Subreen A. Khatib

Obesity-Associated Alterations in Inflammation, Epigenetics, and Mammary Tumor Growth Persist in Formerly Obese Mice

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.

Energy balance modulation impacts epigenetic reprogramming, ERα and ERβ expression and mammary tumor development in MMTV-neu transgenic mice

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.

Leptin Signaling Mediates Obesity-Associated CSC Enrichment and EMT in Preclinical TNBC Models

Obesity is associated with poor prognosis in triple-negative breast cancer (TNBC). Preclinical models of TNBC were used to test the hypothesis that increased leptin signaling drives obesity-associated TNBC development by promoting cancer stem cell (CSC) enrichment and/or epithelial-to-mesenchymal transition (EMT). MMTV-Wnt-1 transgenic mice, which develop spontaneous basal-like, triple-negative mammary tumors, received either a control diet (10% kcal from fat) or a diet-induced obesity regimen (DIO, 60% kcal from fat) for up to 42 weeks (n = 15/group). Mice were monitored for tumor development and euthanized when tumor diameter reached 1.5 cm. Tumoral gene expression was assessed via RNA sequencing (RNA-seq). DIO mice had greater body weight and percent body fat at termination than controls. DIO mice, versus controls, demonstrated reduced survival, increased systemic metabolic and inflammatory perturbations, upregulated tumoral CSC/EMT gene signature, elevated tumoral aldehyde dehydrogenase activity (a CSC marker), and greater leptin signaling. In cell culture experiments using TNBC cells (murine: E-Wnt and M-Wnt; human: MDA-MB-231), leptin enhanced mammosphere formation, and media supplemented with serum from DIO versus control mice increased cell viability, migration, invasion, and CSC- and EMT-related gene expression, including Foxc2, Twist2, Vim, Akt3, and Sox2. In E-Wnt cells, knockdown of leptin receptor ablated these procancer effects induced by DIO mouse serum. These findings indicate that increased leptin signaling is causally linked to obesity-associated TNBC development by promoting CSC enrichment and EMT. Implications: Leptin-associated signals impacting CSC and EMT may provide new targets and intervention strategies for decreasing TNBC burden in obese women. Mol Cancer Res; 16(5); 869–79. ©2018 AACR.

Resveratrol inhibits obesity-associated adipose tissue dysfunction and tumor growth in a mouse model of postmenopausal claudin-low breast cancer

Adipose tissue dysregulation, a hallmark of obesity, contributes to a chronic state of low‐grade inflammation and is associated with increased risk and progression of several breast cancer subtypes, including claudin‐low breast tumors. Unfortunately, mechanistic targets for breaking the links between obesity‐associated adipose tissue dysfunction, inflammation, and claudin‐low breast cancer growth have not been elucidated. Ovariectomized female C57BL/6 mice were randomized (n = 15/group) to receive a control diet, a diet‐induced obesity (DIO) diet, or a DIO + resveratrol (0.5% wt/wt) diet. Mice consumed these diets ad libitum throughout study and after 6 weeks were orthotopically injected with M‐Wnt murine mammary tumor cells, a model of estrogen receptor (ER)‐negative claudin‐low breast cancer. Compared with controls, DIO mice displayed adipose dysregulation and metabolic perturbations including increased mammary adipocyte size, cyclooxygenase‐2 (COX‐2) expression, inflammatory eicosanoid levels, macrophage infiltration, and prevalence of crown‐like structures (CLS). DIO mice (relative to controls) also had increased systemic inflammatory cytokines and decreased adipocyte expression of peroxisome proliferator‐activated receptor gamma (PPARγ) and other adipogenesis‐regulating genes. Supplementing the DIO diet with resveratrol prevented obesity‐associated increases in mammary tumor growth, mammary adipocyte hypertrophy, COX‐2 expression, macrophage infiltration, CLS prevalence, and serum cytokines. Resveratrol also offset the obesity‐associated downregulation of adipocyte PPARγ and other adipogenesis genes in DIO mice. Our findings suggest that resveratrol may inhibit obesity‐associated inflammation and claudin‐low breast cancer growth by inhibiting adipocyte hypertrophy and associated adipose tissue dysregulation that typically accompanies obesity.

Reducing the burden of obesity-associated cancers with anti-inflammatory long-chain omega-3 polyunsaturated fatty acids

Todays world population has an unprecedented risk of dying from the consequences of being overweight and obese. Chronic diseases such as cardiovascular disease, type 2 diabetes, and cancer are often accelerated because of excessive adiposity. Various biological mechanisms are implicated in the obesity-cancer link, particularly local and systemic inflammation as well as altered growth factor signaling pathways. In order to combat obesity-induced inflammation and the resulting increases in cancer risk and progression, the identification of safe and effective mechanism-based interventions is imperative. Notably, long chain omega-3 polyunsaturated fatty acids (PUFAs) modulate the secretion of pro-inflammatory cytokines, prostaglandins and other inflammatory mediators, restore insulin sensitivity, and can prevent or delay tumorigenesis. Delineating the precise mechanisms by which omega-3 PUFAs suppress obesity-induced inflammation will help identify promising key mechanistic targets and intervention strategies to break the obesity-cancer link.

Abstract 2689: The protumorigenic, proinflammatory effects of obesity are reversed by weight loss via bariatric surgery, but not a low-fat diet

Background: Obesity negatively impacts basal-like breast cancer (BLBC) prognosis, but the reversibility of these pro-cancer effects via weight loss remains unclear. However, there is consistent evidence suggesting that weight loss via bariatric surgery reduces breast cancer risk. This may be related to the reductions in metabolic perturbations and inflammation that follow bariatric surgery, effects that could be mediated by epigenetic reprogramming and/or changes in the gut microbiome. Purpose: We previously demonstrated that mammary tumor growth and inflammation remain elevated in formerly obese mice, in concordance with aberrant methylation of inflammation-related genes. Here we aim to determine the differential effects of surgical versus non-surgical weight loss on inflammation, DNA methylation, the gut microbiome, and tumor burden in a mouse model of BLBC. Methods: Mice were fed a low fat control (n=25) or high fat diet-induced obesity (DIO, n=75) regimen for 15 weeks to model chronic obesity. DIO mice were then randomized to remain on DIO (Obese) or receive either a surgical (sleeve gastrectomy) or dietary (low fat control diet) weight loss intervention, resulting in formerly obese (FOb)-Surg or FOb-Diet mice, respectively. The Control mice were maintained on the low fat diet throughout study. Four weeks after weight stabilization in the FOb mice, all mice were orthotopically injected with E0771 mammary tumor cells, which model BLBC. Stool samples were collected at baseline and prior to tumor cell injection. Results: The average weight and percent body fat of the FOb-Surg and FOb-Diet mice were equivalent to Control and significantly lower than Obese mice at study endpoint. Average tumor weight in FOb-Surg mice was statistically equivalent to Control mice, but tumor weight in FOb-Diet mice was significantly greater than Control mice and statistically equivalent to Obese mice. Furthermore, FOb-Surg mice had significantly lower serum tumor necrosis factor alpha, mammary gland interleukin-6 expression, and tumor-infiltrating adipocyte area in comparison to FOb-Diet. To further define the effects of surgical versus non-surgical weight loss, characterization of the gut microbiota as well as global mammary tissue gene expression and DNA methylation via paired RNA sequencing and reduced representation bisulfide sequencing is in progress. Conclusions: Our results suggest that the strong anti-cancer benefits seen with bariatric surgery may be related to a significant reduction in systemic and local inflammation, which did not occur with non-surgical weight loss. Identification of the mechanisms mediating the protective effects of bariatric surgery against breast cancer could help identify new targets and strategies for breaking the obesity-cancer link. Citation Format: Laura W. Bowers, Emily L. Rossi, Subreen A. Khatib, Steven Doerstling, Alfor Lewis, Randy J. Seeley, Stephen D. Hursting. The protumorigenic, proinflammatory effects of obesity are reversed by weight loss via bariatric surgery, but not a low-fat diet [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2689. doi:10.1158/1538-7445.AM2017-2689

Abstract 2614: Surgical weight loss via sleeve gastrectomy, but not a low-fat diet, reverses the pro-tumorigenic effects of obesity

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 5245: Sulindac decreases basal-like mammary tumor burden and proinflammatory mediators in obese mice

Background: A hallmark of the metabolic dysregulation associated with obesity is a pro-inflammatory environment perpetuated by pro-inflammatory mediators including adipokines and growth factor signaling. We previously showed that inflammation and basal-like breast cancer (BLBC) growth are increased in chronically obese mice and persist following weight normalization. Purpose: We tested the hypothesis that Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), can reduce chronic obesity-related inflammation and subsequent BLBC growth. Methods: Mice were administered a control diet (10 kcal% fat) or diet-induced obesity regimen (DIO, 60 kcal% fat). After 15 weeks on diet, DIO mice either continued on DIO diet or were switched to the low fat control diet to induce gradual weight loss, resulting in Formerly Obese (FOb) mice. Half of the mice in all three groups (Control, FOb, DIO, n = 17/group) were randomized to receive Sulindac supplementation at 160 ppm in the diet, which remained constant across diets. Twelve weeks after initiating Sulindac supplementation and weight loss in the FOb groups, all mice were orthopically injected with E0771 cells, a model of basal-like breast cancer. Five mice/group were killed, and their tissue collected and stored at a 4-week intermediate time-point after injection, while 12 mice/group continued in a survival study; these mice were killed when tumor size reached 1.2 cm in diameter in any direction. Results: Sulindac supplementation in DIO mice significantly reduced serum insulin and leptin to levels statistically equivalent to both control and FOb mice. Interestingly, body weight, body fat percentage, and ex vivo visceral white adipose weight were unchanged between all supplemented and non-supplemented counterparts (i.e. DIO vs. DIO+Sulindac). At the 4-week intermediate time point, Sulindac supplementation in DIO mice (but not in control or FOb mice) significantly reduced mean tumor weight relative to their non-supplemented counterparts. In the survival arm of the study, Sulindac significantly increased tumor latency in DIO and FOb groups (but not controls) in comparison to their non-supplemented counterparts. Conclusions: Sulindac supplementation significantly reduced pro-inflammatory mediators insulin and leptin in DIO mice, and increased tumor latency in DIO and FOb mice. Furthermore, Sulindac supplementation did not modulate body weight or fat depots, suggesting that Sulindac indeed offsets some of the pro-tumorigeneic effects of obesity rather than targeting obesity directly. Ongoing analyses of inflammatory surrogates, including circulating cytokines and prostaglandins, mammary gland crown-like structures and cyclooxygenase-2 levels, will help to determine if Sulindac9s effects are mediated through its anti-inflammatory activity. Citation Format: Subreen A. Khatib, Emily L. Rossi, Laura W. Bowers, Andrew J. Dannenberg, Stephen D. Hursting. Sulindac decreases basal-like mammary tumor burden and proinflammatory mediators in obese mice. [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 5245.