Background Fatty acid synthase (FASN) expression is associated with a more aggressive breast cancer phenotype and is regulated downstream of receptor tyrosine kinase (RTK) signaling pathways. Recently, post transcriptional regulation of lipogenic transcripts have been demonstrated as being mediated downstream of serine-arginine rich protein kinase 2 (SRPK2), which acts to phosphorylate serine-arginine rich splicing factors (SRSFs), resulting in RNA binding and various RNA regulatory processes. Though post-transcriptional regulation of FASN has been studied previously, the upstream mediators of these pathways have not been elucidated. Methods Western blotting and RT-qPCR were utilized to demonstrate alterations in FASN and mRNA expression upon modulation of the IGF-1-mTORC1-SRPK2 pathway by small molecule inhibitors or RNAi mediated silencing. RNA stability was accessed by using the transcriptional inhibitor actinomycin-D followed by RT-qPCR. Further, we employed RNA-immunoprecipitation to demonstrate the direct binding of SRSF-1 to FASN transcripts. Results In the current study, we demonstrated an IGF-1 induced increase in FASN mRNA and protein expression that was attenuated by mTORC1 inhibition. This mTORC1 inhibition also resulted in decreases in total and nuclear p-SRPK2 in response to IGF-1 exposure. Upon SRPK2 knockdown and inhibition, we observed a decrease in FASN protein and mRNA stability, respectively, in response to IGF-1 exposure that was specific to triple negative and HER2+ breast cancer cell lines. As we explored further, IGF-1 exposure resulted in an altered localization of eGFP expressed SRSF-1, pEGFP-SRSF-1 that was rescued upon both SRPK2 knockdown and mTORC1 inhibition. Further, we observed an increase binding of SRSF-1 to FASN RNA upon IGF-1 exposure, which was abrogated by SRPK2 knockdown. Conclusion These current findings establish a potential IGF-1-mTORC1-SRPK2-FASN axis in breast cancer, which could be a potential therapeutic target for cancers that overexpress FASN and components of the IGF-1R pathway.

e12548 Background: Almost 40% of women with breast cancer are obese at diagnosis. Obesity is associated with a worse prognosis in triple negative breast cancer (TNBC). Preclinical studies have shown that leptin is an important factor associated with TNBC by promoting cancer stem cell (CSC) enrichment and/or epithelial-to-mesenchymal transition (EMT). Transcription factors SNAIL, TWIST and ZEB are critical components in enhancing EMT in cancer cells. The specific mechanism(s) by leptin regulates SNAIL, TWIST and ZEB expression remain unclear, limiting the development of effective interventions to improve outcomes in obese TNBC patients. Recent studies have demonstrated that miR200c, downstream of leptin receptor signaling, regulates the expression of SNAIL1, TWIST and ZEB. We will test the hypothesis that leptin contributes to obesity-induced EMT/CSC in TNBC through modulation of miR200c. Methods: Ob-R (leptin receptor) expression was suppressed in TNBC MDA-MB-231 and E-Wnt cells using shRNA (Ob-R null). Ob-R and Ob-R null cells were exposed to sera pooled from lean or obese women, as well as lean sera supplemented with leptin, after which expression of SNAIL, TWIST, ZEB and miR200c was measured by qPCR, while activation of the JAK-STAT pathway was assessed by Western blotting. Results: TNBC cells exposed to obese and high leptin conditions demonstrated increased expression of EMT markers compared to levels expressed under lean conditions. The Ob-R WT and null cells were used to determine the specific role of leptin signaling in regulating expression of SNAIL, TWIST and ZEB through miR200c. Conclusions: Both obese and high leptin conditions result in increased expression of EMT regulators, suggesting that effective targeting of this pathway may provide clinical benefit in the obese breast cancer patient. Elucidating the specific mediators of this pathway will guide development of novel and more potent medical therapies.

Purpose: The objective of this study is to determine the impact of exposure to obesity-related systemic factors on fatty acid synthase enzyme (FASN) expression in breast cancer cells. Methods: MCF-7 breast cancer cells were exposed to sera from patients having obesity or not having obesity and subjected to quantitative reverse transcription polymerase chain reaction (RT-qPCR). Subsequent MTT and colony-forming assays using both MCF-7 and T-47D cells exposed to sera and treated with or without FASN inhibitor, TVB-3166, were used. MCF-7 cells were then treated with insulin and the sterol regulatory element–binding protein (SREBP) processing inhibitor, betulin, prior to analysis of FASN expression by quantitative RT-qPCR and western blot. Insulin-induced SREBP-FASN promoter binding was analyzed by chromatin immunoprecipitation with an anti-SREBP antibody. Results: In response to sera exposure (body mass index [BMI] >30) there was an increase in FASN expression in breast cancer cells. Furthermore, treatment with the FASN inhibitor, TVB-3166, resulted in a decreased breast cancer cell survival and proliferation while increasing apoptosis upon sera exposure (BMI >30). Insulin-exposed MCF-7 cells exhibited an increased FASN messenger RNA and protein expression, which is abrogated upon SREBP inhibition. In addition, insulin exposure induced enhanced SREBP binding to the FASN promoter. Conclusions: Our results implicate FASN as a potential mediator of obesity-induced breast cancer aggression and a therapeutic target of patients with obesity-induced breast cancer.

Background: Studies have shown that obesity is associated with a worse breast cancer prognosis. Besides the effect of different stages of diagnosis and co-morbidities, recent data from our published in vitro and retrospective studies suggests that this phenomenon may occur because the obese state promotes a more aggressive cancer phenotype through the cyclooxygenase (COX-2) pathway and its production of prostaglandin E2 (PGE2). The metabolization of omega-3 fatty acids decreases the production of PGE2, and have been shown to have potential benefit to cancer patients by decreasing inflammation-related signaling. Our previous clinical trial showed mixed results in the effect of omega-3 PUFA supplements on PGE2 production in post-menopausal obese women. This led us to the hypothesis that the ratio of omega-3 to omega-6 PUFAs have differential effects on cell types within the tumor microenvironment, impacting cancer cell phenotype. Approach: In vitro experiments, including wound-healing assays to determine motility, and clonogenic assays to determine overall survival, were performed to determine if exposure to higher ratios of omega-6 to omega-3 fatty acids lead to a more aggressive cancer phenotype. MCF-7 breast cancer cells were treated with the following fatty acid ratios of omega-6 (arachidonic acid (AA)) to omega-3 (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)): 46:1, 20:1, 10:1, and 1.3:1. The wound-healing assays showed greater motility with higher ratios of omega-6 to omega-3 fatty acids conditions and the clonogenic assays showed greater survival with the higher ratios. Conclusion: These data indicate that lowering ratios of omega-6 to omega-3 fatty acids may lessen the aggressiveness of breast cancer cells and be beneficial to some patients. Studies are on-going to investigate the impact of PUFA ratios on cancer cell phenotype directly, including proliferation and invasion, as well as the indirect effects from modulation of the other cells within the tumor microenvironment, including the macrophages and adipocytes. Citation Format: Winikka L, Quach D, Harlow B, Brenner A, Munoz N, Tiziani S, deGraffenried L. The ratio of omega-3 to omega-6 PUFAs impact cancer cell phenotype in the tumor microenvironment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-03-12.

Background: The overexpression of the IGF-1R is correlated with an overall worse prognosis for breast cancer patients. IGF-1R contributes to an aggressive phenotype through its downstream signaling cascades, including the classical PI3K-Akt- mTORC1 pathway. Recent studies suggest another important target of IGF-1R regulation is the fatty acid synthase enzyme (FASN), leading to increased endogenous fatty acid synthesis. The importance of FASN activity to breast cancer progression is illustrated by the success of recent clinical trials investigating the efficacy of FASN inhibitors in refractory breast cancer. Previous studies in our lab and others have identified SREBP and SRPK2 as key IGF-1R targets regulating FASN expression. FASN expression can lead to excess palmitate, which can serve as a substrate for palmitoylation shown to aid in the membrane localization and function of various receptor tyrosine kinases, including IGF-1R. However, the role of FASN induced localization and activation of IGF-1R in breast cancer has not been investigated and is critical for a better understanding of the role of FASN in breast cancer progression. Objective: The goal of this study is to investigate a potential feedforward signaling loop between IGF-1R and FASN and its contribution to breast tumorigenesis. Methods: The impact of suppression of mTOR, SREBP, and SRPK2 on FASN gene expression in response to IGF-1 stimulation in MCF-7 and T47D ER+ breast cancer cells over a course of time was evaluated using qPCR. The role of FASN in IGF-1R stabilization and signaling was determined in MCF-7 cells pretreated with 2-bromopalmitate (2-BP, a palmitoylation inhibitor) or the FASN inhibitor, TVB-3166. Colony formation and migration assays were performed for phenotypic analysis of breast cancer cells in response to IGF-1R and SRPK2 modulation. Results: Inhibition of mTORC1 resulted in an attenuation of FASN expression in both MCF-7 and T47D cells upon IGF-1 exposure. Additionally, inhibition of palmitoylation and/or FASN resulted in a decreased stabilization and activation of IGF-1R in MCF-7 breast cancer cells. Further elucidation of specific mechanisms of post-transcriptional regulation of FASN through SRPK2 as well as effects of SRPK2 and IGF-1R inhibition on breast cancer cell migration and survival are on-going. Conclusion: The results of this study suggest that a potential mechanism of breast cancer progression is through an autoregulatory loop between IGF-1R signaling and FASN activity, which can be effectively limited using the new class of FASN inhibitors. These data support the rationale for continued clinical studies evaluating the efficacy of FASN inhibitors in breast cancers driven, at least in part, by IGF-1 signaling. Citation Format: Bryan McClellan, Brittany Harlow, Christopher Jolly, Linda deGraffenried. A potential FASN -IGF1R signaling loop in breast 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 2455.

Background: Obesity is associated with prostate cancer progression and mortality. Previous studies in our laboratory suggest that obesity drives prostate cancer progression in part by increasing macrophage recruitment and by polarizing macrophages in the tumor microenvironment into a tumor promoting M2/TAM phenotype. Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has been used for several decades in transplant patients, and in the last several years has been shown to be an effective disease suppressor in certain cancer types. Intriguingly, mTOR has been shown to be especially important for M2 polarization and stabilization. Hypothesis: Based upon published data and our preliminary studies, we hypothesize that rapamycin will selectively target obesity-polarized macrophages and will provide a survival benefit to the obese prostate cancer patient. Methods: To address this hypothesis, we used our in vitro model of obesity-induced macrophage polarization that includes two different prostate cancer cell lines, macrophages, and sera from obese and non-obese men. qPCR was used to measure expression levels of markers associated with an M2/TAM phenotype. MTT assays were conducted to measure cell viability, and flow cytometry and Western blot analyses were used to determine cell cycle status and apoptosis. Results: Obese conditions increased expression of M2/TAM markers in macrophages and rapamycin selectively decreased viability of obesity-activated M2/TAMs compared to M1 macrophages. Conclusions: Our in vitro study suggests that obesity promotes a tumor-associated phenotype in macrophages and that the mTOR pathway is involved in the survival of M2/TAM macrophages. This study offers a novel mechanistic approach to treat obese patients with prostate cancer. Citation Format: Gloria C. Galván, Tommy Pham, Brittany Harlow, Christian Johnson, Riddhi Patodia, Duan Quach, Michael A. Liss, Linda A. deGraffenried. Rapamycin selectively targets obesity-polarized macrophages in the prostate tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4004. doi:10.1158/1538-7445.AM2017-4004

Background: Approximately 40% of American women suffer from obesity, and about 1 in 8 women will be diagnosed with breast cancer during her lifetime. The concurrence of these trends has the potential to be particularly detrimental, as obesity confers a worse prognosis for both pre- and postmenopausal breast cancer patients. While the precise mechanisms by which obesity impacts breast cancer prognosis remain to be discovered, evidence suggests that obesity upregulates components of cancer-associated fibroblast (CAF) and senescent cell secretomes, both causally associated with carcinogenesis. Specifically, obesity has been shown to induce expression of the secretory products prostaglandin E2, high mobility group protein B1, interleukin (IL)-6, IL-1 beta, and matrix metalloproteinase-1, the expression of which can be modulated by omega-3 fatty acid-induced signaling. However, studies have yet to determine whether obesity imparts these proinflammatory phenotypes in any individual cell type in the breast tumor microenvironment as well as whether these phenotypes can be modulated by administration of omega-3 fatty acids. This said, we hypothesize that fatty acids modulate the ability of obese conditions to induce a proinflammatory senescent- or CAF-like fibroblast phenotype and thus impact breast cancer progression. Because fibroblasts constitute 80% of the tumor stromal mass, it is of the utmost importance to study obesity-stimulated changes in this cellular compartment and their effects on tumor progression. Methods: Fibroblasts were exposed to sera derived from lean or obese women with and without omega-3 fatty acid supplementation and assessed for changes in expression of proinflammatory genes, including IL-1α, IL-6, and IL-8, as well as nuclear localization of p65, a subunit of the NF-kB transcription factor, which transcribes about 75% of genes related to the senescent secretome. Conditioned media (CM) from these fibroblasts were then applied to breast cancer cells to assess measures of cancer cell aggressiveness. Finally, as an ex vivo study, breast tissue samples from lean and obese women and mice were compared for concentration of proinflammatory, senescent fibroblasts by immunohistochemical analyses. Results: Gene and protein expression analyses demonstrated that obese conditions induced a proinflammatory phenotype in fibroblasts, an effect at least partially modulated by omega-3 fatty acids. These phenotypic changes were not without pathological consequence: CM from obesity-stimulated fibroblasts impacted in vitro measures of breast cancer cell aggressiveness to a greater degree than lean sera-stimulated fibroblasts. Conclusions: While correlative, these data contribute to the identification of a link between obesity and proinflammatory, senescent phenotypes and will ultimately allow for elucidation of the means by which obese conditions confer a worse prognosis for breast cancer patients. In addition, these findings will contribute to our understanding of crosstalk within the tumor microenvironment and inform our pursuit for the development of novel therapeutic targets. Citation Format: Brittany Harlow, Albert Davalos, Andrew Brenner, Christopher Jolly, Stefano Tiziani, Stephen Hursting, Linda deGraffenried. Omega-3 fatty acids modulate the ability of obese conditions to induce of a proinflammatory phenotype in fibroblasts [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-08-02.

Background: Prostate cancer remains the second leading cause of cancer related deaths in men and obesity has been shown to severely impact its prognosis. Previous studies in our laboratory have shown that CD4+ T cells polarized under obese conditions are prostate cancer promoting and that this is due in part to suppression of IFNγ and induction of IL-6 in the T cells. Objective: The goal of this study is to determine the role of IL-10 in mediating the impact of obese conditions on T cell phenotype. Methods: A series of in vitro studies were conducted to evaluate the effects of obesity on T cell function and polarization. Splenic CD4+ T-cells from 10-week-old, male, C57BI/6 mice were isolated and stimulated with anti-CD3+/CD28+ antibodies under obese conditions. Gene expression was measured using qPCR and AKT phosphorylation was measured using Western blot analysis. In vivo T-cell function was compared between obese and non-obese male mice. Deletion assays were used to determine the role of IL-10 in mediating the effects observed on T cell phenotype in response to obese conditions. Results: For both the in vitro and in vivo studies, obese conditions suppressed the expression of Th1 biomarkers (IFNγ, IL-2, and T-bet) and upregulated the expression of Th2 biomarkers (IL-6, IL-10, and GATA-3) in CD4+ T cells. Likewise, obese conditions in vitro and in vivo were associated with decreased AKT phosphorylation. Studies are on-going to determine whether inhibiting the IL-10 pathway restores proper T cell activation under obese conditions. Conclusion: Obesity induces T cell dysfunction characterized by a Th1 to Th2 phenotypic shift, resulting in a pro-tumorigenic phenotype, potentially mediated by paracrine IL-10 signaling. These results are important in understanding the mechanisms by which obesity promotes a more aggressive disease, thereby identifying potential targets for improving outcomes. The results from these studies will provide a better understanding of the role of IL-10 as a novel target for preventing prostate cancer in obese subjects and improving overall survival. Citation Format: Margaret Kappel, Michael Adkison, Alejandra De Angulo, Peyton Travis, Brittany Harlow, Christopher Jolly, Linda deGraffenried. The role of IL-10 in obesity-induced T-cell polarization in the prostate tumor microenvironment [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 2793.