Advanced age and obesity are major risk factors for breast cancer progression, including triple-negative breast cancer (TNBC). In this study, we interrogated (i) whether these factors interact to promote TNBC progression and (ii) whether weight loss mitigates the separate and combined effects of aging and obesity on TNBC. We demonstrate that aging and diet-induced obesity interact to promote TNBC growth in mice. Transcriptomic analysis revealed the suppression of antitumor immunity in tumors from aged and/or obese mice. Weight loss via intermittent calorie restriction reduced tumor growth and restored immune-related gene signatures to reverse the protumor effects of aging and/or obesity. Using publicly available genomic datasets from murine studies of obesity, weight loss, and TNBC, we identified a consensus transcriptomic signature of obesity-driven immunosuppression that predicted the survival of patients with breast cancer. This consensus signature was also suppressed by aging, obesity, and their combination. Intermittent calorie restriction reversed the effects of aging and/or obesity on the consensus signature. We conclude that aging and obesity interact to limit antitumor immunity and enhance TNBC progression and that these adverse effects can be disrupted by weight loss. PREVENTION RELEVANCE:Advanced age and obesity are important risk factors for the development and progression of breast cancers, including TNBC. We demonstrate that the suppression of signatures of antitumor immunity is a common feature of accelerated tumor progression in TNBC. We show that weight loss achieved through calorie restriction can restore such markers. See related Spotlight, p. 437.

Obesity promotes triple-negative breast cancer (TNBC), and effective interventions are urgently needed to break the obesity-TNBC link. Epidemiologic studies indicate that bariatric surgery reduces TNBC risk, while evidence is limited or conflicted for weight loss via low-fat diet (LFD) or calorie restriction (CR). Using a murine model of obesity-driven TNBC, we compared the antitumor effects of vertical sleeve gastrectomy (VSG) with LFD, chronic CR, and intermittent CR. Each intervention generated weight and fat loss and suppressed tumor growth relative to obese mice (greatest suppression with CR). VSG and CR regimens exerted both similar and unique effects, as assessed using multiomics approaches, in reversing obesity-associated transcript, epigenetics, secretome, and microbiota changes and restoring antitumor immunity. Thus, in a murine model of TNBC, bariatric surgery and CR each reverse obesity-driven tumor growth via shared and distinct antitumor mechanisms, and CR is superior to VSG in reversing obesity's procancer effects.

Differentially methylated CpG dinucleotides at ESR1 and near ESR2

Introduction:Advanced age and obesity are independent risk and progression factors for triple negative breast cancer (TNBC), which presents significant public health concerns for the aging population and its increasing burden of obesity. Due to parallels between advanced age- and obesityrelated biology, particularly adipose inflammation, we hypothesized that advanced age and obesity each accelerate mammary tumor growth through convergent, and likely interactive, mechanisms. Methods:To test this hypothesis, we orthotopically transplanted murine syngeneic TNBC cells into the mammary glands of young normoweight control (7 months), young diet-induced obese (DIO), aged normoweight control (17 months), and aged DIO female C57BL/6J mice. Results:Here we report accelerated tumor growth in aged control and young DIO mice, compared with young controls. Transcriptional analyses revealed, with a few exceptions, overlapping patterns of mammary tumor inflammation and tumor immunosuppression in aged control mice and young DIO mice, relative to young controls. Moreover, aged control and young DIO tumors, compared with young controls, had reduced abundance ofcytotoxic CD8 T cells. Finally, DIO in advanced age exacerbated mammary tumor growth, inflammation and tumor immunosuppression. Discussion:These findings demonstrate commonalities in the mechanisms driving TNBC in aged and obese mice, relative to young normoweight controls. Moreover, we found that advanced age and DIO interact to accelerate mammary tumor progression. Given the US population is getting older and more obese, age- and obesity-related biological differences will need to be considered when developing mechanism-based strategies for preventing or controlling breast cancer.

<p>Diet Composition Comparison</p>

Reduced representation bisulfite sequencing mapping statistics

<p>Diet Composition Comparison</p>

Ensembl Regulatory Build of the ESR2 genomic landscape

Ensembl Regulatory Build of the ESR1 genomic landscape

Advanced age and obesity are two major risk factors for breast cancer (BC) mortality. This presents a significant public health concern as the number of older individuals and incidence of obesity are increasing worldwide. In our mouse models of BC, we have demonstrated that, similar to obesity, advanced age accelerates mammary tumor growth. Mechanistically, obesity and advanced age suppress tumor gene expression relating to antitumor immunity and reduce tumoral abundance of cytotoxic CD8+ T cells. Thus, advanced age- and obesity-related enhancement of mammary tumor growth is explained, in part, through the development of an immunosuppressive tumor microenvironment. Given the aging of our populations and the increasing prevalence of obesity, interventions capable of reversing the tumor-promoting effects of advanced age and obesity are needed. We have previously demonstrated that weight loss by intermittent calorie restriction (ICR), in which mice are placed on a 5:2 calorie restriction (CR) regimen (5 days 14% CR, 2 nonconsecutive days 70% CR per week), attenuates tumor growth and immunosuppression in formerly obese mice. This project tests if ICR will provide similar benefit to aged and aged obese mice. Cohorts of young control (5 mos), young diet-induced obese (DIO; 5 mos), aged control (15 mos), and aged DIO (15 mos) mice were generated and subsequently randomized to either remain on their baseline diet or switch to the ICR intervention. Following 9 weeks on ICR or baseline diet, serum samples were collected and then tumor development induced by orthotopic transplantation of E0771 cancer cells into the 4th mammary fat pad of mice. At tumor endpoint mammary tumors were collected and weighed. To determine if ICR alters systemic inflammation in each experimental group, cytokine levels were measured in serum samples collected prior to tumor inoculation. Multiple inflammatory cytokines were downregulated following ICR intervention in young DIO, aged control, and aged DIO mice, including CCL7, CCL10, and CCL24. Downregulation of inflammatory cytokines was correlated with decreased tumor burden in young DIO, aged control, and aged DIO mice placed on ICR, compared with their respective non-intervention controls. Ongoing analyses are investigating if ICR increases the abundance of cytotoxic CD8+ T cells within the tumor microenvironment of young DIO, aged control, and aged DIO mice. These findings demonstrate that ICR may be effective in reversing obesity- and advanced age-related enhancement of mammary tumor growth in mouse models of breast cancer. More research is needed to test if these preclinical findings translate to obese and aged humans with BC. Identifying dietary interventions that may attenuate obesity- and age-related tumor growth has the potential to improve both patient outcomes and quality of life. This research was supported by R35CA197627 to S. Hursting. Citation Format: Dalton M. Craven, Laura A. Smith, Michael F. Coleman, Elaine M. Glenny, Stephen D. Hursting. Intermittent calorie restriction reverses the adverse effects of obesity and advanced age on tumor growth in a mouse model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2575.

Abstract Although obesity is a known risk factor for breast cancer incidence, metastasis, and mortality, the mechanisms underlying this link remain elusive. This study therefore aims to uncover the relationship between obesity-associated adipose tissue and breast cancer metastasis by identifying specific inflammatory mediators in the adipose secretome. It is well known that obesity induces hypoxic conditions in the adipose tissue, stimulating an inflammatory response that ultimately creates a favorable environment for cancer cell growth and survival. The link between obesity-associated adipose inflammation and cancer metastasis, however, remains poorly described. We have previously shown that injection of basal-like E0771's, and claudin-low metM-Wnt-Lung and metM-Wnt-Liver metastatic breast cancer cell lines in mice fed a diet-induced obesity (DIO, 60% kcal from fat) diet displayed larger tumors with increased metastatic potential compared to lean mice fed a control (10% kcal from fat) diet. When diets were supplemented with Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), the enhancing effects of DIO on tumor growth and metastasis were mitigated. Gene expression microarray analysis of non-tumor bearing mammary fat pads from these mice revealed increased mammary expression of inflammation-related genes in tissues from DIO relative to control mice. Moreover, gene expression analysis of the tumor microenvironment from these mice revealed a signature of DIO-induced immunosuppression that was normalized by Sulindac supplementation. To further investigate the relationship between obesity-associated inflammation and breast cancer metastasis, we cultured our metastatic metM-Wnt-Lung, and metM-Wnt-Liver, and claudin-low, non-metastatic M-Wnt breast cancer cell lines with conditioned media (FCM) from the mammary fat pads of mice fed control, DIO, or DIO+Sulindac diets. We then conducted phenotypic assays to characterize differences in cell growth, proliferation, and metastatic potential. After normalizing the FCM concentrations to fat pad mass, we saw increased cell viability and migration in cells treated with FCM from DIO relative to control mice. Luminex assays identified several pro-inflammatory factors associated with the pro-cancer effects of FCM from DIO versus control mice, and additional characterization studies utilizing high-throughput proteomic screening and immune-depletion approaches are on-going. Taken together, our findings suggest an important role for the adipose secretome, and specifically inflammatory mediators, in obesity driven breast cancer progression. Citation Format: Lydia K. Eisenbeis, Shannon B. McDonell, Laura A. Smith, Alyssa J. Cozzo, Michael F. Coleman, Stephen D. Hursting. The role of obesity-associated adipose tissue inflammation in breast cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3971.

Advanced age and obesity are major risk factors for breast cancer (BC) mortality that are independently associated with aggressive disease and therapeutic resistance. Despite this, preclinical models of BC mainly utilize young lean mice. The mechanisms underlying age- and obesity-related BC progression are currently unknown, highlighting a critical gap in our understanding of BC biology. To address this gap, we generated aged (16 mos) and young (6 mos) cohorts of lean and obese mice by placement on either low-fat control (10% kcal from fat) or diet-induced obesity (DIO; 60% kcal from fat) diet, respectively, beginning at 8-weeks of age. This resulted in four experimental groups: aged lean, aged DIO, young lean, young DIO. Mice were orthotopically injected with one of two triple negative BC cell lines (E0771 or Met-MWNTLung), which represent aggressive BC subtypes associated with poor prognosis in humans. Outcomes included primary tumor weight and lung metastasis quantification. Biochemical and molecular analyses included serum cytokines and gene expression within tumor and adjacent normal mammary tissue. Our TNBC models indicate tumor growth is similarly increased in young DIO and aged lean mice, with both significantly higher than young lean mice. Moreover, the combination of advanced age and obesity in aged DIO mice significantly increased tumor growth relative to all other experimental groups. Preliminary pathology analyses in Met-MWNTLung-bearing mice reveal that advanced age enhances lung metastasis beyond that of young mice regardless of diet. Increased tumor burden in both the aged lean and young DIO mice is accompanied by increased serum proinflammatory cytokines (IL-1b, CXCL13, CCL11, among others). Consistent with these findings, Gene Set Enrichment Analysis (GSEA) of Met-MWNTLungtumor and adjacent normal mammary microarray data demonstrated enrichment of inflammatory pathways, including IFNγ response, IFNα response, IL-2/STAT5 signaling, and IL-6/JAK/STAT3 signaling in aged lean and young DIO mice relative to young lean mice. Interestingly, tumor-adjacent mammary from aged lean and young DIO mice was also enriched for markers of EMT, consistent with age- and DIO-associated phenotypic switching of cells within the tumor-adjacent microenvironment. Taken together, our findings reveal that obesity and advanced age augment BC progression and promote inflammation within the local (mammary & tumor) and systemic (serum) microenvironments. Ongoing analyses are investigating the role of cellular senescence, a stress-induced cell cycle arrest in which cells acquire a proinflammatory secretome. Knowledge gained from this study will help to elucidate the mechanisms underlying advanced age- and obesity-related BC progression, which is urgently needed to develop new strategies for reducing adverse TNBC outcomes associated with advanced age and obesity in human populations.Citation Format: Laura A. Smith, Magdalena A. Rainey, Nishita T. Sheth, Stephanie A. Montgomery, Stephen D. Hursting. Advanced age and obesity separately and interactively potentiate triple negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2843.

Abstract Breast cancer (BC) is the most common noncutaneous cancer among US women, and ~20% of these cancers overproduce the growth-promoting tyrosine kinase receptor, HER2. Although there are targeted treatments available for HER2-overexpressing BC, there remains an urgent need to identify new treatment strategies since this disease is associated with poor prognosis, resistance to therapy, and high risk of recurrence. We have established that calorie restriction (CR; 30% reduction in total energy relative to ad libitum-fed controls) has significant tumor suppressive effects across all breast cancer subtypes and has potential to be utilized as an adjunctive therapy. This study investigates the mechanisms by which CR decreases HER2-overexpressing BC progression. To mimic CR in vitro, murine MMTV-neu cells that overexpress neu, the rodent homolog of HER2, were treated with media containing reduced serum (1%), reduced glucose (1mM), or reduced serum and reduced glucose (1%/1mM) compared to control media (10% serum, 25mM glucose). MTT assays demonstrated that both serum restricted medias (1% and 1%/1mM) significantly decreased cellular viability (p<0.05) compared to control. To investigate the mechanism of this alteration, western blotting analysis of proteins associated with HER2 BC progression was conducted. In MMTV-neu cells treated with serum restricted medias (1% and 1%/1mM), there was a significant decrease in activity of tumor suppressor protein p21. Both p21 and phospho-21 expression were decreased with serum restriction, as well as the ratio of phospho-p21 relative to p21 (p<0.05). Phosphorylated p21 is localized to the cytoplasm and allows BC cells to evade apoptosis and proliferate uncontrollably. These results suggest that short-term CR, achieved specifically through serum restriction alone or in combination with glucose restriction, is associated with decreased phosphorylation and cytoplasmic localization of p21, which may be responsible for modulating anti-proliferative activities. This finding is significant because clinical studies have found that increased cytoplasmic p21 in HER2-overexpressing BC predicted reduced survival in patients at 5 years. Utilizing CR or pharmacologic regimens that mimic CR in conjunction with existing therapies may prevent or reverse cytoplasmic p21 localization in this cancer subtype, highlighting the importance of this investigation. Studies are ongoing in MMTV-neu cells transfected with plasmids encoding mutated p21 proteins that are constitutively expressed in either the cytoplasm or the nucleus. This will confirm the extent to which localization of p21 is responsible for CR-induced alterations in HER2-overexpressing BC cell proliferation. Citation Format: Magdalena A. Rainey, Laura A. Smith, Ciara H. O'Flanagan, Stephen D. Hursting. Short-term calorie restriction alters expression of tumor suppressor p21 in HER2-overexpressing breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2396.

Prevalence of obesity, an established risk factor for many cancers, has increased dramatically over the past 50 years in the United States and across the globe. Relative to normoweight cancer patients, obese cancer patients often have poorer prognoses, resistance to chemotherapies, and are more likely to develop distant metastases. Recent progress on elucidating the mechanisms underlying the obesity-cancer connection suggests that obesity exerts pleomorphic effects on pathways related to tumor development and progression and, thus, there are multiple opportunities for primary prevention and treatment of obesity-related cancers. Obesity-associated alterations, including systemic metabolism, adipose inflammation, growth factor signaling, and angiogenesis, are emerging as primary drivers of obesity-associated cancer development and progression. These obesity-associated host factors interact with the intrinsic molecular characteristics of cancer cells, facilitating several of the hallmarks of cancer. Each is considered in the context of potential preventive and therapeutic strategies to reduce the burden of obesity-related cancers. In addition, this review focuses on emerging mechanisms behind the obesity-cancer link, as well as relevant dietary interventions, including calorie restriction, intermittent fasting, low-fat diet, and ketogenic diet, that are being implemented in preclinical and clinical trials, with the ultimate goal of reducing incidence and progression of obesity-related cancers.

Abstract Purpose: Approximately 20% of breast cancers (BC) among US women overexpress HER2, an oncoprotein that stimulates increased cellular proliferation and survival. HER2 acts through two major signaling pathways, PI3K/Akt and Raf/MAPK, and its overexpression is associated with aggressive disease, resistance to therapy, and poor prognosis. Our laboratory has previously demonstrated in preclinical models that calorie restriction (CR) modulates HER2 BC pathogenesis, decreasing incidence and increasing latency in transgenic MMTV-HER2/neu mice. Our current study aims to determine the impact of CR on HER2 signaling and cancer cell phenotypes, including proliferation, cell cycle progression, and apoptosis. Methods: In vitro experiments were performed using a BC cell line derived from the MMTV-HER2/neu mouse model. To mimic CR, cells were treated with nutrient restricted media containing either reduced glucose (1mM), reduced serum (1%) or both (1mM/1%). The impact of CR on proliferation and survival was measured using growth curve experiments, and flow cytometric analysis of cell cycle and apoptosis. HER2 signaling proteins were assessed by western blotting. Results: Relative to cells grown in control media (25mM glucose/10% BCS): a) cells grown in glucose restricted (1mM) + serum restricted (1%) media grew 87% slower (p<0.01); b) cells grown in serum-restricted media (1%) with control glucose levels (25mm) grew 86% slower (p<0.01); and c) cells grown in glucose restricted media (1mM) with control serum levels (10%) grew 67% slower (p<0.05). Serum restricted groups, regardless of glucose levels, also exhibited increased G1 cell cycle arrest following 24 hours of treatment and increased percentage of apoptotic cells following 72 hours of treatment compared to control. In contrast, glucose restriction alone did not significantly affect apoptosis compared to control; however, it induced a G2/M arrest, possibly explaining reduction in growth. Western blotting analysis revealed that serum restricted cells had reduced levels of pAkt (Ser473) but no differences in Akt, pERK, or ERK, compared to the control and 1mM groups following 24 hours of CR treatment. Conclusions: In vitro models of CR, specifically reduced serum proteins alone or in combination with glucose, reduced activation of PI3K/Akt in HER2 BC cells. This altered signaling was associated with reduced tumorigenic potential, as evidenced by decreased growth and increased apoptosis. These findings suggest that combining a nutrient restriction regimen or pharmacologic mimetic of CR with HER2-targeted therapy, which also inhibits PI3K/Akt signaling, may act synergistically. Future experiments will test the hypothesis that CR modulates the efficacy of HER2 targeted therapies through in vitro and in vivo models of HER2 BC. Citation Format: Laura A. Smith, Magdalena A. Rainey, Nishita T. Sheth, Ciara H. O'Flanagan, Laura W. Bowers, Stephen D. Hursting. Calorie restriction reduces PI3K/Akt signaling and tumorigenic potential in HER2-overexpressing breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 379.

Calorie restriction (CR) extends lifespan and has been shown to reduce age-related diseases including cancer, diabetes, and cardiovascular and neurodegenerative diseases in experimental models. Recent translational studies have tested the potential of CR or CR mimetics as adjuvant therapies to enhance the efficacy of chemotherapy, radiation therapy, and novel immunotherapies. Chronic CR is challenging to employ in cancer patients, and therefore intermittent fasting, CR mimetic drugs, or alternative diets (such as a ketogenic diet), may be more suitable. Intermittent fasting has been shown to enhance treatment with both chemotherapy and radiation therapy. CR and fasting elicit different responses in normal and cancer cells, and reduce certain side effects of cytotoxic therapy. Findings from preclinical studies of CR mimetic drugs and other dietary interventions, such as the ketogenic diet, are promising for improving the efficacy of anticancer therapies and reducing the side effects of cytotoxic treatments. Current and future clinical studies will inform on which cancers, and at which stage of the cancer process, CR, fasting, or CR mimetic regimens will prove most effective.

Abstract The association between obesity and breast cancer risk and prognosis is well established in estrogen receptor (ER)-positive disease but less clear in HER2-positive disease. Here, we report preclinical evidence suggesting weight maintenance through calorie restriction (CR) may limit risk of HER2-positive breast cancer. In female MMTV-HER2/neu transgenic mice, we found that ERα and ERβ expression, mammary tumorigenesis, and survival are energy balance dependent in association with epigenetic reprogramming. Mice were randomized to receive a CR, overweight-inducing, or diet-induced obesity regimen (n = 27/group). Subsets of mice (n = 4/group/time point) were euthanized after 1, 3, and 5 months to characterize diet-dependent metabolic, transcriptional, and epigenetic perturbations. Remaining mice were followed up to 22 months. Relative to the overweight and diet-induced obesity regimens, CR decreased body weight, adiposity, and serum metabolic hormones as expected and also elicited an increase in mammary ERα and ERβ expression. Increased DNA methylation accompanied this pattern, particularly at CpG dinucleotides located within binding or flanking regions for the transcriptional regulator CCCTC-binding factor of ESR1 and ESR2, consistent with sustained transcriptional activation of ERα and ERβ. Mammary expression of the DNA methylation enzyme DNMT1 was stable in CR mice but increased over time in overweight and diet-induced obesity mice, suggesting CR obviates epigenetic alterations concurrent with chronic excess energy intake. In the survival study, CR elicited a significant suppression in spontaneous mammary tumorigenesis. Overall, our findings suggest a mechanistic rationale to prevent or reverse excess body weight as a strategy to reduce HER2-positive breast cancer risk. Cancer Res; 77(9); 2500–11. ©2017 AACR.

Abstract Using a murine model of basal-like breast cancer, we tested the hypothesis that chronic obesity, an established breast cancer risk and progression factor in women, induces mammary gland epigenetic reprogramming and increases mammary tumor growth. Moreover, we assessed whether the obesity-induced epigenetic and protumor effects are reversed by weight normalization. Ovariectomized female C57BL/6 mice were fed a control diet or diet-induced obesity (DIO) regimen for 17 weeks, resulting in a normal weight or obese phenotype, respectively. Mice on the DIO regimen were then randomized to continue the DIO diet or were switched to the control diet, resulting in formerly obese (FOb) mice with weights comparable with control mice. At week 24, all mice were orthotopically injected with MMTV-Wnt-1 mouse mammary tumor cells. Mean tumor volume, serum IL6 levels, expression of proinflammatory genes in the mammary fat pad, and mammary DNA methylation profiles were similar in DIO and FOb mice and higher than in controls. Many of the genes found to have obesity-associated hypermethylation in mice were also found to be hypermethylated in the normal breast tissue of obese versus nonobese human subjects, and nearly all of these concordant genes remained hypermethylated after significant weight loss in the FOb mice. Our findings suggest that weight normalization may not be sufficient to reverse the effects of chronic obesity on epigenetic reprogramming and inflammatory signals in the microenvironment that are associated with breast cancer progression. Cancer Prev Res; 9(5); 339–48. ©2016 AACR.

Abstract Background: Obesity is associated with increased incidence of basal-like breast cancer (BLBC), the most aggressive and lethal breast cancer subtype. Epidemiological data is conflicting regarding whether weight loss offers protection against BLBC in obese women; only interventions that typically result in significant sustained weight loss, such as bariatric surgery, produce a consistent anti-cancer benefit. Purpose: We sought to determine the differential effects of surgical and non-surgical weight loss interventions on inflammation, metabolic hormones and tumor burden in a mouse model of pre-menopausal breast cancer. Methods: Mice were fed a low fat control or high fat diet-induced obesity (DIO) regimen for 15 weeks to model chronic obesity. Diet-induced obese mice (n = 75) were randomized to receive either a surgical weight loss intervention (sleeve gastrectomy) or dietary weight loss intervention (switch to low fat control diet), resulting in formerly obese (FOb)-Surg or FOb-Diet mice, respectively. Additionally, a subset of mice remained on the DIO diet (Obese, n = 25), with another subset of normoweight control mice (Con, n = 25) maintained on a low fat diet throughout the study. FOb-Surg and FOb-Diet mice lost a nearly identical amount of weight and body fat; both groups had significantly lower weight and percent body fat than Con. Four weeks after weight stabilized, all mice on study were orthotopically injected with E0771 mammary tumor cells, which model BLBC. Results: At study endpoint, the average tumor weight in FOb-Surg mice was statistically equivalent to normoweight control mice. However, the average tumor weight in FOb-Diet mice was significantly greater than controls and statistically equivalent to the Obese mice. Furthermore, FOb-Surg mice had statistically lower levels of serum interleukin-6 and insulin compared to FOb-Diet, suggesting that in obese mice sleeve gastrectomy, relative to diet-induced weight loss, more effectively reduced obesity-associated inflammation, hyperinsulinemia and mammary tumor growth. Conclusion: Our results suggest that the strong anti-cancer benefits seen with bariatric surgery may be related to a significant reduction in systemic inflammation and growth factor signaling, which did not occur with non-surgical weight loss despite an equivalent amount of weight and body fat loss in FOb-Diet mice. Identifying the mechanisms underlying the protective effects of bariatric surgery against breast cancer could help identify new targets and strategies for breaking the obesity-cancer link. Citation Format: Emily L. Rossi, Laura W. Bowers, Subreen A. Khatib, Laura A. Smith, Steven S. Doerstling, Alfor Lewis, Randy J. Seeley, Stephen D. Hursting. Surgical weight loss via sleeve gastrectomy, but not a low-fat diet, reverses the pro-tumorigenic effects of obesity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2614.

Abstract Background: Breast tissue remodeling occurs with age and is marked by a progressive increase in stromal tissue. Advancing age is also associated with increased senescence among stromal cells that promote a protumorigenic microenvironment by adopting a senescence-associated secretory phenotype (SASP). In previous studies, calorie restriction has been effective in cancer prevention through inhibiting cellular senescence and SASP. However, obesity has shown to be cancer promoting with inconsistent findings on its role in cellular senescence. Purpose: We tested the hypothesis that calorie restriction is protective to the protumorigenic, age-related changes that occur within the mammary microenvironment while diet-induced obesity accelerates these changes. Methods: Six week old female mice were randomized to receive either a low-fat control regimen (10 kcal% fat), a 30% calorie restricted (CR) regimen relative to control, or a high-fat diet induced obesity (DIO, 60 kcal% fat) regimen, resulting in control, CR, and DIO mice respectively. A subset of mice was sacrificed at 1, 3, 5, 12 and 20 months following diet initiation with mammary fat pads (MFP) harvested and serum stored for further analysis. H&E staining was analyzed using a digital algorithm to quantify the composition of adipose, epithelia and stoma in the MFP tissue. RNA was extracted from MFP sections and rt-PCR was preformed to analyze gene expression of CDKN2a, the gene encoding p16(INK4a) a well known marker for senescence. Results: CR significantly decreased body weight and decreased serum IGF-1, insulin, leptin and estradiol and significantly increased adiponectin relative to control and DIO mice at all time points. MFP composition remained relatively stable from 1 and 12-month time points. However, there was an apparent non-significant trend from 12 and 20-month time points, with increasing stromal tissue and a decrease in adipose tissue, which was most drastic in CR mice. Gene expression analysis showed an increase in CDKN2a expression in control mice relative to CR mice at 5 and 20-month time points. Additionally, control and CR mice demonstrated an increase in CDKN2a expression with age from 5 months to 20-months. Conclusions: Stromal composition of murine MFP displayed an age-related increase that is consistent with findings in human breast tissue. Despite this uniform increase, we see a decrease in the age-related acquisition of a senescence phenotype in CR mice relative to control supporting the notion that CR is protective to this protumorigenic change that occurs with advancing age. Ongoing analysis of gene expression in DIO mice will determine the role of obesity in MFP cellular senescence and confirmation of senescent phenotypes via β-galactosidase staining and analysis of SASP associated gene expression. Citation Format: Laura A. Smith, Emily L. Rossi, Laura W. Bowers, Emma H. Allot, Sarah Dunlap, Liza Makowski, Bentley Midkiff, Melissa Troester, Stephen D. Hursting. Metabolic and microenvironment changes in the mammary of calorie restricted, normal weight, and obese mice throughout the lifespan. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4090.