bluuebunny/crossref_metadata_2025 huggingface dataset:
12260 Assessing The Impact Of Saliva Collection Devices On Analyte Recovery
Authors: Marshall David , Dolan Megan , Mctaggart Malcolm , Keevil Brian George
Published: October 2024

Disclosure: D. Marshall: None. M. Dolan: None. M. Mctaggart: None. B.G. Keevil: None.

Introduction: Analysis of saliva is gathering momentum, often conveying advantages over regular venepuncture, offering a convenient way of collecting multiple samples throughout the day with relative ease from the comfort of a patient’s home or workplace. The advent of more sensitive mass spectrometry equipment has opened the possibility of measuring analytes previously unsuitable, offering a multitude of steroid hormones for diagnosis and monitoring of a range of endocrine disorders. Traditionally, samples are collected onto a Sarstedt Salivette device comprising of a synthetic swab enclosed within a polypropylene tube. For certain analytes, the Salivette device is known to impede analyte recovery, resulting in the requirement for a passive drool sample which can be difficult and unpleasant to collect for some patients. Methods Previous work has been undertaken showing the SalivaBio Oral Swab (SOS) to have less of an impact on analyte recovery. Here, through addition of low, medium and high spikes and subsequent LC-MS/MS analysis of 19 different steroid hormones (11-dehydrocorticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 11-hydroxyandrostenedione, 11-hydroxytestosterone, 11-ketoandrostenedione, 11-ketotestosterone, 17-hydroxyprogesterone, 18-hydroxycortisol, 18-oxocortisol, 21-deoxycortisol, aldosterone, androstenedione, corticosterone, cortisol, cortisone, dexamethasone, testosterone and tetrahydro11-deoxycortisol) we aimed to assess a broader repertoire of analytes and whether saliva collection devices impact on analyte recovery. Results With an acceptability criterion of 10% difference from baseline passive drool samples, Salivette devices were acceptable for 12 out of 18 analytes, SOS were acceptable for 16 analytes. Both devices were unacceptable for measurement of androstenedione, testosterone and 11-deoxycorticosterone. When 10% is deemed as an acceptable difference, the SOS performs better than the Salivette device for the steroid hormones investigated in this study. This may have implications for research studies, patient diagnosis and monitoring of endocrine disorders where saliva collection devices were previously deemed unacceptable due to issues with analyte recovery.

Presentation: 6/2/2024
12543 Biochemical And Structural Studies Of Human 17-Hydroxysteroid Dehydrogenase Type 1 Mutations Engineered To Convert An NADPH-dependent Reductase To An NAD-dependent Oxidase
Authors: Motl N , Harris K L , Mizrachi D , Auchus R J , Scott E
Published: October 2024

Disclosure: N. Motl: None. K.L. Harris: None. D. Mizrachi: None. R.J. Auchus: None. E. Scott: None.

Background: In intact cells, the hydroxysteroid dehydrogenases (HSDs) strongly favor either NADPH-dependent ketosteroid reduction or NAD+-dependent hydroxysteroid oxidation. For ketosteroid reductases, mutation of the arginine 38 residue in the cofactor-binding domain, which forms a salt-bridge with the 2-phosphate of NADP(H), attenuates the reductive preference but does not eliminate NADPH binding. We sought to engineer mutations of human 17βHSD1 that lack NADP(H) binding, analogous to the oxidative HSDs. Methods: Wild-type 17βHSD1 and mutations of L37 and/or R38 in the cofactor-binding domain were expressed in E. coli and purified to homogeneity in one step. The purified proteins were assayed for NAD+- and NADP-dependent estradiol oxidation to measure cofactor affinity. Crystals were grown in the presence of NAD+ and/or estrone, and structures were determined using molecular replacement. Results: For wild-type 17βHSD1, the Km for NAD+ was 7-times higher than for NADP+ (44 vs 6.2 μM, respectively). Mutations R38D and R38I raised the Km for NAD+ < 4-fold but the Km for NADP+ >60-fold to favor NAD+ utilization. For mutations L37D/R38I and L37D/R38D, the Km for NAD+ was 100-120 μM, but NADP+ could not support catalysis at concentrations up to 4 mM. The crystal structures of 17βHSD1, wild-type and mutations R38D or L37D/R38D soaked with NAD+, were solved to 1.79, 1.75, and 1.80 Å, respectively. All structures displayed a near-identical overall fold. While the NAD density for the mutant structures was much weaker than for the wild type enzymes, some differences in NAD positioning were observed. The R38D mutation had minor NAD repositioning. However, in the L37D/R38D structure the NAD cofactor is repositioned slightly further and 37D is angled towards the cofactor, rather than away from it as observed in the other structures. The positively-charged R38 which interacts with the NADP phosphate oxygen in the wild type enzyme (PDB: 1QYV) is lost in both mutants. Instead the oxygen atoms of both mutated residues orient towards where the cofactor phosphate oxygen would be present, likely interfering with NADP binding and contributing to the observed switch of cofactor preference. Conclusion: Mutation of L37D combined with a second, hydrophobic or negatively-charged substitution at R38 precluded NADP(H) binding and converted 17βHSD1 to a purely oxidative HSD similar to 17βHSD2. NADP(H) exclusion in the L37D/R38D mutant appears to be due to 1) loss of the phosphate-R38 interaction and 2) the position and negatively charged character of oxygen atoms of 37D/38D conflicting with positioning of the positively charged NADP phosphate. Keywords: hydroxysteroid dehydrogenase, nicotinamide cofactor, estrogen, x-ray crystallography

Presentation: 6/2/2024
9273 Characterizing the Genomic Effects of Low-Dose Estrogen in Breast Cancer Cells
Authors: Kim Hyung Bum , Nandu Tulip Sunil , Lee Kraus W
Published: October 2024

Disclosure: H. Kim: None. T.S. Nandu: None. W.L. Kraus: None.

Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. The predominant nuclear receptor for estrogens, estrogen receptor-alpha (ERɑ), functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the major naturally-occurring estrogen. The physiological levels of E2 are regulated by production, sequestration, and degradation, which can limit the amount of bioavailable E2. However, many studies examining the genomic effects of E2-regulated signaling in cell-based assays used supraphysiological levels of hormone. While this has allowed us to understand the extremes of E2 signaling, it limits our ability to examine cellular functions which operate at lower hormone concentrations. The genomic actions of ligand-bound ERɑ are mediated through transcriptional enhancer formation and function. Alterations in protein and DNA components dictate different enhancer molecular subtypes that specify distinct regulatory mechanisms and downstream cellular phenotypes. How the amount of available hormone affects these genomic processes is unknown. In order to understand the genomic effects of physiological low-dose levels of E2, we have treated MCF-7 ERɑ-positive human breast cancer cells with a range of E2 concentrations and examined the global regulation of gene expression using a multi-omics approach (RNA-seq, ERɑ ChIP-seq, H3K27ac ChIP-seq, PRO-seq, ATAC-seq). Interestingly, we identified gene sets that are preferentially expressed maximally at picomolar or nanomolar concentrations. We are using these genes sets to explore the mechanisms of estrogen-regulated gene expression at physiologically relevant of E2 concentrations. Analysis of nascent RNA transcription using PRO-seq indicates regulation at the transcriptional level. Preliminary ERɑ chromatin binding assays show that while many loci require high levels of E2 for maximum ERɑ binding, others show maximum ERɑ binding at lower doses of E2. Further analyses show distinct enhancer features, such as chromatin accessibility and H3K27ac enrichment, may play a role in determining the response to E2 dosage. We are now determining the molecular mechanisms of dosage sensitivity using a multipronged approach integrating genomics, molecular biology, and cell-based assays. With these studies, we hope to understand a long overlooked physiological aspect of E2 with a comprehensive and global analysis of signal-dependent genomic responses.

Presentation: 6/3/2024
8530 Characterizing the Genomic Effects of Low-Dose Estrogen in Breast Cancer Cells
Authors: Kim H , Nandu T S , Kraus W L
Published: October 2024

Disclosure: H. Kim: None. T.S. Nandu: None. W.L. Kraus: None.

Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. The predominant nuclear receptor for estrogens, estrogen receptor-alpha (ERɑ), functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the major naturally-occurring estrogen. The physiological levels of E2 are regulated by production, sequestration, and degradation, which can limit the amount of bioavailable E2. However, many studies examining the genomic effects of E2-regulated signaling in cell-based assays used supraphysiological levels of hormone. While this has allowed us to understand the extremes of E2 signaling, it limits our ability to examine cellular functions which operate at lower hormone concentrations. The genomic actions of ligand-bound ERɑ are mediated through transcriptional enhancer formation and function. Alterations in protein and DNA components dictate different enhancer molecular subtypes that specify distinct regulatory mechanisms and downstream cellular phenotypes. How the amount of available hormone affects these genomic processes is unknown. In order to understand the genomic effects of physiological low-dose levels of E2, we have treated MCF-7 ERɑ-positive human breast cancer cells with a range of E2 concentrations and examined the global regulation of gene expression using a multi-omics approach (RNA-seq, ERɑ ChIP-seq, H3K27ac ChIP-seq, PRO-seq, ATAC-seq). Interestingly, we identified gene sets that are preferentially expressed maximally at picomolar or nanomolar concentrations. We are using these genes sets to explore the mechanisms of estrogen-regulated gene expression at physiologically relevant of E2 concentrations. Analysis of nascent RNA transcription using PRO-seq indicates regulation at the transcriptional level. Preliminary ERɑ chromatin binding assays show that while many loci require high levels of E2 for maximum ERɑ binding, others show maximum ERɑ binding at lower doses of E2. Further analyses show distinct enhancer features, such as chromatin accessibility and H3K27ac enrichment, may play a role in determining the response to E2 dosage. We are now determining the molecular mechanisms of dosage sensitivity using a multipronged approach integrating genomics, molecular biology, and cell-based assays. With these studies, we hope to understand a long overlooked physiological aspect of E2 with a comprehensive and global analysis of signal-dependent genomic responses.

Presentation: 6/3/2024
7071 Cushing disease presenting with Dilated Cardiomyopathy
Authors: Majeed Hafsa , Nguyen Christina , Galagan Robert , Lovre Dragana
Published: October 2024

Disclosure: H. Majeed: None. C. Nguyen: None. R. Galagan: None. D. Lovre: None.

Background: Hypercortisolism manifests with numerous ways due to the wide distribution of glucocorticoid receptors. Here we describe a case of Cushing’s disease presenting with reversible non-ischemic cardiomyopathy. Case Presentation: A 30-year-old male presented to ER with palpitations, dyspnea, and atypical chest pain. His exam was notable for a BMI of 30.26 kg/m2, HR 76 bpm and BP of 200/110 mm Hg. His EKG revealed Left ventricular hypertrophy. Echocardiogram (ECHO) showed an EF of 25-30% with diffuse hypokinesis. An angiogram demonstrated normal coronary anatomy. He was started on Lisinopril, Carvedilol, and Spironolactone for non-ischemic cardiomyopathy. He also complained of a 40 lb weight gain, loss of libido, hot flashes and breast tenderness. He was referred to endocrinology. Initial evaluation demonstrated total testosterone (TT) of 171 ng/dl (286-1511 ng/dl), FSH 3.8 IU/L (2-12 IU/L), LH 5.6 IU/L (2-9 IU/L) consistent with secondary hypogonadism. Further pituitary workup revealed AM cortisol level 31.22 mcg/dl (4.3-22.4mcg/dl), ACTH 79 pg/ml (7.2 - 63.3pg/ml), IGF-1 257 ng/mL (88-246 ng/mL), Prolactin 23.7 ng/dl (2.1- 17.7ng/dl), TSH 0.54 mIU/l (0.35-3.74mIU/l) and FT4 0.6 ng/dl (0.76-1.46 ng/dl). A 24hr urine cortisol was 167 ug(0-50ug/24h) and midnight salivary cortisol 0.160 ug/dl (<0.010-0.090 ug/dl). An overnight 1mg dexamethasone suppression test (DST) resulted in AM cortisol of 20.95 mcg/dl (<1.8mcg/dl) and 8mg DST cortisol fell from 34 mcg/dl to 4.16 mcg/dl. A pituitary MRI revealed a left 6 mm x 4 mm microadenoma. He was treated with levothyroxine pre-op for central hypothyroidism. He underwent transsphenoidal resection of the pituitary adenoma. Post-op AM cortisol was 1.1 mcg/dl. His steroid withdrawal syndrome was treated with a prolonged course of tapered hydrocortisone. Two months post-op ECHO showed EF of 55-60%. His BP normalized on carvedilol alone. Eighteen months post-op he no longer required any hormone replacement. Repeat ACTH 24.4 pg/ml, Total Testosterone 584ng/dl, TSH 0.57 mIU/l and FT4 0.93 and 24hr urine cortisol was 24ug/dl, MN salivary cortisol <0.10 ug/dl confirming remission of his cushing's disease and resolution of secondary hypogonadism and hypothyroidism. Conclusion: Prolonged hypercortisolism can cause abnormal cardiac remodeling resulting in cardiomyopathy. Surgical remission of Cushing’s disease reverses hypercortisolism which may restore normal cardiac function.

Presentation: 6/2/2024
8599 Development of an androgen receptor transgenic mouse that displays the complete attributes of Metabolic Syndrome
Authors: Alvarado C , Beitel L K , Paliouras M , Trifiro M A
Published: October 2024

Disclosure: C. Alvarado: None. L.K. Beitel: None. M. Paliouras: None. M.A. Trifiro: None.

The mouse androgen receptor (AR) shares over 90% homology with the human ortholog, but it lacks the CAG encoded poly-Q tract; instead, it contains a mixed glutamine/histidine tract. To study AR functionality in a more structured fashion, we created a knock-in mouse model that has only the equivalent of human poly-Q tract while maintaining the remaining mouse receptor sequence intact. Our humanized androgen receptor mouse (AR-19Q) displays a phenotype that is best described as Metabolic Syndrome (MetS). The mice gain excess weight, have hypertension, hyperglycemia, pancreatic islet cell abnormalities, and progressive non-alcoholic fatty liver disease (NAFLD). Features of MetS begin at approximately 4 months and progress till 12-14 months. Obesity: AR-19Q mice are significantly heavier, with a 35% gain of weight vs C57BL/6 mice. However, these mice are not hyperphagic at any time and eat identical quantities of chow. To assess obesity status, and found AR-19Q mice to have a significantly higher depositions of total white adipose visceral, lingual and subcutaneous vs age-matched controls. NAFLD: The progression of NAFLD is observed in AR-19Q mice. AR-19Q Histological analysis and immunohistochemistry reveal microvesicular steatosis, superseded by macrovesicular steatosis with hepatocyte balloon degeneration, scattered lobular inflammation (macrophages and lymphocytes) (6-9 months) and further superseded by perisinusoidal fibrosis (12 months). Biochemical blood analysis confirms. that there is a significant increase in alanine aminotransferase (ALT) levels, indicative of liver damage. With time classical NASH hepatitis and subsequent NASH fibrosis ensues; with several mice developing hepatocellular carcinoma (overall 20% of mice). Gene expression profiling AR-19Q mice for fatty liver genes, we observe an upregulation of inflammation genes Il-6, Il-10 and Tnf. In addition, increased Lpl (lipoprotein lipase) is indicative of increased blood triglyceride levels. On the other hand, down-regulation of G6pc (glucose-6-phosphatase) could be linked to glycogen storage disease. Down-regulation of Slc2a2 and Slc2a4, that encode GLUT2 and GLUT4, should have an influence on glucose levels, glucose homeostasis and insulin sensitivity, as insulin promotes glucose uptake through GLUT4. Type 2 Diabetes: Glucose intolerance is observed by 3-4 months followed by fasting hyperglycemia. Pancreases show advanced islet hyperplasia, concurrent with insulin resistance and type 2 diabetes. Pancreases of middle-age AR-19Q mice present with a significant islet hyperplasia, suggesting peripheral insulin resistance. We propose the use of the AR-19Q mice as a model to study the metabolic dysfunction/ deficiency leading to the MetS. The model will allow us to a better understanding into the mechanisms responsible for T2D and NAFLD-related disorders.

Presentation: 6/2/2024
7861 Estrogen Receptor Alpha Regulates the Sex Difference in Jet Lag in Mice
Authors: Venegas Maria Alicai , Westray Nicholas , Pendergast Julie S
Published: October 2024

Disclosure: M.A. Venegas: None. N. Westray: None. J.S. Pendergast: None.

The circadian system aligns 24-hour cycles of internal biological processes with the environmental light-dark cycle. Abrupt shifts in the timing of the light-dark cycle that occur during travel across time zones misalign internal circadian clocks with the environment and cause jet lag. The symptoms of jet lag subside when circadian clocks resynchronize to the new light-dark cycle. Female mice in proestrus resynchronize to shifted light-dark cycles faster than males. The objective of this study was to investigate the estrogen signaling mechanisms underlying this sex difference in jet lag in mice. We measured wheel-running activity rhythms in C57BL/6J male and female wild-type (WT) and ERα knockout (ERαKO) mice. Female WT mice had 2-fold greater daily activity than male mice. Disabling the function of ERα ablated the sex difference in daily activity; both male and female ERαKO mice had markedly reduced activity compared to WT mice. Next, we advanced the timing of the light-dark cycle by 6 hours to simulate eastward travel. Female WT mice resynchronized to the new light-dark cycle faster than WT males. This sex difference was abolished in ERαKO mice. The rate of resynchronization was slower in female ERaKO mice compared to WT females and was indistinguishable from males. We next investigated 2 putative mechanisms underlying the accelerated rate of resynchronization in WT females. First, mice with faster endogenous clocks resynchronize to new light-dark cycles faster. However, we found that the velocity of the endogenous clock (circadian period) did not differ between WT and ERαKO male and female mice. Second, mice with greater phase advances to light exposure in the late night also resynchronize faster. To determine the magnitude of phase advances, we housed mice in constant darkness and then exposed them to a 15-minute light pulse during the late subjective night. We found no differences in the magnitudes of phase shifts between WT and ERαKO male and female mice. Together these studies show that ERα regulates the rate of resynchronization to shifted light-dark cycles in female mice but does not do so by altering conventional circadian rhythm parameters. Uncovering the mechanisms underlying the sex difference in resynchronization to shifted light-dark cycles can be used to develop strategies to alleviate jet lag and shift work symptoms.

Presentation: 6/3/2024
6676 Exposure to Neighborhood Violence Rewires Glucocorticoid Receptor Binding and Drives Proliferation and Invasiveness in Lung Tumor Samples
Authors: Heath Hannah , Yoo Jin Young , Akter Sabrina , Jain Atharv , Sharma Vani , McGee Hannah , Soliman Aiman , Mohamed Abeer , Matthews Alicia K , Winn Robert , Erdogan Zeynep Madak , Kim Sage
Published: October 2024

Disclosure: H. Heath: None. J. Yoo: None. S. Akter: None. A. Jain: None. V. Sharma: None. H. McGee: None. A. Soliman: None. A. Mohamed: None. A.K. Matthews: None. R. Winn: None. Z. Madak Erdogan: None. S. Kim: None.

Background: Despite the lower rates and frequency of smoking, Black men experience a higher incidence of lung cancer compared to white men. This health disparity is even more prevalent in Black men from Chicago, suggesting local neighborhood factors impacting tumorigenesis. Preliminary data indicate that, compared to white men, Black men are more likely to reside in neighborhoods with high levels of violence and have elevated levels of hair cortisol. Objective: To understand the link between lung tumorigenesis and exposure to neighborhood violence, our objective is to investigate the chromatin recruitment of the receptor for cortisol, called the glucocorticoid receptor (GR), and to correlate this recruitment with target gene expression levels. Methods: Utilizing CUT & RUN, we identified the gene binding sites of GR using 15 lung tumor and corresponding healthy tissue samples from lung cancer patients living in Chicago. GR recruitment to chromatin was correlated with the neighborhood homicide rate obtained using patients’ zip codes. Spatial transcriptome profiles for these 15 lung tumor samples were obtained from the Space Ranger pipeline and gene expression hot spots of GR target genes were visualized using Loupe Browser (10x Genomics). Results: GR recruitment to regulatory regions of chromatin increases with greater zip code level violence rates. In patients from high violence neighborhoods, tumor samples had higher magnitude of binding compared to normal neighboring tissue. In tumor samples from patients living in high violence compared to low violence neighborhoods, GR binding is seen in genes that are associated with increased tumor aggressiveness. Hot-spot analysis of these genes found strong co-expression of genes associated with proliferation and invasion in samples from high violence but not low violence neighborhoods. Conclusion: Exposure to neighborhood violence may impact tumor biology via increased GR recruitment to genes associated with proliferation and invasion.

Presentation: 6/2/2024
12565 Expression Of Sex Hormone Receptors In Levator Ani Muscle Tissue From Patients With Pelvic Organ Prolapse
Authors: You T , Halle A , Xu W , Potluri T , Nguyen H H , Bulun S E , Julia Geynisman-Tan J T , Zhao H
Published: October 2024

Disclosure: T. You: None. A. Halle: None. W. Xu: None. T. Potluri: None. H. Nguyen: None. S.E. Bulun: None. J. Julia Geynisman-Tan: None. H. Zhao: None.

Introduction: Women with pelvic floor disorders usually have weakened pelvic floor muscles, which may cause urinary incontinence, fecal incontinence, and pelvic organ prolapse (POP). Histologically, the pelvic floor musculature in POP patients displays increased fibrosis and muscle atrophy. However, the underlying molecular mechanism for POP has not been fully investigated. We and others found that estrogen causes pelvic skeletal muscle fibrosis and inguinal hernias in male animal models. Additionally, clinical trials from the Women's Health Initiative showed women receiving estrogen-only or estrogen plus progestin treatment had statistically significant increases in the risk for urinary incontinence. However, the role of estrogen, progesterone, androgen, and their receptor expression in pelvic floor muscle is not well studied. Methods: TruCut biopsy needles were used to obtain levator ani (LA, the major pelvic floor muscle) biopsies from four groups of patients undergoing surgery: (i) 5 premenopausal women with stage II/III POP (age 38-52 years), (ii) 5 postmenopausal women with stage III POP (59-75 years), (iii) 5 age and parity matched premenopausal controls, and (iv) 5 age and parity matched postmenopausal controls. The control patients did not have any POP (stage 0) or only had sole stress urinary incontinence. H&E staining, Masson’s trichrome staining, and immunohistochemistry (IHC) staining for estrogen receptor α (ERα), progesterone receptor (PGR), and androgen receptor (AR) were performed in LA muscle tissue. Standard patient demographics (age, parity, and diagnosis) were collected. Results: We used H&E staining to morphologically assess stromal cells and myofibers obtained from LA muscle biopsies. Masson’s trichrome staining showed extracellular matrix deposition in the LA muscle from women with or without POP. In addition, we found ERα was present in the stromal cells from nearly every LA biopsy using IHC staining. ERα expression (H-Score) was similar in four groups of patients. ERα IHC staining was absent in LA myofibers. AR was highly expressed in stromal cells in all LA muscles and weakly expressed in myofibers in ∼70% of LA tissues. AR expression (H-Score) was significantly decreased in postmenopausal women with stage III pelvic organ prolapse. PGR was only expressed in a small portion of the LA sample (30%), with PGR exclusively expressed in the stroma of the LA muscle. Conclusion: Our findings indicate that sex hormone receptors (ERα, AR, and PGR) are present in LA muscle stroma, providing the foundation to further define the roles of sex steroid hormones in pelvic organ prolapse.

Presentation: 6/3/2024
7615 Genomic Consequences of GRHL2 Overexpression in ER+ Breast Cancer Cells
Authors: Allen Kaelyn O , Zheng Christy , Solodin Natalia , Ozers Mary S , Warren Christopher L , Messa Paige E , Nicol Noah R , Vogt Daniel T , Meyer Mark B , Alarid Elaine T
Published: October 2024

Disclosure: K.O. Allen: None. C. Zheng: None. N. Solodin: None. M.S. Ozers: Owner/Co-Owner; Self; Proteovista, LLC. C.L. Warren: None. P.E. Messa: None. D.T. Vogt: None. E.T. Alarid: None.

Breast cancer progression is characterized by the presence and prevalence of metastatic disease. At a genomic level, metastasis can be facilitated via rearrangement of the chromatin landscape to expose oncogenic genes for transcription. Grainyhead like protein 2 (GRHL2) is a nuclear transcription factor implicated in epithelial cell differentiation. The GRHL2 motif has been found near activated estrogen receptor (ER) binding sites, and elevated levels of GRHL2 have been linked to worse prognosis in hormone receptor positive breast cancer patients. In its capacity to contribute to a more severe disease state when overexpressed, GRHL2 may function as either a transcription or pioneer factor to aid in the expression of metastasis-associated genes. Our lab previously showed that GRHL2 genomic binding precedes ER binding at co-regulated binding sites. Also, overexpression of GRHL2 in MCF7 cells for 24 hours did not appreciably alter gene expression by RNA-Seq. These data taken together indicate that GRHL2 may be exhibiting pioneer factor activity. However, the specific role of GRHL2 overexpression in hormone positive breast cancer progression remains unknown. In order to examine the mechanism of GRHL2 action, GRHL2 genomic binding was investigated using a tetracycline-inducible MCF7 model that allows controlled overexpression of GRHL2. ChIP studies were performed to determine GRHL2 binding intensity at specific and genome wide GRHL2 binding sites. Specific focus was given to established GRHL2 binding sites near oncogenic genes responsible for epithelial to mesenchymal transition (EMT), dormancy, and proto-oncogenic behavior. A similar analysis examined H3K27 acetylation to validate active gene transcription. An orthogonal technology, Specificity and Affinity for Protein (SNAP) microarrays, which characterizes direct GRHL2 binding to tiled genomic regions identified in ChIP-Seq analyses from multiple labs, was utilized to discriminate direct and indirect GRHL2 binding sites. The requirement for GRHL2 transactivation function in regulation of EMT related genes was further interrogated using transactivation GRHL2 dominant negative mutants. This work aims to distinguish the transcriptional and non-transcriptional activities of GRHL2 in breast cancer cells on a genome wide level and thereby, provide a deeper understanding of how overexpressed GRHL2 contributes to hormone positive breast cancer.

Presentation: 6/3/2024
9246 Mineralocorticoid Receptor Regulation of the Molecular Clock in Cardiomyocytes Is Modulated by Time of Day
Authors: Kanki Monica , Heanue Seamus , Fuller Peter J , Cole Timothy James , Clyne Colin D , Young Morag Jennifer
Published: October 2024

Disclosure: M. Kanki: None. S. Heanue: None. P.J. Fuller: None. T.J. Cole: None. C.D. Clyne: None. M.J. Young: None.

The onset of cardiovascular disease events peaks in the early morning, which coincides with the diurnal surge in aldosterone and cortisol. Aldosterone-induced mineralocorticoid receptor (MR) activation in cardiomyocytes has established roles in cardiac pump function and electrophysiology and inappropriate MR activation promotes cardiovascular pathophysiology in experimental models of hypertension and heart failure and in the clinic. Recently, we identified a novel role for MR in regulating the circadian clock signalling network in cardiac cells and showed that time of day modulates MR activation in the heart. Whether time-of-day-dependent MR regulation of circadian clocks in cardiomyocytes modulates temporal expression of key functional pathways is unknown. Therefore, we sought to identify cardiac responsiveness to MR stimulation at lights on (7AM) versus lights off (7PM) in ≥8-week old male wildtype (WT) and cardiomyocyte-MR knockout (MyoMRKO) mice. RNA-seq analyses revealed that aldosterone (50 ug/kg) administered at 7PM but not at 7AM significantly enriched transcription of genes associated with 52 biological processes including “Circadian rhythm”, “Reg. of blood pressure”, “Ion transport” and “Reg. of the force of heart contraction” in WT hearts (p<0.05 versus 7PM vehicle-injected WT mice). These outcomes were absent in hearts isolated from MyoMRKO mice administered aldosterone at 7AM or 7PM. Analysis by RT-qPCR revealed that aldosterone versus vehicle administered at 7AM increased expression of circadian Period (Per) and Thyrotroph embryonic factor (Tef) genes in WT hearts, which was lost in MyoMRKO mice. Whole transcriptome analysis also detected significant enrichment in “Cell-cell signalling”, “Positive regulation of cell differentiation”, “Positive regulation of glucose metabolic process”, “Regulation of sodium ion transport”, and “Inflammatory response" processes between 7AM and 7PM in WT hearts, which was also lost in MyoMRKO mice. Our data show that MR transcription of genes associated with circadian rhythms and ion transport is temporally regulated, and demonstrates specificity for cardiomyocyte-MR regulation of circadian clocks. Taken together, we highlight that the role for cardiomyocyte-MR regulation of cardiac electrophysiology, heart rate variability, and metabolic processes is time-of-day-dependent. Understanding temporal coordination of MR activation in physiology and pathophysiology may inform new strategies for optimising MR-directed therapies in patients with heart disease.

Presentation: 6/2/2024
7763 Mineralocorticoid Receptor Regulation of the Molecular Clock in Cardiomyocytes Is Modulated by Time of Day
Authors: Kanki Monica , Heanue Seamus , Fuller Peter J , Cole Timothy James , Clyne Colin D , Young Morag Jennifer
Published: October 2024

Disclosure: M. Kanki: None. S. Heanue: None. P.J. Fuller: None. T.J. Cole: None. C.D. Clyne: None. M.J. Young: None.

The onset of cardiovascular disease events peaks in the early morning, which coincides with the diurnal surge in aldosterone and cortisol. Aldosterone-induced mineralocorticoid receptor (MR) activation in cardiomyocytes has established roles in cardiac pump function and electrophysiology and inappropriate MR activation promotes cardiovascular pathophysiology in experimental models of hypertension and heart failure and in the clinic. Recently, we identified a novel role for MR in regulating the circadian clock signalling network in cardiac cells and showed that time of day modulates MR activation in the heart. Whether time-of-day-dependent MR regulation of circadian clocks in cardiomyocytes modulates temporal expression of key functional pathways is unknown. Therefore, we sought to identify cardiac responsiveness to MR stimulation at lights on (7AM) versus lights off (7PM) in ≥8-week old male wildtype (WT) and cardiomyocyte-MR knockout (MyoMRKO) mice. RNA-seq analyses revealed that aldosterone (50 ug/kg) administered at 7PM but not at 7AM significantly enriched transcription of genes associated with 52 biological processes including “Circadian rhythm”, “Reg. of blood pressure”, “Ion transport” and “Reg. of the force of heart contraction” in WT hearts (p<0.05 versus 7PM vehicle-injected WT mice). These outcomes were absent in hearts isolated from MyoMRKO mice administered aldosterone at 7AM or 7PM. Analysis by RT-qPCR revealed that aldosterone versus vehicle administered at 7AM increased expression of circadian Period (Per) and Thyrotroph embryonic factor (Tef) genes in WT hearts, which was lost in MyoMRKO mice. Whole transcriptome analysis also detected significant enrichment in “Cell-cell signalling”, “Positive regulation of cell differentiation”, “Positive regulation of glucose metabolic process”, “Regulation of sodium ion transport”, and “Inflammatory response" processes between 7AM and 7PM in WT hearts, which was also lost in MyoMRKO mice. Our data show that MR transcription of genes associated with circadian rhythms and ion transport is temporally regulated, and demonstrates specificity for cardiomyocyte-MR regulation of circadian clocks. Taken together, we highlight that the role for cardiomyocyte-MR regulation of cardiac electrophysiology, heart rate variability, and metabolic processes is time-of-day-dependent. Understanding temporal coordination of MR activation in physiology and pathophysiology may inform new strategies for optimising MR-directed therapies in patients with heart disease.

Presentation: 6/2/2024
8033 Pioglitazone Regulates Metabolic Comorbidity of Breast Cancer Endocrine Therapy
Authors: Thomas Nisha S
Published: October 2024

Disclosure: N.S. Thomas: None.

The survival rates of women with estrogen receptor-positive (ER+) breast cancer are typically higher when compared to those with ER-negative and triple-negative (TN) breast cancers. This remarkable progress in survivorship during past decades can be attributed to advancements in treatment and enhanced early detection. However, the prevalence of weight gain and increased risk (30%) of type 2 diabetes (T2D) have been observed in breast cancer survivors treated with tamoxifen (TAM) or aromatase inhibitors, particularly in women with elevated BMI. Furthermore, the T2D risk is 19% higher in tamoxifen-treated women compared to matched individuals without cancer, suggesting a potential association between endocrine therapy and diabetes in breast cancer survivors. Our recent research determined that in female obese mice, endocrine therapy disrupted metabolic homeostasis causing glucose intolerance and ectopic fat deposition associated with adipocyte hypertrophy and depletion of adipocyte progenitor cells. We hypothesized that ER signaling maintains adipocyte progenitors to promote healthy adipose expansion during weight gain, which is disrupted with endocrine therapy. Supplementation of pioglitazone, a thiazolidinedione class of drug and agonist for the peroxisome proliferator-activated receptor-γ to endocrine therapy-treated mice promoted proliferation of the adipocyte precursor population within two weeks of treatment. Though pioglitazone did not significantly affect body weight, its protective effect over endocrine therapy was reflected in maintaining estrogen receptor expression, improved insulin sensitivity, reduced subcutaneous adipocyte diameter, decreased visceral fat, diminished liver triglycerides, and less hepatic steatosis. Flow cytometry analysis with mouse adipocyte precursor cells showed that progenitors were greater with either estrogen or pioglitazone treatment and lower after ER antagonism. The loss of progenitor phenotype in endocrine therapy-treated cells was rescued by pioglitazone intervention. The results of this study may help us better understand and prevent the risk for diabetes in breast cancer survivors.

Presentation: 6/2/2024
6401 Exogenous Cushing Syndrome By Over The Counter Substance "AK Forte". Case Report
Authors: Mohamed Eid Mennaallah , Gandhi Shriya
Published: October 2024

Disclosure: M.M. Eid: None. S. Gandhi: None.

Introduction: Cushing syndrome is a condition of hypercortisolism, either due to excess activity of hypothalamic-pituitary-adrenal (HPA) axis or exogenous steroid intake. Case: A 36 year old, female with history of type 2 DM, HTN, HLD and arthritis. She was referred for evaluation of face and neck swelling for 3 months. No known allergy. Irrelevant family history. Daily Medications include Glipizide 2.5mg, Metformin 500mg twice, Lisinopril 2.5mg and Glargine 8 units. She reports weight gain, muscle weakness and fall, easy bruise, irregular menstruation and stretch marks for a year. Denies hirsutism. On exam: normal vital signs, BMI 47.87 with cushingoid features facial plethora, moon face, dorsocervical pad fat, violet abdominal and arm striae, proximal muscle weakness. No thyromegaly. Late night salivary cortisol 0.071ug/dl(N < 0.359),24 hr urine free cortisol < 1ug/dl(N < 45), DHEA-S 6 ug/dl (N 61-337), 17 Hydroxyprogesterone (OH progesterone) < 5ng/dl, 1 mg overnight Dexamethasone suppression test: AM cortisol < 1 ug/dl, ACTH 1.7 pg/ml (N 7.2-63.3). Na 137 mmol/L , K 4.1 mmol/L,TSH 0.51mciu/ml(N 0.3-4), FT4 0.9 ng/dl(N 0.6-1.7), HBA1C 14% (was 5.1% a year ago). CT abdomen pelvis with normal adrenal glands. Endogenous hypercortisolism was excluded with low 24 urinary cortisol, and late-night salivary cortisol and adequate suppression of morning cortisol with dexamethasone. Low DHEA-S and 17 OH progesterone raises the possibility of exogenous hypercortisolism. A Thorough review of medications and substance revealed that she has been taking over the counter (OTC) AK Forte 400mg 2 tablets 2 times/day for 13 months for arthralgia. In April 2023, FDA released a public notification that AK Forte contains unspecified amount of diclofenac, dexamethasone, and methocarbamol not listed on the product label and warned against taking it (2). We recommended discontinuation of OTC substance and kept her on hydrocortisone 20mg morning and 10 mg afternoon to avoid adrenal insufficiency with plan of gradual hydrocortisone tapering till HPA axis recovers. For uncontrolled DM, dulaglutide 0.75mg/week, and premeal insulin were started and glargine dose was increased. Conclusion: We are presenting a case of exogenous Cushing’s syndrome by OTC substance that contains undeclared ingredients. Dexamethasone in AK Forte causes exogenous hypercortisolism and Cushingoid features with HPA axis suppression. We emphasize the importance of thorough medication reconciliation including OTC substances. We encourage reporting adverse events of OTC substances to FDA. Reference: 1-J.Newell-Price,X.Bertagna,A.B.Grossman,andL.K.Nieman, “Cushing’s syndrome,” Te Lancet, vol. 367, no. 9522, pp. 1605-1617, 2006. 2-https://www.fda.gov/drugs/medication-health-fraud/public-notification-ak-forte-contains-hidden-drug-ingredients.

Presentation: 6/2/2024
6636 Primary Hyperaldosteronism Leading to Aldosterone Deficiency
Authors: Haverstick Nathan Grant , Anthony Jeremy Richard
Published: October 2024

Disclosure: N.G. Haverstick: None. J.R. Anthony: None.

Primary Hyperaldosteronism is a disease process that is often missed, and under checked due to poor screening tools. The disease presents with a common problem, uncontrolled hypertension. It also presents with hypokalemia, fatigue and headaches. It can easily be masked with blood pressure medications, CKD, and pain medications. After treatment there are some rare reports of patients developing hypoaldosteronism. Some risk factors are age >50, duration of hypertension >10 years, pre-existing renal impairment and adrenal adenoma size >2 cm. The patient will present with the opposite: hyperkalemia and hypotension. This is due to the atrophy of the previously healthy gland. Usually this resolves within a year. However in our patient she has continued to require long standing mineralocorticoid replacement therapy. This case highlights the importance of early recognition of hyperaldosteronism. A 63 year-old-female with a medical history of hypertension and diabetes mellitus is being followed for 30+ years of uncontrolled hypertension. Patient has been on multiple agents and blood pressure remained elevated after trying triamterene/hctz, metoprolol, irbesartan, amlodipine. After changing her PCP, there was further investigation into the cause. Leading differentials were Primary Hyperaldosteronism vs Renal Artery Stenosis vs Primary Hypertension. She also had multiple labs with her Potassium below 3 mEq/l. Additional labs were obtained and her Aldosterone level 209 ng/dl and Renin <0.167 ng/mL/hr. The patient was referred to Endocrinology who performed a fludrocortisone suppression test, which did not suppress the levels. A CT scan of the abdomen was performed that showed a 1.3 cm right adrenal mass typical with adrenal adenoma. The patient had an adrenalectomy surgery in February 2022. The patient recovered well, however now developed hypotension off medication, and had consistent hyperkalemia with K > 5.5 mEq/l. The patient had now developed aldosterone deficiency. This was due to the long standing hyperaldosteronism that had caused her left adrenal gland to atrophy after 30 years. The patient was started on Fludrocortisone 0.1 mg daily and a low potassium diet. She has remained off any blood pressure medications since her right adrenalectomy. She has continued to require the mineralocorticoid replacement to date. The diagnostic work-up for primary aldosteronism involves screening, confirmatory testing, and localization studies to distinguish unilateral pathology which may be amenable to adrenalectomy from bilateral adrenal hyperplasia. Through literature review, one theory is that chronic suppression of renin may cause atrophy of the zona glomerulosa cells and hypoaldosteronism. Throughout the case report we emphasize on the importance of early diagnostic screening of secondary hypertension as well as the complications from adrenalectomy.

Presentation: 6/2/2024
7324 Regulation of Super Enhancers by Phosphorylated Progesterone Receptors Drives Breast Cancer Stem Cell Expansion
Authors: Gillis N , Lange C A
Published: October 2024

Disclosure: N. Gillis: None. C.A. Lange: None.

Current treatments for estrogen receptor (ER) positive breast cancer largely target rapidly dividing tumor cells. However, aggressive breast cancers often contain long-lived and weakly proliferative cancer stem cells (CSCs) that readily escape treatments aimed at cycling cells. We previously reported that progesterone receptors (PR) and their target genes aid the growth of these CSCs and help them evade anti-estrogen therapy. As proliferation and stemness are directed by separate signaling pathways that lead to the expression of distinct gene programs, it's crucial to understand how PR facilitates this cellular transformation at the genomic level. Establishing a gene expression program that may limit proliferation but encourages stemness requires coordination between transcription factors and their coregulators to alter the nuclear microenvironment and chromatin landscape.To reveal these mechanisms, we profiled PR and ER genomic binding using CUT&RUN in the T47D luminal breast cancer cell line grown in ultra-low attachment (3D) mammosphere conditions. Our analysis of genomic binding profiles displayed a pattern of PR/ER co-occupancy at distant sites more than 10 kilobases away from the nearest transcriptional start site. We used Rank Ordering of Super Enhancer (ROSE) analysis on these PR/ER binding sites to predict which are likely super enhancer elements capable of coordinating the regulation of multi-gene subsets. We employed a genetic approach to directly test the function of novel enhancer sites linked to PR-target genes by disrupting the PR/ER binding with a dCas9-KRAB construct directed to the predicted enhancer elements. We propose that abnormal growth factor signaling in breast cancer cells permits PR/ER complex formation which promotes expression of genes that induce endocrine resistance and CSC expansion through regulation of super enhancer elements. Further studies to elucidate these mechanisms are ongoing. Understanding the intricacies of PR/ER crosstalk is the crucial next step in developing new therapies that target PR-driven processes in ER+ breast cancers.

Presentation: 6/3/2024
8701 Rsk2 Regulates Estrogen Homeostasis in Metabolism and Fertility
Authors: Wright E B
Published: October 2024

Disclosure: E.B. Wright: None.

Rsk2 is a protein kinase activated downstream of the Ras/Raf/MEK/ERK cascade and has numerous substrates in metabolism, transcription, and translation. However, the role for Rsk2 in regulation of metabolic substrates has only been studied in cancer cell lines. In breast cancer, active nuclear Rsk2 associates with ERα and promotes transformation by activating a transcriptional program critical to the ER+ lineage in the mammary gland[1]. Cancer co-opts programs important in development and homeostasis, and in investigating this possibility we identified that Rsk2 is an important mediator of reproduction2. The link between metabolism and fertility is poorly understood, and Rsk2 may act as a sensor linking metabolism to fertility. To investigate this link, we looked to the liver as a central regulator of metabolism. Hepatic ERα is a transcriptional regulator of metabolic programs, but it is unknown whether Rsk2 regulates hepatic ERα activity. To investigate the role of Rsk2 in regulation of hepatic ERα, livers and mammary epithelial cells were collected from WT or Rsk2-KO female mice in estrus or diestrus, age 14.0 ± 1.8 wk. RNAseq analysis was performed on bulk liver as hepatocytes are ERα+ and comprise 60-70% of the liver cell population. RNAseq on mammary gland was performed on purified ERα+ mammary epithelial cells (MEC), as these cells comprise only 10% of the mammary gland. To evaluate liver response to estrogen, cumulative Z-scores of gene expression were compared to an estrogen signature obtained from the livers of mice treated with oral estradiol3. For the mammary gland, cumulative Z-scores were compared against an estrogen signature obtained from MCF7 treated with estradiol4. Livers from WT mice in estrus had a significantly stronger estrogen signature compared to Rsk2-KO mice in estrus (p = 0.0152, n ≥ 6). In contrast to the liver, Rsk2-KO MEC in estrus had a stronger estrogen response compared to WT (p = 0.028, n =3). Therefore, we have identified differential regulation of estrogen response by Rsk2 in the mammary gland and liver. Rsk2 is a negative regulator of estrogen response in the mammary gland, whereas Rsk2 promotes hepatic estrogen response. These data support our hypothesis that Rsk2 is a physiological regulator of metabolism and fertility by regulating estrogen response. References: (1) Ludwik, K. A., et al. Cancer Res. 2018 (78): 2014-2025. (2) Ludwik, K. A. et al. Cell Reports. 2020 (32): 107931. (3) Palierne, G. et al. Mol Endo. 2016 (30): 709-732. (4) Dutertre, M. et al. Cancer Res. 2010 (70): 3760-3770.

Presentation: 6/3/2024