United States

Glycosylation is an important posttranslational modification that modulates protein expression and function. Sialic acid is the most abundant terminal monosaccharide of N-linked glycosylation in mammalian cells, and its biosynthesis is controlled by the rate-limited enzyme UDP-GlcNAc 2-epimerase/ManNac kinase (GNE). There is a positive association between the abundance of terminally sialylated plasma glycoproteins and the risk of type 2 diabetes (T2DM).  The majority of glycosylated plasma proteins are liver-derived, and key hepatic receptors involved in glucose metabolism, such as the insulin receptor (IR) and glucagon receptor (GCGR), are terminally sialylated. Whether and how protein sialylation in the liver influences glucose homeostasis is unknown. To address this question we generated floxed GNE mice and at 4 weeks of age injected males with either AAV8 encoding a liver-specific targeted Cre recombinase or GFP, to yield mice lacking or normally expressing GNE in liver (GNELKO and GNELWT, respectively). Two weeks post AAV8 injection, GNELKO showed a &gt;98% loss of GNE protein selectively in liver and drastic reductions in the sialylation of liver and plasma proteins detected by glycan-specific lectin-based analyses. Whereas body weights/body composition and plasma lipids were unaltered, GNELKO had lower fasting serum glucose and greater glucose tolerance and insulin sensitivity compared to GNELWT; serum insulin or glucose-stimulated insulin secretion were unchanged. In GNELKO mice hyperinsulinemic-euglycemic clamps displayed a loss of insulin suppression of hepatic glucose production, hepatic IRa and IRb subunit expression and Akt and GSK phosphorylation were decreased, and liver glycogen content was lowered &gt;85%.  However, there was also evidence of markedly impaired GCGR function in GNELKO mice, with serum glucagon levels raised 18-fold and attenuation of hepatic cAMP production, CREB phosphorylation and gluconeogenesis in response to glucagon.  Regarding extrahepatic mechanisms, GNELKO mice displayed enhanced peripheral glucose disposal, and this was associated with increased IRb, Akt and GSK phosphorylation in skeletal muscle, and with serum FGF21 levels elevated by 406%. In additional studies, when invoked following 12 weeks of high fat diet feeding, liver GNE silencing caused dramatic improvements in glucose tolerance and insulin sensitivity.  To evaluate the relevance of these findings to humans, liver GNE transcript levels were queried in a publicly available cohort, and they were higher in T2DM patients compared to non-diabetic controls in two independent cohorts (n= 54 and n=9 for Cohort I and n=4 and n=4 for Cohort II, respectively). Collectively these observations reveal that protein sialylation in the liver has dramatic impact on both insulin and glucagon action in the liver, and on peripheral glucose disposal.  Further interrogation of the modulation of glucose control mechanisms by protein sialylation may lead to novel approaches to combatting glucose intolerance in T2DM and other conditions.

ENDO 2022

Liver-specific inhibition of sialic acid biosynthesis improves systemic glucose homeostasis in mice

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
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Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Glycosylation is an important posttranslational modification that modulates protein expression and function. Sialic acid is the most abundant terminal monosaccharide of N-linked glycosylation in mammalian cells, and its biosynthesis is controlled by the rate-limited enzyme UDP-GlcNAc 2-epimerase/ManNac kinase (GNE). There is a positive association between the abundance of terminally sialylated plasma glycoproteins and the risk of type 2 diabetes (T2DM).  The majority of glycosylated plasma proteins are liver-derived, and key hepatic receptors involved in glucose metabolism, such as the insulin receptor (IR) and glucagon receptor (GCGR), are terminally sialylated. Whether and how protein sialylation in the liver influences glucose homeostasis is unknown. To address this question we generated floxed GNE mice and at 4 weeks of age injected males with either AAV8 encoding a liver-specific targeted Cre recombinase or GFP, to yield mice lacking or normally expressing GNE in liver (GNELKO and GNELWT, respectively). Two weeks post AAV8 injection, GNELKO showed a >98% loss of GNE protein selectively in liver and drastic reductions in the sialylation of liver and plasma proteins detected by glycan-specific lectin-based analyses. Whereas body weights/body composition and plasma lipids were unaltered, GNELKO had lower fasting serum glucose and greater glucose tolerance and insulin sensitivity compared to GNELWT; serum insulin or glucose-stimulated insulin secretion were unchanged. In GNELKO mice hyperinsulinemic-euglycemic clamps displayed a loss of insulin suppression of hepatic glucose production, hepatic IRa and IRb subunit expression and Akt and GSK phosphorylation were decreased, and liver glycogen content was lowered >85%.  However, there was also evidence of markedly impaired GCGR function in GNELKO mice, with serum glucagon levels raised 18-fold and attenuation of hepatic cAMP production, CREB phosphorylation and gluconeogenesis in response to glucagon.  Regarding extrahepatic mechanisms, GNELKO mice displayed enhanced peripheral glucose disposal, and this was associated with increased IRb, Akt and GSK phosphorylation in skeletal muscle, and with serum FGF21 levels elevated by 406%. In additional studies, when invoked following 12 weeks of high fat diet feeding, liver GNE silencing caused dramatic improvements in glucose tolerance and insulin sensitivity.  To evaluate the relevance of these findings to humans, liver GNE transcript levels were queried in a publicly available cohort, and they were higher in T2DM patients compared to non-diabetic controls in two independent cohorts (n= 54 and n=9 for Cohort I and n=4 and n=4 for Cohort II, respectively). Collectively these observations reveal that protein sialylation in the liver has dramatic impact on both insulin and glucagon action in the liver, and on peripheral glucose disposal.  Further interrogation of the modulation of glucose control mechanisms by protein sialylation may lead to novel approaches to combatting glucose intolerance in T2DM and other conditions.

technical paper

Congenital unilateral defects of male reproductive tracts have been documented in men, suggesting the potential left-right asymmetry in male reproductive tract development. However, classic textbooks of endocrinology have been teaching us that the development of the paired male reproductive tract is governed by the universal action of the androgen receptor (Ar). During sexual differentiation, testis-derived androgens activate the Ar to promote the stabilization of the bilateral Wolffian ducts, the progenitor for the male reproductive tract. The Wolffian ducts then differentiate into the paired male reproductive tract organs, which include the epididymes, vas deferentes, and seminal vesicles. The Ar is expressed both in the epithelium and mesenchyme of the male reproductive tract during fetal development. Landmark tissue recombinant studies and the observation of normal morphology in the epithelium-specific Ar knockout mouse all support the notion that Ar action in the mesenchyme dictates the morphogenesis of the male reproductive tract. However, no genetic study has been performed to investigate the consequence of the loss of mesenchymal Ar on male reproductive tract development. To test the functional significance of the mesenchymal Ar, we designed a mesenchyme-specific Ar knockout mouse model (ARcKO) to ablate Ar specifically in the mesenchyme prior to the initiation of sexual differentiation. We performed immunohistochemistry of AR to confirm that Ar expression was absent in the mesenchyme while epithelial Ar remained intact in this ARcKO model. Based on the widely accepted notion that mesenchymal Ar regulates epithelial morphogenesis of the male reproductive tract, we expected to see abnormal patterning of the male reproductive tract. Indeed, the epididymis lost its characteristic coiling and became cystic. However, 40% of the 23 collected ARcKO males displayed these abnormalities on both horns; 43% and 17% displayed these abnormal phenotype solely on the left and right horn, respectively. This surprising observation might result from asynchronous activities of Cre on the left and right horns. However, when the Cre was crossed with a reporter (tdTomato), we observed comparable reporter expression on both horns at the onset of sexual differentiation of reproductive tracts. These observations suggest that the developmental programs in the morphogenesis of the left and right male reproductive tracts are asymmetrical and there could be a compensation mechanism for the loss of mesenchymal AR. We are currently in the process of comparing the left and the right horns in Ar knockout male mice to determine any biased signaling. Taken together, our study provides a unique model for not only studying congenital defects of male reproductive tracts but also for investigating the potential asymmetry in the masculinization program.

Bilateral and Unilateral Malformations of Male Reproductive Tracts in Mice with Androgen Receptor Gene Deletion in the Mesenchyme

Despite the frequency of hypospadias (1: 125 live male births) often the etiology is not known. The Lamb Laboratory previously determined that RNA Binding Fox-1 Homolog 2 (RBFOX2) copy number variation (CNV) is associated with clinical cases of upper and lower tract genitourinary (GU) anomalies, including hypospadias. RBFOX2 encodes a RNA binding protein that regulates mRNA splicing. The hypothesis was tested that altered RBFOX2 gene dosage impacts the developing penis transcriptome via mRNA splicing, specifically the mesenchymal to epithelial transition critical for penile urethra formation, resulting in hypospadias.Searches, in publically available and local clinical databases, centered on hypospadias occurance with gene CNV: identified the RBFOX2 locus as a gene dosage sensitve region. To identify developmental pathways regulated by RBFOX2 in the GU system, Rbfox2 haploinsufficient and null mouse models were developed, and penises were collected for RNA-seq, in situ hybridization, and micro-CT analysis of urethral development. In situ hybridization together with re-analysis of published scRNA-seq datasets were employed to define the anatomical regions of Rbfox2-specific expression in the developing mouse penis [Amato & Yao, PNAS 118(25): e2103856118, 2021; Armfield & Cohn, Dev Biol 477: 145-154.2021].Publicly available clinical data identified 17 patients (0.068% n=24,896) with pathogenic RBFOX2 CNVs: 4 were noted to have hypospadias. Results from our cohort of non-syndromic GU birth defect patient samples identified 11 patients (2.4% n=450) with RBFOX2 CNVs: 21% of whom have hypospadias. RNA immunoprecipitation agianst RBFOX2 in human kidney cells validated mRNA transcipt targets previously identified in cardiac cells [Verma 2016]. Micro-CT analysis on fetal Rbfox2-haploinsufficient mice validated the modeled clinical phenotype. Combined in situhybridization and scRNA-seq spatial analysis on mouse samples revealed, after penile sex determination (E15.5), Rbfox2 expression becomes restricted to the penile urethral mesenchyme. Differential gene expression and transcript usage analysis on the Rbfox2-loss mouse penis transcriptome identified mis-expression of gene products associated the estrogen signalling pathway.Alterations in RBFOX2 gene dosage clinically correlate with upper and lower tract genitourinary anomalies, specifically hypospadias. Data from human cell lines and mouse models seemingly indicate the phenotype seems is partially the product of an RBFOX2 role in directing the mesenchymal to epithelial transition. Specifically our data support the a direct role of RBFOX2 in balancing the competing male and female pathways at the level of estrogen and androgen signaling pathways. We are currently using immunofluorescence experiments to define the changes in androgen-receptor, estrogen-receptor, and RBFOX2 patterning throughout perinatal penis development.

RBFOX2 Copy Number Variation Disrupts Urethral Development Causing Hypospadias

The presence of insulin-like growth factor 2 (IGF2) mRNA has been demonstrated in human and mouse spermatozoa. However, the expression of the IGF2 protein in spermatozoa and its function is not known. To accomplish this, we searched for IGF2 protein in spermatozoa of four healthy Caucasian men by Western blot (WB) and immunofluorescence (IM). Semen samples were collected by masturbation into a sterile container after 3-4 days of sexual abstinence. The spermatozoa were separated from leucocytes and epithelial cells by swim-up. The WB demonstrated the presence of the IGF2 protein in each sample examined. IM showed that IGF2 is a cytoplasmic protein with a variable degree of expression in each spermatozoon and localized in the equatorial and post-acrosomal segments. To evaluate the effects of IGF2, porcine Sertoli cells (SCs) were isolated from neonatal Large White pigs (7-15 days) using established methods. SCs were incubated with increasing concentrations (0.33, 3.33, and 10 ng/mL) of recombinant human IGF2 (rhIGF2) for 48 hours. At the end of incubation, the following endpoints were evaluated: a) expression of the GDNF, FGF2, and SCF genes, known to be mitogens that promote gonocyte proliferation and differentiation towards their spermatogonial fate, by real-time PCR (RT-PCR); b) gene (RT-PCR) and protein (WB and IM) expression of the follicle-stimulating hormone receptor (FSHR); and c) proliferation of SCs by flow cytometry. Subsequently, the effects of IGF2 were re-evaluated by pre-treating SCs with NVP-AEW541, a not competitive inhibitor of the insulin-like growth factor 1 receptor (IGF1R), known to be activated by IGF2. For this purpose, the NVP-AEW541 was added to the culture medium at a concentration of 1 µg/mL one hour before the IGF2 and was left for the entire duration of the incubation. The results of this study showed that IGF2 significantly downregulates GDNF gene expression in a concentration-dependent manner (-40.3% at 0.33 ng/mL, -48.2% at 3.33 ng/mL, -55.5%, respectively, p<0.01). Gene expression of FGF2 and SCF was significantly downregulated only by IGF2 at the highest concentration used (-23.6% and -39.1%, respectively, p<0.01). Similarly, IGF2 incubation downregulated FSHR mRNA (-27.1% at 0.33 ng/mL, -45.4.2% at 3.33 ng/mL, -21.1%, respectively, p<0.01) and protein (for both WB and IM) expression. Finally, SC proliferation significantly decreased after incubation with IGF2 (2.82±0.1% in untreated controls, -1.87±0.1% at 0.33 ng/mL, -2.19±0.2% at 3.33 ng/mL, and -2.36±0.1% at 10 ng/mL; p<0.01). In the presence of NVP-AEW541, all these effects were counteracted, thus suggesting that they are mediated by the IGF1R. In conclusion, these results suggest the presence of a paracrine regulatory mechanism between germ cells and SCs by which the former can influence the proliferation of SCs, their sensitivity to FSH, and their function by reducing the secretion of germ cell mitogens.

Sperm-carried IGF2 Downregulates Mitogens Released by Sertoli Cells: A Paracrine Mechanism of Spermatogenetic Regulation?

Introduction: The impact of testosterone (T) on reproductive potential is poorly understood. Masculinizing hormones have demonstrated mixed effects on ovarian histology and data on assisted reproductive outcomes are limited to small case series. There is consensus among major medical societies to encourage fertility preservation counseling prior to initiation of gender-affirming hormone care, however, it is uncertain whether a break in T treatment prior to undergoing oocyte cryopreservation is beneficial. We hypothesized that T treatment would not have an impact on IVF outcomes.Methods: C57BL/6N female mice were implanted with silastic tubing with either 10 mg testosterone enthanate in ethanol (n = 20) or ethanol alone (n = 18) at 10 weeks of age. Four groups of C57BL/6N female mice were used in this study: 1) current T implant 2) current sham implant 3) T cessation 4) control cessation. Biweekly blood samples and vaginal cytology were undertaken to monitor T levels and estrous cycles. For groups 1 and 2, mice were stimulated twelve weeks following implantation. For groups 3 and 4, implants were removed after twelve weeks and mice were stimulated two weeks after explantation. All mice were stimulated with 0.2 mL intraperitoneal CARD HyperOva followed 48 hours later by 7.5 IU intraperitoneal human chorionic gonadotropin (hCG). Oocytes were collected 14 hours after hCG injection and fertilized in vitro with sperm from 12-week-old B6D2F1/J male mice. 2-cell embryos were transferred into the oviducts of pseudopregnant recipient females. The study was powered to detect a 25% difference in 2-cell embryos. Data were analyzed using Chi-squared and unpaired t-tests with Prism 9.0.Results: All T treated mice ceased cycling within one week of implantation and developed clitoromegaly. The current T implant group had fewer total oocytes retrieved (17.00 vs 36.00; p < 0.0001), mature oocytes (13.00 vs 28.10; p = 0.0002) and 2-cell embryos (12.78 vs 26.90; p = 0.0007) as compared to the current sham implant group. There was no significant difference in maturity (76.47% vs 78.06%; p = 0.6937) or fertilization rate (75.16% vs 74.72%; p = 0.9161). Pseudopregnant females who had 2-cell embryos transferred from current T implant mice were less likely to have a live birth than those with transfers from current sham implant mice (25.00% vs 80.00%; p = 0.0196). In contrast, comparison of IVF outcomes in the T cessation group with corresponding control cessation group revealed no significant differences between total oocytes (22.30 vs 30.75; p = 0.0708), mature oocytes (20.80 vs 27.75; p = 0.1288), or 2-cell embryos (20.40 vs 27.38; p = 0.104).Conclusion: In a mouse model of gender-affirming testosterone, active T treatment negatively impacted IVF outcomes. Improved outcomes following cessation of T treatment support reversibility of T impact on reproductive potential.

Active Testosterone Treatment Impairs In Vitro Fertilization (IVF) Outcomes in a Female Mouse Model for Gender-Affirming Testosterone with Improvement in Outcomes Following Cessation of Testosterone Treatment

Background: The cardiovascular safety of testosterone treatment in low testosterone is widely acknowledged to be unclear. Testosterone increases haematocrit, thereby potentially increasing venous thromboembolism risk. The FDA lists cardiovascular risk and stroke as adverse effects of testosterone. Systematic reviews and meta-analyses of published data have limited ability to confirm source data quality and categorisation. Some published meta-analyses have included participants with distinct risk profiles (e.g. cancer, HIV), with serum testosterone atypical for hypogonadism (>12nmol/L), short durations of testosterone treatment, and studies without placebo treatment. Furthermore, subtypes of cardiovascular or cerebrovascular events (e.g. stable angina) during testosterone treatment are seldom published, so have not been analysed previously.Objective: Evaluate frequencies of all-cause mortality, and all cardiovascular or cerebrovascular event subtypes, and analyse efficacy of testosterone monotherapy compared to placebo for men with low testosterone, using individual patient data (IPD) and aggregate data meta-analyses.Methods: MEDLINE, EMBASE, Science Citation Index, CENTRAL and clinical trial registries (PROSPERO CRD42018111005) were searched for placebo-controlled RCTs including men with serum testosterone <12nmol/L. One-stage meta-analyses were performed for studies providing IPD and two-stage meta-analyses were performed to integrate IPD and aggregated data. Primary outcomes were all-cause mortality and cardiovascular and/or cerebrovascular events at 12 months or nearest time point.Results: IPD were obtained from 17 of 35 eligible RCTs (3431/5601 participants) in men with low testosterone. Most participants had functional hypogonadism. Risks of cardiovascular and/or cerebrovascular events were similar between the testosterone and placebo arms (testosterone 120/1601, 7.5%; placebo, 110/1519, 7.2% OR 1.07, 95% CI 0.81-1.42 p=0.62). Frequencies of all cardiovascular or cerebrovascular event subtypes were also similar between testosterone and placebo arms, but testosterone increased risks of oedema and erythrocytosis. No subgroups at higher cardiovascular and/or cerebrovascular event risk were identified. Fewer deaths were recorded in the testosterone arm, but this difference was non-significant (testosterone, 6/1621, 0.4%; placebo, 12/1537, 0.8% OR 0.46, 95% CI 0.17-1.24 p=0.13). Testosterone significantly reduced serum total cholesterol, high-density lipoprotein (HDL), and triglycerides versus placebo. No significant differences in serum low-density lipoprotein (LDL), blood pressure, glycaemic parameters, diabetes incidence or prostate outcomes were observed between groups. Testosterone had positive, albeit varied, effects on quality of life and sexual function.Conclusions: This is the most comprehensive study to date interrogating the safety of testosterone treatment in men with low testosterone. We were unable to find evidence from our IPD meta-analyses that testosterone increases risks of mortality or cardiovascular and/or cerebrovascular events in the short- to medium-term in men with low testosterone, most of whom have various forms of functional hypogonadism. These data provide some reassurance to men with low testosterone and their clinicians about the safety of testosterone in the short-to-medium term although more long-term data are required.Acknowledgement: NIHR TestES Consortium

Adverse Cardiovascular Events and Cause Mortality in Men During Testosterone Treatment: Individual Patient and Aggregate Data Meta-Analyses

Context: Male hypogonadism adversely affects body composition, bone mineral density (BMD), and metabolic health. A previous report showed that pre-treatment testosterone (T) levels of <200 ng/dL is associated with greater improvement in spine BMD with T therapy. However, to date, there is no study investigating whether baseline T levels also influences body composition and metabolic response to T therapy.Objective: To determine if there are differences in the changes in body composition, metabolic profile, and bone turnover markers, in addition to BMD, in response to T therapy in men with baseline T level of < 264 ng/dL compared to those with levels ≥264ng/dLMethods: Secondary analysis of single-arm, open-label clinical trial (NCT01378299) on pharmacogenetics of response to T therapy conducted between 2011-2016 involving 105 men (40-74 years old), with average morning T < 300 ng/dL, given intramuscular T cypionate 200 mg every 2 weeks for 18 months. Subjects were divided into those with baseline T levels of < 264 ng/dL (N=43) and those with ≥264 ng/dL (N=57). T and estradiol (E2) were measured by liquid chromatography/mass spectrometry; serum bone turnover markers (C-telopeptide [CTX], osteocalcin, sclerostin), adiponectin, and leptin by enzyme-linked immunosorbent assay; glycated hemoglobin (HbA1c) by high-performance liquid chromatography, areal BMD, and body composition by dual-energy x-ray absorptiometry (DXA).Results: Men with T < 264 ng/dL showed greater increases in total fat-free mass (FFM) at 18 months compared to those with T ≥ 264 ng/dL (4.2±4.1 vs 2.7±3.8%; p=0.047) and unadjusted appendicular FFM at 6 and 18 months (8.7±11.5 vs 4.4±4.3%, 7.3±11.6 vs 2.4±6.8 %; p=0.033 and p=0.043, respectively). Men with T ≥ 264 ng/dL showed significant decreases in HbA1c at 12 months (-3.1±9.2 vs 3.2±13.9%; p=0.005), fasting glucose at 18 months (-4.2±31.9 vs 13.0±57.3%; p=0.040), LDL at 6 months (-6.4±27.5 vs 12.8±44.1%; p=0.034), leptin at 18 months (-40.2±35.1 vs -27.6±31.0%; p=0.034) compared to those with T < 264 ng/dL. No significant differences in BMD and bone turnover markers were observed.Conclusion: T therapy results in improvement in body composition irrespective of baseline T levels but T <264 ng/dL is associated with greater improvement in FFM, whereas T level of ≥264 ng/dL favors improvement in metabolic profile

Testosterone Therapy Results in Greater Improvement in Body Composition and Metabolic Profile in Hypogonadal Men with Testosterone Levels of < 200 ng/dL Compared To Those with ≥200 ng/dL

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.

Alternative Polyadenylation as a Therapeutic Vulnerability in Prostate Cancer

Background: Phosphorylation of the mineralocorticoid receptor (MR) at serine 843 inhibits its activity. mTOR phosphorylation of ULK1 prevents this MR phosphorylation.
Purpose: To study the relationship between mTOR and MR and its role in the modulation of aldosterone signaling.
Methods: Mouse cortical collecting duct M1 cells stably expressing the rat wild type MR or mutated MR (mu/S843A) and a Gaussia luciferase gene reporter under a hormone response promoter tyrosine aminotransferase (TAT) were incubated with mTOR activator and inhibitors in the presence and absence of aldosterone or corticosterone. Co-immunoprecipitation was performed to study protein interactions with the MR, and a phospho-serine antibody was used to demonstrate MR and ULK1 phosphorylation. Effects of mTOR knockdown in M1 cells by lentiviral transduction with CRISPR/gRNA for Raptor and Rictor were also studied.
Results: mTOR and the adaptor proteins of mTOR complex 1 and 2, Raptor and Rictor, respectively, co-precipitated with the MR of M1-rMR cells. Expression levels of these proteins were not changed by aldosterone. mTOR inhibitors significantly reduced ligand-induced MR reporter gene transactivation, however the mTOR activator MHY1485 had no effect. CRISPR/gRNA gene knockdown of Raptor or Rictor also inhibited MR activation stimulated by ligands, supporting a role of mTOR in maintaining the active MR. The mTOR inhibitor AZD8055 attenuated ligand-mediated MR gene transcription assessed by the reporter gene product and measurement of products of the endogenous MR target genes ACSBG1, LCN2 and Psca. AZD8055 also decreased phospho-ULK1 levels in a dose-dependent manner. We thus assumed that mTOR decreases ULK1 activity and prevents ULK1 phosphorylation of the MR at Serine (S843). Indeed, phosphorylation of the MR was increased in M1-rMR cells treated with AZD8055. However, AZD8055 still inhibited ligand-induced MR activity in cells carrying the mutated MR in which the Serine at 843 was replaced with an Alanine. These results suggest that mTOR has an additional role in MR activity unrelated to the specific phosphorylation at S843 by ULK1. This merits further investigation.

mTOR Interacts with the Mineralocorticoid Receptor to Regulate its Activation by Ligand

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 > 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.

USP19 Deubiquitinating Enzyme Functions as a Novel Co-Chaperone of Hsp90 to Regulate Glucocorticoid Receptor Levels

Critically ill patients are thought to develop maladaptive glucocorticoid-resistance which convinces many clinicians to administer stress doses of glucocorticoids to overcome this state of glucocorticoid-resistance. However, supportive data arises mainly from whole blood cells. It is currently not known if the observed changes in regulators and markers of glucocorticoid signaling and activity are also present in other cell and tissue types with a role in critical illness. We quantified regulators and markers of glucocorticoid signaling and activity in several cell and tissue types in critically ill humans and animals and in healthy controls. We found that throughout critical illness, glucocorticoid activity appeared suppressed in neutrophils, but upregulated in monocytes and skeletal muscle. Also in vital tissues GRα-signaling was altered in a tissue-specific, largely time-independent manner. Increasing systemic glucocorticoid availability increased glucocorticoid activity in adipose tissue, diaphragm and lung, whereas in immune cells and other tissues regulatory pathways counteracted. These data argue against glucocorticoid-treatable generalized glucocorticoid resistance and rather point towards an adaptive response in each specific cell or tissue type to optimally guide the beneficial actions of glucocorticoids to the tissues that need it while protecting collateral undesirable effects in tissue that are harmed by elevated systemic glucocorticoid availability.

Glucocorticoid Receptor Expression and Signaling During Critical Illness, in Relation to the Duration of Illness and the Systemic Glucocorticoid Availability: A Prospective, Observational, Cross-Sectional Human and a Translational Mouse Study

Liver-specific inhibition of sialic acid biosynthesis improves systemic glucose homeostasis in mice

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