Obesity is a risk factor for postmenopausal estrogen receptor alpha (ERa)-positive (ER þ) breast cancer. Molecular mechanisms underlying factors from plasma that contribute to this risk and how these mechanisms affect ERa signaling have yet to be elucidated. To identify such mechanisms, we performed whole metabolite and protein profiling in plasma samples from women at high risk for breast cancer, which led us to focus on factors that were differentially present in plasma of obese versus nonobese postmenopausal women. These studies, combined with in vitro assays, identified free fatty acids (FFA) as circulating plasma factors that correlated with increased proliferation and aggressiveness in ER þ breast cancer cells. FFAs activated both the ERa and mTOR pathways and rewired metabolism in breast cancer cells. Pathway preferential estrogen-1 (PaPE-1), which targets ERa and mTOR sig-naling, was able to block changes induced by FFA and was more effective in the presence of FFA. Collectively, these data suggest a role for obesity-associated gene and metabolic rewiring in providing new targetable vulnerabilities for ER þ breast cancer in postmenopausal women. Furthermore, they provide a basis for preclinical and clinical trials where the impact of agents that target ERa and mTOR signaling cross-talk would be tested to prevent ER þ breast cancers in obese postmenopausal women. Significance: These findings show that obesity-associated changes in certain blood metabolites rewire metabolic programs in cancer cells, influence mammary epithelial cell tumorigenicity and aggressiveness, and increase breast cancer risk.

Scope We studied the impact of dietary supplementation with licorice root components on diet-induced obesity, fat accumulation and hepatic steatosis in ovariectomized C57BL/6 mice as a menopause model. Materials and Methods We evaluated the molecular and physiological effects of dietary licorice root administered to ovariectomized C57BL/6 mice as root powder (LRP), extracts (LRE) or isolated isoliquiritegenin (ILQ) on reproductive (uterus and mammary gland) and non-reproductive tissues important in regulating metabolism (liver, perigonadal, perirenal, mesenteric and subcutaneous fat). Quantitative outcome measures including body weight, fat distribution (MRI), food consumption, bone density and weight (DXA) and gene expression were assessed by the degree of restoration to the premenopausal health state. We characterized histological (H&E and oil red O staining) and molecular properties (expression of certain disease markers) of these tissues, and correlated these with metabolic phenotype as well as blood levels of bioactives. Conclusions Although LRE and ILQ provided some benefit, LRP was the most effective in reducing body weight gain, overall fat deposition, liver steatosis, and expression of hepatic lipid synthesis genes following ovariectomy. Our data demonstrate that licorice root provided improvement of multiple metabolic parameters under conditions of menopausal low estrogen and high-fat diets without stimulating reproductive tissues.

Most breast cancer deaths occur in women with recurrent, estrogen receptor (ER)-α(+), metastatic tumors. There is a critical need for therapeutic approaches that include novel, targetable mechanism-based strategies by which ERα (+) tumors can be resensitized to endocrine therapies. The objective of this study was to validate a group of nuclear transport genes as potential biomarkers to predict the risk of endocrine therapy failure and to evaluate the inhibition of XPO1, one of these genes as a novel means to enhance the effectiveness of endocrine therapies. Using advanced statistical methods, we found that expression levels of several of nuclear transport genes including XPO1 were associated with poor survival and predicted recurrence of tamoxifen-treated breast tumors in human breast cancer gene expression data sets. In mechanistic studies we showed that the expression of XPO1 determined the cellular localization of the key signaling proteins and the response to tamoxifen. We demonstrated that combined targeting of XPO1 and ERα in several tamoxifen-resistant cell lines and tumor xenografts with the XPO1 inhibitor, Selinexor, and tamoxifen restored tamoxifen sensitivity and prevented recurrence in vivo. The nuclear transport pathways have not previously been implicated in the development of endocrine resistance, and given the need for better strategies for selecting patients to receive endocrine modulatory reagents and improving therapy response of relapsed ERα(+) tumors, our findings show great promise for uncovering the role these pathways play in reducing cancer recurrences.

A majority of breast cancer specific deaths in women with ERα (+) tumors occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to resensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and the nuclear transport protein XPO1 in overcoming endocrine resistance. Selinexor (SEL), an XPO1 antagonist, has been evaluated in multiple late stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies. Our transcriptomics analysis showed that 4-Hydroxytamoxifen (4-OHT), SEL alone or their combination induced differential Akt signaling- and metabolism-associated gene expression profiles. Western blot analysis in endocrine resistant cell lines and xenograft models validated differential Akt phosphorylation. Using the Seahorse metabolic profiler, we showed that ERα-XPO1 targeting changed the metabolic phenotype of TAM-resistant breast cancer cells from an energetic to a quiescent profile. This finding demonstrated that combined targeting of XPO1 and ERα rewired the metabolic pathways and shut down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies to improve therapy response in relapsed ERα (+) tumors, our findings show great promise for uncovering the role that ERα-XPO1 crosstalk plays in reducing cancer recurrences.

Estrogen Receptor α (ERα), a member of the large superfamily of nuclear receptors, exerts profound effects on gene expression, cellular response programs, and phenotypic properties of estrogen target cells. Because of these broad and important actions, ERα is considered the single most important predictor of breast cancer prognosis and is the target of endocrine therapies. The importance of kinases in cancer biology is well known, as increased kinase activity through phosphorylation, mutations or increased expression is often observed in clinical samples and is associated with a poorer prognosis. It is believed that, in therapy-resistant breast cancers, control of cellular physiology switches from ERα nuclear-initiated pathways to extranuclear-activated protein kinase pathways, which enable these cells to adopt a more aggressive phenotype. However, the mechanisms underlying the interplay between ERα and protein kinase pathways in cancer, and the processes by which ERα influences these pathways are poorly understood. Our aim in this study was to elucidate how ERα modulated extranuclear- initiated kinase signaling through alteration of the subcellular localization of ERK5. We have previously shown that upon estradiol treatment ERα elicits nuclear localization of ERK5 and Cofilin in ERα-positive breast cancer cells. This event diminishes ERK5 and Cofilin localization to regions of high actin remodeling in the cytoplasm, thereby possibly accounting for the reduced invasiveness and metastatic potential that is characteristic of many ERα-positive vs. ERα-negative breast cancer cells (PMID: 24505128). In our studies involving an endocrine-resistance cell model, we have found that ERK5 and Cofilin become localized to the cytoplasm as resistance progresses. Using several publicly available tumor gene expression databases, we have identified a signature of genes regulating nucleocytoplasmic transport that are differentially upregulated in more aggressive tumors. Additionally, using RPPA data from TCGA, we find that high expression of the signature genes correlates with higher phosphorylation of key signaling molecules like TAZ, indicative of their mislocalization in invasive breast carcinomas. Low expression of signature genes would successfully predict those patients that would respond to endocrine therapy with Luminal B type tumors that are generally more aggressive and harder to manage clinically. We verified our findings in cell line models by modulating levels and activities of these proteins by overexpression, knock-down and use of small molecule inhibitors. These findings provide insight into how ERα regulates extranuclear initiated kinase signaling by modulating nuclear transport of key kinases in breast cancer. They also suggest that therapeutic targeting of nucleocytoplasmic transport machinery in breast cancer might decrease aggressiveness of breast cancers and increase the efficiency of endocrine therapies by sequestering factors in their proper subcellular localizations where they would contribute to effective anti-estrogenic actions of endocrine targeting agents. Citation Format: Madak-Erdogan Z, Chen-Zhao Y, Wrobel K. Estrogen receptor α-regulation of nucleocytoplasmic transport pathways as modulators of breast cancer therapy effectiveness. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-04.

Currently, around 75% of patients with breast tumors test positive for estrogen receptor alpha (ERa) and are treated with endocrine therapies, such as tamoxifen. One-third of the breast tumors eventually become refractory, reducing the survival rate for affected patients. A combination of alternative endocrine therapies and kinase inhibitors is currently used in such patients. However, after an initial period of therapy response, these tumors relapse in a more aggressive form. Further, the alternative therapies are not optimal in terms of pharmacological properties, are poorly tolerated, and have side-effects that severely decrease quality of life of the patient. Thus, there is a critical need for novel, targetable, mechanism-based therapeutic strategies that 1) re-sensitize ERa (+) tumors to endocrine therapies, and 2) include diagnostic methods to select patients likely to benefit from this approach. Our objective in this study is to validate a group of nuclear transport genes as biomarkers for endocrine resistance, and to evaluate their inhibition as a novel means to enhance the effectiveness of endocrine therapies. Our central hypothesis is that high expression of these genes in ERa (+) tumors serve as a viable biomarker for risk of endocrine therapy failure. We focused on XPO1, the main nuclear export protein, which exports ERK5 from the nucleus to the cytoplasm and we used selinexor (KPT-330), the inhibitor of XPO1, which is already used in clinical trials for solid and hematological cancers (clinicalTrials.gov). Our experiments show that estradiol induces nuclear localization of ERK5, which otherwise would contribute to increased invasiveness and metastatic potential in the cytoplasm. Selinexor increases ERK5 nuclear localization in tamoxifen resistant breast cancer cell lines. Our hypothesis is that sequestering ERK5 in the cell nucleus and blocking its recycle into the nucleus by selinexor is directly associated with the improved transcriptional response to endocrine therapies. The nuclear export pathways have not previously been implicated in the development of endocrine resistance, and given the need for better strategies for selecting patients to receive endocrine reagents and improving therapy response of relapsed ERa(+) tumors, our findings show high and significant promise for uncovering the role of these pathways and demonstrating their use in reducing cancer recurrences. Citation Format: Eylem Kulkoyluoglu, Kinga Wrobel, Yiru Chen Zhao, Karen L. Chen, Kadriye Hieronymi, Jamie Holloway, Yosef Landesman, Tania Ray, Partha S. Ray, Alexander E. Lipka, Rebecca L. Smith, Zeynep Madak Erdogan. Targeting nuclear transport pathways to overcome endocrine resistance and recurrence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1812.

Majority of breast cancer specific deaths in women with ERα (+) tumor occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to resensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and XPO1, a nuclear transport protein in overcoming endocrine resistance. Selinexor (SXR), an XPO1 antagonist, has been evaluated in multiple later stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies. Using Cignalfinder to profile kinase signaling pathways, we found that 4-OH-Tam, SXR or their combination induced differential Akt phosphorylation profiles, changing the localization and activity of the kinase. Since we observed dramatic changes in Akt activity we hypothesized that metabolic profile of breast cancer cells would change in the presence of 4-OH-Tam and SXR. Using Seahorse metabolic profiler and cell viability experiments in limited media conditions we showed that tamoxifen resistant cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine as the mitochondrial fuel source. In order to examine metabolic pathways that might result in the observed phenotype we performed transcriptomics and GC/MS whole metabolite profiling and identified aminoacid metabolism pathways to be upregulated when cells were treated with SXR+4-OH-Tam. We demonstrated that combined targeting of XPO1 and ERα rewires metabolic pathways and shuts down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodelling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα(+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences. Citation Format: Zeynep Madak Erdogan, Eylem Cotul-Kulkoyluoglu, Kinga Wrobel, Sunati Sahoo, Barbara Haley, Yosef Landesman. Combined targeting of estrogen receptor alpha and nuclear transport pathways remodel metabolic pathways to induce autophagy and overcome endocrine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3733.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver diseases in the United States. Simple and benign steatosis can gradually develop into the more serious conditions of nonalcoholic steatohepatitis (NASH) and cirrhosis. Population studies have demonstrated that men and postmenopausal women have higher incidences of NAFLD compared to premenopausal women, suggesting a protective role for estrogens. The decrease in estrogens due to the onset of menopause make postmenopausal women more susceptible to weight gain, fat redistribution to abdominal areas, dyslipidemia, hypertension and insulin resistance, all of which are major hallmarks of metabolic syndrome and are associated with NAFLD. Pathway preferential estrogen 1 (PaPE-1) is a novel estrogen receptor ligand that has been shown to favorably affect metabolic tissues without stimulating reproductive tissues. Though its effects on weight gain and fat accumulation have been previously investigated, its effects on liver transcriptome and plasma metabolites have yet to be determined. We use transcriptomics and metabolomics analysis to characterize the effects of PaPEs in two different mouse models: diet-induced obesity (DIO) and leptin-deficient (ob/ob) mice. PaPE-1 significantly decreased liver weight and lipid accumulation in both DIO and ob/ob models. Integrated pathway analysis using transcriptomics and metabolomics data showed that PaPE-1 treatment lowered genes associated with inflammation, collagen deposition, and fatty acid metabolism. On the other hand, PaPE-1 significantly increased expression of mitochondrial genes, particularly ones associated with electron transport chain and ribosome synthesis suggesting an increase in energy expenditure and mTOR signaling activity. In addition, PaPE-1 treatment restored insulin sensitivity in hepatocytes. Overall, our data show that PaPE-1 provides a metabolic benefit without stimulation of reproductive tissues.