Metabolomic profiles are increasingly being used to identify responders to dietary interventions. Advances using this approach are particularly needed to personalize and enhance the effectiveness of dietary weight loss interventions. Using obese Diversity Outbred (DO) mice that model genetic and phenotypic heterogeneity of human populations, we aimed to identify urinary metabolite signatures associated with responsiveness to calorie restriction (CR)-mediated weight loss. DO mice (150 males, 150 females) were fed a high-fat diet for 12 weeks to induce obesity, then urine was collected and an 8-week CR regimen (30% decrease in energy intake) initiated. At study completion, mice were rank-ordered according to their percent body weight change, with mice in the extreme quartiles deemed CR responders (n = 67) versus nonresponders (n = 67). Targeted semi-quantitative metabolomics identified elevated glutamic acid and hydroxyproline as key urinary metabolites that distinguish CR responders from CR nonresponders, independent of sex. Three urinary metabolites (glutamic acid, hydroxyproline, and putrescine) distinguished male CR responders from nonresponders. Six metabolites (glutamic acid, hydroxyproline, dopamine, histamine, lysine, and spermine) distinguished female CR responders from nonresponders. Multivariate receiver operating characteristic analyses integrated these metabolites to reveal potential sex specific and sex-independent associations of CR-mediated weight loss. Further, pathway analysis identified several metabolic pathways, including arginine and proline metabolism, and alanine, aspartate, and glutamate biosynthesis, that distinguished CR responders from nonresponders and could be indicative of metabolic reprogramming to enhance insulin sensitivity and energy metabolism.

Predictive analytics encompassing metabolomic profiles are increasingly being used to forecast responders to dietary interventions. Advances using this approach are particularly needed to personalize and enhance the effectiveness of dietary weight loss interventions. Using obese Diversity Outbred (DO) mice that model genetic and phenotypic heterogeneity of human populations, we aimed to identify urinary metabolite signatures predictive of responsiveness to calorie restriction (CR)-mediated weight loss. DO mice (150 males, 150 females) were fed a high-fat diet for 12 weeks to induce obesity, then urine was collected and an 8-week CR regimen (30% decrease in energy intake) initiated. At study completion, mice were rank-ordered according to their percent body weight change, with mice in the extreme quartiles deemed CR responders (n=67) versus nonresponders (n=67). Targeted semi-quantitative metabolomics identified elevated glutamic acid and hydroxyproline as key urinary metabolites that distinguish CR responders from CR nonresponders, independent of sex. Three urinary metabolites (glutamic acid, hydroxyproline, and putrescine) distinguished male CR responders from nonresponders. Six metabolites (glutamic acid, hydroxyproline, dopamine, histamine, lysine, and spermine) distinguished female CR responders from nonresponders. Multivariate receiver operating characteristic analyses integrated the common metabolites and sex-specific metabolites to reveal moderate (males) to robust (females, males plus females) prediction models of CR-mediated weight loss. Further, pathway analysis identified several metabolic pathways, including arginine and proline metabolism, and alanine, aspartate, and glutamate biosynthesis, that distinguished CR responders from nonresponders and could be indicative of metabolic reprogramming to enhance insulin sensitivity and energy metabolism.

Calorie restriction (CR) is a well-established weight loss strategy, albeit with variation in response. Using genetically heterogeneous mice, we sought to identify metabolic predictors of resistance to CR-induced weight loss. Diversity outbred (DO) mice (150 males and 150 females) were fed a high-fat diet for 12 wk to generate diet-induced obesity (DIO), then underwent CR for 8 wk. Body weight and composition, blood glucose, and plasma levels of nine metabolic hormones were assessed at baseline, following DIO, and following CR. In response to each dietary intervention, the mice displayed substantial heterogeneity across all outcomes, often with sexual dimorphism. Among the metabolic markers, leptin changed the most in response to each dietary intervention. Logistic regression found that resistance to CR-induced weight loss in obese mice was associated with lower glucose levels in males, and with lower levels of insulin, resistin, homeostatic model assessment for insulin resistance (HOMA-IR), and plasminogen activator inhibitor-1 and higher levels of ghrelin in females. Moreover, lower leptin levels predicted resistance to CR-induced weight loss in obese mice, regardless of sex. These preclinical findings provide proof-of-principle that the genetic and phenotypic heterogeneity of DO mice can be leveraged to identify mechanistic predictors that may enhance the personalization of weight loss interventions.NEW & NOTEWORTHY Using a population of obese diversity outbred (DO) mice, we interrogated plasma predictors of resistance to calorie restriction-induced weight loss in nonresponders versus responders to the diet intervention. Lower leptin levels significantly predicted resistance in both sexes. Sexually dimorphic predictors included lower levels of glucose in males and insulin, resistin, and plasminogen activator inhibitor-1 (PAI-1) in females. Hence, genetically and phenotypically heterogeneous diversity outbred mice may be useful for identifying metabolic predictors for personalizing weight loss interventions.

Obesity is an established risk and progression factor for triple-negative breast cancer (TNBC), but preclinical studies to delineate the mechanisms underlying the obesity-TNBC link as well as strategies to break that link are constrained by the lack of tumor models syngeneic to obesity-prone mouse strains. C3(1)/SV40 T-antigen (C3-TAg) transgenic mice on an FVB genetic background develop tumors with molecular and pathologic features that closely resemble human TNBC, but FVB mice are resistant to diet-induced obesity (DIO). Herein, we sought to develop transplantable C3-TAg cell lines syngeneic to C57BL/6 mice, an inbred mouse strain that is sensitive to DIO. We backcrossed FVB-Tg(C3-1-TAg)cJeg/JegJ to C57BL/6 mice for ten generations, and spontaneous tumors from those mice were excised and used to generate four clonal cell lines (B6TAg1.02, B6TAg2.03, B6TAg2.10, and B6TAg2.51). We characterized the growth of the four cell lines in both lean and DIO C57BL/6J female mice and performed transcriptomic profiling. Each cell line was readily tumorigenic and had transcriptional profiles that clustered as claudin-low, yet markedly differed from each other in their rate of tumor progression and transcriptomic signatures for key metabolic, immune, and oncogenic signaling pathways. DIO accelerated tumor growth of orthotopically transplanted B6TAg1.02, B6TAg2.03, and B6TAg2.51 cells. Thus, the B6TAg cell lines described herein offer promising and diverse new models to augment the study of DIO-associated TNBC.

PDF file - 59K, Increased sensitivity of RAP80-/- MEFs to DNA damaging agents

PDF file - 276K, Histopathological examination of tumor tissues

PDF file - 870K, Increased chromosomal instability in RAP80-/- iMEFs after DNA damage

PDF file - 34K, Comparison of the IR sensitivity between WT and RAP80-/- mice

PDF file - 518K, RAP80-/- MEFs undergo early senescence

PDF file - 96K, Generation of RAP80 null Mice and Real-time qRT-PCR analysis

Introduction: Breast cancer (BC) is a leading cause of cancer-related death in women, with the poorest prognosis found in the basal-like subtype. Although obesity is an established risk factor for basal-like BC, existing mouse models to study the interaction of basal-like BC and diet-induced obesity (DIO) are limited. The C3(1)/SV40 large T-antigen (TAg) transgenic mouse is an established, human-relevant basal-like mammary cancer model developed on an obesity-resistant FVB genetic background. The goal of this study was to create and characterize a transplantable C3TAg model on an obesity-sensitive C57BL/6J (B6) genetic background. Methods: Male C3TAg transgenic FVB mice were backcrossed with female wild-type B6 mice for 10 generations. Spontaneous mammary tumors from these female B6:C3TAg mice were dissociated and subcloned, generating 4 distinct cell lines differing in their expression of metabolic genes. A pilot orthotopic transplant study using the 4 candidate cell lines (at 3 cell concentrations of each line) in 96 B6 mice identified cell line 2.51 as an attractive model for further evaluation based on in vivo growth characteristics and initial genomic analyses. To assess the impact of DIO in this model, 61 female B6 mice were randomized to receive either control (n=30) or DIO (n=31) diet regimens for 24 weeks, then were orthotopically injected into the 4th mammary fat pad with cell line 2.51 (2.5x105 cells/mouse). The mice continued on their respective diets, were monitored for tumor development, and were euthanized 4 weeks post-injection. Tumors were collected, weighed and flash frozen for genomic, immunologic and biochemical analyses. Results: All 4 cell lines tested in the pilot study generated basal-like mammary tumors when orthotopically transplanted. Tumor gene expression profiling (via Affymetrix microarray) revealed inflammation and stem-like gene expression profiles discordant between cell lines tested, with tumors induced by cell line 2.51 showing striking enrichment in immune signatures relative to the other cell lines. DIO, relative to control diet, significantly accelerated transplanted 2.51 cell tumor progression, as indicated by increased tumor mass in mice from DIO relative to control diet-fed animals. Genomic, biochemical, and immunological assessment of the tumor-enhancing effects of DIO in this model are ongoing. Conclusions: Herein, we report the development and characterization of a novel transplantable C3TAg mammary tumor model in C57BL/6J mice, highly responsive to energy balance. This work represents a promising new tool for preclinical studies of the interactions between diet, obesity, immunity and basal-like breast cancer. Funding: This work was supported by R35CA197627 to SDH. Citation Format: Meredith S. Carson, Elaine M. Glenny, Violet A. Kiesel, Ashlee Taylor, Daniel Roth, Jody Albright, Melissa VerHague, John E. French, Michael F. Coleman, Stephen D. Hursting. Characterization of a novel transplantable model of obesity-driven basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1336.

Introduction: Breast cancer (BC) is the second leading cause of cancer-related mortality among American women and is exacerbated by obesity. The basal-like (BL) subtype accounts for 10-15% of all BCs and is a particularly aggressive disease due to its extreme intrinsic diversity compared with other subtypes. Paradoxically, obesity promotes immune checkpoint inhibition (ICI) response in some cancers. Further, obesity promotes altered intestinal microbial composition, and recent clinical studies of fecal microbiota transplants suggest causal links between the microbiome and immunotherapy responses. Hence, we evaluated the correlation of tumoral and cecal microbiota with anti-Programmed Cell Death Protein 1 (PD1) immunotherapy response in a murine model of BLBC and obesity. Methods: In an initial study, normoweight female C57BL/6J mice (n=8/group) received orthotopic injection of one of two novel C3TAg BLBC cell lines (1.02 and 2.51) syngeneic to C57BL/6J derived from a spontaneous tumor excised from B6-C3TAg transgenic mice. Gene expression profiling (Affymetrix arrays) with Gene Set Enrichment Analysis (GSEA) was performed on the resulting transplanted tumors. In a second study, female C57BL/6J mice were divided into a diet-induced obesity (DIO) group (n=30), promoted by high-fat diet feeding, and a lean control group (n=30). After 24 weeks, 2.51 cells were orthotopically injected into the fourth mammary fat pad of all mice, followed by treatment with anti-PD1 or IgG control antibodies (n=15/diet group). Tumor and cecal DNA was extracted and subjected to 16S rRNA amplicon sequencing. Results: GSEA from the initial study revealed that orthotopically transplanted line 2.51 tumors, relative to line 1.02 tumors, had lower levels of stromal remodeling as indicated by significant enrichment of gene sets related to extracellular matrix proteoglycans, collagen chain trimerization, and collagen biosynthesis and modification. Gene sets related to insulin and IGF-1 signaling were also lower in line 2.51 tumors than line 1.02 tumors. The in-life portion of the second study revealed a heterogeneous response to ICI therapies among lean and obese mice. To understand this diversity of response, 16S rRNA sequencing analysis of tumoral and cecal microbiota in response to DIO and ICI therapy is underway. Discussion: These data reveal striking differences in tumor microenvironment and nutrient sensing as being potentially important determinants of differential tumor growth in BLBC. Ultimately, this research may contribute to the development of precision nutrition approaches using pro-/pre-biotics to promote microbial communities, targeting the tumor microenvironment, which may improve immunotherapy response and disrupt the obesity-associated exacerbation of breast cancer. Financial Support: This work was supported by R35CA197627 to SDH. Citation Format: Tori L. McFarlane, Meredith S. Carson, Elaine M. Glenny, Violet A. Kiesel, Ashlee Taylor, Daniel Roth, Jody Albright, Melissa VerHague, John E. French, Michael F. Coleman, Stephen D. Hursting. Microbial determinants of immune checkpoint inhibition response in a murine model of obesity and metastatic basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 612.

Obesity is routinely considered as a single disease state, which drives a "one-size-fits-all" approach to treatment. We recently convened the first annual University of North Carolina Interdisciplinary Nutrition Sciences Symposium to discuss the heterogeneity of obesity and the need for translational science to advance understanding of this heterogeneity. The symposium aimed to advance scientific rigor in translational studies from animal to human models with the goal of identifying underlying mechanisms and treatments. In this review, we discuss fundamental gaps in knowledge of the heterogeneity of obesity ranging from cellular to population perspectives. We also advocate approaches to overcoming limitations in the field. Examples include the use of contemporary mouse genetic reference population models such as the Collaborative Cross and Diversity Outbred mice that effectively model human genetic diversity and the use of translational models that integrate -omics and computational approaches from pre-clinical to clinical models of obesity. Finally, we suggest best scientific practices to ensure strong rigor that will allow investigators to delineate the sources of heterogeneity in the population with obesity. Collectively, we propose that it is critical to think of obesity as a heterogeneous disease with complex mechanisms and etiologies, requiring unique prevention and treatment strategies tailored to the individual.

Abstract Introduction: In women in the United States, breast cancer (BC) is the second leading cause of cancer-related death. BC is a heterogeneous disease, characterized by several subtypes based on molecular markers including several hormone receptors. Transcriptomic analysis further stratifies the disease. The basal-like subtype, often triple-negative for hormone receptors, has the poorest prognosis partially due to the absence of targeted therapies. Obesity, a preventable cause of cancer, is an established risk factor for breast cancer, including basal-like breast cancers. Mouse models to study the interaction of BC and obesity, particularly the more aggressive basal-like BC, are crucial. The C3(1)/SV40 large T-antigen (TAg) transgenic mouse model is an established model of basal-like mammary cancer that recapitulates several key features of human breast cancer. However, this model was generated in FVB mice, which is not an ideal genetic background for studying obesity, immunology and other aspects of cancer research. The goal of this study was to develop and characterize a C3TAg transgenic mouse model on a C57BL/6 background. Therefore, the current study backcrossed C3TAg transgenic FVB mice with C57BL/6 mice, a model most commonly used to study obesity, for 10 generations to create a transplantable C3TAg model in C57BL/6 mice. Methods: Cell Line Generation: C3TAg transgenic FVB mice were backcrossed with C57BL/6 mice for 10 generations. Tumors from 6-month-old heterozygous female mammary tissue were harvested, dissociated, and subcloned by limited dilution. A total of 156 clonal lines were established from which a subset of 12 lines displaying differences in morphology, growth and expression of metabolic and EMT genes (by qPCR). In vivo: 96 female C57BL/6J mice were randomized into 12 groups (n=8/group) and orthotopically injected with three different cell densities (5*104, 1.5*105, 5*105 cells/mouse) from one of the four selected lines. Mice tumor growth was assessed using electronic calipers. In vitro: Proliferation and cellular respiration were assessed and molecular subtyping was conducted using Affymetrix microarray data. Results: All tested cell lines formed tumors in vivo, with macroscopic characteristics of basal-like tumors including central necrosis. In vitro growth and metabolism of tested lines showed modest dissimilarity; in contrast, tumor growth rates and metastatic outgrowth were starkly dissimilar among the tested lines. Transcriptomic data clustered these cell lines with human BC tumors. Conclusions: Here we report for the first time a transplantable C3TAg model in C57BL/6 mice with highly effective tumor engraftment and growth, and with variable phenotypes for tumor growth and metastasis. Thus, this model may serve as an attractive model for the study of the interactions of basal-like breast cancer and obesity. This work was supported by R35CA197627 to SH. Citation Format: Meredith S. Carson, Michael F. Colman, Daniel Roth, Jody Albright, Melissa VerHague, John E. French, Stephen D. Hursting. Characterization of a novel transplantable C3TAg breast cancer model in C57BL/6J mice [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 2918.

Abstract The prevalence of obesity, an established risk and prognostic factor for basal-like breast cancer (BLBC), has risen dramatically in the US and many other countries over the past 4 decades. Obesity is also associated with increased resistance to several classes of cancer chemotherapeutic agents used for advanced BLBC. Unfortunately the mechanisms underlying chemotherapy resistance, and the inter-individual differences driving resistance, are poorly understood. Population-based experimental mouse models for determining inter-individual genetic differences in chemotherapy pharmacokinetics and treatment-related efficacy or resistance in mammary gland cancer are not well-defined. Our aim is to develop a population-based experimental model based on outcrosses between 30 different Collaborative Cross line females and hemizygous male B6.FVB-Tg(C3-1-TAg) (greater than N15) congenic mice to produce 30 Collaborative Cross (CC) recombinant inbred line intercross (CC RIX) mouse lines to investigate the interactive effects between diet-induced obesity (DIO) and genetic susceptibility to chemotherapy in a genetically heterogeneous mouse model of BLBC. We report here that thirty genetically-different CCRIL X hemizygous B6.FVB-Tg(C3-1-TAg) congenic female and male CC RIX mice observed longitudinally for occurrence of basal-like mammary tumors showed significant differences in both age at first mammary tumor observation and the fraction of females and males in each RIX bearing tumors. Litter size and the number of females and males of each RIX varied across the 30 CC RIX line. The study was well-powered, with at least X males and Y females for each CC RIX line evaluated longitudinally. We observed that 25 of 30 female CC RIX lines presented with BLMC, with distinct times-to-tumor with each CC RIX. The penetrance of the fraction of each CC RIX line (birth cohort) presenting with BLMC varied greatly in females (25-100%). Surprisingly, 11 of 30 male CC RIX lines presented with BLMC, with penetrance of BLMC varying in males between 10-75%. In conclusion, we present preliminary data to support development of an experimental mouse model for BLBC based on the random mating of 8 genetically-diverse homozygous inbred founder lines followed by in-breeding to greater than 95% homozygosity carrying 45 million single-nucleotide and structural variants. Studies are underway using these CC RIX lines to evaluate the genetic underpinnings of obesity-associated resistance to carboplatin in C3-TAg-driven mammary tumors. Citation Format: John Edgar French, William Pressel, Jody Albright, Melissa VerHague, Stephen D. Hursting. A population-based mouse model for an experimental basal-like mammary 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 2256.

We evaluated the impact of genetic variance on biomarker levels in a population of workers in the automotive repair and refinishing industry who were exposed to respiratory sensitizers 1,6-hexamethylene diisocyanate (HDI) monomer and one of its trimers, HDI isocyanurate. The exposures and respective urine and plasma biomarkers 1,6-diaminohexane (HDA) and trisaminohexyl isocyanurate (TAHI) were measured in 33 workers; and genome-wide microarrays (Affymetrix 6.0) were used to genotype the workers’ single-nucleotide polymorphisms (SNPs). Linear mixed model analyses have indicated that interindividual variations in both inhalation and skin exposures influenced these biomarker levels. Using exposure values as covariates and a false discovery rate < 0.10 to assess statistical significance, we observed that seven SNPs were associated with HDA in plasma, five were associated with HDA in urine, none reached significance for TAHI in plasma, and eight were associated with TAHI levels in urine. The different genotypes for the 20 significant SNPs accounted for 4- to 16-fold changes observed in biomarker levels. Associated gene functions include transcription regulation, calcium ion transport, vascular morphogenesis, and transforming growth factor beta signaling pathway, which may impact toxicokinetics indirectly by altering inflammation levels. Additionally, in an expanded analysis using a minor allele cutoff of 0.05 instead of 0.10, there were biomarker-associated SNPs within three genes that have been associated with isocyanate-induced asthma: ALK, DOCK2, and LHPP. We demonstrate that genetic variance impacts the biomarker levels in workers exposed to HDI monomer and HDI isocyanurate and that genetics can be used to refine exposure predictions in small cohorts when quantitative personal exposure and biomarker measurements are included in the models.

Obesity has reached epidemic levels worldwide. It is associated with major chronic diseases, including cancer and cardiovascular disease. The etiology of obesity-associated cancer is poorly understood. New population-based research mouse models are required to better understand prevention and to increase the translational potential of mouse models to humans. The Collaborative Cross (CC) recombinant inbred lines of mice are a population-based model created from an 8-way multiparental advanced generation intercross with a 12% minor allele frequency (MAF) and more than 45 million SNP and CNV, including a significant number of private (unique) alleles. The Diversity Outbred (DO) mice were created from early CC incipient lines (generations G2:F2-5) by random selection and 175 randomly selected breeding pairs. The genetic diversity of the DO and CC RIL mice is ideal for forward and reverse genetics approaches, respectively, to identify potential causally related risk variants and identify etiology based upon causal relationships. Here, we present data from two cohorts (from different breeding generations) of 50 female and 50 male DO mice each (total 100 each sex). After 12-wks high-fat (60% kcal fat) diet we observed significant differences in weight gain during diet-induced obesity DIO. After switching diets for 8-wks of 30% caloric restriction of a low-fat (10% kcal fat) diet, we observed significant interindividual differences in weight loss. Body weight, lean and fat mass differences), increases in 4-hr fasted blood glucose and insulin levels, insulin resistance (HOMA-IR and increased blood branched chain amino acids levels (BCAA) were observed. Four interacting quantitative trait loci/gene (Chst10, Bmiq5, Ath8, and Tcq14) related to metabolism, body mass index, cardiovascular disease, and total cholesterol levels, respectively, have been identified. Regression analysis suggests significant interactions between fasted blood glucose and fat mass, HOMA-IR and fat mass, and BCAA and fat mass in DIO and CR outcomes. Results to date show significant amounts of interindividual variability of each of the quantified phenotypes in response to both diet interventions were demonstrated in female and male DO mice. These data suggest potential avenues of research on obesity and cancer relationships and pathways for intervention. Citation Format: Melissa VerHague, Jody Albright, Salvador Favela, Kerri Barron, Michael F. Coleman, Katie Meyer, John E. French, Stephen D. Hursting. Diet-induced obesity and caloric restriction weight loss in Diversity Outbred (DO) mice: An experimental preclinical translational model for the investigation of pathways for prevention of obesity and cancer [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A45.

DNA methylation is an essential epigenetic process in mammals, intimately involved in gene regulation. Here we address the extent to which genetics, sex, and pregnancy influence genomic DNA methylation by intercrossing 2 inbred mouse strains, C57BL/6N and C3H/HeN, and analyzing DNA methylation in parents and offspring using whole-genome bisulfite sequencing. Differential methylation across genotype is detected at thousands of loci and is preserved on parental alleles in offspring. In comparison of autosomal DNA methylation patterns across sex, hundreds of differentially methylated regions are detected. Comparison of animals with different histories of pregnancy within our study reveals a CpG methylation pattern that is restricted to female animals that had borne offspring. Collectively, our results demonstrate the stability of CpG methylation across generations, clarify the interplay of epigenetics with genetics and sex, and suggest that CpG methylation may serve as an epigenetic record of life events in somatic tissues at loci whose expression is linked to the relevant biology.