Elizabeth S. Neil

Effect of Nonmotorized Wheel Running on Mammary Carcinogenesis: Circulating Biomarkers, Cellular Processes, and Molecular Mechanisms in Rats

The objective of this experiment was to identify circulating growth factors, hormones, and cellular and molecular mechanisms that account for the effects of physical activity on mammary carcinogenesis. A total of 120 female Sprague-Dawley rats were injected with 1-methyl-1-nitrosourea (50 mg/kg) and 7 days thereafter were randomized to either a physically active or a sedentary control group. Individually housed rats were given free access to a nonmotorized, computer-controlled activity wheel and running behavior was reinforced by food reward. Rats self-determined their daily intensity and duration of running. Sedentary control rats received the same amount of food as the physically active rats to which they were paired. Physical activity reduced mammary cancer incidence (P = 0.015) and cancer multiplicity (P = 0.01). Physical activity induced changes in plasma insulin, insulin-like growth factor-I, and corticosterone, suggesting that mechanisms regulating glucose homeostasis were affected. Western blot analyses of mammary carcinomas revealed that proteins involved in cell proliferation were reduced (P < 0.001) and those involved in apoptosis via the mitochondrial pathway were elevated (P < 0.001) by physical activity. The hypothesis that these effects were mediated by activation of AMP-activated protein kinase, and down-regulation of protein kinase B, which collectively down-regulate the activity of the mammalian target of rapamycin, was evaluated. Evidence in support of this hypothesis was found in the Western blot analyses of mammary carcinomas, mammary gland, liver, and skeletal muscle. Collectively, these findings provide a rationale for additional studies of energy-sensing pathways in the elucidation of mechanisms that account for the inhibition of carcinogenesis by physical activity. (Cancer Epidemiol Biomarkers Prev 2008;17(8):1920–9)

Effects of Energy Restriction and Wheel Running on Mammary Carcinogenesis and Host Systemic Factors in a Rat Model

Limiting energy availability via diet or physical activity has health benefits; however, it is not known whether these interventions have similar effects on the development of cancer. Two questions were addressed as follows: (i) Does limiting energy availability by increasing physical activity have the same effect on mammary carcinogenesis as limiting caloric intake? and (ii) Are host systemic factors, implicated as risk biomarkers for breast cancer, similarly affected by these interventions? Female Sprague Dawley rats were injected with 50-mg 1-methyl-1-nitrosourea per kg body weight at 21 days of age and randomized to one of five groups (30 rats per group) as follows: (i) sham running wheel control; (ii) restricted fed to 85% of the sham control; (iii and iv) voluntary running in a motorized activity wheel (37 m/min) to a maximum of 3,500 m/d or 1,750 m/d; and (v) sedentary ad libitum fed control with no access to a running wheel. The three energetics interventions inhibited the carcinogenic response, reducing cancer incidence (P = 0.01), cancer multiplicity (P < 0.001), and cancer burden (P < 0.001) whereas prolonging cancer latency (P = 0.004) although differences among energetics interventions were not significant. Of the plasma biomarkers associated with the development of cancer, the energetics interventions reduced bioavailable insulin-like growth factor-1 (IGF-1), insulin, interleukin-6, serum amyloid protein, TNF-α, and leptin and increased IGF-binding protein 3 (IGFBP-3) and adiponectin. Plasma-fasting glucose, C-reactive protein, estradiol, and progesterone were unaffected. The plasma biomarkers of greatest value in predicting the carcinogenic response were adiponectin > IGF-1/IGFBP-3 > IGFBP-3 > leptin > IGF-1. Cancer Prev Res; 5(3); 414–22. ©2012 AACR.

Excess weight gain accelerates 1-methyl-1-nitrosourea-induced mammary carcinogenesis in a rat model of premenopausal breast cancer.

In contrast to the null effects generally reported, high-risk premenopausal women (Gail score ≥1.66) enrolled in the Breast Cancer Prevention P-1 Trial were recently reported to be at increased risk for breast cancer when overweight (HR = 1.59) or obese (HR = 1.70). To investigate this clinical observation in a preclinical setting, ovary-intact female rats were intraperitoneally injected with 50 mg/kg 1-methyl-1-nitrosourea at 21 days of age to simulate premenopausal women with increased risk. Two commercially available strains of Sprague–Dawley rat (Taconic Farms) were used, which are dietary resistant (DR) or dietary susceptible (DS) to excess weight gain when fed a purified diet containing 32% kcal from fat, similar to levels consumed by the typical American woman. DS rats were approximately 15.5% heavier than DR rats at study termination and plasma leptin indicated a marked difference in adiposity. DS rats had higher incidence (26% increase), multiplicity (2.5-fold increase), and burden (5.4-fold increase) of mammary carcinomas with a concomitant reduction in cancer latency (16% earlier detection) compared with DR rats (P < 0.001 for all analyses), and displayed a higher proportion of hormone receptor negative tumors compared with DR rats [OR = 1.78; 95% confidence interval (CI), 0.83–3.81]. Circulating levels of several breast cancer–risk factors, including leptin, adiponectin:leptin ratio, insulin, insulin-like growth factor (IGF)-1, IGF-1:IGF-1 binding protein-3 ratio, and calculated insulin resistance (HOMA-IR) were negatively impacted in DS rats (P < 0.05 for all analyses). These findings support further investigation of the effects of excess weight in high-risk premenopausal women and demonstrate a useful preclinical model for rapid evaluation of mechanistic hypotheses. Cancer Prev Res; 7(3); 310–8. ©2014 AACR.

Inherent aerobic capacity-dependent differences in breast carcinogenesis

Although regular physical activity is associated with improvement in aerobic capacity and lower breast cancer risk, there are heritable sets of traits that affect improvement in aerobic capacity in response to physical activity. Although aerobic capacity segregates risk for a number of chronic diseases, the effect of the heritable component on cancer risk has not been evaluated. Therefore, we investigated breast carcinogenesis in rodent models of heritable fitness in the absence of induced physical activity. Female offspring of N:NIH rats selectively bred for low (LIAC) or high (HIAC) inherent aerobic capacity were injected intraperitoneally with 1-methyl-1-nitrosurea (70 mg/kg body wt). At study termination 33 weeks post-carcinogen, cancer incidence (14.0 versus 47.3%; P < 0.001) and multiplicity (0.18 versus 0.85 cancers per rat; P < 0.0001) were significantly decreased in HIAC versus LIAC rats, respectively. HIAC had smaller visceral and subcutaneous body fat depots than LIAC and activity of two proteins that regulated the mammalian target of rapamycin, protein kinase B (Akt), and adenosine monophosphate-activated protein kinase were suppressed and activated, respectively, in HIAC. Although many factors distinguish between HIAC and LIAC, it appears that the protective effect of HIAC against breast carcinogenesis is mediated, at least in part, via alterations in core metabolic signaling pathways deregulated in the majority of human breast cancers.

Effect of low or high glycemic load diets on experimentally induced mammary carcinogenesis in rats

SCOPE High glycemic load diets have been associated with increased breast cancer risk in population-based studies, but the evidence is mixed. This investigation determined whether diets differing in glycemic load affected the carcinogenic process using a preclinical model. METHODS AND RESULTS Human diets, formulated to differ 2-fold in glycemic load, were evaluated in the 1-methyl-nitrosourea-induced (37.5 mg/kg) mammary carcinogenesis model. Cancer incidence (23.3 versus 50.0%, p = 0.032), multiplicity, (0.40 versus 1.03, p = 0.030) and burden, (0.62 versus 1.19 g/rat, p = 0.037) were reduced in the low versus high glycemic load diets, respectively. However, the low glycemic protective effect was attenuated when two purified diets that differed in resistant starch and simulated the glycemic effects of the human diets were fed. Protection was associated with alterations in markers of cell growth regulation. CONCLUSION Our findings show that human low or high glycemic load dietary patterns differentially affect the carcinogenic response in a nondiabetic rodent model for breast cancer. However, factors that are associated with these patterns, in addition to dietary carbohydrate availability, appear to account for the differences observed.

Premenopausal Obesity and Breast Cancer Growth Rates in a Rodent Model.

Obese premenopausal women with breast cancer have poorer prognosis for long term survival, in part because their tumors are larger at the time of diagnosis than are found in normal weight women. Whether larger tumor mass is due to obesity-related barriers to detection or to effects on tumor biology is not known. This study used polygenic models for obesity and breast cancer to deconstruct this question with the objective of determining whether cell autonomous mechanisms contribute to the link between obesity and breast cancer burden. Assessment of the growth rates of 259 chemically induced mammary carcinomas from rats sensitive to dietary induced obesity (DS) and of 143 carcinomas from rats resistant (DR) to dietary induced obesity revealed that tumors in DS rats grew 1.8 times faster than in DR rats. This difference may be attributed to alterations in cell cycle machinery that permit more rapid tumor cell accumulation. DS tumors displayed protein expression patterns consistent with reduced G1/S checkpoint inhibition and a higher threshold of factors required for execution of the apoptotic cell death pathway. These mechanistic insights identify regulatory targets for life style modifications or pharmacological interventions designed to disrupt the linkage between obesity and tumor burden.

Beneficial Effects of Common Bean on Adiposity and Lipid Metabolism

In developed countries which are at the epicenter of the obesity pandemic, pulse crop consumption is well below recommended levels. In a recent systematic review and meta-analysis of 21 randomized controlled clinical trials, pulse consumption was associated with improved weight control and reduced adiposity, although the underlying mechanisms were a matter of speculation. Common bean (Phaseolus vulgaris L.) is the most widely consumed pulse crop and was the focus of this investigation. Using outbred genetic models of dietary induced obesity resistance and of dietary induced obesity sensitivity in the rat, the impact of bean consumption was investigated on the efficiency with which consumed food was converted to body mass (food efficiency ratio), body fat accumulation, adipocyte morphometrics, and patterns of protein expression associated with lipid metabolism. Cooked whole bean as well as a commercially prepared cooked bean powders were evaluated. While bean consumption did not affect food efficiency ratio, bean reduced visceral adiposity and adipocyte size in both obesity sensitive and resistant rats. In liver, bean consumption increased carnitine palmitoyl transferase 1, which is the rate limiting step in long chain fatty acid oxidation and also resulted in lower levels of circulating triglycerides. Collectively, our results are consistent with the clinical finding that pulse consumption is anti-obesogenic and indicate that one mechanism by which cooked bean exerts its bioactivity is oxidation of long chain fatty acids.

Abstract 3601: Excess weight gain accelerates 1-methyl-1-nitrosourea (MNU)-induced mammary carcinogenesis in ovary-intact Sprague Dawley (Levin) rats fed 31.8% dietary calories from fat.

Though population data for breast cancer indicate that adult weight gain is associated with increased risk in postmenopausal women, the generally accepted lack of effect of obesity on premenopausal breast cancer risk has recently been challenged (Cecchini et al Cancer Prev Res; 5(4); 583-92, 2012). In that publication from the National Surgical Adjuvant Breast and Bowel Project (NSABP), BMI >30 was significantly associated with increased risk of invasive breast cancer in high-risk premenopausal women (HR= 1.70). As the effects of excess weight gain on breast carcinogenesis in women prior to menopause remain poorly understood, the Levin strains of outbred Sprague Dawley rats that are dietary obesity sensitive (DS) or resistant (DR) were used to investigate this issue. The Levin rats, which are available from Taconic Farms, were selectively outbred for >20 generations for resistance (DR) or susceptibility (DS) to weight gain when fed a purified diet of similar fat content as the typical diet consumed by women in the United States (31.8% fat by kcal). DS rats gain more weight than DR rats and develop metabolic syndrome after several weeks, including dyslipidemia and insulin resistance. Ovary-intact DS (n=101) and DR (n=103) rats were injected with 50 mg/kg of 1-methyl-1-nitrosourea (MNU) at 21-24 days of age, and began ad libitum feeding of the purified diet the following day. The experiment was terminated 9 weeks post-carcinogen at which point DS rats were approximately 17% heavier than their DR counterparts (220.3 ± 22.1 g vs. 187.9 ± 14.6 g, p Supported by PHS grant CA52626 from the National Cancer Institute. Citation Format: Shawna B. Matthews, John N. McGinley, Zongjian Zhu, Weiqin Jiang, Elizabeth Neil, Angie Neil, Henry J. Thompson. Excess weight gain accelerates 1-methyl-1-nitrosourea (MNU)-induced mammary carcinogenesis in ovary-intact Sprague Dawley (Levin) rats fed 31.8% dietary calories from fat. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3601. doi:10.1158/1538-7445.AM2013-3601

Abstract 593: Mechanisms for inhibition of rodent mammary carcinogenesis by wheel running and/or dietary energy restriction

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Our laboratory has reported the inhibitory effects of combined physical activity (PA) and dietary energy restriction (DER) on mammary carcinogenesis while the associated mechanisms are rarely investigated. The objective of this experiment was to determine the cellular regulation of mammary cancer by PA and/or DER. Female Sprague Dawley rats were injected with 1-methyl-1-nitrosourea (50 mg/kg) and 7 days thereafter randomized to: 1) sedentary control, 2) PA at wheel running high (WR-HIGH, 3.4 km per day); or the energy balance equivalents of PA: 3) PA at wheel running low (WR-LOW, 1500 revolutions per day + DER), or 4) DER. In order to investigate the effects of physical activity at a constant intensity, rats were given free access to a motorized activity wheel (55 m/min) with reinforcement of running behavior using a food reward system. The duration of this experiment was 8 weeks. Overall, PA and/or DER reduced mammary cancer incidence, cancer multiplicity, and tumor burden and prolonged cancer latency (p<0.05) in comparison to the sedentary control group. PA, DER, or their combination differentially affected host systemic factors including bioavailable IGF-1 and adipokines (leptin and adiponectin) and cell autonomous mechanisms including modulation of FoxO and down regulation of Akt/mTOR signaling. Mediation appears to be via inhibition of cell proliferation and induction of apoptosis. Supported by PHS grant CA100693 from the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 593. doi:1538-7445.AM2012-593