bluuebunny/crossref_metadata_2025_split huggingface dataset:
ODP441 NErbB-2 and Androgen Receptor Regulate a Gene Signature Involved in Immune Response and Favorable Outcome in TNBC
Authors: Elizalde Patricia , Roldan Deamicis Agustina , Oakley Robert , Madera Santiago , Cordo Russo Rosalía , Schillaci Roxana , Fresno Cristóbal , Cidlowski John , Proietti Cecilia 
Published: November 2022

Abstract
Triple negative breast cancer (TNBC) has poor prognosis and neither established biomarkers nor therapeutic targets. On the one hand, the androgen receptor (AR), a steroid hormone receptor (SR) which is expressed in 10-53% of TNBC and proved to be critical for BC proliferation, has been proposed as a new target in TNBC. On the other, we and others have shown that membrane ErbB-2 migrates to the nucleus (nuclear ErbB-2, NErbB-2) where it binds DNA at HER-2 associated sequences (HAS) to regulate BC proliferation and migration. Since we have previously shown a functional interplay between growth factors and SR signaling pathways in BC, we propose the existence of an interaction between AR and ErbB-2 which is involved in NErbB-2+/AR+ BC growth. The experimental model used was the human TNBC cell line MDA-MB-453 which displays high expression levels of AR and NErbB-2. We have previously shown that dihydrotestosterone (DHT)-activated AR induces Src-mediated ErbB-2 rapid activation and its migration to the nucleus where it binds to HAS sites in the DNA. By coimmunoprecipitation and WB studies, we have now shown that this crosstalk involves ErbB-2 and AR physical association which is inhibited by the AR antagonist enzalutamide. By microarray and bioinformatics analysis we have also previously identified a set of differentially expressed genes (DEGs) in the presence of DHT and NErbB-2 eviction to define an independent predictor of better clinical outcome in TNBC. By using the Tumor Immune Estimation Resource (TIMER) we found a significant association of the expression of the gene signature and the abundance of tumor immune infiltrates, in accordance with the involvement of the gene signature in the immune response and interferon pathways. We have validated the regulation of the gene signature in MDA-MB-453 cells and found that in the presence of DHT, eviction of NErbB-2 significantly induces the mRNA expression of the genes CXCL10, HLA-A, NMI, STAT1 and TAP1, members of said gene signature. We have also identified AREs and HAS sites in STAT1, NMI and TAP1 and studied AR and ErbB-2 recruitment to the chromatin by ChIP assays. We found that DHT induces ErbB-2 binding to its HAS sites in STAT1 in regulatory regions. Our findings evidence that DHT-activated AR induces ErbB-2 rapid activation and its migration to the nucleus where it binds to HAS sites in regulatory regions of a set of genes involved in the immune response in TNBC.
Presentation: No date and time listed
ODP443 Targeting NAD+ availability modulates 11β- Hydroxysteroid dehydrogenase 1 activity in mouse and human cell lines and primary cells
Authors: Kabli Ali , Elhassan Yasir , Heising Silke , Heaselgrave Samuel , Hardy Rowan , Hodson David , Morgan Stuart , Lavery Gareth 
Published: November 2022

Abstract
Four nicotinamide adenine dinucleotide coenzymes – NAD+, NADH, NADP+, and NADPH – are the central catalysts of metabolism. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is NADPH-dependent reductase that catalyses the reduction of the inactive glucocorticoid cortisone) to active cortisol (11-dehydrocorticosterone to corticosterone in rodents). 11β-HSD1 is in the endoplasmic reticulum (ER) lumen, and the level of NADPH in the ER regulates enzyme activity. Little is known about the biochemical mechanisms controlling cytosolic/ER NAD(P)(H) crosstalk. Here we examined how perturbed cellular NAD+ availability modulates 11β-HSD1 enzyme activity in the ER across a range of mouse and human cell types. 11β-HSD1 activity was measured in a series of transformed and primary mouse and human liver (HepG2), muscle, and Human Dermal Fibroblast cells (HDFn) depleted for NAD+ using FK866 to inhibit nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. To replete NAD+ we supplemented cells with 0.5mM of NAD+ precursors nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). FK866 (100nM, 48h) was effective at depleting NAD+ by approximately 90% in C2C12 myotubes and 50% in HepG2 liver cells. In each cell line, 11β-HSD1 reductase activity was decreased by FK866 compared to untreated controls (80% in C2C12 P&lt;0. 0001, 55% in HepG2, P=0. 03 and 65% in HDFn P=0. 002). Restoring NAD+ level with NR (0.5mM, 6h) in C2C12 and (0.5mM, 24h) in HepG2 and HDFn rescued 11β-HSD1 enzyme activity compared with no precursor treatment (60% in C2C12 P=0. 0007, 52% in HepG2 P=0. 03 and 68% in HDFn P=0. 0006). Human dermal fibroblasts treated with TNFa (10nM, 24h) boosted 11β-HSD1 expression 4-fold, however this was not able to overcome the FK866 induced drop-in reductase activity. These data show that depletion of NAD+ and impaired 11β-HSD1 reductase activity in the ER is a generalised phenomenon across cell types. This suggests a crosstalk between cytosolic and ER NAD(P)(H) pools that can respond rapidly to changes in NAD+ precursor availability. This could provide new insight into the regulation of cellular glucocorticoid metabolism and ER NAD(P)(H) homeostasis.
Presentation: No date and time listed
OR07-1 Estrogen-regulated Enhancer RNAs Contain a Functional Motif that Controls Enhancer Assembly and Function in Breast Cancer Cells
Authors: Hou Tim , Kraus W Lee 
Published: November 2022

Abstract
Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor. Upon binding to endogenous and exogenous ligands, ERα dimerizes and binds to regulatory regions across the genome known as enhancers. Enhancers act as nucleation sites for the recruitment of coregulatory proteins and ultimately regulate target gene transcription. We have previously used global run-on sequencing (GRO-seq) to detect transcription at active enhancers that generates enhancer RNAs (eRNAs). eRNAs are poorly characterized noncoding RNAs and are thought to regulate enhancer function and gene expression. To better understand the functions of these eRNAs, we annotated the estrogen-regulated eRNA transcriptome in ERα-positive breast cancer cells using precision nuclear run-on of capped RNA (PRO-cap) and polyA-depleted RNA-seq. We then cloned selected eRNAs, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9. eRNAs promoted expression of cognate, but not heterologous, target genes by increasing ERα recruitment and simulating p300-catalyzed H3K27 acetylation at the enhancer. We identified a ∼40 nucleotide functional eRNA regulatory motif (FERM) present in many estrogen-regulated eRNAs that is necessary and sufficient to promote gene expression by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation. The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers, to exert its effect on enhancer assembly and dynamics. Overexpression of a targeted eRNA controlling expression of an oncogene increased cell proliferation, implicating eRNAs in regulating breast cancer cell growth. Together, we provide a genome-wide annotation of estrogen-regulated eRNAs, demonstrate the mechanisms by which targeted eRNAs promote target gene expression in vivo, identify the FERM element within eRNAs that is critical for regulating target gene expression, and show the biological implications of oncogenic eRNAs in breast cancer growth. Collectively, our results demonstrate the biological and mechanistic roles of eRNAs in breast cancer.
This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K.
Presentation: Saturday, June 11, 2022 11:30 a.m. - 11:45 a.m.
OR07-6 Disrupting DNA-binding Domain Dimerization In The Androgen Receptor In Mice Results In a Partial Androgen Insensitivity Model
Authors: Claessens Frank , Libert Claude , Palvimo Jorma , El Kharraz Sarah 
Published: November 2022

Abstract
The androgen receptor (AR) plays a crucial role in the development and maintenance of the male phenotype, as shown in patients with androgen insensitivity syndrome (AIS). The AR is a nuclear receptor that needs to homodimerize to execute its role as transcription factor. Dimerization can occur through three different modes: via the DNA-binding domain (DBD), via the ligand-binding domain and via an interaction between the LBD and the aminoterminal domain. Dimerization via the DBD is very well known and occurs through the D-box located in the second zinc finger. DBD dimerization is generally accepted for all steroid receptors, but some of them show that D-box mutations do not inactivate them completely. However, for the AR, no monomeric transactivation capacity was described yet. To study the role of D-box independent activity of the AR, we introduced a double point mutation in the D-box that is predicted to disrupt DBD dimerization (ARDmon). These two point mutations were found in separate patients suffering from partial AIS. In vitro studies with this mutant suggest that the remaining activity depends on the androgen response element under investigation. We show that ligand binding was not affected by the D-box mutation. Furthermore, BioID assays indicated no changes in coregulatory interactions. When introducing the corresponding mutation in mice, this led to a phenotype that is intermediate between global ARKO and wild type (WT) males. Based on their anogenital distance (AGD) and the absence of nipples at early age, the ARDmon/Y mice were identified as males. Follow-up of their AGD and body weight, however, revealed clear differences with both WT males and females. Furthermore, hypospadias was observed in the ARDmon/Y mice. Testes were smaller in size compared to the WT males and first analysis showed almost no expression of well-known AR regulated genes. Reminiscent structures of the epididymis were found.
In summary, the ARDmon mouse model shows an intermediate AIS phenotype. Via orchidectomy in combination with androgen replacement therapy, we will now investigate remaining androgen responses in the ARDmon/Y mice and elucidate response elements on which the AR can act in a D-box independent way. Additionally, we generated the first rodent PAIS model with a genetic cause of hypospadias.
Presentation: Saturday, June 11, 2022 12:45 p.m. - 1:00 p.m.
OR26-1 Alternative Polyadenylation as a Therapeutic Vulnerability in Prostate Cancer
Authors: Dehm Scott , Miller Jeffery , Munro Sarah , Van Etten Jamie , Tietz Kiel 
Published: November 2022

Abstract
Prostate cancer is the second leading cause of male cancer death in the United States. While localized disease can be cured by radiation or surgery, metastatic prostate cancer presents a clinical challenge. Metastatic prostate cancer can initially be controlled by endocrine therapies that target the androgen receptor (AR), however, these tumors will inevitably develop resistance. This stage of the disease, termed castration-resistant prostate cancer (CRPC), is responsible for the majority of prostate cancer-specific deaths. Truncated AR variant (AR-V) proteins are broadly enriched in CRPC cell lines and clinical samples, and can function as ligand-independent, constitutively active transcription factors. We found that blocking an alternative poly(A) site located in AR intron 3 reduced expression of multiple AR-V mRNA and protein species and increased expression of full-length (FL) AR mRNA and protein in 22Rv1 and LNCaP95 CRPC cells. We found the cleavage and polyadenylation specificity factor (CPSF) component, CPSF1, regulates selection of this alternative poly(A) site based on the finding that knockdown of CPSF1 also reduced expression of AR-Vs and increased expression of FL-AR in 22Rv1 and LNCaP95 cells. Further, knockdown of CPSF1 inhibited growth of these CRPC cell lines, as well as androgen-dependent LNCaP cells. To define how CPSF1 regulates alternative polyadenylation of AR and to determine novel pathways that are manipulated by CPSF1 in prostate cancer, we identified CPSF1-dependent gene expression using RNA-seq and CPSF1-dependent poly(A) site usage using poly(A)-ClickSeq (PAC-Seq). Gene set enrichment analysis of RNA-seq data revealed the Glycolysis Hallmark gene was positively regulated by CPSF1, and nominated multiple differentially-expressed genes encoding regulators of glycolysis as CPSF1 targets. These results highlight AR independent pathways that are also regulated by CPSF1 in prostate cancer. We are currently integrating RNA-Seq and PAC-seq datasets to define novel CPSF1 regulated pathways that can be targeted therapeutically in prostate cancer. This research will determine trans-acting factors and cis-regulatory elements that drive alternative polyadenylation of AR mRNA transcripts to promote CRPC progression. This research will also identify AR independent targets of CPSF1 that could be exploited for development of new therapies for CRPC.
Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.
OR26-3 USP19 Deubiquitinating Enzyme Functions as a Novel Co-Chaperone of Hsp90 to Regulate Glucocorticoid Receptor Levels
Authors: Bedard Nathalie , Coyne Erin , Huynh Julie , Jacq Xavier , Rozycka Ewelina , Schorova Lenka , Young Jason , Wing Simon 
Published: November 2022

Abstract
Ubiquitin specific protease 19 (USP19) is a deubiquitinating enzyme involved in metabolism. Its expression is upregulated in skeletal muscle in many conditions of muscle wasting. Genetic inactivation of USP19 (USP19KO) in mice protects from muscle atrophy and diet induced obesity and insulin resistance. We previously reported that the protection against muscle atrophy in USP19KO is due at least in part to reduction in muscle glucocorticoid receptor (GR) levels and signaling. Here, we report that USP19KO mice show &gt; 50% decrease in GR protein level in multiple tissues with no observed changes in GR mRNA levels. Overexpression of the endoplasmic reticulum or cytosol-localized isoforms of USP19 in cells resulted in increased GR protein levels, while knockdown by siRNA or knockout by CRISPR-Cas9 resulted in decreased GR protein levels. Intriguingly, GR was not more ubiquitinated in the absence of USP19. Furthermore, expression of a catalytically inactive form of USP19 also increased GR protein levels and exposure of cells to a small molecule inhibitor of USP19 enzymatic activity had no effects on levels of GR. Deletion analysis revealed that the N-terminal CS domains of USP19, which are structurally similar to the p23 co-chaperone of HSP90, were required for increasing levels of GR. Since HSP90 is known to regulate GR folding and stability, this suggested that USP19 may function as a p23 like co-chaperone. Indeed, overexpression of USP19 not only increased GR levels, but translocation to the nucleus and transcriptional activity of a reporter gene. Using a novel BRET assay, we observed that USP19 interacts indirectly with GR through binding with HSP90. This interaction with HSP90 is critically dependent on the CS2 domain of USP19 and can be competed by expression of the p23 co-chaperone of HSP90. We have therefore identified a novel co-chaperone-like mechanism through which USP19 regulates GR protein stability independent of its catalytic activity. Targeting the interaction of USP19 with the GR-HSP90 complex could be a strategy to decrease GR levels, reduce glucocorticoid signalling and prevent muscle atrophy.
Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.
PSUN66 Statin Intake as a Cause of Decreased Steroid Hormone Secretion
Authors: Mostovaya Maria , Kalinchenko Svetlana , Vorslov Leonid 
Published: November 2022

Abstract

Materials and methods
The study enrolled 2 men aged 58 years (history of acute cerebral circulation disorder at 47 and 57 years) and 64 years (history of acute myocardial infarction at 62 years), who complained of shortness of breath, daytime sleepiness, decreased libido, weakened erections, increased blood pressure. Both patients had been taking atorvastatin 10 mg for more than three months.
We used high-performance liquid chromatography (HPLC) to determine steroid hormones in saliva: Agilent 1200 chromatograph, AB Sciex 3200 MD mass spectrometer (Sciex, USA). Saliva was collected in a Salivette container.
A significant decrease in pregnenolone levels of 440.0 pg/ml and 300 pg/ml, respectively, was detected in the saliva analyses). The norm for pregnenolone was 575-665 pg/ml.

Conclusion
Statins lead to decreased pregnenolone secretion, most likely due to inhibition of HMG-CoA reductase, a key enzyme that converts 3-hydroxy-3-methyl glutaryl-CoA to mevalonate, a cholesterol precursor. The decrease in cholesterol synthesis leads to a decrease in pregnenolone and a decrease in steroid hormone secretion, both in the adrenal glands and gonads, which clinically manifests as hypogonadism and adrenal fatigue. All patients taking statins should have their salivary steroid profile evaluated. If statins cannot be withdrawn, hormone replacement therapy with sex hormones is necessary.
Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.
LBSAT238 Teprotumumab Reduces Orbital Fat And Muscle Volume In Patients With Thyroid Eye Disease
Authors: Ugradar Shoaib 
Published: November 2022

Abstract
Teprotumumab, a novel IGF-1R antibody has been shown to reduce proptosis in patients with thyroid eye disease (TED) in 2 randomized controlled trials. At present, it is unclear whether the reduction in proptosis is related to a decrease in extraocular muscle or orbital fat tissue. In this prospective longitudinal study, we reviewed the changes to orbital fat and muscle, and compared them, in consecutive patients with TED, who had completed treatment with teprotumumab (8 infusions). The primary outcome was change in orbital fat and muscle volume within 6 weeks of starting and completing therapy. Secondary outcome measures included change in proptosis and the clinical activity score (CAS). All patients had either CT or MRI with ≤ 1 mm slice thickness. Three-dimensional volumetric analysis of the orbital fat and muscle volume was performed using the previously validated 3D image analysis software, MIMICS (Materialise, Leuven, Belgium). Hounsfield units of fat and muscle were defined manually. All extraocular muscle tissue and fat between the orbital septum (eyelid) and the orbital outlet of the optic canal was included. A 3D model created by voxel addition was expressed in millimeters cubed. Segmentation and measurements were performed independently by two graders to review repeatability and observer variability. In line with previous clinical trials, the orbit with the most proptosis was defined as the study orbit and the contralateral orbit was designated the fellow orbit. Twenty-one patients (15 females and 6 males) met the inclusion criteria. Mean (SD) age was 55 (16), while mean (SD) duration of TED was 71 (46) months. There was a reduction of 3 mm in both; study and fellow orbits following treatment (p &lt; 0. 01 for both). Compared to baseline, there was a mean (SD) CAS reduction of 4 (1) in the study orbit and 2.4 (1.1) in the fellow orbit (p &lt; 0. 01 for both). Following therapy, mean (SD) reduction in muscle tissue was 1691 mm3 (1358) in the study orbit and 1714 mm3 (1927) in the fellow orbit (p &lt; 0. 01 when compared to baseline for each). The mean (SD) reduction of fat volume was 1382 mm3 (1318) in the study orbit and 933 mm3 (1699) in the fellow orbit (p &lt; 0. 01 when compared to baseline for each). There was no difference between fat and muscle reduction for the study orbit (p = 0.4) or fellow orbit (p = 0.2). There was a significant correlation between a reduction in proptosis and reduction in orbital soft tissue (fat and muscle) volume. The results of this study suggest that following treatment with teprotumumab, there is a significant decrease in orbital fat and muscle volume. This reduction in soft tissue volume likely drives the reduction in proptosis.
Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.
LBSAT244 Prospective, Blinded, Multicenter Head-to-head Clinical Comparison Of Two "TSI" Assays In Patients With Autoimmune Thyroid Disease
Authors: Lupo Mark Armin , Kahaly George Jean 
Published: November 2022

Abstract

Objective
This study evaluated the clinical performance of the thyrotropin receptor Antibody (TSHR-Ab) bridge assay (Siemens TSI assay) versus the cell-based bioassay for the measurement of thyroid stimulating immunoglobulins (TSI, Quidel) (in patients with autoimmune thyroid disease (AITD).

Methods
Two-hundred two subjects (median age 54 years, 162 female, 80%), with well-documented thyroid disorders and controls were prospectively enrolled in a consecutive, unselected manner. Antibody measurements were performed using the Bridge (Siemens Immulite) and stimulating (TSAb or TSI) and blocking (TBAb) TSHR-Ab cell-based bioassays (Quidel Thyretain) each according to the manufacturers’ specfications. The testing laboratories were blinded to any clinical data.

Results
The three assays were negative in controls (n=10), patients with euthyroid nodular disease (n=11) and non-autoimmune thyrotoxicosis (n=21). In patients with Graves’ disease (GD), independent of disease duration or activity, TSHR-Ab positivity was present in 70/113 (62%) and 88 (78%) for the Bridge and TSAb bioassay, respectively (p&lt;0. 001). Concordant positive results in the Bridge and TSAb bioassay were noted only in 67/88 (76%) patients with GD and TSAb positivity. Of these 21 discordant findings of TSAb positive and Bridge negative patients, four were new diagnosis of untreated overt Graves’ hyperthyroidism. Nine patients were euthyroid on low dose methimazole - two of these nine were taken off methimazole shortly after enrollment and remain in remission however the other seven required anti-thyroid drug (ATD) for at least 12 months post-enrollment. Three patients were euthyroid without treatment (follow-up data not available). Four had subclinical hyperthyroidism due to GD and one with GD and subsequent spontaneous hypothyroidism had moderate Graves’ orbitopathy (GO). Patients with GO were TSHR-Ab positive in 23/25 (92%) and 20 (80%) in the TSAb bioassay and Bridge assays, respectively (p&lt;0. 001). In 160 patients with AITD, TBAb were present in 32 (20%), HT 11/27 (41%) and GD 21/133 (16%). 28/32 (88%) of the TBAb positive samples were also Bridge assay positive but TSAb bioassay negative.

Conclusions
Thyroid Stimulating Immunoglobulin (TSI or TSAb) is the key clinical biomarker for GD. We found that the TSAb functional bioassay was more sensitive than the Bridge TSH-R-Ab test not only in cases of subclinical GD but also new overt GD hyperthyroidism, as well as in euthyroid patients on low dose ATD which impacts decisions regarding discontinuing therapy. TSAb was also a better biomarker in patients with GO. In patients with AITD and TSAb negative, the Bridge assay was positive in the presence of blocking TSHR-Ab measured in a TBAb bioassay.
Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.