Alex J. Freemerman

Cafeteria diet is a robust model of human metabolic syndrome with liver and adipose inflammation: comparison to high-fat diet.

Obesity has reached epidemic proportions worldwide and reports estimate that American children consume up to 25% of calories from snacks. Several animal models of obesity exist, but studies are lacking that compare high‐fat diets (HFD) traditionally used in rodent models of diet‐induced obesity (DIO) to diets consisting of food regularly consumed by humans, including high‐salt, high‐fat, low‐fiber, energy dense foods such as cookies, chips, and processed meats. To investigate the obesogenic and inflammatory consequences of a cafeteria diet (CAF) compared to a lard‐based 45% HFD in rodent models, male Wistar rats were fed HFD, CAF or chow control diets for 15 weeks. Body weight increased dramatically and remained significantly elevated in CAF‐fed rats compared to all other diets. Glucose‐ and insulin‐tolerance tests revealed that hyperinsulinemia, hyperglycemia, and glucose intolerance were exaggerated in the CAF‐fed rats compared to controls and HFD‐fed rats. It is well‐established that macrophages infiltrate metabolic tissues at the onset of weight gain and directly contribute to inflammation, insulin resistance, and obesity. Although both high fat diets resulted in increased adiposity and hepatosteatosis, CAF‐fed rats displayed remarkable inflammation in white fat, brown fat and liver compared to HFD and controls. In sum, the CAF provided a robust model of human metabolic syndrome compared to traditional lard‐based HFD, creating a phenotype of exaggerated obesity with glucose intolerance and inflammation. This model provides a unique platform to study the biochemical, genomic and physiological mechanisms of obesity and obesity‐related disease states that are pandemic in western civilization today.

Metabolic Reprogramming of Macrophages GLUCOSE TRANSPORTER 1 (GLUT1)-MEDIATED GLUCOSE METABOLISM DRIVES A PROINFLAMMATORY PHENOTYPE

Background: GLUT1 is the main glucose transporter in certain immune cells. Results: Overexpressing GLUT1 in macrophages results in increased glucose uptake and glucose utilization. Conclusion: Driving glucose uptake and metabolism through GLUT1 induces a proinflammatory response that is dependent upon glycolysis and reactive oxygen species. Significance: Understanding how macrophage substrate metabolism impacts inflammation is crucial to develop novel therapeutics for obesity and diabetes. Glucose is a critical component in the proinflammatory response of macrophages (MΦs). However, the contribution of glucose transporters (GLUTs) and the mechanisms regulating subsequent glucose metabolism in the inflammatory response are not well understood. Because MΦs contribute to obesity-induced inflammation, it is important to understand how substrate metabolism may alter inflammatory function. We report that GLUT1 (SLC2A1) is the primary rate-limiting glucose transporter on proinflammatory-polarized MΦs. Furthermore, in high fat diet-fed rodents, MΦs in crown-like structures and inflammatory loci in adipose and liver, respectively, stain positively for GLUT1. We hypothesized that metabolic reprogramming via increased glucose availability could modulate the MΦ inflammatory response. To increase glucose uptake, we stably overexpressed the GLUT1 transporter in RAW264.7 MΦs (GLUT1-OE MΦs). Cellular bioenergetics analysis, metabolomics, and radiotracer studies demonstrated that GLUT1 overexpression resulted in elevated glucose uptake and metabolism, increased pentose phosphate pathway intermediates, with a complimentary reduction in cellular oxygen consumption rates. Gene expression and proteome profiling analysis revealed that GLUT1-OE MΦs demonstrated a hyperinflammatory state characterized by elevated secretion of inflammatory mediators and that this effect could be blunted by pharmacologic inhibition of glycolysis. Finally, reactive oxygen species production and evidence of oxidative stress were significantly enhanced in GLUT1-OE MΦs; antioxidant treatment blunted the expression of inflammatory mediators such as PAI-1 (plasminogen activator inhibitor 1), suggesting that glucose-mediated oxidative stress was driving the proinflammatory response. Our results indicate that increased utilization of glucose induced a ROS-driven proinflammatory phenotype in MΦs, which may play an integral role in the promotion of obesity-associated insulin resistance.

Dysregulation of fatty acid synthesis and glycolysis in non-Hodgkin lymphoma

The metabolic differences between B-NHL and primary human B cells are poorly understood. Among human B-cell non-Hodgkin lymphomas (B-NHL), primary effusion lymphoma (PEL) is a unique subset that is linked to infection with Kaposis sarcoma-associated herpesvirus (KSHV). We report that the metabolic profiles of primary B cells are significantly different from that of PEL. Compared with primary B cells, both aerobic glycolysis and fatty acid synthesis (FAS) are up-regulated in PEL and other types of nonviral B-NHL. We found that aerobic glycolysis and FAS occur in a PI3K-dependent manner and appear to be interdependent. PEL overexpress the fatty acid synthesizing enzyme, FASN, and both PEL and other B-NHL were much more sensitive to the FAS inhibitor, C75, than primary B cells. Our findings suggest that FASN may be a unique candidate for molecular targeted therapy against PEL and other B-NHL.

Impact of Tumor Microenvironment and Epithelial Phenotypes on Metabolism in Breast Cancer

Purpose: Cancer cells have altered metabolism, with increased glucose uptake, glycolysis, and biomass production. This study conducted genomic and metabolomic analyses to elucidate how tumor and stromal genomic characteristics influence tumor metabolism. Experimental Design: Thirty-three breast tumors and six normal breast tissues were analyzed by gene expression microarray and by mass spectrometry for metabolites. Gene expression data and clinical characteristics were evaluated in association with metabolic phenotype. To evaluate the role of stromal interactions in altered metabolism, cocultures were conducted using breast cancer cells and primary cancer-associated fibroblasts (CAF). Results: Across all metabolites, unsupervised clustering resulted in two main sample clusters. Normal breast tissue and a subset of tumors with less aggressive clinical characteristics had lower levels of nucleic and amino acids and glycolysis byproducts, whereas more aggressive tumors had higher levels of these Warburg-associated metabolites. While tumor-intrinsic subtype did not predict metabolic phenotype, metabolic cluster was significantly associated with expression of a wound response signature. In cocultures, CAFs from basal-like breast cancers increased glucose uptake and basal-like epithelial cells increased glucose oxidation and glycogen synthesis, suggesting interplay of stromal and epithelial phenotypes on metabolism. Cytokine arrays identified hepatocyte growth factor (HGF) as a potential mediator of stromal–epithelial interaction and antibody neutralization of HGF resulted in reduced expression of glucose transporter 1 (GLUT1) and decreased glucose uptake by epithelium. Conclusions: Both tumor/epithelial and stromal characteristics play important roles in metabolism. Warburg-like metabolism is influenced by changes in stromal–epithelial interactions, including altered expression of HGF/Met pathway and GLUT1 expression. Clin Cancer Res; 19(3); 571–85. ©2012 AACR.

Role of HGF in obesity-associated tumorigenesis: C3(1)-TAg mice as a model for human basal-like breast cancer.

Obesity is associated with basal-like breast cancer (BBC), an aggressive breast cancer subtype. The objective of this study was to determine whether obesity promotes BBC onset in adulthood and to evaluate the role of stromal–epithelial interactions in obesity-associated tumorigenesis. We hypothesized that hepatocyte growth factor (HGF) plays a promoting role in BBC, which express the HGF receptor, c-Met. In C3(1)-TAg mice, a murine model of BBC, we demonstrated that obesity leads to a significant increase in HGF secretion and an associated decrease in tumor latency. By immunohistochemical analysis, normal mammary gland exhibited obesity-induced HGF, c-Met and phospho-c-Met, indicating that the activation of the cascade was obesity-driven. HGF secretion was also increased from primary mammary fibroblasts isolated from normal mammary glands and tumors of obese mice compared to lean. These results demonstrate that obesity-induced elevation of HGF expression is a stable phenotype, maintained after several passages, and after removal of dietary stimulation. Conditioned media from primary tumor fibroblasts from obese mice drove tumor cell proliferation. In co-culture, neutralization of secreted HGF blunted tumor cell migration, further linking obesity-mediated HGF-dependent effects to in vitro measures of tumor aggressiveness. In sum, these results demonstrate that HGF/c-Met plays an important role in obesity-associated carcinogenesis. Understanding the effects of obesity on risk and progression is important given that epidemiologic studies imply a portion of BBC could be eliminated by reducing obesity.

High XRCC1 Protein Expression Is Associated with Poorer Survival in Patients with Head and Neck Squamous Cell Carcinoma

Purpose: We evaluated X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) protein in head and neck squamous cell carcinoma (HNSCC) patients in association with outcome. Experimental Design: XRCC1 protein expression was assessed by immunohistochemical (IHC) staining of pretreatment tissue samples in 138 consecutive HNSCC patients treated with surgery (n = 31), radiation (15), surgery and radiation (23), surgery and adjuvant chemoradiation (17), primary chemoradiation (51), and palliative measures (1). Results: Patients with high XRCC1 expression by IHC (n = 77) compared with patients with low XRCC1 expression (n = 60) had poorer median overall survival (OS; 41.0 months vs. OS not reached, P = 0.009) and poorer progression-free survival (28.0 months vs. 73.0 months, P = 0.031). This association was primarily due to patients who received chemoradiation (median OS of high- and low-XRCC1 expression patients, 35.5 months and not reached respectively, HR 3.48; 95% CI: 1.44–8.38; P = 0.006). In patients treated with nonchemoradiation modalities, there was no survival difference by XRCC1 expression. In multivariable analysis, high XRCC1 expression and p16INK4a-positive status were independently associated with survival in the overall study population (HR = 2.62; 95% CI: 1.52–4.52; P < 0.001 and HR = 0.21; 95% CI: 0.06–0.71; P = 0.012, respectively) and among chemoradiation patients (HR = 6.02; 95% CI: 2.36–15.37; P < 0.001 and HR = 0.26; 95% CI: 0.08–0.92, respectively; P = 0.037). Conclusions: In HNSCC, high XRCC1 protein expression is associated with poorer survival, particularly in patients receiving chemoradiation. Future validation of these findings may enable identification of HNSCC expressing patients who benefit from chemoradiation treatment. Clin Cancer Res; 17(20); 6542–52. ©2011 AACR.

Weight Loss Reversed Obesity-Induced HGF/c-Met Pathway and Basal-Like Breast Cancer Progression

Epidemiologic studies demonstrate that obesity is associated with an aggressive subtype of breast cancer called basal-like breast cancer (BBC). Using the C3(1)-TAg murine model of BBC, we previously demonstrated that mice displayed an early onset of tumors when fed obesogenic diets in the adult window of susceptibility. Obesity was also shown to elevate mammary gland expression and activation of hepatocyte growth factor (HGF)/c-Met compared to lean controls, a pro-tumorigenic pathway associated with BBC in patients. Epidemiologic studies estimate that weight loss could prevent a large proportion of BBC. We sought to investigate whether weight loss in adulthood prior to tumor onset would protect mice from accelerated tumorigenesis observed in obese mice. Using a life-long model of obesity, C3(1)-TAg mice were weaned onto and maintained on an obesogenic high-fat diet. Obese mice displayed significant elevations in tumor progression, but not latency or burden. Tumor progression was significantly reversed when obese mice were induced to lose weight by switching to a control low-fat diet prior to tumor onset compared to mice maintained on obesogenic diet. We investigated the HGF/c-Met pathway known to regulate tumorigenesis. Importantly, HGF/c-Met expression in normal mammary glands and c-Met in tumors was elevated with obesity and was significantly reversed with weight loss. Changes in tumor growth could not be explained by measures of HGF action including phospho-AKT or phospho-S6. Other mediators associated with oncogenesis such as hyperinsulinemia and a high leptin:adiponectin ratio were elevated by obesity and reduced with weight loss. In sum, weight loss significantly blunted the obesity-responsive pro-tumorigenic HGF/c-Met pathway and improved several metabolic risk factors associated with BBC, which together may have contributed to the dramatic reversal of obesity-driven tumor progression. Future research aims to evaluate the role of obesity and the HGF/c-Met pathway in basal-like breast cancer progression.

Obesity-Mediated Regulation of HGF/c-Met Is Associated with Reduced Basal-Like Breast Cancer Latency in Parous Mice

It is widely thought that pregnancy reduces breast cancer risk, but this lacks consideration of breast cancer subtypes. While a full term pregnancy reduces risk for estrogen receptor positive (ER+) and luminal breast cancers, parity is associated with increased risk of basal-like breast cancer (BBC) subtype. Basal-like subtypes represent less than 10% of breast cancers and are highly aggressive, affecting primarily young, African American women. Our previous work demonstrated that high fat diet-induced obesity in nulliparous mice significantly blunted latency in C3(1)-TAg mice, a model of BBC, potentially through the hepatocyte growth factor (HGF)/c-Met oncogenic pathway. Experimental studies have examined parity and obesity individually, but to date, the joint effects of parity and obesity have not been studied. We investigated the role of obesity in parous mice on BBC. Parity alone dramatically blunted tumor latency compared to nulliparous controls with no effects on tumor number or growth, while obesity had only a minor role in further reducing latency. Obesity-associated metabolic mediators and hormones such as insulin, estrogen, and progesterone were not significantly regulated by obesity. Plasma IL-6 was also significantly elevated by obesity in parous mice. We have previously reported a potential role for stromal-derived hepatocyte growth factor (HGF) via its cognate receptor c-Met in the etiology of obesity-induced BBC tumor onset and in both human and murine primary coculture models of BBC-aggressiveness. Obesity-associated c-Met concentrations were 2.5-fold greater in normal mammary glands of parous mice. Taken together, our studies demonstrate that, parity in C3(1)-TAg mice dramatically reduced BBC latency compared to nulliparous mice. In parous mice, c-Met is regulated by obesity in unaffected mammary gland and is associated with tumor onset. C3(1)-TAg mice recapitulate epidemiologic findings such that parity drives increased BBC risk and potential microenvironmental alterations in c-Met signaling may play a role in etiology.

Glucose metabolism is linked to the inflammatory status of macrophages

Background Macrophages infiltrate adipose tissue at the onset of weight gain and directly contribute to adipose inflammation, insulin resistance, and obesity [1]. The type of fuel substrate utilized by macrophages is central to the formation of obesity, a global epidemic [2]. Our goal is to understand the role of macrophage glucose metabolism in the promotion of inflammation and insulin resistance during high fat diet-induced obesity. We hypothesize that macrophages with blunted or elevated glucose metabolism will display limited or exaggerated immune responses, and modulate susceptibility to insulin resistance and obesity, respectively.

Abstract 4871: Obesity-mediated regulation of HGF/c-Met and reduced basal-like breast cancer latency in parous mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Epidemiologic and experimental data have shown that a full term pregnancy reduces breast cancer risk. While a full term pregnancy reduces risk for estrogen receptor positive (ER+) and luminal breast cancers, parity is associated with increased risk of basal-like breast cancer (BBC) subtype. BBC represents <10% of breast cancers, are highly aggressive, affecting primarily young and African-American women. Experimental studies have examined parity and obesity individually, but to date, the independent and joint effects of parity and obesity have not been dissected. Our previous work demonstrated that high fat diet-induced obesity significantly blunted BBC latency in nulliparous C3(1)-Tag mice, potentially through the hepatocyte growth factor (HGF)/c-Met oncogenic pathway. Since obesity and post-partum weight gain are sometimes difficult to parse out, we investigated the role of obesity in parous mice on BBC. Methods: Seven week old female C3(1)-TAg mice were placed with male mice for breeding. Males were removed at pregnancy and pups were removed immediately after birth. The mothers were then randomly assigned to diet groups to model post-partum obesity. Mice were fed control low fat diet (10% kcal from fat) or high fat diet (45% or 60% kcal from fat) at 10 weeks until sacrifice. Mice were monitored for fat accretion, tumor onset, and tumor progression. Plasma measures of cytokines and metabolic parameters were assessed. Immunohistochemical analyses for HGF, c-Met and F4/80 macrophage markers were performed. Results: Mice on both 45% and 60% diets gained significantly greater fat mass compared to mice remaining lean on 10% diet. Compared with nulliparous mice fed the same diets, parity induced significant decreases in latency in C3(1)-TAg mice fed 10% and 45% diets. 60% diet reduced latency in parous mice to the same extent as nulliparous mice fed 60% diet. Tumor burden and tumor aggressiveness were not regulated by obesity in parous mouse. Obesity-associated metabolic mediators and hormones such as insulin, estrogen, and progesterone were not significantly regulated however, leptin levels were elevated in 45%-fed mice compared to other groups. Plasma IL-6 was significantly elevated by obesity in parous mice. Importantly, the HGF/c-Met axis in normal mammary gland was elevated significantly by obesity and correlated with reduced tumor latency. Conclusions: In summary, our studies demonstrate that, similar to epidemiologic reports, parity in C3(1)-TAg mice alone dramatically reduced BBC latency compared to nulliparous mice. Obesity in parous mice did not reduce latency further than obesity alone, indicating that obesity and parity may work through similar pathways. Finally, obesity induced c-Met expression in normal mammary gland in parous mice, similar to our reports in nulliparous mice, implicating an interaction between the HGF/c-Met signaling pathway and obesity in the etiology of BBC. Citation Format: Sneha Sundaram, Alex J. Freemerman, Erin L. Kirk, Joseph A. Galanko, Kirk K. McNaughton, Katharine M. Bendt, David B. Darr, Melissa A. Troester, Liza Makowski. Obesity-mediated regulation of HGF/c-Met and reduced basal-like breast cancer latency in parous mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4871. doi:10.1158/1538-7445.AM2014-4871