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.

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.