The majority of breast cancers are estrogen receptor (ERα) positive making endocrine therapy a mainstay for these patients. Unfortunately, resistance to endocrine therapy is a common occurrence. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and its expression is commensurate with tumor grade and resistance to numerous therapies. MethodsThe effect of the FASN inhibitor TVB-3166 on ERα expression and cell growth was characterized in tamoxifen resistant cell lines, xenografts, and patient explants. Subcellular localization of ERα was assessed using a combination of immuno uorescence and subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB-3166 with or without the addition of palmitate or BAPTA. ResultsTVB-3166 treatment leads to a marked inhibition of proliferation in tamoxifen-resistant cells compared to the parental cells. Additionally, TVB-3166 signi cantly inhibited tamoxifen-resistant breast tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition signi cantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB-3166 treatment is mediated through the induction of endoplasmic reticulum stress. ConclusionOur preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered.

PurposeThe majority of breast cancers are estrogen receptor (ERα) positive making endocrine therapy a mainstay for these patients. Unfortunately, resistance to endocrine therapy is a common occurrence. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and its expression is commensurate with tumor grade and resistance to numerous therapies. MethodsThe effect of the FASN inhibitor TVB-3166 on ERα expression and cell growth was characterized in tamoxifen resistant cell lines, xenografts, and patient explants. Subcellular localization of ERα was assessed using a combination of immunofluorescence and subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB-3166 with or without the addition of palmitate or BAPTA. ResultsTVB-3166 treatment leads to a marked inhibition of proliferation in tamoxifen-resistant cells compared to the parental cells. Additionally, TVB-3166 significantly inhibited tamoxifen-resistant breast tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB-3166 treatment is mediated through the induction of endoplasmic reticulum stress. ConclusionOur preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered.

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.

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: 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.