Abstract 1847: Effect of rapamycin and metformin on skin tumor promotion in mice overweight and obese

Abstract The prevalence of obesity in the US has risen drastically over the past few decades and is an established risk factor for several human cancers. Insights into the mechanisms underlying this link are urgently needed to develop strategies for treatment and prevention. Various studies have shown that calorie restriction (CR) during tumor promotion in the two-stage model of skin carcinogenesis causes a significant reduction in tumorigenesis. In addition, diet induced obesity (DIO) leads to insulin resistance, increased circulating levels of IGF-1 and increased susceptibility to tumor development. Current data indicate that signaling through the IGF-1R and EGFR as well as several downstream signaling pathways plays an important role in dietary energy balance effects on tumor promotion during epithelial carcinogenesis. The current study was designed to evaluate the ability of rapamycin (an established mTORC1 inhibitor) to inhibit or reverse the effects of overweight/obesity on skin tumor promotion in mice. In initial experiments using female FVB mice on an AIN76A diet, rapamycin (5-1000 nmol per mouse) was found to be a potent inhibitor of skin tumor promotion by TPA. In this regard, rapamycin reduced both tumor multiplicity and incidence in a dose dependent manner. A dose of 5 nmol rapamycin given 30 min before each TPA treatment reduced the number of papillomas per mouse by approximately 50%. Rapamycin treatment inhibited TPA-induced mTORC1 activation as well as downstream signaling proteins p70S6KT389, prS6S240/244 and p4EBP1T37/46. Multiple treatment experiments were performed to evaluate the effects of rapamycin (5-200 nmol per mouse) on TPA-induced epidermal hyperproliferation (as assessed by epidermal hyperplasia and epidermal labeling index). Rapamycin significantly inhibited TPA-induced epidermal hyperproliferation in a dose dependent manner. In addition, immunohistochemical analyses of the skin from mice in this multiple treatment experiment revealed that rapamycin significantly decreased the number of infiltrating macrophages, T-cells, neutrophils, and mast cells seen in the dermis following TPA treatment. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited their growth. Two-stage skin carcinogenesis studies are currently underway to further evaluate the impact of rapamycin treatment on mice made overweight (AIN76A) or obese (DIO diet; 60 Kcal % fat) via dietary energy balance manipulation. In addition, groups of mice are also being treated with metformin. These ongoing studies will clarify whether suppression of the mTORC1 signaling pathway is a viable strategy for reversing the effects of obesity on tumor development in this well established model of epithelial carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1847. doi:10.1158/1538-7445.AM2011-1847</jats:p

Abstract 966: Dietary energy balance modulates IGF-1R and EGFR signaling and crosstalk during tumor promotion

Abstract Negative energy balance (calorie restriction, CR) inhibits, while positive energy balance enhances tumor promotion during two-stage skin carcinogenesis. Biochemical studies have demonstrated that CR reduced, while diet-induced obesity (DIO) increased activation of the insulin-like growth factor-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR), as well as downstream signaling (e.g., Akt and mTOR) during tumor promotion. Additional studies performed using the liver IGF-1 deficient (LID) mouse model demonstrated that a 75% reduction in circulating IGF-1 attenuated growth factor signaling through the IGF-1R and the EGFR similar to mice on CR diets. These findings suggest that dietary energy balance, primarily through its effects on levels of circulating IGF-1, modulates epithelial carcinogenesis and tumor promotion through diet-induced changes in signaling and crosstalk between the IGF-1R and the EGFR, which subsequently alters signaling to downstream effectors. To determine the impact of IGF-1 on IGF-1R and EGFR signaling and crosstalk, Western blot analyses, immunoprecipiation and qPCR analyses were performed on C50 cells (nontumorigenic keratincyte cell line) stimulated with IGF-1. IGF-1 increased activation of the IGF-1R and the EGFR, as well as signaling to downstream effectors (i.e., Akt and mTOR). Immunoprecipitation experiments demonstrated an increase in association between the IGF-1R and the EGFR following IGF-1 stimulation. Furthermore, IGF-1 induced changes in expression levels of the EGFR, as well as EGFR ligands (i.e., HB-EGF, amphiregulin, TGF-α). Additional experiments performed using C50 cells stably transfected with EGFR shRNA or cells pretreated with PD153035 (EGFR inhibitor) demonstrated reduced IGF-1 mediated activation of the EGFR as well as reduced downstream signaling to Akt and mTOR. In vivo studies were performed to evaluate the effect of dietary energy balance on IGF-1R and EGFR crosstalk. For these experiments, ICR mice were maintained on a CR and DIO regimen for 15 weeks, after which they were treated with a single application of 3.4 nmol TPA. CR reduced, while DIO increased the interaction between the IGF-1R and the EGFR in the epidermis of TPA treated mice. Furthermore, dietary energy balance modulated EGFR and EGFR ligand expression levels. Taken together, these data suggest that levels of IGF-1 can modulate signaling through both the IGF-1R and the EGFR. Furthermore, diet-induced changes in IGF-1R/EGFR crosstalk subsequently modulate activation of downstream signaling to Akt and mTOR, thus contributing, at least in part, to the effect of dietary energy balance on skin tumor promotion. Supported by NIH grants CA37111 and CA129409. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 966.</jats:p

Abstract 5085: Formerly obese mice display increased mmtv-wnt-1 tumor growth despite normalization of body weight: The role of mTOR

Abstract Obesity, an established breast cancer risk factor in postmenopausal women, is also associated with poor prognosis and shorter disease-free and overall survival in pre-and postmenopausal breast cancer patients. It is not known if restoration of normal weight in obese individuals reverses the negative effects of obesity on mammary tumor progression. Since increased signaling through the mammalian target of rapamycin (mTOR) pathway has been implicated in breast cancer, and obesity can activate this pathway, we hypothesized that mTOR signaling remains activated and tumor growth high following weight loss in diet-induced obesity (DIO) mice, and that mTOR inhibition would offset this effect. Ovariectomized (OVX) C57BL/6 mice were randomized to diet regimens that induce a lean (calorie restricted; n=20), overweight (control; n=20), or DIO (n=30) phenotype. Following 17 weeks of diet treatment, quantitative magnetic resonance was performed on all mice, and the DIO mice were switched to the control diet. At week 20, all mice were injected with 5×104 syngeneic MMTV-Wnt-1 mammary tumor cells in the 4th mammary fat pad. Two weeks after tumor injection, 10 mice/diet group received RAD001 (10 mg/kg/body weight) and 10/group received placebo by oral gavage 2x/week for 6 weeks. The remaining 10 DIO mice received a higher dose of RAD001 (15 mg/kg/body weight). Tumor growth was measured 2x/week and serum levels of IGF-1 were measured at study endpoint. DIO mice were significantly heavier (47.4 ± 1.1) at week 17 than control (33.7 ± 0.6) and lean mice (25.3 ± 0.4); % body fat was also higher (p&amp;lt;0.001) in DIO (54.4%) compared to the control (41.7%) and lean (32.7%) mice. However, DIO mice lost weight after they were switched to the control diet, and their body weights matched the controls at the time of tumor cell injection. Tumor growth in formerly DIO mice was enhanced (p&amp;lt;0.05) compared to the control and lean mice. RAD001 was effective at decreasing tumor growth in all diet groups (p&amp;lt;0.01). The effect of RAD001 was diminished in the formerly DIO mice compared to control and lean mice, but this partial resistance was overcome with the higher RAD001 dose. RAD001 significantly increased serum IGF-1 levels in all groups. Gene expression microarray, Western blot, and immunohistochemical analyses of mTOR and other key growth and survival pathways are ongoing to identify pathways underlying the enhanced tumor growth in formerly obese mice. In conclusion, our results suggest that the growth-enhancing effects of obesity on mammary cancer may persist even after weight loss, due at least in part to activation of mTOR signaling that can be offset by RAD001 treatment. These findings suggest that a combination of dietary and pharmacologic interventions, such as mTOR inhibitors, may be required to break the obesity-cancer link. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5085.</jats:p

Perspective on This Article from Rapamycin Is a Potent Inhibitor of Skin Tumor Promotion by 12-<i>O</i>-Tetradecanoylphorbol-13-Acetate

Perspective on This Article from Rapamycin Is a Potent Inhibitor of Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetat

Supplementary Figure 1 from Energy Balance Modulates Mouse Skin Tumor Promotion through Altered IGF-1R and EGFR Crosstalk

PDF file - 2.8MB, Cell Proliferation and Tumor Promotion</p

Supplementary Table 1 from Energy Balance Modulates Mouse Skin Tumor Promotion through Altered IGF-1R and EGFR Crosstalk

PDF file - 88K, Effect of Dietary Energy Balance on Tumor Promotion and Tumor Progression Using the Two-Stage Skin Carcinogenesis Protocol</p

Supplementary Table 2 from Energy Balance Modulates Mouse Skin Tumor Promotion through Altered IGF-1R and EGFR Crosstalk

PDF file - 96K, Effect of Dietary Energy Balance on Body Mass and Serum Protein Levels</p

Abstract P5-06-05: Palmitate induces a senescent-like phenotype in fibroblasts resulting in altered phenotypes in cells of the breast tumor microenvironment

Abstract Background: Obese breast cancer patients face a worse prognosis, including an increased risk of recurrence and mortality. While the causative mechanisms have yet to be fully uncovered, emerging evidence implicates palmitate, increased in the obese state, in development of cellular senescence, an inflammatory state associated with proliferation, metastasis, and tumor-associated neutrophil (TAN) polarization, among other measures of carcinogenesis. However, studies have yet to investigate the impact of palmitate on induction of senescence in breast fibroblasts, and no studies have assessed the effect of senescent fibroblasts on neutrophil polarization in the breast tumor microenvironment. This said, we hypothesize that palmitate alters breast cancer cell gene expression and neutrophil phenotype via induction of a senescent-like phenotype in fibroblasts. Methods: HCA2, IMR-90, and human mammary fibroblasts were exposed to palmitate or vehicle in media supplemented with 2% charcoal-stripped fetal bovine serum, after which the cells were measured through qPCR for expression of IL-1a, IL-6 and IL-8, some of the most prominent members of the senescence-associated secretory phenotype. Palmitate-exposed fibroblasts were also subjected to chromogenic staining for senescence-associated beta-galactosidase and immunoenzymatic BrdU analysis, two well-established measures of senescence. Next, in order to study the influence of these fibroblasts on other cells of the breast tumor microenvironment, we assessed their impact on polarization of DMSO-differentiated HL-60 neutrophils by using flow cytometry to measure neutrophil expression of CD54 and CD95, differentially expressed on N1 and N2 neutrophils. Finally, we employed PCR arrays to assess the impact of palmitate-exposed fibroblasts on the expression of 84 genes in MCF-7 and 231 breast cancer cells, measuring activation of pathways related to apoptosis, cell cycle, DNA damage, senescence, telomere maintenance, metabolism, angiogenesis, and the endothelial-to-mesenchymal transition. Results and Conclusions: Palmitate induced pro-inflammatory gene expression and SA-beta-gal activity and decreased BrdU incorporation in fibroblasts. Palmitate also exhibited non-cell-autonomous effects, as palmitate-exposed fibroblasts induced phenotypic changes in both neutrophils and breast cancer cells. These findings are among the first to implicate palmitate-induced fibroblast senescence in the stimulation of non-cell-autonomous changes in the breast tumor microenvironment and will ultimately inform our understanding of the mechanistic connection between the obesity-associated factor palmitate and breast tumorigenesis. Citation Format: Brittany Susanne Harlow, Albert Davalos, Bryan McClellan, Andrew Brenner, Christopher Jolly, Stefano Tiziani, Steve Hursting, Linda deGraffenried. Palmitate induces a senescent-like phenotype in fibroblasts resulting in altered phenotypes in cells of the breast tumor microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-05.</jats:p