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In this study, we used a bioinformatic approach to identify genes whose expression is dysregulated in human prostate cancers. One of the most dramatically downregulated genes identified encodes CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis. Importantly, lower CYP27A1 transcript levels were associated with shorter disease-free survival and higher tumor grade. Loss of CYP27A1 in prostate cancer was confirmed at the protein level by immunostaining for CYP27A1 in annotated tissue microarrays. Restoration of CYP27A1 expression in cells where its gene was silenced attenuated their growth in vitro and in tumor xenografts. Studies performed in vitro revealed that treatment of prostate cancer cells with 27-hydroxycholesterol (27HC), an enzymatic product of CYP27A1, reduced cellular cholesterol content in prostate cancer cell lines by inhibiting the activation of sterol regulatory-element binding protein 2 and downregulating low-density lipoprotein receptor expression. Our findings suggest that CYP27A1 is a critical cellular cholesterol sensor in prostate cells and that dysregulation of the CYP27A1/27HC axis contributes significantly to prostate cancer pathogenesis. Cancer Res; 77(7); 1662–73. ©2017 AACR.

Disruption of systemic homeostasis by either chronic or acute stressors, such as obesity(1) or surgery(2), alters cancer pathogenesis. Cancer patients, particularly those with breast cancer, can be at increased risk for cardiovascular disease due to treatment toxicity and changes in lifestyle behaviors(3–5). While elevated risk and incidence of cardiovascular events in breast cancer is well-established, whether such events impact cancer pathogenesis is not known. Here, we show that myocardial infarction (MI) accelerates breast cancer outgrowth and cancer-specific mortality in mice and humans. In mouse models of breast cancer, MI epigenetically reprogrammed Ly6C(high) monocytes in the bone marrow reservoir to an immunosuppressive phenotype that was maintained at the transcriptional level in monocytes in both the circulation and tumor. In parallel, MI increased circulating Ly6C(high) monocyte levels and recruitment to tumors, and depletion of these cells abrogated MI-induced tumor growth. Furthermore, early-stage breast cancer patients who experienced cardiovascular events after cancer diagnosis had increased risk of recurrence and cancer-specific death. These preclinical and clinical results demonstrate that MI induces alterations to systemic homeostasis, triggering cross-disease communication that accelerates breast cancer.

Seasonally spawning fish rely on a dynamic and complex hormonal interplay to regulate cycles of gonadal development and the regression. Thyroid hormones have been shown to be a key player during gonadal development, and can regulate the activity of a number of essential reproductive hormones. Apoptosis is a vital cellular process that contributes to the hormonal control of gonadal development and regression, but the roles of thyroid hormones on gonadal apoptosis in goldfish have not been explored. The present study examines the role of acute T3 exposure on caspase 3-dependent apoptosis in dispersed goldfish gonadal tissue in vitro. We examined the levels of caspase 3 activity in early, mid, and late recrudescent gonadal tissue after exposure to physiological doses of T3 for up to 24 h. Acute treatment with T3 did not alter basal caspase 3 activity in goldfish gonads in vitro in these reproductive stages. This initial study suggests that transient increases in T3 levels are unlikely to directly contribute to basal caspase 3-dependent apoptosis in the gonadal tissue of goldfish, although we cannot rule out an interaction of T3 with other hormones involved in the control of apoptosis in the testis and ovary.

There is an urgent need for more effective strategies to treat ovarian cancer. Elevated cholesterol levels are associated with a decreased progression-free survival time (PFS) while statins are protective. 27-Hydroxycholesterol (27HC), a primary metabolite of cholesterol, has been shown to modulate the activities of the estrogen receptors (ERs) and liver x receptors (LXRs) providing a potential mechanistic link between cholesterol and ovarian cancer progression. We found that high expression of CYP27A1, the enzyme responsible for the synthesis of 27HC, was associated with decreased PFS, while high expression of CYP7B1, responsible for 27HC catabolism, was associated with increased PFS. However, 27HC decreased the cellular proliferation of various ovarian cancer cell lines in an LXR-dependent manner. Intriguingly, ID8 grafts were unable to effectively establish in CYP27A1−/− mice, indicating involvement of the host environment. Tumors from mice treated with 27HC had altered myeloid cell composition, and cells from the marrow stem cell lineage were found to be responsible for the effects in CYP27A1−/− mice. While inhibition of CYP27A1 or immune checkpoint did not significantly alter tumor size, their combination did, thereby highlighting this axis as a therapeutic target.

Breast cancer is a leading cause of death among women in the US with mortality most often due to metastasis. Bone is a common primary site of metastasis, and it is known that local bone quality dictates the susceptibility of this tissue to colonization. Estrogen-related receptor alpha (ERRα) is a nuclear receptor expressed in a variety of tissues and is involved in development, metabolic regulation, bone physiology, and immune cell differentiation. ERRα is expressed in macrophages, and there is a growing body of literature suggesting that macrophages are involved in both bone physiology as well as metastatic progression. Therefore, we hypothesized that the activity of this receptor within macrophages would have important patho-physiological consequences. Using RAW264.7 cells as a model, we first explored the expression of genes key to macrophage function. RAW264.7 cells polarized into the ‘M1’ state with lipopolysaccharide and IFNγ for 24hrs. Interestingly, when ERRα was overexpressed in these cells, we observed a significant reduction in interleukin 6 (IL-6) and RANKL expression, but an increase in the classic M1 markers, NOS2 and CD82. Conversely, when cells were treated with XCT-790, a small molecule antagonist and degrader of ERRα, IL-6 mRNA was increased, while NOS2 and CD86 were reduced. IL-6 and RANKL are known to be involved in bone resorption, and IL-6 has been implicated in cancer cell migration and the promotion of tumor progression. Furthermore, the production of nitric oxide by NOS2 can impair T cell expansion thus facilitating immune-escape by cancer cells. Thus, we speculated that macrophage activation of ERRα may promote breast cancer metastasis to the bone. We first confirmed that the bone-tropic MDA-MB-231 breast cancer cells migrate towards macrophages. We found that this migration was attenuated when IL-6 was depleted. Cholesterol has recently been identified as an agonist for ERRα. Therefore, we placed mice on a normal or high cholesterol diet and grafted with MDA-MB-231 cells. Contrary to our data indicating that ERRα activation would be protective, mice on a high cholesterol diet developed significantly more bone lesions. Hence, the effects of cholesterol on promoting breast cancer metastasis to the bone could not be explained solely by its ERRα activity. In conclusion, we have found that ERRα functions to attenuate cytokine release and decrease NOS2 expression in M1 polarized macrophages. Although cholesterol has been described as an ERRα agonist, a high cholesterol diet increased metastasis to the bone, contrary to what would be expected based on the ERRα inhibition of IL-6 and NOS2. Our data suggest that further studies are required to better elucidate the role of ERRα, prior to developing this nuclear receptor as a therapeutic target.

Despite the development of various therapeutic strategies, breast cancer persists as the second leading cause of cancer-related death among women in the United States. While endocrine modulation and monoclonal antibody therapy have proved to be indispensable modes of intervention for hormone receptor (HR)-positive and HER-2 positive patients, the triple negative breast cancer (TNBC) patient population do not respond to these therapies. As TNBC is considered one of the most challenging subtypes of breast cancer to treat, there is a significant need for the development of targeted therapeutics. Due to their well-known amenability to small-molecule modulation, we investigated whether any nuclear receptors beyond those that are traditionally studied in breast cancer (e.g. ER, PR, and AR), may represent a novel target in the TNBC patient population. Analysis of clinical data revealed that expression of the orphan nuclear receptor TLX (NR2E1) was positively correlated with relapse-free survival, distant metastasis-free survival, and overall survival in both ER-negative and basal-like breast cancer patients. Therefore, we hypothesized that TLX could influence the pathophysiology of TNBC. To interrogate this hypothesis, we established TNBC cells with stable expression of TLX in order to identify direct regulatory targets, as well as the precise physiological mechanism(s) TLX may be regulating. To date, our work has revealed that TLX inhibits proliferation, slows migration, alters chemosensitivity, and impairs cell cycle progression in TNBC cells. In agreement with these findings, our work has also revealed that TLX is capable of modulating the expression of several genes that are known to regulate the processes of growth, migration, and cell cycle. Taken together, our early work supports our hypothesis, and provides valuable insight into the potential pro-survival function of TLX in TNBC. Ongoing work will continue to probe the mechanisms by which TLX impacts breast cancer biology, and establish whether the growth-inhibitory effects translate to in vivo models. As prior work has demonstrated that TLX’s transcriptional activity can be regulated by both synthetic and natural ligands, the results of our work would provide the foundational data necessary for the development of a TLX-based therapy for a patient population with limited therapeutic options and a poor prognostic outlook.

Breast cancer remains one of the leading causes of cancer mortality in the US. Elevated cholesterol is a major risk factor for breast cancer onset and recurrence, while cholesterol-lowering drugs, such as statins, are associated with a good prognosis. Previous work in murine models showed that cholesterol increases breast cancer metastasis, and the pro-metastatic effects of cholesterol were due to its primary metabolite, 27HC. In our prior work, myeloid cells were found to be required for the pro-metastatic effects of 27HC, but their precise contribution remains unclear. Here we report that 27HC impairs T cell expansion and cytotoxic function through its actions on myeloid cells, including macrophages, in an LXR-dependent manner. Many oxysterols and LXR ligands had similar effects on T cell expansion. Moreover, their ability to induce the LXR target gene ABCA1 was associated with their effectiveness in impairing T cell expansion. Interestingly, the enzyme responsible for the synthesis of 27HC, CYP27A1, is highly expressed in myeloid cells, suggesting that 27HC may have important autocrine or paracrine functions in these cells, a hypothesis supported by our finding that breast cancer metastasis was reduced in mice with a myeloid specific knockout of CYP27A1. Pharmacologic inhibition of CYP27A1 reduced metastatic growth and improved the efficacy of checkpoint inhibitor, anti-PD-L1. RNA sequencing of 27HC-treated macrophages and GSEA analysis provide further mechanistic insight, revealing an enrichment of MYC and NOTCH signaling pathways, both of which are documented pathways involved in tumor-associated macrophages. Taken together, our work suggests that targeting the CYP27A1 axis in myeloid cells may present therapeutic benefits and improve the response rate to immune therapies in breast cancer. Delineating the link between LXR and the known mechanisms of tumor-associated macrophages is important to fully understand 27HC-driven cancer metastasis. This work was supported by grants to ERN from the National Cancer Institute of the National Institutes of Health (R01CA234025) and METAvivor. This abstract is also being presented as PO050. Citation Format: Liqian Ma, Lawrence Wang, Adam T. Nelson, Chaeyeon Han, Sisi He, Madeline A. Henn, Karan Menon, Joy J. Chen, Amy E. Baek, Anna Vardanyan, Sayyed Hamed Shahoei, Sunghee Park, David J. Shapiro, Som G. Nanjappa, Erik R. Nelson. 27-Hydroxycholesterol acts on myeloid immune cells to induce T cell dysfunction, promoting breast cancer progression [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR006.

Vitellogenin (Vtg) is an egg-yolk precursor protein that is synthesized in the liver of oviparous species and taken up from the circulation by the ovary. It is well known that Vtg is induced by circulating estrogens. However, other endocrine factors that regulate the expression of Vtg are less well characterized; factors that might play significant roles, especially in seasonal spawners such as the goldfish which require increased quantities of Vtg for the development of hundreds of follicles. In this regard, thyroid hormones have been shown to cycle with the reproductive season. Therefore, we hypothesized that the thyroid hormones might influence the synthesis of Vtg. Treatment of female goldfish with triiodothyronine (T3) resulted in increased Vtg, an observation that was absent in males. Furthermore, T3 failed to induce Vtg in cultured hepatocytes of either sex. Interestingly however, T3 consistently up-regulated the expression of the estrogen receptor alpha (ERα). The T3 mediated upregulation of ERα requires the presence of both thyroid receptor (TR) α-1 and TRβ. When goldfish or cultured hepatocytes were treated with T3 followed by estradiol, there was a synergistic increase in Vtg, a response which is dependent on the presence of ERα. Therefore, by upregulating ERα, T3 serves to prime the liver to subsequent stimuli from estradiol. This cross-talk likely reveals an important physiologic mechanism by which thyroid hormones, whose circulating levels are high during early gonadal recrudescence, facilitate the production of large amounts of Vtg required for egg development.

// Xiaobin Zheng 1 , Neal Andruska 1,6 , Michael J. Lambrecht 2 , Sisi He 3 , Amadeo Parissenti 4 , Paul J. Hergenrother 2 , Erik R. Nelson 3,5 and David J. Shapiro 1,5,6 1 Department of Biochemistry University of Illinois, Urbana, IL, USA 2 Department of Chemistry, University of Illinois, Urbana, IL, USA 3 Department of Molecular Integrative Physiology, University of Illinois, Urbana, IL, USA 4 Cancer Research Program, Advanced Medical Research Institute of Canada, Sudbury, ON, Canada 5 University of Illinois Cancer Center, Urbana, IL, USA 6 College of Medicine, University of Illinois, Urbana, IL, USA Correspondence to: David J. Shapiro, email: // Keywords : ERα biomodulator, ATP depletion, unfolded protein response (UPR), MDR1/P-glycoprotein/ABCB1, OVCAR-3 ovarian cancer Received : May 02, 2016 Accepted : July 10, 2016 Published : July 24, 2016 Abstract Ovarian cancers often recur and tumors acquire resistance to chemotherapy due to overexpression of the ATP-dependent efflux pump, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1). Nontoxic small molecule inhibitors targeting MDR1 have remained largely elusive. Instead, in a novel application of our recently described estrogen receptor α (ERα) biomodulator, BHPI, we targeted MDR1’s substrate, ATP. BHPI depletes intracellular ATP and nearly blocks MDR1-mediated drug efflux in ovarian cancer cells by inducing toxic hyperactivation of the endoplasmic reticulum stress sensor, the unfolded protein response (UPR). BHPI increased sensitivity of MDR1 overexpressing multidrug resistant OVCAR-3 ovarian cancer cells to killing by paclitaxel by >1,000 fold. BHPI also restored doxorubicin sensitivity in OVCAR-3 cells and in MDR1 overexpressing breast cancer cells. In an orthotopic OVCAR-3 xenograft model, paclitaxel was ineffective and the paclitaxel-treated group was uniquely prone to form large secondary tumors in adjacent tissue. BHPI alone strongly reduced tumor growth. Notably, tumors were undetectable in mice treated with BHPI plus paclitaxel. Compared to control ovarian tumors, after the combination therapy, levels of the plasma ovarian cancer biomarker CA125 were at least several hundred folds lower; moreover, CA125 levels progressively declined to undetectable. Targeting MDR1 through UPR-dependent ATP depletion represents a promising therapeutic strategy.

Obesity leads to an increased risk for type 2 diabetes, heart disease, stroke, and cancer. The causal link between obesity and these pathologies has recently been identified as chronic low-grade systemic inflammation initiated by pro-inflammatory macrophages in visceral adipose tissue. Current medications based on small-molecule drugs yield significant off-target side effects with long-term use, and therefore there is a major need for targeted therapies. Here we report that nanoscale polysaccharides based on biocompatible glucose polymers can efficiently target adipose macrophages in obese mice. We synthesized a series of dextran conjugates with tunable size linked to contrast agents for positron emission tomography, fluorophores for optical microscopy, and anti-inflammatory drugs for therapeutic modulation of macrophage phenotype. We observed that larger conjugates efficiently distribute to visceral adipose tissue and selectively associate with macrophages after regional peritoneal administration. Up to 63% of the injected dose remained in visceral adipose tissue 24 h after administration, resulting in2-fold higher local concentration compared to liver, the dominant site of uptake for most nanomedicines. Furthermore, a single-dose treatment of anti-inflammatory conjugates significantly reduced pro-inflammatory markers in adipose tissue of obese mice. Importantly, all components of these therapeutic agents are approved for clinical use. This work provides a promising nanomaterials-based delivery strategy to inhibit critical factors leading to obesity comorbidities and demonstrates a unique transport mechanism for drug delivery to visceral tissues. This approach may be further applied for high-efficiency targeting of other inflammatory diseases of visceral organs.

As the most common cancer in women, one in eight will develop invasive breast cancer over their lifetime making it the second most common cause of cancer-related death among women. Of the many known risk factors for developing breast cancer, obesity stands out as prominent and modifiable. Interestingly, elevated cholesterol is highly associated with obesity and has emerged as an independent risk factor for breast cancer onset and recurrence. This indicates that cholesterol also contributes to the breast cancer pathogenicity of obesity. This review highlights our current understanding of the mechanisms by which cholesterol impacts breast cancer. Key preclinical studies have been highlighted, including the discussion of homeostatic control of cholesterol levels, signaling by cholesterol metabolites through the estrogen receptors, cholesterol formation of lipid rafts and subsequent signaling, and the potential roles of cholesterol in creating a pro-inflammatory tumor microenvironment. Future directions and avenues for therapeutic exploitation are also considered.

There is an urgent need to develop new therapeutic or lifestyle strategies for treating ovarian cancer due to its high mortality and recurrence rate. In this regard, we found that cholesterol appears to be clinically important for ovarian cancer survival, highlighting it and its metabolism as potential therapeutic targets for improving the lifespan of patients. In a cohort of 153 patients, we found high total cholesterol or high LDL cholesterol was associated with high tumor grade, which in turn was associated with poor survival. Additionally, patients prescribed cholesterol lowering drugs such as statins, cholesterol absorption inhibitors or fiber were associated with significantly improved overall survival. We did not observe significant differences between statins and other cholesterol lowering drugs, indicating the effects observed were most likely due to cholesterol lowering rather than other unintended pharmacologic properties of statins. Cytochrome P450 enzymes catalyze the first steps in cholesterol metabolism pathways. Among them, CYP27A1 is highly expressed in myeloid cells and oxidizes cholesterol into 27-hydroxychoelsterol (27HC), which is the most abundant oxysterol and an endogenous signaling molecule. Analysis of publicly available databases indicated that low tumoral CYP27A1 expression was associated with better progression-free survival and overall survival. Conversely, high tumoral CYP7B1 expression, the enzyme that metabolites 27HC, was associated with improved progression-free survival. Therefore, we hypothesized that CYP27A1/27HC might be mediating the effects of cholesterol on cancer survival. Indeed, we found ovarian tumors failed to grow in CYP27A1−/− mice, eventually completely regressing, while treatment with exogenous 27HC was able to sustain tumor growth in CYP27A1−/− mice, indicating that CYP27A1/27HC are important for ovarian cancer progression. Analysis of tumors and lymphoid tissues suggested that 27HC was associated with compromised immunosurveillance. Specifically, CYP27A1/27HC-axis altered the recruitment of 1) monocytic-MDSCs, an immunosuppressive subtype of myeloid cells, 2) antigen-presenting myeloid cells, and 3) CD4+ T cells. Thus, the CYP27A1/27HC-axis presents an alternative path for cancer associated immunosuppression, offering a potentially novel therapeutic target. We tested the therapeutic utility of targeting this axis and found that a small molecule CYP27A1 inhibitor significantly improved the efficacy of anti-PDL1 checkpoint inhibition in a preclinical model. Collectively, our data suggest that cholesterol/27HC-axis modulates the ovarian tumor microenvironment and promotes cancer progression, revealing a novel therapeutic target for the treatment of ovarian cancer. Supported by: NIH R01CA234025 (E.R.N.), a Cancer Scholars for Translational and Applied Research (C*STAR) Award (S.H.) and NIH T32EB019944 (S.H.). Citation Format: Sisi He, Georgina Cheng, Edward Roy, Marta Spain, Ronald Kimball, Nikolas Snyder, Melina Salgado, Debby Vannoy, Betsy Barnick, Liqian Ma, Varsha Vembar, Anna Vardanyan, Amy Baek, Joanna Burdette, Erik R. Nelson. Cholesterol and its metabolism impact ovarian cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2821.

Breast cancer continues to be the second most common cancer-related mortality among women, providing strong rationale for the development of new therapeutic approaches. Cholesterol and its metabolism have been implicated in the progression of breast cancer. Specifically, elevated circulating cholesterol is a poor prognostic, while patients taking cholesterol-lowering drugs such as statins display increased recurrence-free survival time. In addition to cholesterol, various downstream metabolites play direct roles in promoting breast cancer growth and metastasis. Given the demonstrated importance of cholesterol and its metabolites in breast cancer pathophysiology, we hypothesized that proteins involved in the regulation of cholesterol homeostasis would play a role in cancer progression. A bioinformatics-based screen identified small heterodimer partner (SHP; NR0B2) as being associated with an increased time to recurrence. However, manipulation of this negative regulator of cholesterol metabolism within breast cancer cells did not alter proliferation or migration, suggesting that its protective role is likely conveyed through the tumor microenvironment. Macrophages were found to express SHP, and manipulation of SHP within macrophages resulted in altered expression of molecules associated with antigen presentation. Considering the clinical data indicating a protective role for SHP, it was somewhat paradoxical that its loss within macrophages resulted in an increased expansion of T cells. Upon further investigation, we found that this expansion was skewed towards regulatory T cells (Tregs). While immune therapies have revolutionized the treatment of certain cancers, their utility in breast cancer has been limited, especially outside of triple-negative disease. It has been postulated that this may be due to the highly immune-suppressive activities of certain myeloid and T-cell populations. Thus, reducing Treg infiltration or activity likely represents a rational way to enhance immune therapies. In this regard, SHP-knockout mice bred with the MMTV-PyMT model of mammary cancer displayed significantly enhanced tumor growth compared to SHP-replete mice. Likewise, orthotopic mammary tumor grafts grew at an increased rate in mice where SHP was selectively knocked out in cells of the myeloid lineage (SHPfl/fl;LysMCre), compared to controls. Importantly, treatment with a small-molecule agonist of SHP significantly enhanced the efficacy of anti-PD-L1 therapy in blocking the growth of an orthotopically grafted tumor, as well as in a model of metastatic mammary cancer. Collectively, our data highlight SHP as a modulator of Tregs, a cell population that has thus far been therapeutically intractable. By limiting Treg expansion and thus facilitating an anticancer immune response, SHP may represent a unique way to enhance the efficacy of immune checkpoint blockade. Funding: DOD BCRP BC171214 and NCI R01CA234025 (ERN), Lipstic Labex ANR-11-LABX-0021 (LA), Chateaubriand Fellowship (SHS). Citation Format: Sayyed Hamed Shahoei, Adam T. Nelson, Madeline A. Henn, Ashley E. Mathews, Joy J. Chen, Varsha Vembar, Liqian Ma, Lionel Apetoh, Erik R. Nelson. Macrophage-expressed small heterodimer partner impairs expansion of regulatory T cells and enhances immune checkpoint inhibition [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A93.

Ovarian cancer continues to have a high mortality and recurrence rate. Hence, there is an urgent need to develop new therapeutic or lifestyle strategies. In this regard, epidemiologic studies have implicated elevated cholesterol as a negative prognostic factor. Conversely, ovarian cancer patients prescribed cholesterol-lowering drugs (HMGCoA-R inhibitors; statins) exhibit significantly increased progression-free survival (PFS). In a cohort of 134 patients, we found high LDL cholesterol (≥130 mg/dL) is also associated with high tumor grade at diagnosis. Therefore, cholesterol appears to be clinically important for the progression of ovarian cancer. These observations highlight the potential relevance of previous work implicating the cholesterol metabolite, 27-hydroxychoelsterol (27HC), as a ligand for the estrogen and liver x receptors, providing a putative mechanism for the actions of cholesterol. CYP27A1 encodes the first enzyme in the alternative pathway of cholesterol catabolism, producing 27HC. Analysis of TCGA and other publicly available databases indicated that low CYP27A1 expression was associated with increased PFS. Conversely, high CYP7B1 expression, the enzyme that metabolizes 27HC, was associated with increased PFS. Therefore, we hypothesized that CYP27A1 is involved in ovarian cancer pathophysiology via 27HC signaling. To directly investigate the role of CYP27A1 in ovarian cancer progression, we monitored the growth of tumors grafted into the ovarian bursa area of wild-type and CYP27A1 knockout (KO) mice. Strikingly, we found that ovarian tumors failed to thrive in CYP27A1 KO mice, eventually completely regressing. Treatment with exogenous 27HC was able to sustain tumor growth in CYP27A1 KO mice, indicating that it was indeed a deficiency in 27HC that was mediating the antitumor effects in these mice. Somewhat paradoxically, we found 27HC had growth inhibitory effects on in vitro proliferation of ovarian cancer cells. However, analysis of tumors indicated that 27HC treatment was associated with the enrichment of certain myeloid populations, especially M-MDSCs. M-MDSCs are a myeloid-immune cell type known to be protumorigenic, at least in part through their suppression of cytotoxic CD8+ T cells. This would suggest that inhibition of CYP27A1 might improve the efficacy of immune checkpoint inhibition. Therefore, we tested the therapeutic utility of combining a small-molecule inhibitor of CYP27A1 with anti-PD-L1. Indeed, combining these two approaches significantly decreased ovarian tumor growth in a preclinical model. Collectively, our preliminary data indicate that cholesterol/27HC-axis modulates myeloid cells within tumor microenvironment and promotes cancer progression, revealing a novel therapeutic target for ovarian cancer treatments. Supported by NIH NCI R00CA172357 (E.R.N.), NIH NCI R01CA234025 (E.R.N.), a Cancer Scholars for Translational and Applied Research (C*STAR) Award (S.H.) and NIH T32EB019944 (S.H.). Citation Format: Sisi He, Liqian Ma, Georgina Cheng, Betsy Barnick, Marta Spain, Ronald Kimball, Amy E. Baek, Joanna Burdette, Erik R. Nelson. The impact of cholesterol and its metabolites on ovarian tumor microenvironment and cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B105.

Elevated cholesterol has been identified as a major risk factor of breast cancer onset and recurrence, while cholesterol-lowering drugs are associated with a good prognosis. Previous work in murine models has found that cholesterol increases breast cancer metastasis. However, the prometastatic effects of cholesterol were due to its primary metabolite, 27-hydroxycholesterol (27HC). 27HC is a ligand of both the estrogen receptor (ER) and liver X receptor (LXR). Intriguingly, the prometastatic effects of 27HC require the presence of myeloid-immune cells. This cell type has been implicated in suppressing acquired immunity, allowing cancer cells to escape immune surveillance. Therefore, we hypothesize that 27HC acts on myeloid cells to facilitate immune escape of cancer cells. To characterize the immunomodulatory capacity of 27HC, we cocultured vehicle- or 27HC-treated bone marrow-derived macrophages (BMDMs) with T cells. In support of our hypothesis, we found that 27HC-treated BMDMs inhibited T cell expansion in a dose-dependent manner. Additionally, cocultured BMDMs treated with 27HC resulted in decreased granzyme B expression and secretion in CD8+ cytotoxic T cells. Subsequent experiments showed that T cells activated by 27HC-treated macrophages had significantly reduced ability to eliminate cancer cells. Although 27HC modulated the expression of MHCII and CD80/86, the immunosuppressive effect of 27HC-treated BMDMs persisted even without a direct contact between BMDMs and T cells, suggesting that the T cell inhibition is not limited to cell-cell interaction. To determine the mechanisms by which 27HC-treated BMDMs suppress T-cell activity, we first evaluated the relative contributions of the two receptors known to bind 27HC: the ERs and LXRs. To this end, BMDMs were exposed to pharmacologic antagonists of, or siRNA against the ERs or LXRs. These BMDMs were subsequently cocultured with activated T cells to study their impact on T-cell proliferation. Interestingly, 27HC’s immunosuppressive capacity on preactivated T cells required LXR in BMDMs, but not ER. The involvement of LXR was further validated by surveying a panel of related oxysterols and LXR ligands, as the inhibition of T-cell expansion is directly proportional to their ability to activate LXR. Therefore, our results suggest that 27HC affects the microenvironment of breast cancer by altering the activity of antigen-presenting cells at least in part through the modulation of LXR. This in turn reduces the expansion and function of T cells, ultimately resulting in immune escape and tumor progression. Our ongoing work is aimed at elucidating the 27HC-induced signaling pathway in BMDMs. Collectively, these data provide further support to target the cholesterol-27HC axis as an adjuvant with other immune therapy. This is especially relevant given the prevalence of hypercholesterolemia as well as metastatic breast cancer. This work was supported by the grants from the NCI and DOD-BCRP to ERN (R00CA172357, R01CA234025, BC171214). Citation Format: Liqian Ma, Chaeyeon Han, Lawrence Wang, Amy E. Baek, David J. Shapiro, Som G. Nanjappa, Erik R. Nelson. 27-hydroxycholesterol acts on myeloid cells to inhibit both T cell expansion and cytotoxic activity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B86.

It has become clear that cholesterol metabolism and homeostasis play significant roles in the progression of breast cancer. Specifically, elevated circulating cholesterol is a poor prognostic, while patients taking cholesterol-lowering drugs such as statins display increased recurrence-free survival time. Preclinical and clinical work has established that in addition to cholesterol, various downstream metabolites play direct roles in promoting breast cancer growth and metastasis. Given the demonstrated importance of cholesterol and its metabolites in breast cancer pathophysiology, we hypothesized that proteins involved in the regulation of cholesterol homeostasis would play a role in cancer progression. Therefore, we performed an informatics screen to identify those regulatory proteins associated with breast cancer progression. We focused on nuclear receptors due to their well-defined ligand-binding pocket and thus their proclivity to drug intervention. Our screen revealed that increased expression of Small Heterodimer Partner (SHP; NR0B2) was associated with an increased time to recurrence. However, manipulation of SHP within breast cancer cells did not alter proliferation or migration, suggesting that its protective role is likely conveyed through the tumor microenvironment. Macrophages were found to express SHP, and manipulation of SHP within macrophages resulted in altered expression of molecules associated with antigen presentation. Considering the clinical data indicating a protective role for SHP, it was somewhat paradoxical that its loss within macrophages resulted in an increased expansion of T cells. Upon further investigation, we found that this expansion was skewed towards regulatory T cells (Tregs). On the other hand, overexpression of SHP resulted in decreased expansion of Tregs. The immune-suppressive activity of the resulting Tregs was confirmed in subsequent assays. While immune therapies have revolutionized the treatment of certain cancers, their utility in breast cancer has been limited, especially outside of triple-negative disease. It has been speculated that this may be due to the highly immune-suppressive activities of certain myeloid and T cell populations. Thus, reducing Treg infiltration or activity likely represents a rational way to enhance immune therapies. In this regard, SHP-knockout mice bred with the MMTV-PyMT model of mammary cancer displayed significantly enhanced tumor growth compared to SHP-replete mice. Likewise, orthotopic mammary tumor grafts grew at an increased rate in mice where SHP was selectively knocked out in cells of the myeloid lineage (SHPfl/fl;LysMCre), compared to controls. Importantly, treatment with a small molecule agonist of SHP significantly enhanced the efficacy of anti-PD-L1 therapy in blocking the growth of an orthotopically grafted tumor, as well as in a model of metastatic mammary cancer. Collectively, our data strongly support a role for SHP in reducing the progression of breast cancer by limiting Treg expansion, thereby facilitating an anti-cancer immune response. As this nuclear receptor is amenable to small molecule intervention, SHP may represent a unique way to enhance the efficacy of immune checkpoint blockade. This study was funded in part by awards to ERN from the DOD BCRP (BC171214) and NCI (R01CA234025), to LA from the French National Research Agency Lipstic Labex (ANR-11-LABX-0021), and a STEM Chateaubriand Fellowship to SHS from the Embassy of France in the United States. Citation Format: Erik R. Nelson, Sayyed Hamed Shahoei, Adam T Nelson, Madeline A Henn, Ashley E Mathews, Joy J Chen, Varsha Vembar, Liqian Ma, Lionel Apetoh. The small heterodimer partner in macrophages reduces expansion of regulatory T cells and enhances immune checkpoint inhibition in breast cancer [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 P6-05-01.

Approximately 30% of metastatic breast cancers harbor estrogen receptor α (ERα) mutations associated with resistance to endocrine therapy and reduced survival. Consistent with their constitutive proliferation, T47D and MCF7 cells in which wild-type ERα is replaced by the most common mutations, ERαY537S and ERαD538G, exhibit partially estrogen-independent gene expression. A novel invasion/dissociation/rebinding assay demonstrated that the mutant cells have a higher tendency to dissociate from invasion sites and rebind to a second site. Compared to ERαD538G breast tumors, ERαY537S tumors exhibited a dramatic increase in lung metastasis. Transcriptome analysis showed that the ERαY537S and ERαD538G mutations each elicit a unique gene expression profile. Gene set enrichment analysis showed Myc target pathways are highly induced in mutant cells. Moreover, chromatin immunoprecipitation showed constitutive, fulvestrant-resistant, recruitment of ERα mutants to the Myc enhancer region, resulting in estrogen-independent Myc overexpression in mutant cells and tumors. Knockdown and virus transduction showed Myc is necessary and sufficient for ligand-independent proliferation of the mutant cells but had no effect on metastasis-related phenotypes. Thus, Myc plays a key role in aggressive proliferation-related phenotypes exhibited by breast cancer cells expressing ERα mutations.

Oxysterols are derivatives of cholesterol and an important regulator of cholesterol metabolism, in part due to their role as ligands for nuclear receptors, such as the liver X receptors. Oxysterols are also known to be ligands for the RAR-related orphan receptors, involved in normal T cell differentiation. However, increasing evidence supports a role for oxysterols in the progression of several diseases. Here, we review recent developments in oxysterol research, highlighting the biological functions that oxysterols exert through their target nuclear receptors: the liver X receptors, estrogen receptors, RAR-related orphan receptors and the glucocorticoid receptor. We also bring the regulation of the immune system into the context of interaction between oxysterols and nuclear receptors, discussing the effect of such interaction on the pro-inflammatory function of macrophages and the development of T cells. Finally, we examine the impact that oxysterols have on various disease models, including cancer, Alzheimer's disease and atherosclerosis, stressing the role of nuclear receptors if previously identified. This review underscores the need to consider the multifaceted roles of oxysterols in terms of multiple receptor engagements and selective modulation of these receptors.

Recent studies suggest that exposure to some plasticizers, such as Bisphenol A (BPA), play a role in endocrine/metabolic dispruption and can affect lipid accumulation in adipocytes. Here, we investigated the adipogenic activity and nuclear receptor interactions of four plasticizers approved for the manufacturing of food-contact materials (FCMs) and currently considered safer alternatives. Differentiating 3T3-L1 mouse preadipocytes were exposed to scalar concentrations (0.01–25 μM) of DiNP (Di-iso-nonyl-phthalate), DiDP (Di-iso-decyl-phthalate), DEGDB (Diethylene glycol dibenzoate), or TMCP (Tri-m-cresyl phosphate). Rosiglitazone, a well-known pro-adipogenic peroxisome proliferator activated receptor gamma (PPARγ) agonist, and the plasticizer BPA were included as reference compounds. All concentrations of plasticizers were able to enhance lipid accumulation, with TMCP being the most effective one. Accordingly, when comparing in silico the ligand binding efficiencies to the nuclear receptors PPARγ and retinoid-X-receptor-alpha (RXRα), TMPC displayed the highest affinity to both receptors. Differently from BPA, the four plasticizers were most effective in enhancing lipid accumulation when added in the mid-late phase of differentiation, thus suggesting the involvement of different intracellular signalling pathways. In line with this, TMCP, DiDP, DiNP and DEGDB were able to activate PPARγ in transient transfection assays, while previous studies demonstrated that BPA acts mainly through other nuclear receptors. qRT-PCR studies showed that all plasticizers were able to increase the expression of CCAAT/enhancer binding protein β (Cebpβ) in the early steps of adipogenesis, and the adipogenesis master gene Pparγ2 in the middle phase, with very similar efficacy to that of Rosiglitazone. In addition, TMCP was able to modulate the expression of both Fatty Acid Binding Protein 4/Adipocyte Protein 2 (Fabp4/Ap2) and Lipoprotein Lipase (Lpl) transcripts in the late phase of adipogenesis. DEGDB increased the expression of Lpl only, while the phthalate DiDP did not change the expression of either late-phase marker genes Fabp4 and Lpl. Taken together, our results suggest that exposure to low, environmentally relevant doses of the plasticizers DiNP, DiDP, DEGDB and TMCP increase lipid accumulation in 3T3-L1 adipocytes, an effect likely mediated through activation of PPARγ and interference at different levels with the transcriptional cascade driving adipogenesis.

Background: Few studies have investigated the effects of exercise on modulation of host factors in cancer patients. We investigated the efficacy of chronic aerobic training on multiple host-related effector pathways in patients with solid tumours. Patients and Methods: Paired peripheral blood samples were obtained from 44 patients with solid tumours receiving cytotoxic therapy and synthetic erythropoietin (usual care; n=21) or usual care plus supervised aerobic training (n=23) for 12 weeks. Samples were characterised for changes in immune, cytokine and angiogenic factors, and metabolic intermediates. Aerobic training consisted of three supervised cycle ergometry sessions per week at 60% to 100% of peak oxygen consumption (VO2peak), 30–45 min per session, for 12 weeks following a nonlinear prescription. Results: The between-group delta change in cardiopulmonary function was +4.1 ml kg −1 min−1, favouring aerobic training (P

Estrogen receptors (ERs) mediate the actions of the steroidal estrogens, and are important for the regulation of several physiological and pathophysiological processes, including reproduction, bone physiology, cardiovascular physiology and breast cancer. The unique pharmacology of the ERs allows for certain ligands, such as tamoxifen, to elicit tissue- and context-specific responses, ligands now referred to as selective estrogen receptor modulators (SERMs). Recently, the cholesterol metabolite 27-hydroxychoelsterol (27HC) has been defined as an endogenous SERM, with activities in atherosclerosis, osteoporosis, breast and prostate cancers, and neural degenerative diseases. Since 27HC concentrations closely mirror those of cholesterol, it is possible that 27HC mediates many of the biological effects of cholesterol. This paper provides an overview of ER pharmacology and summarizes the work to date implicating 27HC in various diseases. Wherever possible, we highlight clinical data in support of a role for 27HC in the diseases discussed.

Although significant advances in the treatment of breast cancer have been made, in particular in the use of endocrine therapy, de novo and aquired resistance to therapy, and metastatic recurrence continue to be major clinical problems. Given the high prevalence of breast cancer, new life-style or chemotherapeutic approaches are required. In this regard, cholesterol has emerged as a risk factor for the onset of breast cancer, and elevated cholesterol is associated with a poor prognosis. While treatment with cholesterol lowering medication is not associated with breast cancer risk, it does appear to be protective against recurrence. Importantly, the cholesterol axis represents a potential target for both life-style and pharmacological intervention. This review will outline the clinical and preclinical data supporting a role for cholesterol in breast cancer pathophysiology. Specific focus is given to 27-hydroxycholesterol (27-OHC; (3β,25R)-Cholest-5-ene-3,26-diol)), a primary metabolite of cholesterol that has recently been defined as an endogenous Selective Estrogen Receptor Modulator. Future perspectives and directions are discussed.

Cholesterol is a risk factor for breast cancer although the mechanisms by which this occurs are not well understood. One hypothesis is that dyslipidemia results in increased cholesterol content in cell membranes, thus impacting upon membrane fluidity and subsequent signaling. In addition, studies demonstrate that the metabolite, 27-hydroxycholesterol (27HC), can function as an estrogen, increasing the proliferation of estrogen receptor (ER)-positive breast cancer cells. This was unexpected because 27HC and other oxysterols activate the liver X receptors (LXR), resulting in a reduction of intracellular cholesterol. Resolution of this paradox will require dissection of the molecular mechanisms by which ER and LXR converge in breast cancer cells. Regardless, the observation that 27HC influences breast cancer provides a rationale for strategies that target cholesterol metabolism.

Our understanding of the molecular mechanisms underlying the pharmacological actions of estrogen receptor (ER) ligands has evolved considerably in recent years. Much of this knowledge has come from a detailed dissection of the mechanism(s) of action of the Selective Estrogen Receptor Modulators (SERMs) tamoxifen and raloxifene, drugs whose estrogen receptor (ER) agonist/antagonist properties are influenced by the cell context in which they operate. These studies have revealed that notwithstanding differences in drug pharmacokinetics, the activity of an ER ligand is determined primarily by (a) the impact that a given ligand has on the receptor conformation and (b) the ability of structurally distinct ER-ligand complexes to interact with functionally distinct coregulators. Exploitation of the established relationships between ER structure and activity has led to the development of improved SERMs with more favorable therapeutic properties and of tissue-selective estrogen complexes, drugs in which a SERM and an ER agonist are combined to yield a blended activity that results in distinct clinical profiles. Remarkably, endogenous ligands that exhibit SERM activity have also been identified. One of these ligands, 27-hydroxycholesterol (27HC), has been shown to manifest ER-dependent pathological activities in the cardiovascular system, bone and mammary gland. Whereas the physiological activity of 27HC remains to be determined, its discovery highlights how cells have adopted mechanisms to allow the same receptor ligand complex to manifest different activities in different cells, and also how these processes can be exploited for new drug development.

Obesity and altered lipid metabolism are risk factors for breast cancer in pre- and post-menopausal women. These pathologic relationships have been attributed in part to the impact of cholesterol on the biophysical properties of cell membranes and to the influence of these changes on signaling events initiated at the membrane. However, more recent studies have indicated that the oxysterol 27-hydroxycholesterol (27HC), and not cholesterol per se, may be the primary biochemical link between lipid metabolism and cancer. The enzyme responsible for production of 27HC from cholesterol, CYP27A1, is expressed primarily in the liver and in macrophages. In addition, significantly elevated expression of this enzyme within breast tumors has also been observed. It is believed that 27HC, acting through the liver X receptor in macrophages and possibly other cells, is involved in maintaining organismal cholesterol homeostasis. It has also been shown recently that 27HC is an estrogen receptor agonist in breast cancer cells and that it stimulates the growth and metastasis of tumors in several models of breast cancer. These findings provide the rationale for the clinical evaluation of pharmaceutical approaches that interfere with cholesterol/27HC synthesis as a means to mitigate the impact of cholesterol on breast cancer pathogenesis. Cancer Res; 74(18); 4976–82. ©2014 AACR.

Cholesterol is essential for maintaining membrane fluidity in eukaryotes. Additionally, the synthetic cascade of cholesterol results in precursor molecules important for cellular function such as lipid raft formation and protein prenylation. As such, cholesterol homeostasis is tightly regulated. Interestingly, it is now known that some cholesterol precursors and many metabolites serve as active signaling molecules, binding to different classes of receptors including the nuclear receptors. Furthermore, many cholesterol metabolites or their nuclear receptors have been implicated in the regulation of the immune system in normal physiology and disease. Therefore, in this focused review, cholesterol homeostasis and nuclear receptors involved in this regulation will be discussed, with particular emphasis on how these cascades influence the immune system.

Breast cancer continues to be the most commonly diagnosed cancer among women, with the majority of mortality being associated with the metastatic spread of this disease. In terms of onset, genetic drivers such as mutations in BCR1/2 account for only ~10% of all cases. Thus, environmental factors, including the diet are significant contributors to breast cancer onset and progression. In this regard, elevated circulating cholesterol levels are associated with a poor prognosis, while cholesterol lowering medication (statins) appear protective. We have previously shown that a high cholesterol diet increased both primary tumor growth and metastasis in preclinical models. Interestingly, it was found that the metastatic effects of cholesterol were primarily mediated by its metabolite, 27-hydroxychoelsterol (27HC). In addition to its effects on cancer cells, 27HC also required neutrophils and gamma delta T cells for its pro-metastatic effects. Therefore, we hypothesized that the consumption of foods with high cholesterol, and potentially oxidized cholesterol products, promote breast cancer progression. Bacon is a common food in the US and is prepared by frying in its own fat. We mimicked the preparation process by using a controlled pan-frying procedure. The used oil was collected and processed into food pellets (5% lipid from bacon frying fat, 5% lipid from soybean oil). A control diet was formulated as 5% fat from rendered pork lard, 5% lipid from soybean oil. We also included a diet with no expected cholesterol, where fat content was matched with soybean oil (10%). In order to determine the relative contribution of cholesterol to observed changes in metastatic colonization and outgrown, we also included groups where mice were treated with ezetimibe, a cholesterol uptake inhibitor. Mice were placed on their respective diets for 4 weeks prior to intravenous engraftment with Met1 cancer cells. There were no significant differences in weight gain observed between the experimental groups. 5 weeks post-engraftment, metastatic burden was assessed by ex vivo imaging. Compared to the no cholesterol control diet, there was a significant increase in metastatic burden in the lard group. Intriguingly, the bacon oil diet increased metastatic burden compared to both the lard and no cholesterol groups, and this increase was attenuated when mice were treated with ezetimibe. Ongoing work is aimed at assessing the circulating cholesterol and oxysterol concentrations, and evaluating changes in the metastatic microenvironment between mice on the different diets. Collectively, this study indicates that while cholesterol consumption increases metastatic progression, its preparation prior to consumption can also have significant impacts. Our work provides further rationale for diets low in cholesterol for breast cancer patients. This work was supported by the grants from the NCI, AICR and DOD-BCRP to ERN, and Arnold O. Beckman Research Award to WGH. Citation Format: Cheng Chen, Joy J. Chen, Liqian Ma, William G. Helferich, Erik R. Nelson. Consumption of oil derived from frying bacon increases breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1893.

This abstract was withdrawn by the authors. Citation Format: Katzenellenbogen JA, Min J, Kim SH, Laws MJ, Zhao Y, Ziegler Y, Nelson ER, Shahoei SH, Chu D, Park BH, Katzenellenbogen BS. Withdrawn [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-06.

Cholesterol and Cancer Obesity and high cholesterol levels are associated with an increased risk of breast cancer in post-menopausal women. Nelson et al. (p. 1094 ) found that a specific metabolite of cholesterol, 27-hydroxycholesterol (27HC), promoted tumor growth and metastasis in mouse models of mammary cancer by serving as a partial agonist for the estrogen receptor and the liver X receptor. The most aggressive human breast cancers were found to express the highest level of the enzyme that converts cholesterol to 27HC, suggesting that 27HC produced within tumors (in addition to circulating 27HC) may contribute to tumorigenesis.

Estrogens, steroid hormones critically involved in reproductive processes of vertebrates, signal primarily through their intracellular estrogen receptors (ERs). The ERs belong to a superfamily of nuclear receptors that act as ligand inducible transcription factors. Herein, we review what is known about ER structure, subtypes, mechanism(s) of action and auto-regulation by estrogens. Focus is placed on the ER in fish but comparisons are made to mammals and other vertebrates. Finally, we provide context and a proposed model integrating our knowledge on autoregulation of the receptor and its functions in the liver. Future areas of study are suggested, along with cautions when designing experiments, especially for the detection of endocrine disruptors.

Estrogen therapy and hormone therapy are effective options for the prevention and treatment of osteoporosis, although because of their significant side effect profile, long term use for these applications is not recommended. Whereas SERMs (Selective Estrogen Receptor Modulators) exhibit a more favorable side effect profile, the currently available medicines in this class are substantially less effective in bone than classical estrogens. However, the results of substantial efforts that have gone into defining the mechanisms that underlie the pharmacology of estrogens, antiestrogens and SERMs have informed the development of the next generation of SERMs and have led to the development of TSECs (Tissue Selective Estrogen Complexes), a new class of ER-modulator. Further, the recent determination that the oxysterol 27-hydroxycholesterol functions as an endogenous SERM has highlighted an unexpected link between hypercholesterolemia and bone biology and must be considered in any discussions of ER-pharmacology. This review considers the most recent progress in our understanding of ER pharmacology and how this has and will be translated into new medicines for the treatment and prevention of osteoporosis.

// Erik R. Nelson 1,5 , Shenduo Li 4 , Margaret Kennedy 4 , Sturgis Payne 4 , Kelly Kilibarda 4 , Jeffrey Groth 4 , Michelle Bowie 4 , Edgardo Parilla-Castellar 4 , Gustaaf de Ridder 4 , Paul Kelly Marcom 4 , Matthew Lyes 4 , Bercedis L. Peterson 2 , Michael Cook 3 , Salvatore V. Pizzo 4 , Donald P. McDonnell 5 and Robin E. Bachelder 4 1 Department of Molecular and Integrative Physiology, University of Illinois at Urbana Champaign, Urbana and University of Illinois Cancer Center, University of Illinois at Chicago, Chicago, IL, USA 2 Department of Biostatistics and Bioinformatics, Duke University School of Medicine, Durham, NC, USA 3 Department of Immunology, Duke University School of Medicine, Durham, NC, USA 4 Department of Pathology, Duke University Medical Center, Durham, NC, USA 5 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Correspondence to: Robin E. Bachelder, email: // Keywords : Precursor-N-cadherin, triple-negative breast cancer, invasion, metastasis, chemotherapy resistance Received : August 16, 2016 Accepted : October 07, 2016 Published : October 28, 2016 Abstract Background: Although most triple-negative breast cancer (TNBC) patients initially respond to chemotherapy, residual tumor cells frequently persist and drive recurrent tumor growth. Previous studies from our laboratory and others’ indicate that TNBC is heterogeneous, being composed of chemo-sensitive and chemo-resistant tumor cell subpopulations. In the current work, we studied the invasive behaviors of chemo-resistant TNBC, and sought to identify markers of invasion in chemo-residual TNBC. Methods: The invasive behavior of TNBC tumor cells surviving short-term chemotherapy treatment in vitro was studied using transwell invasion assays and an experimental metastasis model. mRNA expression levels of neural cadherin (N-cadherin), an adhesion molecule that promotes invasion, was assessed by PCR. Expression of N-cadherin and its precursor form (pro-N-cadherin) was assessed by immunoblotting and flow cytometry. Pro-N-cadherin immunohistochemistry was performed on tumors obtained from patients pre- and post- neoadjuvant chemotherapy treatment. Results: TNBC cells surviving short-term chemotherapy treatment exhibited increased invasive behavior and capacity to colonize metastatic sites compared to untreated tumor cells. The invasive behavior of chemo-resistant cells was associated with their increased cell surface expression of precursor N-cadherin (pro-N-cadherin). An antibody specific for the precursor domain of N-cadherin inhibited invasion of chemo-resistant TNBC cells. To begin to validate our findings in humans, we showed that the percent cell surface pro-N-cadherin (+) tumor cells increased in patients post- chemotherapy treatment. Conclusions: TNBC cells surviving short-term chemotherapy treatment are more invasive than bulk tumor cells. Cell surface pro-N-cadherin expression is associated with the invasive and chemo-resistant behaviors of this tumor cell subset. Our findings indicate the importance of future studies determining the value of cell surface pro-N-cadherin as: 1) a biomarker for TNBC recurrence and 2) a therapeutic target for eliminating chemo-residual disease.

Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.

The estrogen receptors (ERs) belong to the nuclear receptor superfamily, and as such act as ligand inducible transcription factors, mediating the effects of estrogens. However, their pharmacology is complex, having the ability to be differentially activated by ligands. Such ligands possess the ability to behave as either ER-agonists or ER-antagonists, depending on the cellular and tissue context, and have been termed Selective Estrogen Receptor Modulators (SERMs). Several SERMs have been identified with clinical relevance such as tamoxifen and raloxifene. Recently, 27-hydroxycholesterol has been characterized as the first identified endogenous SERM leading to the notion that other endogenous SERMs may exist, each having potential pathophysiological functions. This, coupled with the historic pharmaceutical interest as well as growing concern over chemicals in the environment with the ability to behave like SERMs, has increased the demand for assays to detect SERM-like activity. Here, we describe a common, straightforward in vitro assay investigating the induction of classic ER-target genes in MCF7 breast cancer cells, allowing one to identify ligands with SERM-like activity.

The purpose of this study is to investigate the effects of exercise on cancer progression, metastasis, and underlying mechanisms in an orthotopic model of murine prostate cancer. C57BL/6 male mice (6–8 wk of age) were orthotopically injected with transgenic adenocarcinoma of mouse prostate C-1 cells (5 × 105) and randomly assigned to exercise ( n = 28) or a non-intervention control ( n = 31) groups. The exercise group was given voluntary access to a wheel 24 h/day for the duration of the study. Four mice per group were serially killed on days 14, 31, and 36; the remaining 38 mice (exercise, n = 18; control, n = 20) were killed on day 53. Before death, MRI was performed to assess tumor blood perfusion. Primary tumor growth rate was comparable between groups, but expression of prometastatic genes was significantly modulated in exercising animals with a shift toward reduced metastasis. Exercise was associated with increased activity of protein kinases within the MEK/MAPK and PI3K/mTOR signaling cascades with subsequent increased intratumoral protein levels of HIF-1α and VEGF. This was associated with improved tumor vascularization. Multiplex ELISAs revealed distinct reductions in plasma concentrations of several angiogenic cytokines in the exercise group, which was associated with increased expression of angiogenic and metabolic genes in the skeletal muscle. Exercise-induced stabilization of HIF-1α and subsequent upregulation of VEGF was associated with “productive” tumor vascularization with a shift toward suppressed metastasis in an orthotopic model of prostate cancer.

A number of studies have provided evidence for a link between thyroid hormones and physiological or pathophysiological conditions associated with reproduction. Most of the information available is based on clinical observations in human or research in mammals. There are also a number of studies in non-mammalian species, primarily investigating thyroid and reproductive endocrinology in isolation. The findings demonstrate that hyperthyroidism or hypothyroidism are associated with altered fertility due to changes in the levels and activities of hormones of the brain-pituitary-gonadal axis. There appears to be a consistent pattern based on a number of studies in mammalian and non-mammalian species, linking thyroid with reproduction. Results obtained in goldfish suggest that increased levels of thyroid hormones may reduce overall reproductive function. Since thyroid hormones influence metabolism and are known to stimulate growth in most species, it is likely that increased thyroid hormone levels may divert energy from reproduction and promote somatotropic functions. This is particularly important in oviparous species such as fish since energy investment in females during reproductive season is very significant, and increasing thyroid hormone levels after ovulation may be a contributing factor in promoting growth response. Thyroid hormones will likely work in concert with other hormones to influence reproduction in fish and other vertebrates.

Osteoporosis and age-related bone loss are important public health concerns. Therefore, there is a high level of interest in the development of medical interventions and lifestyle changes that reduce the incidence of osteoporosis and age-related bone loss. Decreased bone mineral density is associated with high cholesterol, and patients on statins have increased bone mineral densities, strongly implicating cholesterol as a negative regulator of bone homeostasis. In this study, using both molecular and pharmacological approaches, we have been able to demonstrate that the primary cholesterol metabolite, 27-hydroxycholesterol, through its actions on both estrogen receptors and liver X receptors, decreases osteoblast differentiation and enhances osteoclastogenesis, resulting in increased bone resorbtion in mice. Induction of the short heterodimer partner protein by estrogens in osteoblasts can attenuate the liver X receptor-mediated actions of 27-hydroxycholesterol in bone. These data establish a mechanistic link between cholesterol and bone quality, highlight an unexpected target of estrogens in osteoblasts, and define a signaling axis, the therapeutic exploitation of which is likely to yield novel antiosteoporotic drugs.

The purpose of this study is to investigate the mechanism by which the cholesterol metabolite, 27-hydroxycholesterol (27HC) promotes breast cancer metastasis. Breast cancer is the most commonly diagnosed cancer and among the leading causes of death in women in the United States. Elevated cholesterol is identified as a major risk factor of breast cancer onset and recurrence, while cholesterol-lowering drugs are associated with a good prognosis. Previous work has found that many of the effects of cholesterol on breast cancer progression are due to the actions of its primary metabolite, 27HC. 27HC is a ligand of both the estrogen receptor (ER) and liver X receptor (LXR). Intriguingly, the pro-metastatic effects of 27HC require the presence of myeloid-immune cells. This cell type has been implicated in suppressing acquired immunity, allowing cancer cells to escape immune-surveillance. Therefore, we hypothesize that 27HC suppresses the immune system to promote metastasis. To elucidate the immunomodulatory capacity of 27HC, we co-cultured vehicle- or 27HC-treated bone marrow-derived macrophages (BMDMs) with activated, CFSE-prelabeled T cells. As a readout of immune activation, we measured the resulting proliferation of T cells by flow cytometry. In support of our hypothesis, we found that 27HC-treated BMDMs inhibited T cell expansion in a dose dependent manner. In order to determine the mechanisms by which 27HC-treated BMDMs suppress T cell proliferation, we first evaluated the relative contributions of two receptors known to bind 27HC: the ERs and LXRs. To this end, BMDMs were treated with various combinations of pharmacologic agonists or antagonists of the ERs or LXRs. These treated BMDMs were subsequently co-cultured with activated T cells to study their impact on T cell proliferation. Interestingly, a combination treatment of suboptimal doses of an ER antagonist and LXR agonist moderately reduced T cell expansion, similar to 27HC. Therefore, our results suggest that 27HC affects the microenvironment of breast cancer by altering the activity of antigen-presenting cells through the modulation of the ER and LXR. This in turn reduces the expansion of T cell function, ultimately resulting in tumor progression. Our ongoing work is aimed at further elucidating the crosstalk between the ERs and LXRs and identifying the downstream targets of 27HC involved in mediating this immune-suppressive phenotype. Our work is of considerable importance given the prevalence of metastatic breast cancer and that the cholesterol axis is highly amenable to therapeutic targeting. This work was supported by the National Cancer Institute of the National Institutes of Health (R00CA172357, E.R.N.). Citation Format: Liqian Ma, Amy E. Baek, Erik R. Nelson. Mechanisms by which 27-hydroxycholesterol promotes breast cancer metastasis [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 2133.

Ovarian cancer continues to have a high mortality and recurrence rate. Hence, there is an urgent need to develop new therapeutic or lifestyle strategies. In this regard, epidemiological studies have implicated elevated cholesterol as a negative prognostic factor. Conversely, ovarian cancer patients prescribed cholesterol lowering drugs (HMGCoA-R inhibitors; statins) exhibit significantly increased progression free survival (PFS). Therefore, cholesterol appears to be clinically important for the progression of ovarian cancer. Considering the potential mechanisms by which cholesterol impacts ovarian cancer progression, it was noteworthy that CYP27A1 encodes the first enzyme in the alternative pathway of cholesterol catabolism, producing 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol that has recently been shown to have the capacity to activate the estrogen receptors and liver x receptors. Our bioinformatics analysis of human tumoral mRNA expression indicated that low CYP27A1 expression was associated with increased PFS. Conversely, high CYP7B1 expression, the enzyme that metabolites 27HC, was associated with increased PFS. Therefore, we hypothesized that CYP27A1 is involved in ovarian cancer pathophysiology via 27HC signaling. To directly investigate the role of CYP27A1 in ovarian cancer progression, we monitored the growth of tumors grafted into the ovarian bursa of wildtype and CYP27A1 knockout (KO) mice. Strikingly, we found that ovarian tumors failed to thrive in CYP27A1 KO mice, eventually completely regressing. Importantly, treatment with exogenous 27HC was able to sustain tumor growth in CYP27A1 KO mice. In exploring the potential mechanisms by which this occurs, we found 27HC had very little effect on ovarian cancer cell proliferation in vitro, however, was associated with the enrichment of certain myeloid populations within tumors. Specifically, treatment with exogenous 27HC significantly enhanced the infiltration of M-MDSCs, while significantly fewer M-MDSCs were found in tumors from CYP27A KO mice. M-MDSCs are a myeloid-immune cell type known to be pro-tumorigenic, at least in part through their suppression of cytotoxic CD8+ T cells. This would suggest that inhibition of CYP27A1 might improve the efficacy of immune checkpoint inhibition. Therefore, we tested the therapeutic utility of combining a small molecule inhibitor of CYP27A1 with anti-PD-L1. Indeed, combining these two approaches significantly decreased ovarian tumor growth in a preclinical model. Collectively, our preliminary data strongly indicate that CY27A1 expression is critical for sustaining ovarian cancer progression and is a viable target in combination with checkpoint inhibition. This work was supported by the NIH R00CA172357 (E.R.N.), a Cancer Scholars for Translational and Applied Research (C*STAR) Award (S.H.) and NIH T32EB019944 (S.H.). Citation Format: Sisi He, Amy E. Baek, Liqian Ma, Joanna Burdette, Erik R. Nelson. Host CYP27A1 expression is essential for ovarian cancer progression [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 199.

With upwards of 90% of mortalities associated with breast cancer being attributed to metastasis, there is an urgent need to better understand what drives this stage of disease. In this regard, it has been reported that an elevated concentration of circulating cholesterol is an independent risk factor for breast cancer recurrence. Supporting the relationship between cholesterol and breast cancer progression, the use of cholesterol lowering drugs (statins) has been demonstrated to improve recurrence-free survival. These clinical observations suggest that cholesterol influences the metastatic progression of breast cancer. Therefore, in this study we directly tested the impact of cholesterol on breast cancer metastasis, and interrogated the downstream mechanisms by which it may be doing so. In strong support of our hypothesis, increased metastasis was observed when mice were placed on a high cholesterol diet. These findings highlighted recent work by us and others demonstrating that the primary metabolite, 27-hydroxycholesterol (27HC), was able to modulate the activities of two hormone receptors: the estrogen and the liver X receptors. Thus, we pursued a series of experiments to determine whether 27HC was responsible for the metastatic actions of cholesterol. 27HC is synthesized from cholesterol by the enzyme CYP27A1. The genetic ablation of CYP27A1 completely attenuated the effects of a high cholesterol diet, implicating 27HC as a primary mediator of cholesterol. Treatment with exogenous 27HC robustly increased metastasis. Importantly, genetic or pharmacologic inhibition of CYP27A1 also reduced the basal metastatic burden, indicating that this enzyme may be a suitable target for the prevention and/or treatment of metastatic breast cancer. Intriguingly, we found that the pro-metastatic effects of 27HC also required the presence of myeloid cells, which was demonstrated by their ablation using clodronate-loaded liposomes. In the absence of myeloid cells, the ability of 27HC to promote metastasis was dramatically reduced. Whilst interrogating the distal metastatic site, we found that this oxysterol enriched Ly6G+/CD11b+ polymorphonuclear-neutrophils (PMNs) and γδ T cells. The induction of metastasis by 27HC was lost in models where either PMNs or γδ T cells were ablated. We further demonstrate that 27HC treatment results in a decrease in cytotoxic CD8+ T cells, suggesting that the net result of the 27HC enriched γδ T cells and PMNs is local immune suppression. Collectively, our results demonstrate that cholesterol increases metastasis via the actions of its metabolite 27HC, which exerts its effects through γδ T cells and PMNs to suppress acquired immunity. Since 27HC acts on the host environment to promote breast cancer progression, these results strongly support the immediate translational potential of targeting the 27HC pathway for the prevention and treatment of metastasis. Citation Format: Amy E. Baek, Sisi He, Hannah B. McDowell, Erik R. Nelson. The cholesterol metabolite 27-hydroxycholesterol promotes breast cancer progression by affecting immune responses [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 997.

In 1924, Otto Warburg first postulated that cancer was caused by altered metabolism, specifically the reliance of cancer cells on glycolysis. Since then, this altered metabolism has been recognized as a "hallmark of cancer," with numerous studies supporting this. It is now clear that pathologies resulting in perturbed homeostasis at the level of the organism, such as obesity, diabetes, and hypercholesterolemia, are also associated with tumor progression. Since aerobic exercise is known to alter systemic homeostasis, we reasoned that it may also impact tumor progression. In this regard, emerging epidemiologic evidence indicates an inverse correlation between exercise and the onset of many forms of cancer, including breast cancer. Intriguingly, several studies have found that self-reported physical activity is associated with decreased recurrence. We have now directly tested the impact of exercise on the progression of breast cancer, using several murine models of mammary cancer, finding that exercise significantly reduces tumor growth and metastasis. Subsequent work has been focused on delineating the molecular mechanisms by which exercise hinders the progression of breast cancer. We will discuss these results and present our current understanding of how exercise influences tumor pathophysiology. Collectively, our work suggests that aerobic exercise may offer an effective complementary adjunct therapy with current standard of care. Citation Format: Erik R. Nelson. The impact of aerobic exercise on breast cancer progression [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 SY29-03.

Mutations of the FLT3 receptor tyrosine kinase consisting of internal tandem duplications (ITD) have been detected in blasts from 20% to 30% of patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. FLT3/ITD results in constitutive autophosphorylation of the receptor and factor-independent survival in leukemia cell lines. The C-28 methyl ester of the oleane triterpenoid (CDDO-Me) is a multifunctional molecule that induces apoptosis of human myeloid leukemia cells. Here, we report that CDDO-Me blocks targeting of NFκB to the nucleus by inhibiting IκB kinase β–mediated phosphorylation of IκBα. Moreover, CDDO-Me blocked constitutive activation of the signal transducer and activator of transcription 3. We report the potent and selective antiproliferative effects of CDDO-Me on FLT3/ITD-positive myeloid leukemia cell lines and primary AML cells. The present studies show that CDDO-Me treatment results in caspase-3–mediated induction of apoptosis of FLT3/ITD-expressing cells and its antiproliferative effects are synergistic with PKC412, a FLT3-tyrosine kinase inhibitor currently in clinical trials. Taken together, our studies indicate that CDDO-Me greatly enhanced the efficacy of the FLT3 inhibitor PKC412, suggesting that combining two separate pathway inhibitors might be a viable therapeutic strategy for AML associated with a FLT3/ITD mutation. Mol Cancer Res; 8(7); 986–93. ©2010 AACR.

Estrogens work by binding to and activating specific estrogen receptors (ERs). Although mammals have two major nuclear ER subtypes (ERalpha and ERbeta), three subtypes have been shown in teleost fish (ERalpha, ERbeta-I, and ERbeta-II). 17beta-Estradiol stimulates the production of an egg yolk precursor protein (vitellogenin) in the liver of oviparous species, including the goldfish. However, the functional involvement of the ER subtypes in this process is not fully understood. Here, using primary goldfish hepatocytes, we test the hypothesis that all three ER subtypes are functionally involved in the liver of goldfish by using RNA interference to specifically knock-down the different ER subtypes. The results suggest that ERalpha is induced by estradiol through activation of the ERbeta subtypes. This induction serves to sensitize the liver to further stimulation by estradiol. The knock-down results were supported by use of ER subtype specific antagonists. Sensitization by up-regulation of ERalpha is likely to be important for seasonal spawners such as goldfish, to bring about a change from somatic growth to reproductive development, and vitellogenesis. The novel data presented in this study provide strong support for the hypothesis that the goldfish ER subtypes play functional roles in the regulation of vitellogenin and ERalpha and provide a framework for the better understanding of ER signaling in fish and other vertebrates.

Thyroid hormones are important regulators of vertebrate growth and development, and are under the control of the hypothalamic-pituitary-thyroid axis. Nuclear thyroid receptors (TRs), which act as inducible transcription factors, mediate cellular functions of thyroid hormones. The molecular structure of several subtypes of TRs have been elucidated in vertebrate species, including N-terminal truncations as well as C-terminal variations in the domain responsible for binding hormone. In this paper, we review current information on the thyroid receptors studied in the vertebrate species with emphasis on recent findings in goldfish concerning functional significance of the thyroid receptor subtypes.

While considerable information is available on the physiological effects of estrogen, much less is known about the regulation of estrogen receptor (ER) subtypes, particularly in non-mammalian vertebrates. Using goldfish as primary experimental model, we investigated sex- and tissue-specific homologous regulation of ER subtypes (ERα, ERβI, and ERβII) by estradiol in vivo, in the liver and gonads. Treatment with estradiol, significantly upregulated transcript levels for all three types of ERs (ERα, ERβI, and ERβII) in the goldfish ovary and testis. In the goldfish liver, treatment with estradiol significantly increased ERα, ERβI transcript levels without affecting ERβII. In all cases increased ER transcript level was correlated with increased ER protein level determined by Western blot analysis, although we are not able to distinguish between ER subtypes. The results provide strong support for the hypothesis that homologous regulation of ERs is tissue- and gender-specific, and may be a mechanism for estrogen-mediated regulation of reproduction in goldfish. Mol. Reprod. Dev. 74: 1105–1112, 2007. © 2007 Wiley-Liss, Inc.

The estrogen receptors (ERs) belong to the nuclear receptor superfamily, and as such act as ligand inducible transcription factors, mediating the effects of estrogens. However, their pharmacology is complex, having the ability to be differentially activated by ligands. Such ligands possess the ability to behave as either ER-agonists or ER-antagonists, depending on the cellular and tissue context, and have been termed Selective Estrogen Receptor Modulators (SERMs). Several SERMs have been identified with clinical relevance such as tamoxifen and raloxifene. Recently, 27-hydroxycholesterol has been characterized as the first identified endogenous SERM leading to the notion that other endogenous SERMs may exist, each having potential pathophysiological functions. This, coupled with the historic pharmaceutical interest as well as growing concern over chemicals in the environment with the ability to behave like SERMs, has increased the demand for assays to detect SERM-like activity. Here, we describe a common, straightforward in vitro assay investigating the induction of classic ER-target genes in MCF7 breast cancer cells, allowing one to identify ligands with SERM-like activity.

Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains a first-line treatment for estrogen receptor 1 (ESR1) positive breast cancer. However, tumor resistance limits the duration of response. The clinical efficacy of fulvestrant, a selective ER degrader (SERD) that triggers receptor degradation, has confirmed that ESR1 often remains engaged in endocrine therapy resistant cancers. Recently developed, selective ER modulators (SERMs)/SERD hybrids (SSHs) that facilitate ESR1 degradation in breast cancer cells and reproductive tissues have been advanced as an alternative treatment for advanced breast cancer, particularly in the metastatic setting. RAD1901 is one SSH currently being evaluated clinically that is unique among ESR1 modulators in that it readily enters the brain, a common site of breast cancer metastasis. In this study, RAD1901 inhibited estrogen activation of ESR1in vitroandin vivo, inhibited estrogen-dependent breast cancer cell proliferation and xenograft tumor growth, and mediated dose-dependent downregulation of ESR1 protein. However, doses of RAD1901 insufficient to induce ESR1 degradation were shown to result in the activation of ESR1 target genes and in the stimulation of xenograft tumor growth. RAD1901 is an SSH that exhibits complex pharmacology in breast cancer models, having dose-dependent agonist/antagonist activity displayed in a tissue-selective manner. It remains unclear how this unique pharmacology will impact the utility of RAD1901 for breast cancer treatment. However, being the only SERD currently known to access the brain, RAD1901 merits evaluation as a targeted therapy for the treatment of breast cancer brain metastases.