Sarah M. Dunlap

Leptin deficiency suppresses MMTV-Wnt-1 mammary tumor growth in obese mice and abrogates tumor initiating cell survival

Obesity increases both the risk and mortality associated with many types of cancer including that of the breast. In mice, obesity increases both incidence of spontaneous tumors and burden of transplanted tumors. Our findings identify leptin, an adipose secreted cytokine, in promoting increased mammary tumor burden in obese mice and provide a link between this adipokine and cancer. Using a transplantable tumor that develops spontaneously in the murine mammary tumor virus-Wnt-1 transgenic mice, we show that tumors transplanted into obese leptin receptor (LepRb)-deficient (db/db) mice grow to eight times the volume of tumors transplanted into lean wild-type (WT) mice. However, tumor outgrowth and overall tumor burden is reduced in obese, leptin-deficient (ob/ob) mice. The residual tumors in ob/ob mice contain fewer undifferentiated tumor cells (keratin 6 immunopositive) compared with WT or db/db mice. Furthermore, tumors in ob/ob mice contain fewer cells expressing phosphorylated Akt, a growth promoting kinase activated by the LepRb, compared with WT and db/db mice. In vivo limiting dilution analysis of residual tumors from ob/ob mice indicated reduced tumor initiating activity suggesting fewer cancer stem cells (CSCs). The tumor cell populations reduced by leptin deficiency were identified by fluorescence-activated cell sorting and found to express LepRb. Finally, LepRb expressing tumor cells exhibit stem cell characteristics based on the ability to form tumorspheres in vitro and leptin promotes their survival. These studies provide critical new insight on the role of leptin in tumor growth and implicate LepRb as a CSC target.

Obesity, metabolic dysregulation, and cancer: a growing concern and an inflammatory (and microenvironmental) issue

Obesity is an established risk and progression factor for many cancers. In the United States more than one‐third of adults, and nearly one in five children, are currently obese. Thus, a better understanding of the mechanistic links between obesity and cancer is urgently needed to identify intervention targets and strategies to offset the procancer effects of obesity. This review synthesizes the evidence on key biological mechanisms underlying the obesity–cancer association, with particular emphasis on obesity‐associated enhancements in growth factor signaling, inflammation, and perturbations in the tumor microenvironment. These interrelated pathways and processes represent mechanistic targets for disrupting the obesity–cancer link.

Calorie restriction and cancer prevention: a mechanistic perspective

Calorie restriction (CR) is one of the most potent broadly acting dietary interventions for inducing weight loss and for inhibiting cancer in experimental models. Translation of the mechanistic lessons learned from research on CR to cancer prevention strategies in human beings is important given the high prevalence of excess energy intake, obesity, and metabolic syndrome in many parts of the world and the established links between obesity-associated metabolic perturbations and increased risk or progression of many types of cancer. This review synthesizes findings on the biological mechanisms underlying many of the anticancer effects of CR, with emphasis on the impact of CR on growth factor signaling pathways, inflammation, cellular and systemic energy homeostasis pathways, vascular perturbations, and the tumor microenvironment. These CR-responsive pathways and processes represent targets for translating CR research into effective cancer prevention strategies in human beings.

Insulin-like growth factor-binding protein 2-driven glioma progression is prevented by blocking a clinically significant integrin, integrin-linked kinase, and NF-κB network

Insulin-like growth factor-binding protein 2 (IGFBP2) is increasingly recognized as a glioma oncogene, emerging as a target for therapeutic intervention. In this study, we used an integrative approach to characterizing the IGFBP2 network, combining transcriptional profiling of human glioma with validation in glial cells and the replication-competent ASLV long terminal repeat with a splice acceptor/tv-a glioma mouse system. We demonstrated that IGFBP2 expression is closely linked to genes in the integrin and integrin-linked kinase (ILK) pathways and that these genes are associated with prognosis. We further showed that IGFBP2 activates integrin β1 and downstream invasion pathways, requires ILK to induce cell motility, and activates NF-κB. Most significantly, the IGFBP2/integrin/ILK/NF-κB network functions as a physiologically active signaling pathway in vivo by driving glioma progression; interfering with any point in the pathway markedly inhibits progression. The results of this study reveal a signaling pathway that is both targetable and highly relevant to improving the survival of glioma patients.

Calorie restriction and rapamycin inhibit MMTV-Wnt-1 mammary tumor growth in a mouse model of postmenopausal obesity

Obesity is an established risk and progression factor for postmenopausal breast cancer. Interventions to decrease caloric intake and/or increase energy expenditure beneficially impact tumor progression in normoweight humans and animal models. However, despite the increasingly high global prevalence of obesity, the effects and underlying mechanisms of these energy balance modulating interventions are poorly characterized in obese individuals. The goal of this study was to better characterize the mechanism(s) responsible for the link between energy balance and breast cancer progression in the postmenopausal obesity context. We compared the effects of calorie restriction (CR), treadmill exercise (EX), and mammalian target of rapamycin (mTOR inhibitor) treatment on body composition, serum biomarkers, cellular signaling, and mammary tumor growth in obese mice. Ovariectomized C57BL/6 mice were administered a diet-induced obesity regimen for 8 weeks, then randomized into three treatment groups: control (semipurified diet fed ad libitum, maintained the obese state); 30% CR (isonutrient relative to control except 30% reduction in carbohydrate calories); and EX (control diet fed ad libitum plus treadmill exercise). Mice were implanted with syngeneic MMTV-Wnt-1 mammary tumor cells at week 12. Rapamycin treatment (5  mg/kg every 48  h) started at week 14. Tumors were excised at week 18. CR and rapamycin (but not EX) significantly reduced final tumor weight compared to control. In follow-up analysis, constitutive activation of mTOR ablated the inhibitory effects of CR on Wnt-1 mammary tumor growth. We conclude that mTOR inhibition may be a pharmacologic strategy to mimic the anticancer effects of CR and break the obesity-breast cancer progression link.

Leptin receptor maintains cancer stem-like properties in triple negative breast cancer cells

Despite new therapies, breast cancer continues to be the second leading cause of cancer mortality in women, a consequence of recurrence and metastasis. In recent years, a population of cancer cells has been identified, called cancer stem cells (CSCs) with self-renewal capacity, proposed to underlie tumor recurrence and metastasis. We previously showed that the adipose tissue cytokine LEPTIN, increased in obesity, promotes the survival of CSCs in vivo. Here, we tested the hypothesis that the leptin receptor (LEPR), expressed in mammary cancer cells, is necessary for maintaining CSC-like and metastatic properties. We silenced LEPR via shRNA lentivirus transduction and determined that the expression of stem cell self-renewal transcription factors NANOG, SOX2, and OCT4 (POU5F1) is inhibited. LEPR-NANOG signaling pathway is conserved between species because we can rescue NANOG expression in human LEPR-silenced cells with the mouse LepR. Using a NANOG promoter GFP reporter, we showed that LEPR is enriched in NANOG promoter active (GFP+) cells. In lineage tracing studies, we showed that the GFP+ cells divide in a symmetric and asymmetric manner. LEPR-silenced MDA-MB-231 cells exhibit a mesenchymal to epithelial transition morphologically, increased E-CADHERIN and decreased VIMENTIN expression compared with control cells. Finally, LEPR-silenced cells exhibit reduced cell proliferation, self-renewal in tumor sphere assays, and tumor outgrowth in xenotransplant studies. Given the emergence of NANOG as a pro-carcinogenic protein in multiple cancers, these studies suggest that inhibition of LEPR may be a promising therapeutic approach to inhibit NANOG and thereby neutralize CSC functions.

Dietary Energy Balance Modulates Epithelial-To-Mesenchymal Transition and Tumor Progression in Murine Claudin-Low and Basal-Like Mammary Tumor Models

Using novel murine models of claudin-low and basal-like breast cancer, we tested the hypothesis that diet-induced obesity (DIO) and calorie restriction (CR) differentially modulate progression of these aggressive breast cancer subtypes. For model development, we characterized two cell lines, “mesenchymal (M)-Wnt” and “epithelial (E)-Wnt,” derived from MMTV-Wnt-1 transgenic mouse mammary tumors. M-Wnt, relative to E-Wnt, cells were tumor-initiating cell (TIC)-enriched (62% vs. 2.4% CD44high/CD24low) and displayed enhanced ALDEFLUOR positivity, epithelial-to-mesenchymal transition (EMT) marker expression, mammosphere-forming ability, migration, invasion, and tumorigenicity (P < 0.001; each parameter). M-Wnt and E-Wnt cells clustered with claudin-low and basal-like breast tumors, respectively, in gene expression profiles and recapitulated these tumors when orthotopically transplanted into ovariectomized C57BL/6 mice. To assess the effects of energy balance interventions on tumor progression and EMT, mice were administered DIO, control, or CR diets for 8 weeks before orthotopic transplantation of M-Wnt or E-Wnt cells (for each cell line, n = 20 mice per diet) and continued on their diets for 6 weeks while tumor growth was monitored. Relative to control, DIO enhanced M-Wnt (P = 0.01), but not E-Wnt, tumor progression; upregulated EMT- and TIC-associated markers including N-cadherin,fibronectin, TGFβ, Snail, FOXC2, and Oct4 (P < 0.05, each); and increased intratumoral adipocytes. Conversely, CR suppressed M-Wnt and E-Wnt tumor progression (P < 0.02, each) and inhibited EMT and intratumoral adipocyte accumulation. Thus, dietary energy balance interventions differentially modulate EMT and progression of claudin-low and basal-like tumors. EMT pathway components may represent targets for breaking the obesity–breast cancer link, particularly for preventing and/or controlling TIC-enriched subtypes such as claudin-low breast cancer. Cancer Prev Res; 5(7); 930–42. ©2012 AACR.

Metformin and Rapamycin Reduce Pancreatic Cancer Growth in Obese Prediabetic Mice by Distinct MicroRNA-Regulated Mechanisms

Metformin treatment is associated with a decreased risk and better prognosis of pancreatic cancer (PC) in patients with type 2 diabetes, but the mechanism of metformin’s PC growth inhibition in the context of a prediabetic state is unknown. We used a Panc02 pancreatic tumor cell transplant model in diet-induced obese (DIO) C57BL/6 mice to compare the effects of metformin and the direct mammalian target of rapamycin (mTOR) inhibitor rapamycin on PC growth, glucose regulation, mTOR pathway signaling, and candidate microRNA (miR) expression. In DIO/prediabetic mice, metformin and rapamycin significantly reduced pancreatic tumor growth and mTOR-related signaling. The rapamycin effects centered on decreased mTOR-regulated growth and survival signaling, including increased expression of let-7b and cell cycle–regulating miRs. Metformin (but not rapamycin) reduced glucose and insulin levels and expression of miR-34a and its direct targets Notch, Slug, and Snail. Metformin also reduced the number and size of Panc02 tumor spheres in vitro and inhibited the expression of Notch in spheroids. Our results suggest that metformin and rapamycin can both inhibit pancreatic tumor growth in obese, prediabetic mice through shared and distinct mechanisms. Metformin and direct mTOR inhibitors, alone or possibly in combination, represent promising intervention strategies for breaking the diabetes-PC link.

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.

Obesity, Independent of p53 Gene Dosage, Promotes Mammary Tumor Progression and Upregulates the p53 Regulator MicroRNA-504

Obesity, prevalent in >35% of US women, is an established risk and progression factor for postmenopausal breast cancer, and strategies to break the obesity-breast cancer link are urgently needed. Approximately 30% of breast cancers carry p53 tumor suppressor gene alterations; however, the effects of obesity on breast cancer progression in relation to p53 gene dosage are unclear. Using murine models of postmenopausal breast cancer, we characterized the interactive effects of diet-induced obesity (DIO) and p53 gene dosage on mammary tumor growth and associated p53-related regulatory mechanisms. Ovariectomized C57BL/6 mice were randomly assigned to receive a DIO or control diet, and (at 10 weeks) orthotopic injection of MMTV-Wnt-1 p53+/− or MMTV-Wnt-1 p53+/+ mammary tumor cells (n = 20 mice per diet and genotype group). DIO and control diets produced distinct phenotypes (mean percent body fat at 10 weeks: 57% and 39%, respectively, P<0.001). Regardless of phenotype, time to first palpable tumor was 57% less for Wnt-1 p53+/− than Wnt-1 p53+/+ tumors. Regardless of tumoral p53 genotype, DIO (relative to control) increased tumor burden, tumor cell proliferation (Ki-67), severity of tumor pathology, local tissue invasion, epithelial-to-mesenchymal transition (EMT) programming, and tumoral microRNA-504 (a negative regulator of p53) expression; and suppressed p53, p21, and estrogen receptor-alpha protein expression. These findings in murine models of postmenopausal breast cancer suggest that obesity may augment procancer effects related to p53 gene alterations. Furthermore, microRNA-504, an obesity-responsive negative regulator of p53 and putative EMT regulator, may represent a novel molecular target for breaking the obesity-breast cancer link.