Androgen Signaling Research Articles

Impact of germline DNA repair gene variants on prognosis and treatment of men with advanced prostate cancer

The clinical importance of germline variants in DNA repair genes (DRGs) is becoming increasingly recognized, but their impact on advanced prostate cancer prognosis remains unclear. A cohort of 221 newly diagnosed metastatic castration-resistant prostate cancer (mCRPC) patients were screened for pathogenic germline variants in 114 DRGs. The primary endpoint was progression-free survival (PFS) on first-line androgen signaling inhibitor (ARSI) treatment for mCRPC. Secondary endpoints were time to mCRPC progression on initial androgen deprivation therapy (ADT) and overall survival (OS). Twenty-seven patients (12.2%) carried a germline DRG variant. DRG carrier status was independently associated with shorter PFS on first-line ARSI [HR 1.72 (1.06–2.81), P = 0.029]. At initiation of ADT, DRG carrier status was independently associated with shorter progression time to mCRPC [HR 1.56, (1.02–2.39), P = 0.04] and shorter OS [HR 1.99, (1.12–3.52), P = 0.02]. Investigating the contributions of individual germline DRG variants on PFS and OS revealed CHEK2 variants to have little effect. Furthermore, prior taxane treatment was associated with worse PFS on first-line ARSI for DRG carriers excluding CHEK2 (P = 0.0001), but not for noncarriers. In conclusion, germline DRG carrier status holds independent prognostic value for predicting advanced prostate cancer patient outcomes and may potentially inform on optimal treatment sequencing already at the hormone-sensitive stage.

P025 - The role of DNA repair genes mutational status in prostate cancer treatment with androgen signaling blockade

P025 - The role of DNA repair genes mutational status in prostate cancer treatment with androgen signaling blockade

Single-dose 177Lu-PSMA-617 followed by maintenance pembrolizumab in patients with metastatic castration-resistant prostate cancer: an open-label, dose-expansion, phase 1 trial

Checkpoint inhibitors have been shown to have limited activity in patients with metastatic castration-resistant prostate cancer. We aimed to determine whether a single dose of lutetium-177 [177Lu]-prostate-specific membrane antigen (PSMA)-617 (177Lu-PSMA-617) followed by maintenance pembrolizumab was safe and could induce durable clinical benefit. We did an open-label, dose-expansion, phase 1 study at the University of California, San Francisco (San Fransisco, CA, USA). Eligible patients were men aged 18 years or older with progressive metastatic castration-resistant prostate cancer who had an Eastern Cooperative Oncology Group performance status of 0 or 1, had progression on one or more androgen signalling inhibitors, and at least three PSMA-avid lesions on 68Ga-PSMA-11 positron emission tomography. In part A, patients were enrolled sequentially to one of three schedules in which a single dose of 177Lu-PSMA-617 (7·4 GBq) was given intravenously 28 days before (schedule 1), concomitant with (schedule 2), or 21 days after (schedule 3) the start of maintenance intravenous pembrolizumab (200 mg every 3 weeks). In part B, 25 patients were enrolled using the recommended phase 2 schedule. The primary endpoint in part A was determination of the recommended phase 2 schedule, and in part B, the objective response rate. The analysis set included all patients who received at least one dose of pembrolizumab or 177Lu-PSMA-617. This study is registered with ClinicalTrials.gov, NCT03805594. Between Aug 8, 2019 and May 7, 2022, 43 male patients were enrolled (n=18 part A [six patients per schedule]; n=25 part B), with a median follow-up of 16·5 months (IQR 12·2-21·9). Schedule 1 was selected as the recommended phase 2 schedule for part B, on the basis of safety and feasibility of administration observed in part A. In part B, 14 (56%; 95% CI 35-76) of 25 patients had a confirmed objective response. Two (5%) of 43 patients had a treatment-related adverse event of grade 3 or worse (grade 3 arthritis in schedule 2, grade 3 pneumonitis in schedule 3). One serious adverse event (one death due to aspiration pneumonia) and no treatment-related deaths were observed. A single priming dose of 177Lu-PSMA-617 followed by pembrolizumab maintenance was safe and had encouraging preliminary activity in patients with metastatic castration-resistant prostate cancer. Prostate Cancer Foundation, National Cancer Institute, Novartis Pharmaceuticals, and Merck.

Toxicological investigation of lilial

Lilial (also called lysmeral) is a fragrance ingredient presented in many everyday cosmetics and household products. The concentrations of lilial in the final products is rather low. Its maximum concentration in cosmetics was limited and recently, its use in cosmetics products was prohibited in the EU due to the classification as reproductive toxicant. Additionally, according to the European Chemicals Agency, it was under assessment as one of the potential endocrine disruptors, i.e. a substance that may alter the function of the endocrine system and, as a result, cause health problems. Its ability to act as an androgen receptor agonist and the estrogenic and androgenic activity of its metabolites, to the best of our knowledge, have not yet been tested. The aim of this work was to determine the intestinal absorption, cytotoxicity, nephrotoxicity, mutagenicity, activation of cellular stress-related signal pathways and, most importantly, to test the ability to disrupt the endocrine system of lilial and its Phase I metabolites. This was tested using set of in vitro assays including resazurin assay, the CHO/HPRT mutation assay, γH2AX biomarker-based genotoxicity assay, qPCR and in vitro reporter assays based on luminescence of luciferase for estrogen, androgen, NF-κB and NRF2 signalling pathway. It was determined that neither lilial nor its metabolites have a negative effect on cell viability in the concentration range from 1 nM to 100 µM. Using human cell lines HeLa9903 and MDA-kb2, it was verified that this substance did not have agonistic activity towards estrogen or androgen receptor, respectively. Lilial metabolites, generated by incubation with the rat liver S9 fraction, did not show the ability to bind to estrogen or androgen receptors. Neither lilial nor its metabolites showed a nephrotoxic effect on human renal tubular cells (RPTEC/TERT1 line) and at the same time they were unable to activate the NF-κB and NRF2 signalling pathway at a concentration of 50 µM (HEK 293/pGL4.32 or pGL4.37). Neither lilial nor its metabolites showed mutagenic activity in the HPRT gene mutation test in CHO-K1 cells, nor were they able to cause double-strand breaks in DNA (γH2AX biomarker) in CHO-K1 and HeLa cells. In our study, no negative effects of lilial or its in vitro metabolites were observed up to 100 µM using different in vitro tests.

Androgen receptor contributes to repairing DNA damage induced by inflammation and oxidative stress in prostate cancer.

Androgen deprivation therapy remains the first-line therapy option for prostate cancer, mostly resulting in the transition of the disease to a castration-resistant state. The lack of androgen signaling during therapy affects various cellular processes, which sometimes paradoxically contributes to cancer progression. As androgen receptor (AR) signaling is known to contribute to oxidative stress regulation, loss of AR may also affect DNA damage level and the response mechanism in oxidant and inflammatory conditions of the prostate tumor microenvironment. Therefore, this study aimed to investigate the role of AR and AR-regulated tumor suppressor NKX3.1 upon oxidative stress-induced DNA damage response (DDR) in the inflammatory tumor microenvironment of the prostate. Intracellular reactive oxygen species (ROS) level was induced by either inflammatory conditioned media obtained from lipopolysaccharide-induced macrophages or oxidants and measured by dichlorodihydrofluorescein diacetate. In addition to this, DNA damage was subsequently quantified by counting gH2AX foci using an immunofluorescence-based Aklides platform. Altered expression of proteins function in DDR detected by western blotting. Cellular levels of ROS and ROS-induced DNA double-strand break damage were analyzed in the absence and presence of AR signaling upon treatment of prostate cancer cells by either oxidants or inflammatory microenvironment exposure. The results showed that AR suppresses intracellular ROS and contributes to DNA damage recognition under oxidant conditions. Besides, increased DNA damage due to loss of NKX3.1 under inflammatory conditions was alleviated by its overexpression. Moreover, the activation of the DDR mediators caused by AR and NKX3.1 activation in androgen-responsive and castration-resistant prostate cancer cells indicated that the androgen receptor function is essential both in controlling oxidative stress and in activating the ROS-induced DDR. Taken together, it is concluded that the regulatory function of androgen receptor signaling has a vital function in the balance between antioxidant response and DDR activation.

Androgen receptor study in children with hypospadias in comparison with children with normal genitalia

Androgen receptors (ARs) have been localized to the penile skin, inner prepuce and urethra. Androgens play an important and vital role in the development of male urethra. Androgen signalling through the AR is critical for normal penile development. The objective of our study was to prospectively assess the AR staining score in children with hypospadias in comparison with children with normal genitalia. All children with hypospadias presenting to the Paediatric urological services for repair formed the study group. Children with normal genitalia and undergoing circumcision either for phimosis or for religious indications formed the controls. A piece of the foreskin was collected during surgery and were stained for immunohistochemistry. AR staining was expressed as an m-quick score. A total of 32 children (group I) underwent primary hypospadias repair and 24 children (group II) underwent circumcision during the study period. The mean m-quick score in patients with hypospadias was 219.96 and that of children undergoing circumcision was 90.04 (p <0.050). AR is overexpressed in patients with hypospadias when compared with patients with normal genitalia. Similarly, AR is significantly overexpressed in patients with proximal hypospadias when compared with distal hypospadias.

Revisiting the roles of cAMP signalling in the progression of prostate cancer.

Prostate cancer is one of the most common cancers in men and one of the top causes of death in men worldwide. Development and function of both normal prostate cells and early-stage prostate cancer cells are dependent on the cross-talk between androgen signalling systems and a variety of other transduction pathways which drive differentiation of these cells towards castration-resistance. One such signalling pathway is the ubiquitous cAMP signalling axis which functions to activate spatially restricted pools of cAMP effectors such as protein kinase A (PKA). The importance of both PKA and cAMP in the development of prostate cancer, and their interactions with the androgen receptor, were the focus of a review by Merkle and Hoffmann in 2010. In this updated review, we revisit this topic with analysis of current PKA-related prostate cancer literature and introduce novel information on the relevance of another cAMP effector, the exchange protein directly activated by cAMP (EPAC).

Cell Plasticity in a Mouse Model of Benign Prostate Hyperplasia Drives Amplification of Androgen-Independent Epithelial Cell Populations Sensitive to Antioxidant Therapy

Benign prostate hyperplasia (BPH) is caused by the nonmalignant enlargement of the transition zone of the prostate gland, leading to lower urinary tract symptoms. Although current medical treatments are unsatisfactory in many patients, the limited understanding of the mechanisms driving disease progression prevents the development of alternative therapeutic strategies. The probasin-prolactin (Pb-PRL) transgenic mouse recapitulates many histopathological features of human BPH. Herein, these alterations parallel urodynamic disturbance reminiscent of lower urinary tract symptoms. Single-cell RNA-sequencing analysis of Pb-PRL mouse prostates revealed that their epithelium mainly includes low-androgen signaling cell populations analogous to Club/Hillock cells enriched in the aged human prostate. These intermediate cells are predicted to result from the reprogramming of androgen-dependent luminal cells. Pb-PRL mouse prostates exhibited increased vulnerability to oxidative stress due to reduction of antioxidant enzyme expression. One-month treatment of Pb-PRL mice with anethole trithione (ATT), a specific inhibitor of mitochondrial ROS production, reduced prostate weight and voiding frequency. In human BPH-1 epithelial cells, ATT decreased mitochondrial metabolism, cell proliferation, and stemness features. ATT prevented the growth of organoids generated by sorted Pb-PRL basal and LSCmed cells, the two major BPH-associated, androgen-independent epithelial cell compartments. Taken together, these results support cell plasticity as a driver of BPH progression and therapeutic resistance to androgen signaling inhibition, and identify antioxidant therapy as a promising treatment of BPH.

THU544 Prostatic Acid Phosphatase Is Negatively Regulated By Androgens And Persists In Castrate-resistant Metastatic Prostate Cancer

Abstract Disclosure: V. Rajagopalan: None. A. Kirschenbaum: None. S. Yao: None. A. Levine: None. Abstract: Prostate cancer (PCa) is the most common cancer and second leading cause of cancer death in American men. Most patients with metastatic PCa respond initially to androgen deprivation therapy (ADT) but the majority will progress to lethal, metastatic, castrate-resistant PCa (mCRPC). The identification of markers of mCRPC that are not regulated or negatively regulated by androgens can serve as markers and therapeutic targets in mCRPC. We hypothesized that prostatic acid phosphatase (PAP), a protein phosphatase and 5’ectonucleotidase that is expressed in both the cytoplasm and transmembrane (TM-PAP) of PCa cells, is negatively regulated by androgen signaling and can serve as a therapeutic target. Methods: Protein expression of PAP was assessed with western blotting in PCa cell lines. TM-PAP is identified by sub-cellular fractionation analysis, confocal microscopy, and flow cytometry. Effects of androgens and anti-androgens on PAP expression were assessed in VCaP cells with dihydrotestosterone (DHT) and Enzalutamide treatment followed by PAP, AR and PSA protein expression determined with Western Blotting. Loss of function analysis of PAP was performed with siRNA-mediated silencing and functional analysis was evaluated with migration and colony formation assays. Transcriptional regulation of PAP was determined by real time qPCR analysis. ChiPseq was done to confirm the binding of AR to PAP promoter. Results: All forms of PAP including TM-PAP are expressed in VCaP cell line. However, we did not detect PAP protein expression in-vitro in the LNCaP, C42B or 22Rv1 cell lines. In VCaP cell lines, we demonstrated that androgens decreased while anti-androgens increased PAP expression in a time and dose dependent fashion. Androgenic regulation of PAP occurred, at least in part, at the transcriptional level. We successfully knocked down TM and secretory PAP expression in VCaP cells using siRNA technology and showed that loss of PAP decreased migration and colony formation. Conclusions: The identification of markers of mCRPC that not regulated or negatively regulated by androgen could help identify recalcitrant tumor cells and mount targeted delivery of therapeutic agents. In the VCaP cell line, derived from a patient with mCRPC, TM and cytosolic PAP is one such protein that is negatively regulated by androgen signaling and may therefore serve as a viable therapeutic target in mCRPC. Presentation: Thursday, June 15, 2023

SAT019 Ligand-independent Signaling And Migration Of Breast Cancer Cells Overexpressing Membrane Androgen Receptor, ZIP9

Abstract Disclosure: P. Thomas: None. The zinc transporter ZIP9 also functions as a membrane androgen receptor (mAR) that mediates androgen-dependent rapid signaling and pro- or anti-apoptotic, proliferative, and migratory responses in fish, rodent, and human cells. Testosterone activation of ZIP9 in two triple-negative breast cancer cell lines, MDA-MB-468 cells and MDA-MB-231 cells transfected with ZIP9 (231-ZIP9), induces G-protein-dependent second messenger signaling and increases in intracellular free zinc. However, the relative importance of these two signaling pathways in the regulation of pro- and anti-tumorigenic responses in these cell lines is unclear because both androgen signaling and zinc status influence tumorigenesis in breast tissues. The effects of overexpression of ZIP9 on free zinc status, G protein- and zinc-dependent regulation of migration, and migration marker expression in 231-ZIP9 cells in the absence of testosterone were compared to those observed in MDA-MB-231 cells transfected with vector alone (231-vect). Migration and invasion of 231-ZIP9 cells were significantly increased after 24-48 hr culture in serum-free media compared to 231-vect cells which was accompanied by a significant increase in free zinc levels. Treatment of 231-vect cells with a zinc ionophore also increased migration, whereas migration of 231-ZIP9 cells was attenuated by treatment with an intracellular zinc chelator, TPEN. Expression of the migration markers, MYL9 and CYR61, was increased in 231-ZIP9 after 24-48 hr culture, whereas expression of apoptotic markers was decreased compared to 231-vect controls. Proximity Ligation Analysis revealed that ZIP9 is closely associated with a stimulatory G protein (Gs) in 231-ZIP9 cells which is consistent with the finding that treatment with the adenylyl cyclase (AC) stimulator forskolin, further increased migration marker expression and migration compared to vehicle controls, whereas the PKA inhibitor, H-89, attenuated these responses as well as the increase in free zinc concentrations. Knockdown of ZIP9 expression in MDA-MB-468 cells which express relatively high levels of ZIP9 with siRNA decreased cell migration and invasion as well as free zinc levels, confirming a role of unliganded ZIP9 in migration in another breast cancer cell line. Treatment of 231-ZIP9 cells with 100 nM testosterone further stimulated CYR61 and MYL9 expression and cell migration which were blocked by co-treatment with a Gs inhibitor and TPEN. The results indicate that high ZIP9 expression in breast cancer cells causes androgen-independent increased cell migration and invasion which in 231-ZIP9 cells is through Gs/mAC/PKA-mediated increases in intracellular zinc concentrations and upregulation of MYL9 and CYR-61 expression. Furthermore, the results suggest that testosterone-dependent signaling through ZIP9 acts through the same pathways to further enhance signaling and migration. Presentation: Saturday, June 17, 2023

SAT051 The Effect Of High Fat Diet On Insulin Signaling In White Adipose Tissue Of Liver Androgen Receptor Knockout In Male Mice

Abstract Disclosure: J.O. Hill-Dick: None. D. Young: None. D. Curry: None. E. Bolarinwa: None. R. Qutab: None. K. Carr: None. C. Falzarano: None. S. Andrisse: None. Insulin resistance affects up to 33% of the US adult population and polycystic ovary syndrome affects up to 10% of reproductive-age adult women. The liver and white adipose tissue play an essential role in the metabolism of insulin and androgen signaling. Hyperandrogenism in females can increase their predisposition to insulin resistance. It has been previously shown that deleting the liver androgen receptor (LivARKO) prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. The goal of this research was to determine if LivARKO prevents high fructose diet (HFrD) induced insulin resistance. It was hypothesized that HFrD LivARKO male mice would display impaired insulin action in white adipose tissue in comparison to the control diet-fed LivARKO female mice, suggesting that AR does not play a significant role in regulating HFrD-induced insulin resistance. Male LivARKO mice were placed on either a control (Research Diets Inc, RDI D12450J) or High Fructose (HFrD, RDI D02022704) diet and sacrificed after 1 month. Half of the mice were given 0.5 U/kg of insulin before being sacrificed to investigate the effects of the diets on insulin signaling. Western blots were used to determine protein expression in tissue from the white adipose tissue (WAT) standardized using BCA assays. LivARKO male mice fed a chow or control diet for 1 month displayed no or low expression of p-AKT in the basal and insulin-stimulated state, suggesting that LivARKO had an indirect effect on insulin-stimulated signaling in WAT. Oddly, LivARKO male mice fed a HFrD displayed increased insulin-stimulated p-AKT compared to basal, suggesting functional insulin-stimulated p-AKT. This increased p-AKT may be a compensatory mechanism as HFrD is known to disrupt glucokinase and glycogen synthase. In normal physiology, on a chow diet, insulin should increase p-AKT. The data shows that in male LivARKO mice, insulin did not increase p-AKT in chow, control, or HFrD, suggesting that the male LivARKO mice are experiencing insulin resistance at the level of p-AKT in WAT. Additionally, HFrD is known to cause insulin resistance, however, it is not known to alter p-AKT levels. Thus, the change in p-AKT levels is presumably associated with the LivARKO. Further research is required into what components of the Control Diet are prompting this difference in insulin action and if it only takes place in the LivARKO mouse model. Presentation: Saturday, June 17, 2023

SAT054 LivARKO Female Mice On 1-month Of High Fat Diet (HFD) Did Not Experience Insulin Resistance At The Level Of p-AKT In WAT

Abstract Disclosure: D.F. Curry: None. E. Bolarinwa: None. J.O. Hill-Dick: None. D. Young: None. R. Qutab: None. K. Carr: None. C. Falzarano: None. S. Andrisse: None. Insulin resistance affects up to a third of the US adult population and polycystic ovary syndrome affects up to 10% of reproductive-age adult women. The liver and white adipose tissue play an essential component in the metabolism of insulin and androgen signaling. Hyperandrogenism in females can increase predisposition to insulin resistance. Andrisse et al 2021 showed that deleting the liver androgen receptor (LivARKO) prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. The goal of this research was to determine if LivARKO prevents high fat diet (HFD) induced insulin resistance.It was hypothesized that (unlike HA-LivARKO) HFD LivARKO female mice would display impaired insulin action (lowered insulin-stimulated p-AKT) in white adipose tissue in comparison to the Control diet (CD) fed LivARKO female mice, suggesting that AR does not play a significant role in regulating HFD-induced insulin resistance. Female LivARKO mice were placed on two diets for 1 month: CD and HFD. The mice were then sacrificed. Half of the mice were given a dose of 0.5 U/kg insulin before sacrificing to investigate the effects of the diets on insulin signaling. Western blots were used to determine protein expression in tissue from the WAT. BCA assays were used to standardize the protein concentration in each sample. Insulin action can be measured molecularly by examining p-AKT Serine 473 (positive regulator, Santa Cruz sc-514032) and p-IRS1 Serine 306 (negative regulator, Santa Cruz sc-33956). If p-AKT S473 levels increase in the presence of insulin, this indicates that insulin is likely functioning properly in the sample. Whereas if p-IRS1 S306 are elevated, this indicates malfunctioning insulin action or insulin resistance. Following a similar pattern as WT mice on a chow diet (White 2014, Fig. 4), insulin increased p-AKT in WAT of LivARKO female mice fed a CD for 1-month in comparison to LivARKO female mice not given insulin (basal). Following the pattern of WT mice on a HFD (White 2014), LivARKO female mice fed a HFD for 1-month, displayed a similar increase in insulin-stimulated p-AKT in the WAT compared to the Con-Ins group. p-IRS-1 was not detected in the WAT of LivARKO female mice fed a control diet or HFD. In conclusion, LivARKO female mice on 1-month of HFD were not experiencing insulin resistance at the level of p-AKT in WAT. Further studies in different energy storage tissues in this model are warranted. Presentation: Saturday, June 17, 2023

FRI024 The Effect Of High Fat Diet On Insulin Signaling In White Adipose Tissue Of Liver Androgen Receptor Knockout In Female Mice

Abstract Disclosure: D. Young: None. Introduction: Insulin resistance affects up to a third of the US adult population and polycystic ovary syndrome affects up to 10% of reproductive-age adult women. The liver and white adipose tissue play an essential component in the metabolism of insulin and androgen signaling. Hyperandrogenism in females can increase predisposition to insulin resistance. Andrisse et al 2021 showed that deleting the liver androgen receptor (LivARKO) prevented female mice from developing hyperandrogenemia-induced insulin resistance. We wanted to know does LivARKO prevent high fructose diet (HFrD) induced insulin resistance? In our study, we placed female LivARKO mice on high fructose diets (HFrD) to determine if they demonstrated blunted insulin resistance. We hypothesized was that HFrD LivARKO female mice would display impaired insulin action in white adipose tissue. We needed to demonstrate that insulin signaling was functional in LivARKO mice on a control and a HFrD. There are six groups in this study: wild type (WT) no treatment, WT treatment-1, WT treatment-2, LivARKO no treatment, LivARKO treatment-1, and LivARKO treatment-2. No treatment is a chow diet, treatment-1 is a control diet, and treatment-2 is HFrD. Methods: Female LivARKO mice were placed on two diets: Control (Con, Research Diets Inc, RDI D12450J and High Fructose (HFrD, RDI D02022704. The mice were sacrificed after 1 month on the diet. Half of the mice were given a dose of 0.5 U/kg insulin before sacrificing to investigate the effects of the diets on insulin signaling. Western blots were used to determine protein expression in tissue from the white adipose tissue. BCA assays were used to standardize the protein concentration in each sample. Insulin action was measured molecularly by examining p-AKT Serine 473 ( Santa Cruz sc-514032) and p-IRS1 Serine 306 ( Santa Cruz sc-33956). If p-AKT S473 levels increase in the presence of insulin, indicating that insulin is likely functioning properly. If p-IRS1 S306 are elevated, indicates malfunctioning insulin or resistance. Results: LivARKO Female mice on a control diet did not express any insulin stimulated p-AKT in WAT, LivARKO Female mice on a high fructose diet displayed increased insulin stimulated p-AKT compared to LivARKO HFrD mice not given insulin. P-IRS1 was increased in the WAT of the Control Insulin group compared to the WAT of the Control Basal group of female LivARKO mice. Once placed on a HFrD, p-IRS1 was not present in the WAT either group of LivARKO mice. Conclusion: In conclusion, the data is inconclusive. HFrD appeared to augment insulin-stimulated p-AKT but lowered p-IRS1 in WAT of female LivARKO mice. There were several limitations to this study. We do not have data for LivARKO on chow or data for WT on control diet to decipher these effects. Further research is required into what components of the Control Diet are prompting this difference in insulin action. Presentation: Friday, June 16, 2023

SAT055 The Effect Of High Fat Diet On Insulin Signaling In White Adipose Tissue Of Liver Androgen Receptor Knockout In Female Mice

Abstract Disclosure: E. Bolarinwa: None. D. Young: None. J.O. Hill-Dick: None. D.F. Curry: None. K. Carr: None. R. Qutab: None. C. Falzarano: None. S. Andrisse: None. Insulin resistance affects up to a third of the US adult population and polycystic ovary syndrome affects up to 10% of reproductive-age adult women. The liver and white adipose tissue play an essential component in the metabolism of insulin and androgen signaling. Hyperandrogenism in females can increase predisposition to insulin resistance. Andrisse et al 2021 showed that deleting the liver androgen receptor (LivARKO) prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. This study places female LivARKO mice on high fructose diets (HFrD) to determine if LivARKO mice demonstrate blunted insulin resistance on HFrD compared to control diet. It was hypothesized that (unlike HA-LivARKO) HFrD LivARKO female mice would display impaired insulin action (lowered insulin-stimulated p-AKT) in white adipose tissue in comparison to the Control diet fed LivARKO female mice, suggesting that AR does not play a significant role in regulating HFrD-induced insulin resistance. Female LivARKO mice were placed on two diets: Control (Con, Research Diets Inc, RDI D12450J) and High Fructose (HFrD, RDI D02022704). After 3 months on the diet, the mice were sacrificed. Half of the mice were given a dose of 0.5 U/kg insulin before sacrificing to investigate the effects of the diets on insulin signaling. Western blots were used to determine protein expression in tissue from the white adipose tissue (WAT). BCA assays were used to standardize the protein concentration in each sample. Insulin action can be measured molecularly by examining p-AKT Serine 473 (positive regulator, Santa Cruz sc-514032) and p-IRS1 Serine 306 (negative regulator, Santa Cruz sc-33956). If p-AKT S473 levels increase in the presence of insulin, this indicates that insulin is likely functioning properly in the sample. Whereas if p-IRS1 S306 are elevated, indicating malfunctioning insulin action or resistance. LivARKO female mice fed a chow or control diet for 3-months displayed no expression of basal or insulin stimulated p-AKT. LivARKO female mice fed a HFrD for 3-months displayed elevated AKT and similar p-AKT in WAT of HFrD Insulin mice compared to HFrD Basal treated mice. p-IRS1 was not detected in Control or HFrD female LivARKO mice. In conclusion, these data suggest that LivARKO indirectly disrupted insulin-stimulated p-AKT in WAT possibly via altering metabolites in the blood. Additionally, HFrD is known to cause insulin resistance, however, it is not known to alter p-AKT levels. Thus, the change in p-AKT levels is presumably associated with the LivARKO. Further research is required on what components of the Control Diet are prompting this difference in insulin action and if it only takes place in the LivARKO mouse model. Presentation: Saturday, June 17, 2023

THU573 Paxillin Regulates Androgen Receptor In Granulosa Cells

Abstract Disclosure: A. Weidner: None. A. Roy: None. K. Vann: None. O. Astapova: None. Paxillin is a ubiquitously-expressed adaptor protein that is integral in focal adhesions and plays an active role in the regulation of cell motility and apoptosis. Recently, paxillin was shown to mediate pro-tumorigenic androgen signaling in prostate cancer cells, where it is necessary for rapid cytoplasmic kinase signaling triggered by activation of membrane-bound androgen receptor. We sought to investigate the relationship between paxillin and androgen receptor (AR) in granulosa cells, where androgen actions, apoptosis and focal adhesions are of known importance, but the role of paxillin is understudied. When paxillin expression was silenced using siRNA in human granulosa-derived KGN cells, AR protein expression was significantly reduced, while its RNA expression was unaffected. Similarly, in granulosa cell-specific paxillin knockout mice, AR protein expression in granulosa cells was reduced. Further, we found that AR and paxillin co-localize in focal adhesions in mouse primary granulosa cells and in KGN cells, and their interaction requires the activity of focal adhesion kinase. Disruption of focal adhesion with a focal adhesion kinase inhibitor also leads to reduced AR protein levels. To further explore the mechanisms by which paxillin regulates AR expression, we created paxillin-null KGN cell lines using CRISPR-Cas9 gene editing. While paxillin loss resulted in reduced AR protein expression early (days) in the gene-editing process, this effect disappeared after weeks of clonal expansion from selected single cells. Expanded paxillin-null KGN clones showed normal AR protein expression, but increased AR mRNA expression, suggesting an adaptive increase in AR gene transcription to compensate for reduced AR protein levels in cells that survived paxillin deletion. Studies of AR protein degradation using cycloheximide to block protein synthesis showed that the half-life of AR was reduced from 6.7 to 4.5 hours (approximately 33%) by paxillin deletion, indicating accelerated AR protein degradation in cells lacking paxillin. Based on our results, we hypothesize that paxillin brings AR into focal adhesions in granulosa cells, thereby sequestering it from proteasomal degradation in the cytoplasm. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as polycystic ovary syndrome. Presentation Date: Thursday, June 15, 2023

OR16-04 Androgen Deprivation Restricts Anti-tumor Immunity In Adrenocortical Carcinoma

Abstract Disclosure: K.M. Warde: None. L. Smith: None. C.J. Stubben: None. P.W. Willet: None. G. Castaneda-Hernandez: None. L. Liu: None. K. Basham: None. Adrenocortical carcinoma (ACC) is a rare cancer that is often diagnosed late and progresses rapidly, resulting in routinely poor outcomes. Through The Cancer Genome Atlas (TCGA) and other genomic studies, homozygous loss of ZNRF3 was newly identified as a frequent genetic alteration in human ACC tumors. ZNRF3 is an E3 ubiquitin ligase that negatively regulates Wnt signaling by degrading Wnt receptors. To examine the role of ZNRF3 in adrenal biology, we generated a Znrf3 conditional knockout (cKO) mouse model. Despite roust early hyperplasia, we found that Znrf3 cKO mice only develop adrenal tumors with advanced aging. Moreover, tumors emerge in a sex dimorphic manner whereby males, who exhibit a high myeloid immune response, predominately get benign tumors and females, who have a dampened myeloid response, develop metastatic ACC. This phenomenon is reflected clinically in patients whereby the incidence of ACC is up to 2.5x higher in women compare to men. In human ACC patients, females exhibit a lower myeloid signature compared to males. Moreover, a low myeloid response signature is associated with shorter progression-free and overall survival. These data suggest a potential relationship between anti-tumor immunity and sex in ACC, yet the underlying mechanisms remain unclear. Sex differences are common in cancer, and although there are many potential factors, gonadal hormones including androgens and estrogens, have been implicated as major biological drivers. Previous studies of the adrenal cortex highlight a specific role for androgens in repressing stem cell recruitment and proliferation. We hypothesized androgens promote anti-tumor immunity to help protect against ACC. To test this hypothesis, we castrated male Znrf3 cKO mice to lower androgen levels. We found that androgen deprivation significantly reduced myeloid cell infiltration, resulting in significantly larger adrenals in castrated as compared to sham controls. RNA-seq analysis revealed a decrease in immune activation pathways and monocyte-derived myeloid cell populations. Additionally, pro-inflammatory cytokines, including TNF, INFG, and IL-6, were significantly inhibited in castrated animals compared to controls. These data support a protective role for androgens in ACC through driving an intra-adrenal myeloid immune response. Consistent with these results, analysis of the TCGA-ACC patient cohort demonstrated that high androgen receptor expression is associated with better overall survival. Taken together, our results reveal an important role for androgens in promoting anti-tumor immunity in a mouse model of ACC, and we highlight the prognostic value of androgen receptor expression in patients. Further understanding of androgen signaling and downstream biological mechanisms that regulate the immune response may ultimately offer novel therapeutics and improve patient outcomes in ACC. Presentation: Friday, June 16, 2023

FRI475 The Impact Of Suppression Of Androgen Signaling On Thyroid Hormone Homeostasis In Prostate Cancer Patients

Abstract Disclosure: K. David: None. G. Devos: None. W. Devlies: None. N. Narinx: None. L. Antonio: None. L. Deboel: None. D. Schollaert: None. D.M. Vanderschueren: None. F.A. Claessens: None. S. Joniau: None. B. Decallonne: None. Next to the classical androgen deprivation therapy (ADT) with GnRH-(ant)agonists, androgen receptor signaling inhibitors (ARSIs) such as apalutamide (APA) have become available for the treatment of prostate cancer (PC) patients in the last years. Hypothyroidism was more frequent in APA-treated patients compared to placebo in the SPARTAN trial (NCT01946204) (8.1% vs 2%). We collected serum of PC patients included in a RCT (ARNEO; NCT03080116) before and after 12 weeks of neo-adjuvant ADT (GnRH-antagonist degarelix (DGX) with APA or placebo). We aimed to explore changes in thyroid homeostasis under deep androgen suppression with combined GnRH-antagonist and ARSI. Of the 88 patients ending the trial, 1 was excluded because of history of hypothyroidism for which he received levothyroxine. 43 and 44 patients were included in the DGX+placebo and DGX+APA group, respectively. Mean age was 66.3y. Sex steroid levels measured by LC-MS/MS decreased significantly to castrate range with median testosterone 10.5 ng/dL and estradiol 3.6 ng/L. TSH levels increased in both treatment groups, but more pronounced in the DGX+APA group (DGX+placebo before: 1.18 mIU/L and after: 1.33 mIU/L, p 0.0351 vs. DGX+APA before: 1.07 mIU/L and after: 1.87 mIU/L, p <0.0001; difference between groups after treatment: p <0.0001). Free T4 and total T4 levels decreased in the DGX+APA group, but remained unaltered in the DGX+placebo group (free T4 after: 16.53 vs 13.87 pmol/L, p<0.0001 and total T4 after: 8.77 vs. 7.78 µg/dL, p 0.0055). Total T3 and free T3 increased in the DGX+APA group, and again did not differ after treatment in the DGX+placebo group. (free T3 after: 5.03 vs 5.41 pmol/L, p 0.0066 and total T3 after: 132.30 vs 149.50 ng/dL, p 0.0003). As a result, the total and free T3/T4 ratio increased in the DGX+APA group with 32% and 35%, respectively. Reverse T3 decreased in both treatment arms, but greater in the DGX+APA group (19.55 vs 14.20 ng/dL, p 0.0157). Thyroxine binding globulin (TBG) increased in both treatment arms, with 6.9% and 8.4%, respectively. In conclusion, interference with androgen signaling alters thyroid homeostasis in PC patients, and is most pronounced under deep androgen suppression with combined GnRH-antagonist and ARSI therapy. The increase in T3 and decrease in rT3 may indicate additional effects of ARSI on deiodinase activity. Longer follow-up trials are needed to confirm these findings and assess the clinical implications of these changes in thyroid homeostasis. Presentation: Friday, June 16, 2023

Castration promotes the browning of the prostate tumor microenvironment

BackgroundAdipose tissue has gained attention due to its potential paracrine role. Periprostatic adipose tissue surrounds the prostate and the prostatic urethra, and it is an essential player in prostate cancer progression. Since obesity is directly related to human tumor progression, and adipose tissue depots are one of the significant components of the tumor microenvironment, the molecular mediators of the communication between adipocytes and epithelial cells are in the spotlight. Although periprostatic white adipose tissue contributes to prostate cancer progression, brown adipose tissue (BAT), which has beneficial effects in metabolic pathologies, has been scarcely investigated concerning cancer progression. Given that adipose tissue is a target of androgen signaling, the actual role of androgen removal on the periprostatic adipose tissue was the aim of this work.MethodsSurgical castration of the transgenic adenocarcinoma of the mouse prostate (TRAMP) was employed. By histology examination and software analysis, WAT and BAT tissue was quantified. 3T3-like adipocytes were used to study the role of Casodex® in modifying adipocyte differentiation and to investigate the function of the secretome of adipocytes on the proliferation of androgen-dependent and independent prostate cancer cells. Finally, the role of cell communication was assayed by TRAMP-C1 xenograft implanted in the presence of 3T3-like adipocytes.ResultsAndrogen removal increases brown/beige adipose tissue in the fat immediately surrounding the prostate glands of TRAMP mice, concomitant with an adjustment of the metabolism. Castration increases body temperature, respiratory exchange rate, and energy expenditure. Also, in vitro, it is described that blocking androgen signaling by Casodex® increases the uncoupling protein 1 (UCP1) marker in 3T3-like adipocytes. Finally, the effect of brown/beige adipocyte secretome was studied on the proliferation of prostate cancer cells in vivo and in vitro. The secretome of brown/beige adipocytes reduces the proliferation of prostate cancer cells mediated partly by the secretion of extracellular vesicles.ConclusionsConsequently, we concluded that hampering androgen signaling plays a crucial role in the browning of the periprostatic adipose tissue. Also, the presence of brown adipocytes exhibits the opposite effect to that of white adipocytes in vitro regulating processes that govern the mechanisms of cell proliferation of prostate cancer cells. And finally, promoting the browning of adipose tissue in the periprostatic adipose tissue might be a way to handle prostate cancer cell progression.EECkevEGf-HMwkJP3j4bZsVideo

Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

Effects of Dibutylphthalate and Steroid Hormone Mixture on Human Prostate Cells.

Phthalates are a family of aromatic chemical compounds mainly used as plasticizers. Among phthalates, di-n-butyl phthalate (DBP) is a low-molecular-weight phthalate used as a component of many cosmetic products, such as nail polish, and other perfumed personal care products. DBP has toxic effects on reproductive health, inducing testicular damage and developmental malformations. Inside the male reproductive system, the prostate gland reacts to both male and female sex steroids. For this reason, it represents an important target of endocrine-disrupting chemicals (EDCs), compounds that are able to affect the estrogen and androgen signaling pathways, thus interfering with prostate homeostasis and inducing several prostate pathologies. The aim of this project was to investigate the effects of DBP, alone and in combination with testosterone (T), 17β-estradiol (E2), and both, on the normal PNT1A human prostate cell-derived cell line, to mimic environmental contamination. We showed that DBP and all of the tested mixtures increase cell viability through activation of both estrogen receptor α (ERα) and androgen receptor (AR). DBP modulated steroid receptor levels in a nonmonotonic way, and differently to endogenous hormones. In addition, DBP translocated ERα to the nucleus over different durations and for a more prolonged time than E2, altering the normal responsiveness of prostate cells. However, DBP alone seemed not to influence AR localization, but AR was continuously and persistently activated when DBP was used in combination. Our results show that DBP alone, and in mixture, alters redox homeostasis in prostate cells, leading to a greater increase in cell oxidative susceptibility. In addition, we also demonstrate that DBP increases the migratory potential of PNT1A cells. In conclusion, our findings demonstrate that DBP, alone and in mixtures with endogenous steroid hormones, acts as an EDC, resulting in an altered prostate cell physiology and making these cells more prone to cancer transformation.