induce phenotypic changes of WAT, alleviation of obesity and systemic metabolic improvements, as well as anti-cancer effects on melanoma and colon cancer via the suppression of leptin (Cao et al. 2010, 2011). However, breast cancer is imbedded in mammary fat and is directly exposed to the changes in leptin and NE. Most animal studies on the stress-cancer relationship impose experimental stressors associated with distress such as restraint stress, social confrontation and social isolation (Stefanski & Ben-Eliyahu 1996, Thaker et al. 2006, Sloan et al. 2010) and demonstrate that stress hormones can modulate multiple components of the tumor microenvironment directly or indirectly to collectively support tumor initiation and progression (Antoni et  al. 2006). E and

NE have been shown to enhance growth and metastatic potential of multiple cancer types (Szpunar et al. 2016). Therefore, we analyzed the amount of both in the tumor, the T-mWAT, mWAT and circulation (serum). E is released predominantly from the adrenal medulla (Bartness et al. 2014), which has not been shown to be activated by EE or the HSA axis (Cao et  al. 2010). Similarly, there was no change in E following HSA axis activation via EE in DIO or ob/ob (Fig. 8). In contrast, EE elevated NE levels in WAT and serum in the ob/ob mice but did not change in WT DIO mice (Fig. 8C and D), which might underlie the accelerated PyMT mammary tumor growth observed only in ob/ob mice. Of note, in our previous study when B16 melanoma cells were implanted to the flank of male ob/ob mice, EE was unable to affect melanoma progression (Cao et  al. 2010). Gender difference and more likely the distance from adipose tissue (in contrast to breast cancer cells implanted to mammary fat) might cause the inconsistency among different types of cancers. Taken together, we propose that EE is a valuable model of eustress that is associated with adaptive responses and benign or beneficial effects on health (Selye 1974, Milsum 1985). EE represents an active and often challenging lifestyle in a more complex environment devoid of elements traditionally thought of as being stressful (Cao & During 2012). We previously publish that EE activates the HSA axis leading to increased NE release to adipose tissue and robust reduction of leptin (Cao et  al. 2010). We speculate that the overall impact on mammary tumor progression depends on the balance between the elevation of NE and the drop of leptin. In animals with leptin signaling, the anti-cancer effect of leptin depletion outweighed the pro-cancer effect of NE and resulted in the inhibition of mammary tumor growth. In the absence of leptin such as ob/ob mice, the effect of elevated NE cannot be offset by the leptin drop and thus lead to acceleration of tumor progression associated with EE, although the mammary tumor growth is slower in ob/ob mice compared to WT mice on HFD independent of living conditions. Monogenic obesity caused by leptin deficiency is extremely rare in humans (Farooqi et  al. 2002, Gibson et  al. 2004). Though homozygous ob/ob mice are useful to investigate the roles of leptin and NE in mediating the EE effects on mammary tumor, the pro-cancer effect observed in ob/ob mice is not a substantial concern because the vast majority of obesity is associated with increased leptin levels in animals and humans.

In addition to leptin, multiple mechanisms likely contribute to the EE’s anti-cancer effects. For example, cancer is associated with inflammation (Coussens & Werb 2002),

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and obesity is associated with chronically inflamed WAT increasing the release of proinflammatory cytokines including TNF-α, C-reactive proteins and several interleukins (IL-1β and IL-6) (Olefsky 2009). Our previous study demonstrates that genetically activating the HSA axis via hypothalamic overexpression of BDNF inhibits inflammation markers in the hypothalamus, the WATs and the implanted EO771 breast cancer in middle age obese mice (Liu et  al. 2014). Here, an overall anti-inflammatory response was also observed in young mice responding to environmental activation of the HSA axis (Figs 1 and 3). Further investigation is required to elucidate the importance of the EE-induced anti-inflammatory on cancer. Furthermore, we recently published that environmental and genetic activation of hypothalamic BDNF modulates T-cell immunity and contributes to the anti-cancer phenotype in a melanoma model in young male mice of normal body weight (Xiao et al. 2016). Our preliminary data showed that EE profoundly modulated immune cells residing in adipose tissue in both lean and obese animals. How EE might affect breast cancer progression via systemic and local immune modulations is another intriguing question to answer in future studies. Furthermore, we are generalizing EE to other breast cancer models and older mice.

In summary, EE was effective at delaying the onset of spontaneous breast cancer in the MMTV-PyMT mouse model as well as attenuating DIO and breast cancer growth when orthotopically transplanted into WT mice fed a HFD. The EE-induced inhibition of breast cancer required leptin. In the absence of leptin, EE promoted breast cancer growth in the ob/ob mouse model, possibly due to the elevated NE levels. Results of this study and future studies will provide novel, dual-purpose therapeutic strategies to both improve systemic metabolism as well as mitigate cancer onset and progression.

Declaration of interestThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

FundingThis work was supported by the NIH grants NCI R01-CA166590, NCI R01-CA163640, NCI R21-CA178227 to L C and the Pelotonia Graduate Fellowship to G D F.

AcknowledgementsThe authors thank Dr Michael Ostrowski of The Ohio State University Department of Cancer Biology and Genetics for providing breeder

MMTV-PyMT mice. The authors also thank him for thoughtful experimental guidance and discussion.

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Received in final form 6 March 2019Accepted 11 March 2019Accepted Preprint published online 11 March 2019

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