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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.

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.

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.