Reversing the Genomic, Epigenetic, and Triple-Negative Breast Cancer-Enhancing Effects of Obesity

Abstract

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.

Figure 1.

Diet regimens and study design. (A) Following the week 15 diet switch, the control mice remained on the same low-fat control diet, and the diet-induced obesity (DIO) mice were randomized to either remain on the DIO diet (obese) or change to 1 of 4 weight loss regimens: non-restricted low-fat diet (LFD), low-fat calorie restricted (LFCR), Mediterranean-style calorie restricted (MCR), or intermittent calorie-restricted (ICR). The control, obese, and LFD mice were fed ad libitum, with the LFD group receiving the same low-fat diet as the control group. The LFCR and MCR groups were chronically 30% calorie restricted, receiving a daily food pellet of their respective diets equal to 70% of the previous week’s average daily control mouse kcal intake. On Mondays and Thursdays, the ICR mice received the high protein 2-day ICR diet in an amount equal to 30% of the previous week’s average daily control mouse kcal intake. On the other days of the week, the ICR mice received the 5-day ICR diet in an amount equal to 87% of the average daily control mouse kcal intake. See Table 1 for the macronutrient and fatty acid content of each diet. (B) At study initiation, the mice were randomized to 2 diets: DIO or control. After 15 weeks, the DIO mice were further randomized to remain on DIO (Obese) or switch to 1 of 4 weight loss diets: non-restricted LFD, LFCR, MCR, and ICR. The mice remained on these diets for 10 weeks and were then orthotopically injected with 3.5×10⁴ E0771 mammary tumor cells. During the 3.5-week period between tumor cell injections and euthanization, the mice continued on the same diets.

Figure 2.

Calorie restriction promotes weight loss and reductions in adipokines in obese mice. (A) Weekly body weights were recorded for the control, obese, nonrestricted low-fat diet (LFD), low-fat calorie-restricted (LFCR), Mediterranean-style calorie-restricted (MCR), and intermittent calorie-restricted (ICR) mice. Final body weights (B) and body fat percentage (C) were measured just prior to euthanasia for all mice and 6 mice/group, respectively. (D) The average kcal/mouse/day consumed by each diet group was calculated for the time period between the end of week 15, when the diet switch occurred, and the end of study. (E) Insulin levels, (F) the leptin-adiponectin ratio, (G) insulin-like growth factor (IGF)-1 levels, (H) transforming growth factor beta 1 (TGF-β1) levels, (I) tumor necrosis factor alpha (TNFα) levels, and (J) interleukin 1 beta (IL-1β) levels were all measured in sera collected 1 week prior to tumor cell injections from 5–8 mice/group. *P<0.05, **P<0.01, ***P<0.001, all relative to the obese mice. ^#P<0.05, ^##P<0.01, ^###P<0.001, all relative to the nonrestricted LFD mice.

Figure 3.

The protumor effects of obesity are reversed by weight loss via calorie restriction, but not a nonrestricted low-fat diet. (A) Tumor cross-sectional area (mm²) was calculated following biweekly palpations and is shown here over time for all control, obese, nonrestricted low-fat diet (LFD), low-fat calorie-restricted (LFCR), Mediterranean-style calorie-restricted (MCR), and intermittent calorie-restricted (ICR) mice. The ICR group is not visible on the graph because its growth curve is almost identical to the LFCR growth curve. (B) Tumor weight was measured ex vivo at the study endpoint in all mice. (C) Incidence of lung metastasis in each diet group was determined by examining the lungs of 6 mice/group for micrometastases. (D) Lung metastasis burden for each mouse was calculated as the percentage of lung sections positive for at least 1 micrometastasis, 6 mice/group assessed. (E) Tumor expression of genes related to the epithelial-to-mesenchymal transition were measured by quantitative RT-PCR array in all groups except control (n=3–5 mice/group). Expression levels in all 4 weight loss groups, relative to the obese group, of the 37 genes that differed between all CR mice combined and the obese group, the nonrestricted LFD group, or both (P<0.05) are shown. *P<0.05, ***P<0.001 relative to the obese mice. ^#P<0.05, ^##P<0.01, ^###P<0.001, relative to the nonrestricted LFD mice.

Figure 4.

Calorie restriction modulates gene expression in normal mammary tissue. (A) Differentially expressed genes (DEG, FDR adjusted P<0.05) were identified by RNAseq for obese mice versus every other group (control, nonrestricted low-fat diet (LFD), low-fat calorie-restricted (LFCR), Mediterranean-style calorie-restricted (MCR), and intermittent calorie-restricted (ICR) mice, n=3–5 mice/group) and all calorie-restricted (CR) mice combined. (B) Overlapping and non-overlapping DEG for the obese versus nonrestricted LFD and obese versus all-CR comparisons were determined. (C) The top ten Ingenuity Pathway Analysis (IPA) canonical pathways (ranked by -log10(p-value)) and (D) the top ten significant IPA functions (ranked by z-score) associated with obese versus all-CR DEG, excluding obese versus nonrestricted LFD DEG, are shown. (E) The top ten IPA canonical pathways (ranked by -log10(p-value)) and (F) the top ten significant IPA functions (ranked by z-score) associated with the obese versus nonrestricted LFD DEG, excluding obese versus all-CR DEG, are shown.

Figure 5.

Obesity reversal decreases global DNA methylation levels in normal mammary tissue. Pairwise comparisons of DNA methylation levels between (A) the obese and nonrestricted low-fat diet (LFD) groups, (B) the obese and low-fat calorie restricted (LFCR) groups, and (C) the obese and Mediterranean-style calorie restricted (MCR) groups are shown, expressed as the mean change in percent methylation and with a differentially methylated region (DMR) defined as a genomic site with an average absolute methylation value change of ≥15% and an FDR adjusted P<0.05 (n=3–5 mice/group). Locations of the DMR for each comparison, expressed as the number of DMR and percentage of DMR in each location, are also shown.

Figure 6.

Methylation of obesity-linked transcription factor binding sites is reduced by Mediterranean-style calorie restricted (MCR) diet. (A-B) Hypergeometric Optimization of Motif EnRichment (HOMER) analysis was used to identify enrichment in transcription factor binding motifs at the differentially methylated regions (DMR) for the obese versus MCR and obese versus nonrestricted low-fat diet (LFD) comparisons. (C) Ingenuity Pathway Analysis (IPA) prediction of upstream regulators was conducted for obese versus nonrestricted LFD, obese versus nonrestricted low-fat calorie restricted (LFCR), obese versus MCR, and obese versus intermittent calorie restricted (ICR) comparisons. (D) The top ten significant IPA functions (ranked by z-score) associated with differentially expressed genes (DEG) that contained ≥1 DMR (DEG+DMR) for the obese versus MCR comparison are shown. (E) Relative expression levels of the overlapping DEG+DMR for the obese versus LFCR and obese versus MCR comparisons are shown.

Figure 7.

Obesity reversal by calorie restriction promotes changes in the expression of mouse mammary tissue genes that are enriched in the breast tissue of calorie-restricted women. (A) The top 100 significant differentially expressed genes (DEG) from 4 pairwise comparisons (low-fat calorie-restricted (LFCR), Mediterranean-style calorie-restricted (MCR), intermittent calorie restricted (ICR), and nonrestricted low-fat diet (LFD) mice versus obese, n=3–5 mice/group) were assessed by Gene Set Enrichment Analysis (GSEA) for enrichment in a gene set from the breast tissue of women who were overweight/obese and randomized to a chronic energy restriction (CER) regimen versus usual diet (control) (GSE66159). GSEA was also used to assess (B) all DEG from the comparison of all-CR mice versus obese mice, excluding any DEG from the comparison of nonrestricted LFD versus obese mice, and (C) all DEG from the comparison of nonrestricted LFD versus obese mice, excluding any DEG from the all-CR versus obese comparison, for enrichment in the same human dataset.