The reversibility of the procancer effects of obesity was interrogated in formerly obese C57BL/6 mice that lost weight via a nonrestricted low-fat diet (LFD) or 3 distinct calorie-restricted (CR) regimens (low-fat CR, Mediterranean-style CR, or intermittent CR). These mice, along with continuously obese mice and lean control mice, were orthotopically injected with E0771 cells, a mouse model of triple-negative breast cancer. Tumor weight, systemic cytokines, and incidence of lung metastases were elevated in the continuously obese and nonrestricted LFD mice relative to the 3 CR groups. Gene expression differed between the obese and all CR groups, but not the nonrestricted LFD group, for numerous tumoral genes associated with epithelial-to-mesenchymal transition as well as several genes in the normal mammary tissue associated with hypoxia, reactive oxygen species production, and p53 signaling. A high degree of concordance existed between differentially expressed mammary tissue genes from obese versus all CR mice and a microarray dataset from overweight/obese women randomized to either no intervention or a CR diet. Assessment of differentially methylated regions in mouse mammary tissues revealed that obesity, relative to the 4 weight loss groups, was associated with significant DNA hypermethylation. However, the anticancer effects of the CR interventions were independent of their ability to reverse obesity-associated mammary epigenetic reprogramming. Taken together, these preclinical data showing that the procancer effects of obesity are reversible by various forms of CR diets strongly support translational exploration of restricted dietary patterns for reducing the burden of obesity-associated cancers.

Prevention Relevance:

Obesity is an established risk and progression factor for triple-negative breast cancer (TNBC). Given rising global rates of obesity and TNBC, strategies to reduce the burden of obesity-driven TNBC are urgently needed. We report the genomic, epigenetic, and procancer effects of obesity are reversible by various calorie restriction regimens.

More than 40% of adult US women are obese (1), and obesity at diagnosis is associated with worse breast cancer–specific survival in both pre- and postmenopausal women, regardless of hormone receptor status (2). Currently, no targeted therapies or specific treatment regimens exist for women with triple-negative breast cancer (TNBC), many of whom are also obese. Several epidemiological studies have examined the impact of postdiagnosis weight loss on breast cancer outcomes, and most have found that weight loss is associated with an increased risk of breast cancer recurrence and/or mortality (3–10). However, these findings are likely capturing a link between cancer progression and unintentional weight loss. On the basis of the data linking obesity with breast cancer progression, as well as results from lifestyle intervention trials in breast cancer survivors (11–13), many clinicians counsel postdiagnosis weight loss for obese patients with breast cancer. However, most women find it challenging to achieve and maintain substantial weight loss, and optimal strategies for losing weight to improve breast cancer outcomes remain unclear.

Several weight loss trials with breast cancer recurrence or survival as the primary outcome are ongoing and, when completed in the coming years, are anticipated to further elucidate the impact of weight loss on obesity-associated breast cancer (14). Bariatric surgery is the only weight-loss intervention in the literature that consistently reduces the obesity-associated increase in breast cancer risk (15). However, bariatric surgery can be costly and poses independent risks, whereas its impact on breast cancer progression and metastases remains unclear. Therefore, low-cost, sustainable alternatives to bariatric surgery are needed. To date though, neither nonsurgical weight loss regimens, including low-fat diets and various forms of calorie restriction (CR) such as intermittent CR, nor healthful dietary patterns such as Mediterranean-style diets, have been well studied as interventions to reverse the procancer effects of obesity on TNBC.

We are using preclinical studies combined with transcriptomic and epigenomic analyses to identify mechanistic targets and nonsurgical intervention strategies for breaking the obesity-breast cancer link. We previously reported that (i) obesity increases spontaneous and transplanted Wnt-driven mammary tumor development and progression in association with upregulation of epithelial-to-mesenchymal transition (EMT) markers (16, 17), whereas CR suppresses Wnt-driven tumor progression and inhibits EMT (16); (ii) obesity reversal in chronically obese mice via a nonrestricted low-fat diet (LFD) fails to attenuate the tumor-promoting effects of obesity in a mouse model of Wnt-driven TNBC (18, 19); and (iii) obesity-elevated tumor growth in both obese and formerly obese (via nonrestricted LFD) mice is associated with aberrant increases in the expression and methylation of a network of proinflammatory genes in the normal mammary tissue (19). In the present study, we assess the impact of weight loss via 4 diet regimens, specifically a nonrestricted LFD and 3 forms of calorie restriction, on tumor growth, lung metastases, tumoral and mammary gland gene expression, and mammary methylation patterns, in the E0771 murine model of TNBC.

Animal study

Animal studies and procedures were approved and monitored by the University of North Carolina Institutional Animal Care and Use Committee. Female, 6–8-week-old C57BL/6 mice were purchased from Charles River Laboratories, Inc. Mice were housed 2/cage and randomized (1:5) to receive either a low-fat (control; 10 kcal% fat) or diet-induced obesity (DIO; 60 kcal% fat) regimen, both fed ad libitum. All diets were from Research Diets, Inc. Body weight and food intake were measured weekly. After 15 weeks, mice receiving the control diet continued on that regimen ad libitum (control, n = 20), whereas mice receiving the DIO regimen were randomized (n = 20/group) to either remain on DIO diet ad libitum (obese) or switch to 1 of 4 weight loss regimens: (i) Nonrestricted LFD; (ii) low-fat, 30% calorie-restricted (LFCR); (iii) Mediterranean-style, 30% calorie-restricted (MCR); or (vi) intermittent calorie-restricted (ICR).

The amount of food for each CR diet provided 70% of the average kcal consumed by the control mice the previous week and was administered as daily aliquots to the CR mice. The ICR regimen consisted of a 13% CR diet for 5 d/wk and a 70% CR, high-protein diet on 2 non-consecutive d/wk. The MCR and ICR diet composition and schedule were modeled after the “daily energy restriction” and “intermittent energy and carbohydrate restriction” diets, respectively, used by Harvie and colleagues (20, 21) in randomized controlled trials. The intermittent restriction regimen was more readily adopted by the women in their trials than the more severe chronic CR regimen, which is challenging for many people to maintain (20, 21). Figure 1A illustrates the schedule for each diet regimen following the week 15 diet switch, and Table 1 lists the nutrient profiles of all diets. Mice in the CR groups continued to be housed 2 per cage, and a cage divider was used to separate cage mates during food consumption. The mice remained on these diets through the end of study.

At 25 weeks after their initial randomization, mice were orthotopically injected in the 4th mammary fat pad with 3.5 × 104 E0771 mammary tumor cells. Figure 1B illustrates the study design. In vivo tumor growth was monitored twice weekly with digital calipers. Body composition was assessed by quantitative magnetic resonance imaging (Echo Medical Systems) in a random sample of 6 mice/group immediately before euthanization. All mice were euthanized when a tumor in 1 mouse from any group reached 1.5 cm in diameter. Tumors and nontumor-bearing 9th mammary glands were excised, weighed, and divided into equal parts, with 1 half flash-frozen in liquid nitrogen and stored at −80°C and the other half formalin-fixed and paraffin-embedded (FFPE). Lungs were excised, examined for evidence of macrometastases (none observed), and underwent FFPE before histological examination.

Serum analytes

One week before tumor injection, blood was collected from all mice by submandibular bleed after fasting 4–6 hours, and serum was processed and stored. In 5–8 mice/group, serum concentrations of a panel of metabolic hormones and growth factors were measured using a BioPlex Pro Mouse Diabetes Panel (Bio-Rad). Adiponectin, IGF-1 and TGF-β1 were measured with Bio-Rad singleplex assays, whereas a panel of cytokines was measured using a BioPLex Pro Mouse Cytokine Panel (Bio-Rad). A Bio-Rad MAGPIX multiplex analyzer was used to read all assays, as previously described (17).

Lung metastases

A veterinary pathologist, blinded to sample identities, examined a random sample of FFPE lung tissue from 6 mice/group for micrometastases, using 10 hematoxylin and eosin-stained sections/FFPE lung tissue sample (100-μm apart). Lung metastasis burden was defined as the percentage of positive sections per mouse.

Quantitative RT-PCR

Total RNA was isolated from flash-frozen tumor tissue and reverse transcribed as previously described (22). Tumor expression of EMT-related genes was assessed in 3–5 mice/group using a Mouse EMT RT2 Profiler PCR Array (Qiagen), and read on a ViiA7 RT-PCR System (Applied Biosystems).

RNA-sequencing analysis

Total RNA and genomic DNA were isolated from randomly selected flash-frozen distal mammary fat pad samples (n = 3–5 mice/group) using TRizol Reagent (Sigma-Aldrich) according to the manufacturer's instructions. RNA quality was assessed by an Agilent 2100 Bioanalyzer. RNA libraries were prepared for sequencing using the Illumina TruSeq Stranded Total RNA Library Preparation Kit and then sequenced using a 2 × 76 bases paired-end protocol on an Illumina HiSeq 2000 (Illumina Inc.). The reads were mapped to mouse genome (mm10) by TopHat (version 2.0.7). The number of fragments in each known gene from RefSeq database (UCSC Genome Browser 2013) was enumerated using HTSeq-count from HTSeq package (version 0.5.3p9). Differential expression was performed using DESeq2. Genes with an FDR-adjusted two-tailed P value of <0.05 were considered differentially expressed. Differentially expressed genes (DEG) were assessed by principle components analysis (PCA) and entered into Ingenuity Pathway Analysis (IPA, Qiagen) to identify significantly modulated canonical pathways, cellular functions, and predicted upstream regulators (P < 0.05). The top functions were ranked by absolute z-score value (among those with P < 0.05), whereas canonical pathways and upstream regulators were ranked by a P value.

DNA methylation analysis

Genome-wide methylation profiles in mammary tissue were determined by reduced representation bisulfite sequencing (RRBS). Genomic DNA (1 mg) was digested with MspI, and enrichment for CpG sites in CG-dense and CpG islands was achieved by selective collection and PCR amplification of MspI fragments with sizes of 40–170 bp. Sequence reads were mapped to the mouse genome (mm10) using Bismark Bisulfite Read Mapper. RNA and DNA library preparation and sequencing were performed at the UNC High-Throughput Sequencing Facility. An integrated analysis of the RNA-Seq and RRBS data was performed via Joint and Individual Variation Explained to identify genes displaying alterations in both DNA methylation and gene expression. Differentially methylated regions (DMR) were defined for each pairwise comparison as genomic sites with an average absolute value change in methylation of ≥15% and an FDR-adjusted two-tailed P value of <0.05.

Gene set enrichment analysis

To compare the gene expression profiles of normal mammary tissue from all weight loss groups, relative to the obese mice, with the transcriptomic changes that occur with human weight loss, we performed gene set enrichment analysis (GSEA) with a microarray dataset (provided by our collaborator M. Harvie) of normal breast tissue samples from premenopausal overweight or obese women who either continued their normal eating habits or underwent dietary energy restriction (864 kcal/d) for 1 menstrual cycle (GEO: GSE66159; refs. 23, 24). For this comparison, we generated several gene sets from our RNA sequencing data. First, for each pairwise comparison with the obese group, we identified genes that were significantly differentially expressed (adjusted P value of <0.05) and subsequently selected the top 100 upregulated genes in each group compared with the obese group. Second, we identified all DEG (adjusted P value <0.05) from a comparison of all CR mice combined versus the obese group, excluding any genes that were also differentially expressed in the nonrestricted LFD versus obese mice comparison. Third, we identified DEG (adjusted P value of <0.05) from a comparison of nonrestricted LFD mice versus obese mice, excluding any genes that were also differentially expressed in the all-CR versus obese mice comparison. Finally, mouse gene symbols were converted to the orthologous human gene symbol using Ensembl BioMart to align with the human microarray data (25). For GSEA analysis, the microarray expression data for the participants in the energy restriction and control groups were tested for enrichment of the gene sets described above that were generated from our mouse RNA sequencing data.

Statistical analysis

Animal study data are presented as mean ± SD. Differences between the experimental groups (excluding analyses of the RNA sequencing and RRBS data, as described previously in those sections) were analyzed by one-way ANOVA, followed by the Tukey's post hoc test, using GraphPad Prism software (GraphPad Software Inc.). A P value of <0.05 was considered significantly different.

Data availability

The RNA sequencing and RRBS data generated in this study are publicly available in the GEO repository (GSE202332).

CR promotes greater body-weight and fat losses than nonrestricted LFD in obese mice

At the time of tumor cell injection, body weights were greater in the obese group than all other groups, were lesser in the 3 CR groups than the nonrestricted LFD group, and did not differ between the control and nonrestricted LFD groups (Fig. 2A). At study endpoint, body weight, and body fat percentage were lower in the CR groups than all other groups, and lower in the nonrestricted LFD group than the obese group (Fig. 2B and C). The nutrient profiles of all diets are listed in Table 1. From week 15 through the study endpoint, the obese mice consumed a greater average kcal/d than all other groups, whereas the 3 CR groups consumed a lower average kcal/d than the control and LFD groups (Fig. 2D).

CR, but not nonrestricted LFD, resolves obesity-induced elevations in IGF-1 and proinflammatory cytokines

Supplementary Table S1 lists the concentrations of all serum analytes measured. Serum insulin levels were higher in the obese group versus all groups except LFCR, and the serum leptin:adiponectin ratio was elevated in the obese group versus all other groups (Fig. 2E and F). Serum IGF-1, TGF-β1, and TNFα concentrations were elevated in obese and nonrestricted LFD groups versus the CR groups, and they did not differ between obese and nonrestricted LFD mice (Fig. 2GI). Serum IL-1β levels were greater in nonrestricted LFD mice versus control, LFCR, and ICR mice and in obese versus LFCR mice (Fig. 2J).

CR, but not nonrestricted LFD, reverses DIO effects on mammary tumor progression and metastasis

Figure 3A illustrates mammary tumor growth over time, demonstrating that the tumors in the obese and nonrestricted LFD mice had the steepest growth curves, particularly relative to the three CR groups. At the final palpation, all CR groups had significantly smaller tumors than the obese and LFD mice. Ex vivo tumor weight was greater in the obese group relative to all groups except nonrestricted LFD. Tumors were also smaller in the CR groups than in the nonrestricted LFD group (Fig. 3B). Lung micrometastases occurred in 83% of mice in both the obese and nonrestricted LFD groups, 17% in the ICR group, and 0% in the other groups (Fig. 3C). The nonrestricted LFD group also had greater lung metastasis burden, relative to the control, LFCR, and MCR groups. Metastasis burden was quantified as the percentage of the lung sections assessed that were positive for ≥1 micrometastatic lesion (Fig. 3D).

CR modifies tumoral expression of EMT-related genes

Tumoral expression of 37 genes in a panel of 84 EMT-related genes differed for all CR mice combined relative to the obese group, the nonrestricted LFD group, or both (P < 0.05). Although only 2 genes, Plek2 and Snai1, differed between the obese and nonrestricted LFD groups, these 2 groups both differed from the LFCR, MCR, and ICR groups in expression of 11, 15, and 18 genes, respectively (Fig. 3E).

CR modifies normal mammary tissue expression of genes related to reactive oxygen species production

To explore whether differences in normal mammary tissue signaling contribute to the differences in tumor progression, we performed RNA sequencing on nontumor-bearing mammary fat pad tissue from a subset of mice in each group. PCA plots of the sequencing data demonstrated within-group similarity in global gene expression among the obese and MCR mice. There was greater dissimilarity among the remaining groups (Supplementary Fig. S1).

We then performed pairwise comparisons between the obese group and each other group, as well as all CR mice combined, to identify DEG. Compared with the obese group, the all-CR group had the greatest number of DEG (3,311) and the nonrestricted LFD group had the lowest number (1,522; Fig. 4A).

For the obese versus all-CR comparison, 2,299 of 3,311 (69%) DEGs were not also differentially expressed between the obese and nonrestricted LFD mice (Fig. 4B), whereas IPA analysis of these DEG indicated an enrichment in several canonical pathways related to metabolism, including “Mitochondrial Dysfunction” and “Sirtuin Signaling.” Most of these pathways had no z-score or higher activity in the CR mice (Fig. 4C). We also found strong evidence of increased reactive oxygen species (ROS) production and metabolism in the mammary tissue of the obese mice, relative to all-CR mice (Fig. 4D). Furthermore, the most significant IPA-predicted upstream regulator for this DEG set was TP53, which also had greater activity in the obese mice (Supplementary Fig. S2A) and is linked with ROS production (26). There was substantial overlap in the DEG linked to TP53, the “Mitochondrial Dysfunction” and “Sirtuin Signaling” pathways, and the “Synthesis of ROS” function (Supplementary Fig. S2B).

For the obese versus nonrestricted LFD comparison, 510 of 1,522 DEGs (34%) did not overlap with the obese versus all-CR comparison. IPA analysis of these DEG indicated an enrichment in the “p53 Signaling” pathway, with greater activity in the nonrestricted LFD than obese mice; no other metabolism-related pathways were among the top 10 pathways (Fig. 4E). Functional analysis suggested increased immune activity in the obese mouse mammary tissue, with 3 of the top 10 enriched functions related to immune response (Fig. 4F). There was no enrichment in any ROS-related functions, at any level of significance. TNF was among the 10 most significant IPA-predicted upstream regulators, but its negative z-score indicated greater activity in the nonrestricted LFD versus obese mice (Supplementary Fig. S2C).

Changes in mammary tissue DNA methylation are diet-dependent

To assess whether reversal of obesity-induced epigenetic reprogramming mediates CR-induced changes in gene expression, we characterized global DNA methylation levels in the same normal mammary tissue samples that underwent RNA sequencing. Pairwise comparisons between the obese mice and each other group revealed 6,693 DMR versus the MCR group and <100 DMR versus the remaining groups (Supplementary Fig. S3A), including no DMR versus the ICR group (Supplementary Fig. S3B). A graph of all methylation differences detected for the obese versus nonrestricted LFD comparison illustrates that only 69 of these differences met our DMR definition, and gene body hypermethylation predominated in the obese mice (Fig. 5A). Among the 56 DMR for the obese versus LFCR comparison and the 6,693 obese versus MCR DMR, the majority were again hypermethylated and within the gene body (Fig. 5B and C). Overall, a pattern of DNA hypermethylation was observed in the mammary tissue of the obese mice, relative to all the weight loss groups, with the possible exception of ICR.

MCR decreases hypermethylation in binding motifs for obesity-linked transcription factors

We then focused on defining the functional relevance of these DMR, first using Hypergeometric Optimization of Motif EnRichment analysis, which enabled the identification of enrichment in specific transcription factor (TF)–binding motifs at the DMR. A single motif was exclusively enriched within the obese versus nonrestricted LFD DMR. In contrast, 152 motifs were exclusively enriched for the obese versus MCR comparison (Fig. 6A; Supplementary Table S2). Six of these motifs corresponded to TFs linked to energy balance, specifically forkhead (Fox)a1 and Foxa2, thyroid hormone receptor beta, hepatocyte nuclear factor 4 alpha, peroxisome proliferator-activated receptor alpha, and retinoid X receptor (Fig. 6B). At the DMR linked to these 6 motifs, there was consistent hypermethylation in the obese versus MCR mice (Supplementary Fig. S4). All 6 differentially methylated TFs were predicted upstream regulators for this comparison, with z-scores indicating greater activity in the MCR mice. Almost all of the TFs were also predicted upstream regulators for the obese versus nonrestricted LFD, LFCR, and ICR comparisons. The obese versus individual CR groups’ comparisons had more overlapping genes and higher significance for each TF than the obese versus nonrestricted LFD comparisons (Fig. 6C).

We next examined the 270 DEG that also contained ≥1 DMR (DEG+DMR) for the obese versus MCR comparison, thereby assessing only DMR that occurred in parallel with differential gene expression. Functional analysis of these DEG+DMR primarily indicated an enrichment in immune response-related genes in the obese versus MCR mice (Fig. 6D; Supplementary Fig. S5A). We determined that 6 DEG+DMR overlapped between the obese versus MCR and obese versus LFCR comparisons. The differential expressions for these DEG+DMR were similar in direction and magnitude and identical in location for both CR groups compared with obese mice (Fig. 6E; Supplementary Table S3). These genes were primarily involved in cell differentiation and lipid metabolism, including a downregulation in sterol regulatory element-binding protein 1 (Srebf1). Srebf1 was also a significant predicted upstream regulator for the obese versus MCR DEG comparison (Supplementary Fig. S5B). Four of these genes, Srebf1, Tripartite motif-containing protein 67, TF-like 5 protein, and Transmembrane 6 superfamily member 1, were also differentially expressed, but not differentially methylated, for the obese versus ICR comparison.

CR response in mouse mammary tissue parallels the CR response in breast tissue of women who are overweight

To determine the relevance of the CR-induced transcriptomic changes to human health, we next used GSEA to compare CR-responsive DEG from our data to normal human breast tissue samples following dietary energy restriction or no intervention (23, 24). The top 100 upregulated genes from each CR group (but not the nonrestricted LFD group) versus the obese group were enriched in the human dietary energy restriction samples relative to the no intervention samples (Fig. 7A). DEG exclusive to the all-CR versus obese comparison were also significantly enriched in the human dietary energy restriction samples relative to control (Fig. 7B). In contrast, DEG exclusive to the nonrestricted LFD versus obese comparison were not enriched in either human phenotype (Fig. 7C).

Given the globally high rates of obesity and breast cancer, uncertainty regarding reversibility of the obesity-breast cancer link represents an important knowledge gap. Our study demonstrates, for the first time, that weight loss via 3 distinct CR regimens, but not a nonrestricted LFD, mitigates obesity's procancer effects in a mouse model of TNBC.

One important limitation of the current study is the use of a single TNBC cell model due to budget constraints. However, the observed elevation in final tumor sizes in obese mice and formerly obese nonrestricted LFD mice, such that there is no statistical difference between the 2 groups, is consistent with our previous findings in different tumor models of breast cancer (18, 19). By comparison, Qin and colleagues and Sundaram and colleagues (27, 28) demonstrated in the C3(1)-TAg transgenic mouse model of TNBC that switching to a nonrestricted LFD reverses the tumor-promoting effects of a high-fat diet (HFD). However, their obesity induction period was substantially shorter than in our study, resulting in minimal weight differences between their control and diet-induced obese mice (27, 28). Thus, the observed effects of their diet switch to a nonrestricted LFD were likely due to the change in dietary fat intake rather than obesity reversal. In another preclinical study, obesity-associated lung metastasis from a transplanted MMTV-PyMT mouse-derived mammary tumor cell line was reversed by weight loss via nonrestricted LFD (29). This study used a tail vein injection model of metastasis, which enabled them to focus on the metastatic microenvironment of the lung. In contrast, our study assessed how obesity and weight loss affect lung micrometastases from orthotopically injected mammary tumor cells, and we demonstrated that metastatic capacity in this model is not reduced by weight loss via nonrestricted LFD following chronic obesity, but is decreased by CR-induced weight loss. Consequently, the findings are complementary, together suggesting that the mammary gland microenvironment is an essential mediator of obesity's lingering pro-metastatic effects after weight loss via LFD.

A number of factors may have contributed to the observed differences in tumor outcomes and metabolic markers between the LFD and CR groups. First, the LFD mice always had nonrestricted access to their food, whereas the CR mice received a daily food aliquot that they typically consumed within an hour, thereby subjecting them to an extended fasting period. A recent study by Das and colleagues (30) suggests that the feeding schedule, and any fasting periods within that schedule, may be at least as important for tumor outcomes as the amount of kcal consumed and/or weight lost. They reported that obese mice fed an HFD on a time-restricted feeding schedule maintained a stable body weight, improved their glucose tolerance, and developed smaller E0771 mammary tumors and fewer lung metastases than obese mice fed a nonrestricted HFD. A second possible contributing factor was that our LFD mice consumed significantly more kcal/d than any of the CR groups during the weight loss period. Third, the LFD mice weighed significantly more than the CR mice, and had greater body fat levels, both at the time of tumor cell injection and at study endpoint.

In our murine model of TNBC, the global mammary gene expression of DIO mice, relative to all-CR mice, indicated extensive metabolic dysfunction, as most of the top differentially enriched pathways were metabolism-related. Functional evaluation also demonstrated enrichment in genes linked to ROS production and oxidative metabolism in obese versus all-CR mice. Typical effects of chronic positive energy balance on adipose tissue physiology include adipocyte hyperplasia and hypertrophy (31), with the latter leading to adipose tissue hypoxia, activating HIF-1α signaling (32), as observed in our obese versus all-CR comparison. HIF-1α signaling, and hypoxic cell death, stimulates inflammation within adipose tissue, which involves infiltration of multiple proinflammatory immune cell types, including M1 macrophages. These immune cells produce inflammatory cytokines like TNFα, which was elevated in the serum of our obese and nonrestricted LFD mice, relative to the CR mice, and those cytokines activate macrophages, attracting additional immune cells (32, 33). Hypoxia and proinflammatory cytokines both induce the production of ROS in the dysfunctional adipose tissue (33).

p53 was the most significant predicted upstream regulator for the obese versus all-CR comparison, with greater activity predicted in obese mice. In contrast, although p53 signaling was a top canonical pathway for the obese versus nonrestricted LFD comparison, greater activity was predicted in LFD mice. p53 is a key TF responsible for sensing and coordinating appropriate cellular responses to metabolic stress. Under oxygen-deprivation conditions, p53 directs cells toward nonoxidative energy generation and typically represses ROS synthesis (26). Evidence for the former in the obese mouse mammary tissue, relative to all-CR mice, was clear in the top 10 canonical pathways for this comparison, which included “oxidative phosphorylation,” “TCA cycle,” and “fatty acid β-oxidation.” Each pathway was more active in CR mice, indicating inhibition of oxidative metabolism in obese mice. The continued elevation in ROS in obese mice, versus CR, despite high levels of p53 activity, may be due to p53’s failure to fully suppress ROS production. ROS can, in turn, activate Wnt and TGFβ signaling pathways in neighboring cancer cells, increasing their stemness and metastatic potential (33). Many stem- and EMT-related genes were upregulated in the obese and nonrestricted LFD mouse tumors, relative to all-CR mice, including Tgfb1, Tgfb3, and Wnt5a.

Previous studies have reported that energy balance affects DNA methylation in white adipose tissue (WAT). Wahl and colleagues (34) established robust associations in men and women between body mass index and changes in subcutaneous WAT DNA methylation, with methylation loci concentrated in genes involved in lipid metabolism, substrate transport, and inflammatory pathways. Similar patterns of differential methylation have been found by others in genes related to similar functions, as well as glycemic control (35–37). Significant weight loss following bariatric surgery reverses this obesity-induced epigenetic reprogramming (37, 38). We therefore examined whether the differential effects observed following CR-induced versus nonrestricted LFD-induced weight loss on obesity-associated mammary tumor growth and lung metastasis may be mediated by epigenetic reprogramming in the normal mammary gland. Overall, our findings indicated that obesity is associated with mammary DNA hypermethylation, which has been linked to proinflammatory pathways (39). Surprisingly, among the pairwise comparisons, there were exponentially more DMR for the obese versus MCR group comparison, relative to all other comparisons not involving the MCR group. Preliminary findings from our recently completed study comparing DNA methylation, inflammatory markers, and tumor progression in obese versus formerly obese mice achieved by bariatric surgery (sleeve gastrectomy), using the same TNBC model, suggest that surgical weight loss may exert similar anti-inflammatory and methylation effects, and comparable reductions in tumor growth, as observed here with MCR.

Hypermethylation at several TF–binding motifs was noteworthy in the mammary tissue from obese versus MCR mice. These TFs, which regulate lipid metabolism, adipocyte differentiation, and/or inflammation (40–44), were all predicted upstream transcriptional regulators for the comparison, with greater activation of each predicted in MCR mice. We also identified any genes that were both differentially expressed and contained a DMR at identical sites for the obese versus LFCR and MCR comparisons. Six genes, each involved in cell differentiation and lipid metabolism, met these criteria, including Srebf1, which is implicated in oxidative stress-induced inhibition of healthy WAT expansion (45). However, Srebf1 and 3 of the other genes were also differentially expressed, but not differentially methylated, in obese versus ICR mice. Consequently, our findings suggest that the anticancer effects of the CR interventions are likely independent of their ability to reverse epigenetic reprogramming.

Comparisons between the 3 CR diets found no significant differences in their ability to reverse obesity's effects on tumor growth, likely due to their similar impact, relative to DIO or LFD, on p53-related metabolic, ROS-regulating, and oncogenic signaling pathways. These findings on tumor growth are consistent with previous animal studies that indicated that an ICR diet might be superior to a nonrestricted diet, and comparable with a chronic CR diet, in reducing mammary tumor growth (46–50). However, the design of these studies differs from ours in several ways, including an ICR regimen that rotated between equal time periods of nonrestricted and 50% CR feeding. Moreover, the CR diets in these studies were initiated at a young age without a prior obesity induction period, and metastatic progression was not assessed.

Several clinical trials have assessed the effects of weight loss via chronic CR versus ICR (the latter with a feeding schedule similar to our study design) on cancer-related biomarkers. Two studies from Harvie and colleagues (20, 21) indicated that weight loss via an ICR diet reduces insulin resistance to a greater degree than a chronic CR regimen, but found no difference in their effects on inflammatory markers. Harvie and colleagues (24) subsequently compared breast tissue gene expression in premenopausal women before and after they followed an ICR diet for 1 menstrual cycle. These data were also compared with breast biopsy data from a previous chronic CR study (23). They found that 11 of 20 ICR subjects had significant changes in genes associated with metabolic pathways, and these changes were similar to the differences observed in the chronic CR study. The authors reported that, in general, the ICR diet induced more subtle and variable changes in breast gene expression than the chronic CR regimen (24). This description fits our findings regarding the effects of chronic versus intermittent CR on mammary gland gene expression, as more variability in gene expression occurred among the ICR mice than within the LFCR and MCR groups. We demonstrated that the gene sets from our 3 CR versus obese group comparisons, but not the nonrestricted LFD versus obese comparison, were all enriched in the human chronic CR gene set from the Harvie and colleagues study (24), providing validation for the mouse to human translatability of our findings.

In sum, we established that weight loss via various forms of CR, including low-fat, Mediterranean-style, and intermittent CR regimens, but not a nonrestricted LFD, reverses obesity's effects on mammary tumor growth and metastasis to the lung in a mouse model of TNBC. The persistence in tumor growth and metastasis observed in the formerly obese mice that were switched to nonrestricted LFD was consistent with our previous work using different models of TNBC (18, 19) and was associated with unresolved obesity-associated abnormalities in mammary tissue metabolism and ROS production. In contrast, each of the CR regimens resolved these metabolic and oxidative perturbations. There was no clear evidence that sustained epigenetic reprogramming was involved. Given that substantial weight loss and maintenance is difficult for most people, further translational exploration of efficacious and sustainable restricted diet regimens are warranted, with the ultimate goal of developing mechanism-based, nonsurgical interventions to reduce the burden of obesity-related breast cancer.

J.S. Parker reports non-financial support from Reveal Genomics outside the submitted work; as well as reports a patent for PAM50 with royalties paid from Veracyte. No disclosures were reported by the other authors.

L.W. Bowers: Conceptualization, formal analysis, funding acquisition, investigation, methodology, writing–original draft, writing–review and editing. S.S. Doerstling: Formal analysis, investigation, methodology, writing–original draft. M.G. Shamsunder: Investigation. C.G. Lineberger: Investigation. E.L. Rossi: Conceptualization, investigation. S.A. Montgomery: Formal analysis, investigation, writing–original draft. M.F. Coleman: Conceptualization, data curation, formal analysis, supervision, validation, investigation, writing–original draft. W. Gong: Formal analysis. J.S. Parker: Data curation, formal analysis, writing–original draft. A. Howell: Resources, writing–review and editing. M. Harvie: Conceptualization, writing–original draft. S.D. Hursting: Conceptualization, resources, writing–original draft, writing–review and editing.

We would like to acknowledge the contributions of Andrew H. Sims, who passed away in 2021, to this project. His assistance with data analysis was much appreciated. This study was supported by a grant from the Breast Cancer Research Foundation and R35 CA197627 (to S.D. Hursting). L.W. Bowers and E.L. Rossi were supported by a grant from the National Cancer Institute (R25CA057726).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/).

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Supplementary data