A novel group of genes that cause endocrine resistance in breast cancer identified by dynamic gene expression analysis.

Breast cancer (BC) is the most common type of cancer diagnosed in women. Among female cancer deaths, BC is the second leading cause of death worldwide. For estrogen receptor-positive (ER-positive) breast cancers, endocrine therapy is an effective therapeutic approach. However, in many cases, an ER-positive tumor becomes unresponsive to endocrine therapy, and tumor regrowth occurs after treatment. While some genetic mutations contribute to resistance in some patients, the underlying causes of resistance to endocrine therapy are mostly undetermined. In this study, we utilized a recently developed statistical approach to investigate the dynamic behavior of gene expression during the development of endocrine resistance and identified a novel group of genes whose time course expression significantly change during cell modelling of endocrine resistant BC development. Expression of a subset of these genes was also differentially expressed in microarray analysis of endocrine-resistant and endocrine-sensitive tumor samples. Surprisingly, a subset of those genes was also differentially genes expressed in triple-negative breast cancer (TNBC) as compared with ER-positive BC. The findings suggest shared genetic mechanisms may underlie the development of endocrine resistant BC and TNBC. Our findings identify 34 novel genes for further study as potential therapeutic targets for treatment of endocrine-resistant BC and TNBC.

A Novel Mouse Model to Analyze Non-Genomic ERα Physiological Actions.

Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents.

Neutralizing Aptamers Block S/RBD-ACE2 Interactions and Prevent Host Cell Infection.

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (K D≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.

Neutralizing Aptamers Block S/RBD-ACE2 Interactions and Prevent Host Cell Infection.

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (KD ≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50 ≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.

27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.

Obesity and Cancer: 27-Hydroxycholesterol, the Missing Link.

Obesity is currently affecting more than 40% of the Americans, and if it progresses with this rate, soon one out of two Americans will be obese. Obesity is an important risk factor for several disorders including cardiovascular disease, the first cause of death in the United States. Cancer follows as the second deadliest disease, and a link between obesity and cancer has been suggested. However, it is very hard to establish an exact connection between obesity and cancers due to the multifactorial nature of obesity. Hypercholesterolemia is a comorbidity of obesity and also linked to several cancers. Recently a cholesterol metabolite 27-hydroxycholesterol (27HC) was found to be an endogenous selective estrogen receptor modulator (SERM), which opened new doors toward several interesting studies on the role of this molecule in biological disorders. It is speculated that 27HC might be the missing link in the obesity and cancer chain. Here, we explored the effects of 27-hydroxycholesterol on obesity and cancers with a focus on the SERM capacity of 27HC.

Vascular Protection by Exercise in Obesity: Inflammasome-associated Mechanisms.

PURPOSE: The nodlike receptor family pyrin domain containing 3 (NLRP3) inflammasome is a critical player in vascular pathology as it regulates caspase-1-mediated interleukin (IL)-1ß processing. Physical activity ameliorates obesity-induced inflammation and vascular dysfunction, but the mechanisms responsible for these positive changes are incompletely understood. Here, the protective effect of physical activity on the inflammasome-associated vascular dysfunction in obesity and its putative mechanisms were investigated. METHODS: Mice were fed a control low-fat diet (LFD) or a high-fat diet (HFD; 45% of calories from fat) and provided with running wheel access (LF-RUN or HF-RUN) or denied wheel access for our sedentary condition (LF-SED or HF-SED). The NLRP3 inflammasome-associated pathway, including NLRP3, caspase-1, and IL-1ß, in mice aorta was examined by RT-qPCR and FLICA and DAB staining. The protein expression of zonula occluden-1 (ZO-1), ZO-2, adiponectin (APN), and adiponectin receptor 1 (AdipoR1) in aortic endothelial cells was determined by immunofluorescence double staining. Intracellular reactive oxidative stress and nitric oxide (NO) production were monitored with fluorescence probes, dihydroethidium, and diaminofluorecein. RESULTS: HFD increased caspase-1 and IL-1ß at mRNA and protein levels in endothelial cells of the aorta, and this was attenuated by voluntary running. HFD decreased ZO-1 and ZO-2 expression and reduced APN and AdipoR1 signaling; these were restored by running. The elevated intracellular superoxide (O2) production observed in HF-SED was ameliorated in HF-RUN. Finally, HF-RUN improved NO production in the aorta compared with HF-SED. CONCLUSIONS: Our findings suggest that voluntary running ameliorates mechanisms associated with vascular dysfunction by suppressing NLRP3 inflammasome, improving NO production, and reducing oxidative stress. Such benefits of physical activity may be, at least in part, associated with APN-AdipoR1 signaling and tight junction protein expression.

Gene expression dynamic analysis reveals co-activation of Sonic Hedgehog and epidermal growth factor followed by dynamic silencing.

Aberrant activation of the Sonic Hedgehog (SHH) gene is observed in various cancers. Previous studies have shown a "cross-talk" effect between the canonical Hedgehog signaling pathway and the Epidermal Growth Factor (EGF) pathway when SHH is active in the presence of EGF. However, the precise mechanism of the cross-talk effect on the entire gene population has not been investigated. Here, we re-analyzed publicly available data to study how SHH and EGF cooperate to affect the dynamic activity of the gene population. We used genome dynamic analysis to explore the expression profiles under different conditions in a human medulloblastoma cell line. Ordinary differential equations, equipped with solid statistical and computational tools, were exploited to extract the information hidden in the dynamic behavior of the gene population. Our results revealed that EGF stimulation plays a dominant role, overshadowing most of the SHH effects. We also identified cross-talk genes that exhibited expression profiles dissimilar to that seen under SHH or EGF stimulation alone. These unique cross-talk patterns were validated in a cell culture model. These cross-talk genes identified here may serve as valuable markers to study or test for EGF co-stimulatory effects in an SHH+ environment. Furthermore, these cross-talk genes may play roles in cancer progression, thus they may be further explored as cancer treatment targets.

Contribution of transcription factor EB to adipoRon-induced inhibition of arterial smooth muscle cell proliferation and migration.

Abnormal vascular smooth muscle cell (SMC) dedifferentiation with increased proliferation and migration during pathological vascular remodeling is associated with vascular disorders, such as atherosclerosis and in-stent restenosis. AdipoRon, a selective agonist of adiponectin receptor, has been shown to protect against vascular remodeling by preventing SMC dedifferentiation. However, the molecular mechanisms that mediate adipoRon-induced SMC differentiation are not well understood. The present study aimed to elucidate the role of transcription factor EB (TFEB), a master regulator of autophagy, in mediating adipoRon's effect on SMCs. In cultured arterial SMCs, adipoRon dose-dependently increased TFEB activation, which is accompanied by upregulated transcription of genes involved in autophagy pathway and enhanced autophagic flux. In parallel, adipoRon suppressed serum-induced cell proliferation and caused cell cycle arrest. Moreover, adipoRon inhibited SMC migration as characterized by wound-healing retardation, F-actin reorganization, and matrix metalloproteinase-9 downregulation. These inhibitory effects of adipoRon on proliferation and migration were attenuated by TFEB gene silencing. Mechanistically, activation of TFEB by adipoRon is dependent on intracellular calcium, but it is not associated with changes in AMPK, ERK1/2, Akt, or molecular target of rapamycin complex 1 activation. Using ex vivo aortic explants, we demonstrated that adipoRon inhibited sprouts that had outgrown from aortic rings, whereas lentiviral TFEB shRNA transduction significantly reversed this effect of adipoRon on aortic rings. Taken together, our results indicate that adipoRon activates TFEB signaling that helps maintain the quiescent and differentiated status of arterial SMCs, preventing abnormal SMC dedifferentiation. This study provides novel mechanistic insights into understanding the therapeutic effects of adipoRon on TFEB signaling and pathological vascular remodeling.

27-Hydroxycholesterol Promotes Adiposity and Mimics Adipogenic Diet-Induced Inflammatory Signaling.

27-Hydroxycholesterol (27HC) is an abundant cholesterol metabolite and has detrimental effects on the cardiovascular system, whereas its impact on adiposity is not well known. In this study, we found that elevations in 27HC cause increased body weight gain in mice fed a high-fat/high-cholesterol diet in an estrogen receptor α-dependent manner. Regardless of diet type, body fat mass was increased by 27HC without changes in food intake or fat absorption. 27HC did not alter energy expenditure in mice fed a normal chow diet and increased visceral white adipose mass by inducing hyperplasia but not hypertrophy. Although 27HC did not augment adipocyte terminal differentiation, it increased the adipose cell population that differentiates to mature adipocytes. RNA sequencing analysis revealed that 27HC treatment of mice fed a normal chow diet induces inflammatory gene sets similar to those seen after high-fat/high-cholesterol diet feeding, whereas there was no overlap in inflammatory gene expression among any other 27HC administration/diet change combination. Histological analysis showed that 27HC treatment increased the number of total and M1-type macrophages in white adipose tissues. Thus, 27HC promotes adiposity by directly affecting white adipose tissues and by increasing adipose inflammatory responses. Lowering serum 27HC levels may lead to an approach targeting cholesterol to prevent diet-induced obesity.

Tricyclic antidepressant amitriptyline inhibits autophagic flux and prevents tube formation in vascular endothelial cells.

Amitriptyline is a tricyclic antidepressant and an inhibitor of lysosomal acid sphingomyelinase (ASM). Amitriptyline is well known for its cardiovascular side effects and toxicity in psychiatric patients. However, the mechanisms underlying the cardiovascular side effects of amitriptyline remain largely undefined. This study aimed to determine the effects of amitriptyline on angiogenic capability of vascular endothelial cells in physiological settings and identify its mechanism of action. The ex vivo aortic ring angiogenesis and in vitro-cultured endothelial cell tube formation assay were used to assess the effects of amitriptyline on endothelial angiogenic capability. It was demonstrated that amitriptyline impaired the angiogenesis of aortic rings, which was similar to that found in aortic rings with haploinsufficiency of the ASM gene. In cultured mouse microvascular endothelial cells (MVECs), amitriptyline impaired the proliferation and tube formation under basal condition, which were accompanied by attenuated angiogenic signalling pathways such as endothelial nitric oxide synthase, Akt and Erk1/2 pathways. Mechanistically, amitriptyline inhibited autophagic flux without affecting autophagosome biogenesis at basal condition. ASM gene silencing or autophagy inhibition mimics the inhibitory effects of amitriptyline on endothelial cell proliferation and tube formation. Collectively, our data suggest that amitriptyline inhibits endothelial cell proliferation and angiogenesis via blockade of ASM-autophagic flux axis. It is implicated that the cardiovascular side effects of amitriptyline may be associated with its inhibitory action on physiological angiogenesis.

Estrogen Receptor Beta-Mediated Modulation of Lung Cancer Cell Proliferation by 27-Hydroxycholesterol.

27-hydroxycholesterol (27HC) is an abundant cholesterol metabolite in human circulation and promotes breast cancer cell proliferation. Although lung is one of the organs that contain high levels of 27HC, the role of 27HC in lung is unknown. In this study, we found that 27HC promotes lung cancer cell proliferation in an estrogen receptor ß (ERß)-dependent manner. The expression of 27HC-generating enzyme CYP27A1 is higher in lung cancer cells than in normal lung cells. Treatment with 27HC increased cell proliferation in ERß-positive lung cancer cells, but not in ERα-positive or ER-negative cells. The effect on cell proliferation is specific to 27HC and another oxysterol, 25-hydroxycholesterol that has a similar oxysterol structure with 27HC. Moreover, among ligands for nuclear receptors tested, only estrogen had the proliferative effect, and the effect by 27HC and estrogen was inhibited by ERß-specific, but not ERα-specific, inhibitors. In addition, the effect by 27HC was not affected by membrane-bound estrogen receptor GPR30. Interestingly, despite the high expression of CYP27A1, endogenously produced 27HC was not the major contributor of the 27HC-induced cell proliferation. Using kinase inhibitors, we found that the effect by 27HC was mediated by the PI3K-Akt signaling pathway. These results suggest that 27HC promotes lung cancer cell proliferation via ERß and PI3K-Akt signaling. Thus, lowering 27HC levels may lead to a novel approach for the treatment of lung cancer.

Physical activity protects NLRP3 inflammasome-associated coronary vascular dysfunction in obese mice.

Activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome mediates the release of pro-inflammatory cytokine interleukin (IL)-1ß and thereby plays a pivotal role in the inflammatory response in vascular pathology. An active lifestyle has beneficial effects on inflammation-associated vascular dysfunction in obesity. However, it remains unclear how physical activity regulates NLRP3 inflammasome-mediated vascular dysfunction in obesity. Therefore, we explored the protective effect of physical activity on NLRP3 inflammasome-associated vascular dysfunction in mouse hearts, and the potential underlying mechanisms. C57BL/6J male mice were randomly divided into four groups: (1) control low-fat diet (LF-SED), (2) LF diet with free access to a voluntary running wheel (LF-RUN), (3) high-fat diet (HF-SED; 45% of calories from fat), and (4) HF-RUN. We examined NLRP3 inflammasome-related signaling pathways, nitric oxide (NO) signaling, and oxidative stress in coronary arterioles to test effects of HFD and physical activity. Voluntary running reduced NLRP3 inflammasome and its downstream effects, caspase-1 and IL-1ß in coronary arteriole endothelium of obese mice in immunofluorescence staining. HF-RUN attenuated HFD-dependent endothelial NO synthase (eNOS) reduction and thus increased NO production compared to HF-SED. HFD elevated intracellular superoxide production in coronary arterioles while voluntary running ameliorated oxidative stress. Our findings provide the first evidence that voluntary running attenuates endothelial NLRP3 inflammasome activation in coronary arterioles of HFD feeding mice. Results further suggest that voluntary running improves obesity-induced vascular dysfunction by preserved NO bioavailability via restored expression of eNOS and reduced oxidative stress.

27-Hydroxycholesterol induces hematopoietic stem cell mobilization and extramedullary hematopoiesis during pregnancy.

Extramedullary hematopoiesis (EMH) is induced during pregnancy to support rapid expansion of maternal blood volume. EMH activation requires hematopoietic stem cell (HSC) proliferation and mobilization, processes that depend upon estrogen receptor α (ERα) in HSCs. Here we show that treating mice with estradiol to model estradiol increases during pregnancy induced HSC proliferation in the bone marrow but not HSC mobilization. Treatment with the alternative ERα ligand 27-hydroxycholesterol (27HC) induced ERα-dependent HSC mobilization and EMH but not HSC division in the bone marrow. During pregnancy, 27HC levels increased in hematopoietic stem/progenitor cells as a result of CYP27A1, a cholesterol hydroxylase. Cyp27a1-deficient mice had significantly reduced 27HC levels, HSC mobilization, and EMH during pregnancy but normal bone marrow hematopoiesis and EMH in response to bleeding or G-CSF treatment. Distinct hematopoietic stresses thus induce EMH through different mechanisms. Two different ERα ligands, estradiol and 27HC, work together to promote EMH during pregnancy, revealing a collaboration of hormonal and metabolic mechanisms as well as a physiological function for 27HC in normal mice.

Nonnuclear Estrogen Receptor Activation Improves Hepatic Steatosis in Female Mice.

Estrogens have the potential to afford atheroprotection, to prevent excess adiposity and its metabolic complications including insulin resistance, and to lessen hepatic steatosis. Cellular responses to estrogens occur through gene regulation by nuclear estrogen receptors (ERs), and through signal initiation by plasma membrane-associated ER. Leveraging the potentially favorable cardiometabolic actions of estrogens has been challenging, because their reproductive tract and cancer-promoting effects adversely impact the risk to benefit ratio of the therapy. In previous works, we discovered that an estrogen dendrimer conjugate (EDC) comprised of ethinyl-estradiol (E2) molecules linked to a poly(amido)amine dendrimer selectively activates nonnuclear ER, and in mice, EDC does not invoke a uterotrophic response or support ER-positive breast cancer growth. In the present investigation, we employed EDC to determine how selective nonnuclear ER activation impacts atherosclerosis, adiposity, glucose homeostasis, and hepatic steatosis in female mice. In contrast to E2, EDC did not blunt atherosclerosis in hypercholesterolemic apoE-/- mice. Also in contrast to E2, EDC did not prevent the increase in adiposity caused by Western diet feeding in wild-type mice, and it did not affect Western diet-induced glucose intolerance. However, E2 and EDC had comparable favorable effect on diet-induced hepatic steatosis, and this was related to down-regulation of fatty acid and triglyceride synthesis genes in the liver. Predictably, only E2 caused a uterotrophic response. Thus, although nonnuclear ER activation does not prevent atherosclerosis or diet-induced obesity or glucose intolerance, it may provide a potential new strategy to combat hepatic steatosis without impacting the female reproductive tract or increasing cancer risk.

Re-adopting classical nuclear receptors by cholesterol metabolites.

Since the first cloning of the human estrogen receptor (ER) α in 1986 and the subsequent cloning of human ERß, there has been extensive investigation of the role of estrogen/ER. Estrogens/ER play important roles not only in sexual development and reproduction but also in a variety of other functions in multiple tissues. Selective Estrogen Receptor Modulators (SERMs) are ER lignds that act as agonists or antagonists depending on the target genes and tissues, and until recently, only synthetic SERMs have been recognized. However, the discovery of the first endogenous SERM, 27-hydroxycholesterol (27HC), opened a new dimension of ER action in health and disease. In addition to the identification of 27HC as a SERM, oxysterols have been recently demonstrated as indirect modulators of ER through interaction with the nuclear receptor Liver X Receptor (LXR) ß. In this review, the recent progress on these novel roles of oxysterols in ER modulation is summarized.

The cholesterol metabolite 27-hydroxycholesterol promotes atherosclerosis via proinflammatory processes mediated by estrogen receptor alpha.

Oxysterols are cholesterol metabolites that serve multiple functions in lipid metabolism, including as liver X receptor (LXR) ligands. 27-hydroxycholesterol (27HC) is an abundant oxysterol metabolized by CYP7B1. How 27HC impacts vascular health is unknown. We show that elevations in 27HC via cyp7b1 deletion promote atherosclerosis in apoe(-/-) mice without altering lipid status; furthermore, estrogen-related atheroprotection is attenuated. In wild-type mice, leukocyte-endothelial cell adhesion is increased by 27HC via estrogen receptor (ER)-dependent processes. In monocytes/macrophages, 27HC upregulates proinflammatory genes and increases adhesion via ERα. In endothelial cells, 27HC is also proadhesive via ERα, and in contrast to estrogen, which blunts NF-κB activation, 27HC stimulates NF-κB activation via Erk1,2 and JNK-dependent IκBα degradation. Whereas 27HC administration to apoe(-/-) mice increases atherosclerosis, apoe(-/-);erα(-/-) are unaffected. Thus, 27HC promotes atherosclerosis via proinflammatory processes mediated by ERα, and it attenuates estrogen-related atheroprotection. Strategies to lower 27HC may complement approaches targeting cholesterol to prevent vascular disease.

The interaction between metabolism, cancer and cardiovascular disease, connected by 27-hydroxycholesterol.

Oxysterols are metabolites of cholesterol that are produced in liver and other peripheral tissues as a means to eliminate cholesterol to bile acid. Recent studies have revealed that the most abundant circulating oxysterol 27-hydroxycholesterol (27HC) is the first identified endogenous selective estrogen receptor modulator. 27HC levels correlate well with that of cholesterol, and also rise progressively with age. 27HC affects estrogen receptor function by the antagonism of estrogen action and also by the direct modulation of the receptor function, and similar to estrogen/estrogen receptors, 27HC has many actions in various tissues. This review article introduces the recent progress in the understanding of the role of 27HC in breast cancer and cardiovascular dysfunction.

27-Hydroxycholesterol promotes cell-autonomous, ER-positive breast cancer growth.

To date, estrogen is the only known endogenous estrogen receptor (ER) ligand that promotes ER+ breast tumor growth. We report that the cholesterol metabolite 27-hydroxycholesterol (27HC) stimulates MCF-7 cell xenograft growth in mice. More importantly, in ER+ breast cancer patients, 27HC content in normal breast tissue is increased compared to that in cancer-free controls, and tumor 27HC content is further elevated. Increased tumor 27HC is correlated with diminished expression of CYP7B1, the 27HC metabolizing enzyme, and reduced expression of CYP7B1 in tumors is associated with poorer patient survival. Moreover, 27HC is produced by MCF-7 cells, and it stimulates cell-autonomous, ER-dependent, and GDNF-RET-dependent cell proliferation. Thus, 27HC is a locally modulated, nonaromatized ER ligand that promotes ER+ breast tumor growth.