Dihydrotestosterone Treatment Research Articles

Consumption of Soya Isoflavones Improved Polycystic Ovary Syndrome (PCOS)-Associated Metabolic Disorders in a Rat Model.

Polycystic ovary syndrome (PCOS) is associated with increased risks for certain metabolic disorders such as insulin resistance, non-alcoholic fatty liver disease (NAFLD), and suppressed ovarian follicular development. This study aimed to examine whether soya isoflavones (ISF) mitigate these PCOS-associated metabolic disorders in a rat model. Weanling Sprague-Dawley female rats were randomly divided into 6 groups and were treated with either 0 or 83 µg/day dihydrotestosterone (DHT) to induce PCOS and fed diets containing 0, 0.5, or 1g ISF/kg diet for 8 weeks. DHT treatment increased food intake, body weight gain (BWG, p<0.001), percentage of primordial follicles (60% vs 50.9%, p<0.05), and accumulation of lipid droplets in the livers. It also elevated serum total cholesterol (TC), free cholesterol (FC), triglycerides, non-esterified fatty acids (NEFA), and leptin, and hepatic TC and NEFA. Additionally, DHT treatment reduced the percentage of primary follicles (13.8% vs 30.2%, p<0.05), ovary weight, and length (p<0.001), as well as insulin sensitivity (p<0.01) compared to the Control. ISF intake at 1g/kg reduced BWG, serum TC, FC, NEFA, leptin, and hepatic triglycerides and DHT-induced insulin resistance (p<0.01). ISF intake at both levels decreased DHT-induced lipid droplet accumulation in the livers, and changes in the percentages of primordial and primary follicles. Dietary soya ISF alleviated DHT-induced BWG, insulin resistance, and hepatic lipid droplet accumulation, as well as suppressed ovarian follicular development. This suggests that consumption of soya foods or ISF supplements may be beneficial for the individuals with PCOS, mitigating the associated metabolic disorders such as diabetes and NAFLD.

Abstract PO1-13-06: Androgen Receptor Isoform V7 (AR-V7): A Promising Biomarker for Prognosticating Breast Cancer Aggressiveness

Abstract Androgen Receptor (AR) is an emerging endocrine therapy target in Breast Cancer (BrCa) with up to 80% expression in clinical cases. AR-V7 is a constitutively activated splice variant of AR having a truncated ligand-binding domain (LBD). This confers ligand-independent transcriptional activity to AR-V7 and makes it susceptible to nonsteroidal antiandrogens such as Bicalutamide or Enzalutamide which target the LBD of AR. Higher levels of AR-V7 in metastatic prostate cancer leads to therapeutic resistance and enhanced metastasis. We assessed AR-full length (AR-FL) and AR-V7 expression in BrCa cell lines of different molecular subtypes. This was extended for a clinical correlation of AR-FL and AR-V7 in validated treatment naïve BrCa patients undergoing surgical intervention at a tertiary care hospital in India. Transcriptomic and proteomic analysis was performed using qRT-PCR and Western Blotting. Immunocytochemistry and immunohistochemistry were used to examine protein expression and localisation in cells and tissues. AR-FL and AR-V7 were variably expressed in cell lines which got elevated with Dihydrotestosterone (DHT) treatment. Cell lines stimulated with Bicalutamide or Enzalutamide showed significant downregulation in AR-FL expression. From a cohort of 82 clinical cases, more than 50% originated from the urban setting, post-menopausal stage, and in the age groups of 61 to 70 years. ER, PR, HER2, and AR were positive in 65%, 55%, 24%, and 67% respectively. We were able to correlate aggressive clinical traits and higher pathological grades with increased expression of AR-V7 in the AR-positive BrCa fraction. This was consistent with the cell line-based preclinical research. This study affiliates AR-V7 as an important prognostic biomarker for predicting breast cancer aggressiveness. It can serve as a screening marker of poor clinical outcomes and aid in appropriate therapeutic intervention. These outcomes, however, seek extensive prospective validation. Citation Format: Tryambak Pratap Srivastava, Joyeeta Talukdar, Rohit Kumar Verma, Subhradip Karmakar. Androgen Receptor Isoform V7 (AR-V7): A Promising Biomarker for Prognosticating Breast Cancer Aggressiveness [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-06.

Role of Adiponectin in the Androgen-Mediated Cardiometabolic Complications in a Rat Model of Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is associated with a high prevalence of obesity, adipose insulin resistance (adipose IR), and insulin resistance (IR). Adipose IR is an early metabolic defect in the development of whole-body IR. Specific evaluation of adipose IR and its consequences on the whole-body metabolic state in women with PCOS has not been thoroughly investigated. Adiponectin is a peptide secreted by adipocytes that increases insulin sensitivity. PEG-BHD1028 is a novel potent peptide adiponectin receptor agonist. Despite that PEG-BHD1028 modulates insulin sensitivity in different models of diabetes, the potential of PEG-BHD1028 to attenuate androgen-mediated adipose IR and obesity remains unknown. Hypothesis: We tested the hypothesis that PEG-BHD1028 attenuates adipose IR and obesity in a rat model of PCOS. Methods: To induce PCOS, four-week-old female Sprague Dawley rats were implanted with dihydrotestosterone (DHT) silastic tubes (7.5 mg) or placebo (PBO) for 90 days (n=4-8/group). During the final three weeks of DHT treatment, rats received PEG-BHD1028 (25 μg/kg/day) subcutaneously. Body weight (BW) by gravimetry, fat and lean mass were measured by Echo-MRI. Fasting plasma insulin was measured by ELISA. Fasting plasma glucose and non-esterified fatty acids (NEFA) were measured with a clinical chemistry analyzer. HOMA-IR, an index of systemic IR, and adipose IR (fasting insulin x fasting NEFA) were calculated. Protein levels of insulin signaling markers including IRS1, p-IRS1 (Ser1101), PI3 kinase p110, Akt, p-Akt (SER473), GLUT4, mTOR, p-mTOR (Ser2481) were quantified in the retroperitoneal fat by Western-blot. Results: DHT significantly (p&lt;0.05) increased BW (352.67± 6.93 vs 250.88 ± 5.26 g), fat mass (20.82 ± 1.48 vs 9.44 ± 1.81 g), and lean mass (316.92 ± 4.15 vs 224.45 ± 4.79 g), compared to PBO group. DHT significantly (p&lt;0.05) increased HOMA-IR and adipose IR by 75% and 80%, respectively, compared to PBO group. DHT downregulated retroperitoneal fat p-IRS-1, IRS-1, p-mTOR, mTOR, GLUT4, PI3 kinase and Akt protein expression by 50% to 70% while upregulating p-Akt by 1.8- fold compared to the PBO group. PEG-BHD1028 significantly decreased BW, fat mass and lean mass while attenuating IR by 34.7% reduction in HOMA-IR and significantly (p&lt;0.05) decreased adipose IR by 51.6% in PCOS rats. Interestingly, PEG-BHD1028 significantly (p&lt;0.05) upregulated p-IRS-1, IRS-1, p-mTOR, mTOR and p-AKT levels by 2.6-, 3-, 2.2-, 4-, 2.6- fold, respectively with no impact on the other insulin signaling markers compared to vehicle-treated group. Conclusion: PCOS rats exhibit obesity and lower adiponectin levels that are associated with both adipose and whole-body insulin resistance. Activating adiponectin receptors with PEG-BHD1028 attenuated PCOS-associated obesity and adipose IR. Significance: Targeting adiponectin signaling could be a novel and effective therapeutic approach to mitigate metabolic dysfunction in females with excess androgens. Supported by NIH-NIGMS grant P20GM121334 (L.L.Y.C. and D.G.R.), NIH-NIGMS grant P20GM121334, P20GM104357, NIH-NHLBI grant P01HL51971, NIH-NIMHD-P50MD017338 and University of Mississippi Medical Center Medical Student Research Program (R.C.) and the Robert M. Hearin Foundation (R.C.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

Sex differences on constitutive long non-coding RNA expression: Modulatory effect of estradiol and testosterone in muscle cells.

Despite the growing awareness of sexual dimorphism between males and females under pathological and physiological conditions, sex bias in biomedical research in animal models and patients is still present nowadays. The main objective of this work was to investigate sex differences in constitutive long non-coding RNA expression in spinal cord and skeletal muscle from wild-type mice. To assess the influence of gender on long non-coding RNAs, we extracted RNA from tissues of male and female mice and analyzed the expression on nine long non-coding RNAs, selected for being among the most commonly studied or exerting an important role in muscle, at 50, 60, and 120 days of age. We observed age- and tissue-dependent significant sex differences, being more prominent in skeletal muscle. We also studied the effect of sex steroid hormones on long non-coding RNA expression in vitro, noticing a modulation of long non-coding RNA levels upon estradiol and dihydrotestosterone treatment in muscle. Taken together, results obtained evidenced sex differences on constitutive long non-coding RNA expression and suggested an influence of steroid hormones complementary to other possible factors. These findings emphasize the importance of including both sexes in experimental design to minimize any potential sex bias.

Dihydrotestosterone induces arterial stiffening in female mice

BackgroundAndrogens are important sex hormones in both men and women and are supplemented when endogenous levels are low, for gender transitioning, or to increase libido. Androgens also circulate at higher levels in women with polycystic ovarian syndrome, a condition that increases the risk for cardiovascular diseases including hypertension and arterial stiffness. Since our previous work shows an important role for the G protein-coupled estrogen receptor (GPER) in arterial stiffness, we hypothesized that other hormones including androgens may impact arterial stiffness in female mice via downregulation of GPER.MethodsThe impact of the non-aromatizable androgen dihydrotestosterone (DHT), the glucocorticoid dexamethasone, and the progestin medroxyprogesterone acetate (all 100 nM for 24 h) on GPER and ERα expression was assessed in cultured vascular smooth muscle cells using droplet digital PCR (ddPCR). To assess the in vivo impact of the DHT-induced downregulation of GPER, female ovary-intact C57Bl/6 mice at 15–16 weeks of age were treated with silastic capsules containing DHT for 4 weeks, one with a dosage expected to mimic human male DHT levels and another to double the expected human concentration (n = 8–9/group).ResultsIn cultured vascular smooth muscle cells, GPER mRNA was decreased by DHT (P = 0.001) but was not impacted by dexamethasone or medroxyprogesterone. In contrast, ERα expression in cultured cells was significantly suppressed by all three hormones (P < 0.0001). In control mice or mice treated with a single or double dose of DHT, a dose-dependent increase in body weight was observed (control 22 ± 2 g, single dose 24 ± 2 g, double dose 26 ± 2 g; P = 0.0002). Intracarotid stiffness measured via pulse wave velocity showed a more than two-fold increase in both DHT-treated groups (control 1.9 ± 0.3 m/s, single dose 4.3 ± 0.8 m/s, double dose 4.8 ± 1.0 m/s). This increase in arterial stiffness occurred independent of changes in blood pressure (P = 0.59). Histological analysis of aortic sections using Masson’s trichrome showed a significant decrease in collagen between the control group (24 ± 5%) and the double dose group (17 ± 3%, P = 0.007), despite no changes in aortic wall thickness or smooth muscle content. Lastly, ddPCR showed that in vivo DHT treatment decreased aortic expression of both GPER (control 20 ± 5, single dose 10.5 ± 5.6, double dose 10 ± 4 copies/ng; P = 0.001) and ERα (control 54 ± 2, single dose 24 ± 13, and double dose 23 ± 12 copies/ng; P = 0.003).ConclusionsThese findings indicate that androgen promotes arterial stiffening and cardiovascular damage in female mice and is associated with decreased estrogen receptor expression. These data are important for transgender men, women using testosterone for fitness or reduced libido, as well as patients with polycystic ovarian syndrome.

DHT and Insulin Upregulate Secretion of the Soluble Decoy Receptor of IL-33 From Decidualized Endometrial Stromal Cells.

Interleukin 33 (IL-33) signaling regulates most of the key processes of pregnancy, including decidualization, trophoblast proliferation and invasion, vascular remodeling, and placental growth. Accordingly, dysregulation of IL-33, its membrane-bound receptor (ST2L, transducer of IL-33 signaling), and its soluble decoy receptor (sST2, inhibitor of IL-33 signaling) has been linked to a wide range of adverse pregnancy outcomes that are common in women with obesity and polycystic ovary syndrome, that is, conditions associated with hyperandrogenism, insulin resistance, and compensatory hyperinsulinemia. To reveal if androgens and insulin might modulate uteroplacental IL-33 signaling, we investigated the effect of dihydrotestosterone (DHT) and/or insulin on the expression of ST2L and sST2 (along with the activity of their promoter regions), IL-33 and sIL1RAP (heterodimerization partner of sST2), during in vitro decidualization of endometrial stromal cells from 9 healthy women. DHT and insulin markedly upregulated sST2 secretion, in addition to the upregulation of its messenger RNA (mRNA) expression, while the proximal ST2 promoter, from which the sST2 transcript originates, was upregulated by insulin, and in a synergistic manner by DHT and insulin combination treatment. On the other hand, sIL1RAP was slightly downregulated by insulin and IL-33 mRNA expression was not affected by any of the hormones, while ST2L mRNA expression and transcription from its promoter region (distal ST2 promoter) could not be detected or showed a negligibly low level. We hypothesize that high levels of androgens and insulin might lead to subfertility and pregnancy complications, at least partially, through the sST2-dependent downregulation of uteroplacental IL-33 signaling.

Androgen signaling stabilizes genomes to counteract senescence by promoting XRCC4 transcription.

Aging is accompanied by a decreased DNA repair capacity, which might contribute to age-associated functional decline in multiple tissues. Disruption in hormone signaling, associated with reproductive organ dysfunction, is an early event of age-related tissue degeneration, but whether it impacts DNA repair in nonreproductive organs remains elusive. Using skin fibroblasts derived from healthy donors with a broad age range, we show here that the downregulation of expression of XRCC4, a factor involved in nonhomologous end-joining (NHEJ) repair, which is the dominant pathway to repair somatic double-strand breaks, is mediated through transcriptional mechanisms. We show that the androgen receptor (AR), whose expression is also reduced during aging, directly binds to and enhances the activity of the XRCC4 promoter, facilitating XRCC4 transcription and thus stabilizing the genome. We also demonstrate that dihydrotestosterone (DHT), a powerful AR agonist, restores XRCC4 expression and stabilizes the genome in different models of cellular aging. Moreover, DHT treatment reverses senescence-associated phenotypes, opening a potential avenue to aging interventions in the future.

Metabolic dysregulation and gut dysbiosis linked to hyperandrogenism in female mice.

Polycystic ovary syndrome (PCOS) is a common endocrine pathology in women. In addition to infertility, women with PCOS have metabolic dysregulation which predisposes them to Type 2 diabetes, cardiovascular disease and non-alcoholic fatty liver disease. Moreover, women with PCOS have changes in their gut microbial community that may be indicative of dysbiosis. While hyperandrogenism is associated with both the development of metabolic dysfunction and gut dysbiosis in females, the mechanisms involved are not well understood. We used dihydrotestosterone (DHT) and ovariectomy (OVX) mouse models coupled with metabolic assessments and 16S rRNA gene sequencing to explore the contributions of hyperandrogenism and oestrogen deficiency to the development of insulin resistance and gut microbial dysbiosis in pubertal female mice. We demonstrated that, while DHT treatment or OVX alone were insufficient to induce insulin resistance during the pubertal-to-adult transition, combining OVX with DHT resulted in insulin resistance similar to that observed in letrozole-treated mice with elevated testosterone and decreased oestrogen levels. In addition, our results showed that OVX and DHT in combination resulted in a distinct shift in the gut microbiome compared to DHT or OVX alone, suggesting that the substantial metabolic dysregulation occurring in the OVX + DHT model was accompanied by unique changes in the abundances of gut bacteria including S24-7, Rikenellaceae and Mucispirillum schaedleri. While hyperandrogenism plays an important role in the development of metabolic dysregulation in female mice, our results indicate that investigation into additional factors influencing insulin resistance and the gut microbiome during the pubertal-to-adult transition could provide additional insight into the pathophysiology of PCOS.

BMP4 participates in the pathogenesis of PCOS by regulating glucose metabolism and autophagy in granulosa cells under hyperandrogenic environment

Polycystic ovary syndrome (PCOS) is a complex reproductive endocrine disease characterized by ovulation dysfunction with multiple etiologies and manifestations, and it is widely believed that the disorders of hyper-androgen and glucose metabolism play a key role in its progression. There has been evidence that bone morphogenetic protein 4 (BMP4) is essential for the regulation of granulosa cells, but whether it regulates metabolism level of granulosa cells under hyperandrogenic environment remains unclear. In this study, Gene Expression Omnibus, clinical data and serum of PCOS patient were collected to detect androgen and BMP4 levels. KGN cells exposed to androgens as a model for simulating PCOS granulosa cells. Lactate/pyruvate kits, and Extracellular Acidification Rate and Oxygen Consumption Rate assay were performed to detect glycolysis and autophagy levels of granulosa cells. Lentivirus infection was used to investigate the effects of BMP4 on granulosa cells. RNA-seq were performed to explore the special mechanism. We found that BMP4 was increased in PCOS patients with hyper-androgen and granulosa cells with dihydrotestosterone treatment. Mechanically, on the one hand, hyperandrogenemia can up-regulate BMP4 secretion and induce glycolysis and autophagy levels. On the other hand, we found that hyperandrogenic-induced YAP1 upregulation may mediate BMP4 to increase glycolysis level and decrease autophagy, which plays a protective role in granulosa cells to ensure subsequent energy utilization and mitochondrial function. Overall, we innovated on the protective effect of BMP4 on glycolysis and autophagy disorders induced by excessive androgen in granulosa cells. Our study will provide guidance for future understanding of PCOS from a metabolic perspective and for exploring treatment options.

GABBR2 as a Downstream Effector of the Androgen Receptor Induces Cisplatin Resistance in Bladder Cancer.

The precise molecular mechanisms responsible for resistance to cisplatin-based chemotherapy in patients with bladder cancer remain elusive, while we have indicated that androgen receptor (AR) activity in urothelial cancer is associated with its sensitivity. Our DNA microarray analysis in control vs. AR-knockdown bladder cancer sublines suggested that the expression of a GABA B receptor GABBR2 and AR was correlated. The present study aimed to determine the functional role of GABBR2 in modulating cisplatin sensitivity in bladder cancer. AR knockdown and dihydrotestosterone treatment considerably reduced and induced, respectively, GABBR2 expression, and the effect of dihydrotestosterone was at least partially restored by an antiandrogen hydroxyflutamide. A chromatin immunoprecipitation assay further revealed the binding of AR to the promoter region of GABBR2 in bladder cancer cells. Meanwhile, GABBR2 expression was significantly elevated in a cisplatin-resistant bladder cancer subline, compared with control cells. In AR-positive bladder cancer cells, knockdown of GABBR2 or treatment with a selective GABA B receptor antagonist, CGP46381, considerably enhanced the cytotoxic activity of cisplatin. However, no additional effect of CGP46381 on cisplatin-induced growth suppression was seen in GABBR2-knockdown cells. Moreover, in the absence of cisplatin, CGP46381 treatment and GABBR2 knockdown showed no significant changes in cell proliferation or migration. These findings suggest that GABBR2 represents a key downstream effector of AR signaling in inducing resistance to cisplatin treatment. Accordingly, inhibition of GABBR2 has the potential of being a means of chemosensitization, especially in patients with AR/GABBR2-positive bladder cancer.

Neuronal Ptpn1 and Socs3 deletion improves metabolism but not anovulation in a mouse PCOS model.

Polycystic ovary syndrome (PCOS) is one of the most common causes of infertility in women. Approximately half of diagnosed individuals also experience metabolic syndrome. Central and peripheral resistance to the hormones insulin and leptin have been reported to contribute to both metabolic and reproductive dysregulation. In PCOS and preclinical PCOS animal models, circulating insulin and leptin levels are often increased in parallel with the development of hormone resistance, however it remains uncertain whether these changes contribute to the PCOS state. In this study we tested whether central actions of protein tyrosine phosphatase 1B (PTP1B) and suppressor of cytokine signaling 3 (SOCS3), negative regulators of insulin and leptin signaling pathways respectively, play a role in the development of PCOS-like phenotype. A peripubertal dihydrotestosterone (DHT) excess PCOS-like mouse model was used, which exhibits both metabolic and reproductive dysfunction. Mice with knockout of the genes encoding PTP1B and SOCS3 from forebrain neurons were generated, and metabolic and reproductive function were compared between knockout and control groups. DHT treatment induced mild insulin resistance but not leptin resistance, so the role of SOCS3 could not be tested. As expected, DHT excess abolished estrous cycles and corpora lutea presence and caused increased visceral adiposity and fasting glucose levels. Knockout mice did not show any rescue of reproductive dysfunction, but did have reduced adiposity compared to control DHT mice. These data suggest that negative regulation of central insulin signaling by PTP1B is not responsible for peripubertal DHT excess-induced reproductive impairments, but may mediate its increased adiposity effects.

Effects of Androgen Treatment on Growth in Patients with 5-α-Reductase Type 2 Deficiency.

Patients with 5-α-reductase type 2 deficiency (5αRD2) require androgen treatment for the growth of normal male external genitalia. Since limited research has been conducted on the effects of androgen treatment on height in individuals with 5αRD2, we investigated the effect of androgen treatment on bone age (BA) and the height status in children with 5αRD2. Of the 19 participants who were followed up for an average of 10.6 years, 12 received androgen treatment. BA and height standard deviation scores (SDS) were compared between the treatment and non-treatment groups, as well as between the dihydrotestosterone (DHT) and testosterone enanthate (TE) treatment groups. Despite the above-average height of the 19 patients with 5αRD2, the height SDS relative to BA (htSDS-BA) was below average, particularly in the androgen treatment group. DHT treatment did not lead to an increase in BA or htSDS-BA, whereas TE treatment resulted in BA advancement and decreased htSDS-BA, especially in the prepubertal period. DHT treatment is more favorable for height than TE treatment in patients with 5αRD2, particularly during the prepubertal period. Therefore, age and the type of androgen used should be carefully considered to minimize the risk of height reduction in these patient groups.

MP20-05 LATROPHILINS AS DOWNSTREAM EFFECTORS OF THE ANDROGEN RECEPTOR INDUCE PROSTATE CANCER PROGRESSION

You have accessJournal of UrologyCME1 Apr 2023MP20-05 LATROPHILINS AS DOWNSTREAM EFFECTORS OF THE ANDROGEN RECEPTOR INDUCE PROSTATE CANCER PROGRESSION Yuki Teramoto, Takuro Goto, Masato Yasui, and Hiroshi Miyamoto Yuki TeramotoYuki Teramoto More articles by this author , Takuro GotoTakuro Goto More articles by this author , Masato YasuiMasato Yasui More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Latrophilins (LPHNs), including LPHN1, LPHN2, and LPHN3, are a group of the G-protein-coupled receptor where a spider venom latrotoxin (LTX) is known to bind. Alterations in their encoded genes (e.g. ADGRL1, ADGRL2, ADGRL3) have been linked to susceptibility to attention deficit hyperactivity disorder (ADHD). Interestingly, ADHD is diagnosed approximately 3 times more often in boys than in girls, and prenatal exposure to excess androgens has been suggested to increase its risk. However, LPHNs remain largely uncharacterized in neoplastic diseases. The present study aimed to determine the functional role of LPHNs in the progression of prostate cancer. METHODS: In human prostate cancer lines, we assessed the effects of androgen receptor (AR) ligands and LPHN ligands (e.g. LTX, FLRT3) or silencing of each LPHN via shRNA virus infection on the expression of LPHNs and the cell growth/expression of related proteins, respectively. In 150 radical prostatectomy cases, we immunohistochemically stained for LPHN1/LPHN2/LPHN3. RESULTS: In AR-positive LNCaP/C4-2/CWR22Rv1 cells, dihydrotestosterone treatment considerably increased the expression levels of LPHN1, LPHN2, and LPHN3, which was at least partially restored by AR inhibitors, bicalutamide and enzalutamide. Chromatin immunoprecipitation assay further revealed the binding of endogenous ARs, including ARv7, to the promoter region of each LPHN in prostate cancer cells. Treatment with LTX or FLRT3 resulted in induction in the cell viability and migration of AR-positive lines, as well as the expression of prostate-specific antigen, and reduction in apoptosis. LTX/FLRT3 also enhanced the expression of phosphorylated forms of JAK2 and STAT3, as well as Bcl-2. Meanwhile, knockdown of LPHN1, LPHN2, or LPHN3 showed opposite effects on all of those described above for LTX/FLRT3 treatment. In addition, similar stimulation by LTX/FLRT3 treatment or inhibition by knockdown of each LPHN was seen in AR-negative PC-3 and DU145 cells. In immunohistochemistry, high LPHN1 (H-score >200; HR=3.706, p=0.006)/LPHN2 (>100; HR=6.023, p=0.002)/LPHN3 (>200; HR=4.265, p=0.004) was associated with a significantly higher risk of biochemical recurrence after prostatectomy even in a multivariate setting. CONCLUSIONS: These findings indicate that LPHNs, as downstream effectors of the ARs, promote the growth of prostate cancer. LPHNs could thus be independent prognosticators in prostate cancer patients undergoing radical prostatectomy, as well as therapeutic targets for both androgen-sensitive and castration-resistant tumors. Source of Funding: NA © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e276 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yuki Teramoto More articles by this author Takuro Goto More articles by this author Masato Yasui More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract P3-11-05: Efficacy of Gonadal steroids on aromatase inhibitor-resistant ER+ breast cancer: Insights from single-cell trajectory analysis of a patient-derived xenograft model

Abstract Background: While estrogen typically promotes the progression of hormone-dependent breast cancer via the activation of estrogen receptor (ER)-α, estrogen-induced suppression of ER+ breast cancer has been clinically observed. Our previous study demonstrated that estrogen increased the percentage of cells expressing IL24, linking to the estrogen-dependent growth inhibition of an ER+ aromatase inhibitor (AI)-resistant tumor (Mori et al. Cancers, 2021). To continue our evaluation, we investigated whether progesterone (P4) and dihydrotestosterone (DHT) would affect the growth of this estrogen-suppressive tumor. Methods: An estrogen-suppressive patient-derived xenograft (PDX) model (named GS3) was established from an AI resistant ER+/HER2– brain metastatic breast cancer. E2 (1mg), P4 (10mg), DHT (12.5mg), or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC) and RNA sequencing of PDX specimens were conducted to decipher molecular changes after each treatment. The single-cell RNAseq analysis was further performed to examine gene expression profiles in individual cells. Results: ERα, ERβ, Progesterone receptor (PR), and androgen receptor (AR) genes in GS3 are wild-type and are not amplified. Measurements of tumor volume showed that E2, E2+P4, and DHT suppressed the growth of GS3. The Tumor growth was not modulated by P4 treatment. IHC indicated that the number of Ki-67+ cells were decreased after E2, E2+P4, and DHT treatments, but were not changed after P4 treatment. PR+ cells appeared after E2 and E2+P4 treatments, and the AR+ cells increased after DHT treatment. Bulk-RNA sequencing indicated that E2 and E2+P4 treatments resulted in comparable gene expression patterns, while those of placebo and P4-treated tumors were similar. GSEA analysis showed that in E2 treatment, the hallmark estrogen response gene sets were upregulated, and the hallmark G2M checkpoint gene set was downregulated. However, in DHT treatment, the hallmark interferon alpha/gamma response gene sets and androgen response gene set were upregulated, and the hallmark TNFA signaling via NFkB gene set was downregulated. Single-cell RNA sequencing analysis of Placebo/E2/DHT samples revealed 9 clusters; cells from E2-treated and DHT-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes, although both E2 and DHT treatments resulted in tumor regression. DHT promoted cell cycle arrest, but it did not increase IL24 expression. The hallmark oxidative phosphorylation and androgen response gene sets were upregulated in all clusters from DHT-treated tumors. Trajectory analysis of single cells revealed that three major branches associated with clusters selective to the treatments of placebo, E2, and DHT were separated from a common branch which are consist of G2M phase cells. Conclusions: E2 and DHT were effective suppressor of GS3, but not P4. Based on the results from IHC, bulk-RNAseq, and Single-cell RNAseq, the mechanism of DHT-induced tumor regression is different from that by E2. Our results suggest that DHT/E2 could be treatment options for patients with relapsed ER+ AI-resistance breast cancer. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Jinhui Wang, Xiwei Wu, Noriko Kanaya, George Somlo, Shiuan Chen. Efficacy of Gonadal steroids on aromatase inhibitor-resistant ER+ breast cancer: Insights from single-cell trajectory analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-05.

Abstract P2-19-02: Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors

Abstract Background: Epithelial-mesenchymal transition (EMT) phenotype is a complex process and plays a central role in tumor progression, aggression, invasion, metastasis, and resistance to therapy. Role of androgen receptor (AR) and androgens in inducing EMT has been well established in experimental systems of prostate cancer. AR is widely expressed in all subtypes of breast cancer (BC) and is considered to have context dependent role based on the steroid hormone microenvironment. Previous studies have shown AR as the only sex steroid receptor detectable in BC metastases and in metastatic lesion. We investigated the role of androgens in driving the EMT phenotype and association of AR induced genes with EMT in BC tumors using public dataset. Methods: We treated AR positive breast cancer cell line MDA-MB-453 with the androgen, dihydrotestosterone (DHT) followed by AR antagonist bicalutamide (Bic) and examined the expression levels of AR induced genes by quantitative real time PCR (qPCR) and AR induced protein GCDFP-15 by western blot. AR driven genes (SEC14L2, C1orf116, FKBP5, UGT2B11, UGT2B28, KCNMA1, PIP and ABCC11) were identified by bioinformatic pipeline using a systematic bioinformatic approach from public microarray data sets derived from AR positive breast cancer cell lines (S Rajarajan et al, SABCS 2021). Mesenchymal phenotype of the cell lines under DHT/Bic was evaluated morphologically and expression levels of EMT markers (ZEB1, Slug, Vimentin) and E-cadherin was estimated by qPCR, western blot and immunofluorescence. Migratory potential of the cell line under treatment was evaluated by wound healing assay and proliferation was examined by the MTT assay. Association of AR induced genes and levels of enzymes involved in intracrinal testosterone metabolism (SRD5A1 &amp; SRD5A3) with EMT in breast tumors was evaluated using transcript data from METABRIC database. EMT scores were derived from previous publications (Byers et al, 2013, Mak et al, 2016) and correlation was tested with AR induced genes. Results: The MDAMB-453 cell line was treated with 10nM DHT for 72hrs and this increased the expression of AR downstream protein GCDFP-15 by 31.47% (p=0.022) when compared to the control. Further, a significant increase in the expression levels of AR driven genes with a fold change &amp;gt;2 was observed in the DHT treated cells (p&amp;lt; 0.05). The expression of GCDP15 protein (p=0.028) and the AR downstream genes (p&amp;lt; 0.05) were significantly repressed upon treatment with bicalutamide. DHT treatment also showed a significant higher expression of EMT master regulator, SLUG (fold change&amp;gt;3, p=0.045) and a wound healing assay showed a 22% increase in migration (p=0.027). The expression of SLUG was significantly repressed (p=0.003) and the migratory ability of the DHT treated cells reduced upon treatment with bicalutamide (p=0.145). We also observed a significant loss of E-cadherin protein (by 23.65%; p=0.024) with DHT treatment and this was completely reversed upon treatment with bicalutamide (p=0.047). No significant change in proliferation was observed among the different treatment conditions. A significant positive correlation was observed between AR induced genes and the EMT scores (p&amp;lt; 0.05). Expression level of SRD5A3 was also positively correlated with EMT score (p=0.043) and AR induced genes (p&amp;lt; 0.05), except SEC14L2. Conclusion: Androgen receptor plays an important role in the biology of breast cancer and is considered a useful marker for prognosis. However, antiandrogen therapies haven’t seen the expected success in clinical trial settings. Our results support the controversial role played by AR in higher androgenic environment within breast cancer and warrant the consideration of the intratumoral levels of sex steroid hormones. Citation Format: Savitha Rajarajan, Snijesh VP, Madhumathy G Nair, Apoorva D, Chandrakala M, Vidya P Nimbalkar, Maalavika Pillai, Mohit Kumar Jolly, Jyothi S Prabhu. Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-02.

Diane-35 and metformin therapy in rats with endometrial lesions induced by dihydrotestosterone exposure.

Cotreatment with metformin and Diane-35 is conventionally used in the clinic to ameliorate ovulatory dysfunction and insulin resistance in women with polycystic ovary syndrome (PCOS). We previously showed that this combination treatment could reverse endometrial hyperplasia and endometrial cancer (EC) in patients with PCOS. Here, we aimed to investigate the influence of dihydrotestosterone (DHT) and this cotreatment on the endometrium, along with the related mechanisms. We treated a DHT-exposed rat model with Diane-35 or metformin alone or their combination and investigated the 3-dimensional (3D) endometrial structure to determine the role of these treatments in reversing endometrial lesions and to clarify the underlying mechanisms. Uterine segments were made transparent with clear, unobstructed brain/body imaging cocktails and a computational analysis protocol and then labeled with 4',6-diamidino-2-phenylindole (DAPI), antiandrogen receptor (AR) antibody, and antiglucose transportation protein 4 (GLUT4) antibody. We visualized and analyzed the endometrial structure, AR expression, and GLUT4 expression under 3D conditions using light sheet microscopy and Imaris software (Bitplane, Zurich, Switzerland). Long-term DHT treatment contributed to hyperandrogenism and insulin resistance in female Wistar rats. After DHT treatment, rats exhibited other PCOS-like characteristics, such as polycystic ovary morphology, hypothalamic-pituitary-ovarian axis disorder, and relative endometrial hyperplasia. After metformin and Diane-35 treatment, the PCOS-like characteristics and endometrial hyperplasia were alleviated. Hyperandrogenism and insulin resistance likely play important roles in the pathophysiological changes of PCOS and lead to PCOS-like characteristics as well as endometrial lesions. Hypoandrogenic and insulin sensitization therapy can alleviate DHT-induced endometrial hyperplasia by regulating AR and GLUT4 expression.

Mitoguardin2 Is Associated With Hyperandrogenism and Regulates Steroidogenesis in Human Ovarian Granulosa Cells.

Polycystic ovary syndrome (PCOS) is an endocrinopathy characterized by hyperandrogenism, anovulation, and polycystic ovaries, in which hyperandrogenism manifests by excess androgen and other steroid hormone abnormalities. Mitochondrial fusion is essential in steroidogenesis, while the role of mitochondrial fusion in granulosa cells of hyperandrogenic PCOS patients remains unclear. In this study, mRNA expression of mitochondrial fusion genes mitoguardin1, -2 (MIGA 1, -2) was significantly increased in granulosa cells of hyperandrogenic PCOS but not PCOS with normal androgen levels, their mRNA expression positively correlated with testosterone levels. Dihydrotestosterone (DHT) treatment in mice led to high expression of MIGA2 in granulosa cells of ovulating follicles. Testosterone or forskolin/ phorbol 12-myristate 13-acetate treatments increased expression of MIGA2 and the steroidogenic acute regulatory protein (StAR) in KGN cells. MIGA2 interacted with StAR and induced StAR localization on mitochondria. Furthermore, MIGA2 overexpression significantly increased cAMP-activated protein kinase A (PKA) and phosphorylation of AMP-activated protein kinase (pAMPK) at T172 but inhibited StAR protein expression. However, MIGA2 overexpression increased CYP11A1, HSD3B2, and CYP19A1 mRNA expression. As a result, MIGA2 overexpression decreased progesterone but increased estradiol synthesis. Besides the androgen receptor, testosterone or DHT might also regulate MIGA2 and pAMPK (T172) through LH/choriogonadotropin receptor-mediated PKA signaling. Taken together, these findings indicate that testosterone regulates MIGA2 via PKA/AMP-activated protein kinase signaling in ovarian granulosa cells. It is suggested mitochondrial fusion in ovarian granulosa cells is associated with hyperandrogenism and potentially leads to abnormal steroidogenesis in PCOS.

OR30-5 Androgen-Regulated Secreted Factors Promote Tumorigenesis and Immune Suppression in Breast Cancer

Abstract Background Breast cancer (BC) is the most diagnosed cancer and the second-leading cause of cancer-associated death in women in the United States. BCs that express the estrogen and/or progesterone receptor (ER/PR) are treated with ER-targeting endocrine therapies that effectively reduce disease burden and extend lifespan. However, up to 50% of patients with ER+BCs will experience recurrence, often at a metastatic site, within 10 years of diagnosis. Novel targeted therapies are needed to treat both metastatic ER+BC resistant to current treatment strategies and chemoresistant ER-BCs.We identified chitinase 3–like-1 (CHI3L1/BRP-39/YKL-40) and prolactin inducible protein (PIP/GCDFP-15) as androgen receptor (AR)-regulated in BC. PIP is reported to be detectable in &amp;gt;90% of BCs, but its function in BC is not well-characterized. CHI3L1 is a glycosyl hydrolase family 18 protein member with reported roles in aging and numerous pathologies. During mammary gland involution, CHI3L1 contributes to a pro-tumorigenic wound-healing-like environment and increases macrophage infiltration. Further, CHI3L1 correlates with tumor-suppressive M2 macrophage infiltration in ER-BC. We previously reported that ER+ cells and patient-derived xenografts with activating mutations in the ESR1 gene encoding ER—a known mechanism of endocrine therapy resistance—had elevated AR and CHI3L1 gene and protein expression. Based on the above and our finding that metastatic BCs biopsies harboring ESR1 mutations exhibited increased AR expression, we hypothesized that AR regulation of CHI3L1 and PIP promote progression to metastatic disease in part through immune suppressive effects. Methods A ConTra(V3) transcription factor binding site analysis was performed on CHI3L1 and PIP promoters. CHI3L1 and PIP were measured via immunohistochemistry in xenograft tumors in mice treated with or without the androgen dihydrotestosterone (DHT). Immunoblotting, immunohistochemistry, and immunofluorescence approaches were also utilized to assess CHI3L1 protein expression in DHT-treated, tamoxifen resistant (TAMR), and long term E2 deprived (LTED) ER+BC cells. CIBERSORT analysis was conducted on the Firehouse Legacy TCGA dataset. Macrophage efferocytosis assays were performed on an IncuCyte Live Cell Imaging system. Results We identified highly conserved AR binding sites in the CHI3L1 and PIP promoters. CHI3L1 protein was elevated in TAMR and LTED BC cells, which was further elevated in BC cells with ESR1 mutations, as well as DHT-treated cells. Further, CHI3L1 and PIP were increased in xenograft tumors after DHT treatment. CIBERSORT analysis revealed that CHI3L1 and PIP high tumors had altered immune profiles, including differential macrophage levels. Finally, CHI3L1 and PIP treatment significantly reduced macrophage efferocytosis of BC cells (p ≤ 0.0001). Conclusions Our work suggests that AR-mediated regulation of CHI3L1 and PIP may alter immune cell infiltration/function in BC and may be important in endocrine therapy resistant ER+BCs. Further characterization and manipulation of these AR-regulated proteins is underway to test the potential to target and reverse their immune suppressive functions. Presentation: Tuesday, June 14, 2022 10:45 a.m. - 11:00 a.m.

Impact of One-Week Administration of Dihydrotestosterone in Rat Anterior Pituitary Gland.

Hyperandrogenism causes dysfunction of the hypothalamic–pituitary–gonadal (HPG) axis in reproductive women. In this study, we examined the effects of dihydrotestosterone (DHT) on characteristic changes in rat anterior pituitary gland samples. DHT was administered to ovary-intact 6-week postnatal female rats for 7 days, after which the anterior pituitary glands were examined and compared with those in control rats. Estrous cyclicity was not drastically disrupted by DHT treatment. Common gonadotropin α subunit (Cga), luteinizing hormone β subunit (Lhb), and follicle-stimulating hormone (FSH) β subunit (Fshb) gene expression levels were not modulated by DHT treatment, while prolactin (Prl) gene expression was significantly repressed by DHT. Gonadotropin-releasing hormone (GnRH) receptor (Gnrh-r) gene expression was significantly inhibited by DHT, whereas pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (Pca1-r) gene expression was increased by DHT. Gene expression levels of the receptors encoded by thyrotropin-releasing hormone (Trh-r) and kisspeptin (Kiss1-r) genes were unchanged. Expression of inhibin α subunit (Inha) and activin βA subunits (Actba) within the pituitary was inhibited by DHT treatment, while activin B subunit (Actbb) and follistatin (Fst) gene expression was unchanged by DHT. In mouse pituitary gonadotroph LβT2 cells, DHT did not modulate the gene expression of Gnrh-r, but it inhibited the expression of Inha and Actba subunits within the LβT2 cells. In rat prolactin-producing GH3 cells, DHT did not modulate prolactin gene expression, but it increased Pac1-r gene expression. The present observations suggest that DHT directly or indirectly affects the anterior pituitary gland and induces characteristic changes in hormone-producing cells.