Decreased Androgen Receptor Expression Research Articles

Synthesis, molecular docking and biological evaluation of 1,2,4-oxadiazole based novel non-steroidal derivatives against prostate cancer

Prostate cancer is one of the most prevalent cancers in men leading to second most death causing cancer in men. Despite the availability of multiple treatment still the prevalence is high for prostate cancer. Steroidal antagonists associated with poor bioavailability, side effects while non-steroidal antagonists show serious side effects like gynecomastia. Therefore, there is a need of potential candidate for the treatment of prostate cancer with better bioavailability, good therapeutic effect and minimal side effects. In the same context, we have designed the series, SP1–SP25 based 3-phenyl-5-styryl-1,2,4-oxadiazole as the core structure. We successfully synthesized all 25 molecules in this series and characterized them using 1H, 13C NMR, and mass spectroscopy. Subsequently, we conducted MTT assays using PC-3 cells and observed that all the compounds exhibited a dose-dependent decrease in cell viability. Notably, compounds SP04, SP16, and SP19 demonstrated a significant decrease in cell viability and exhibited potent activity compared to the other synthesized molecules and standard drug bicalutamide. Among them, SP04 emerged as the one of the most potent compounds with an IC50 value of 238.13 nM and an 89.99 % inhibition of PC-3 cells, compared to synthesized molecules and standard drug bicalutamide.Furthermore, we conducted ROS assays and androgen receptor inhibition assays using the potent compound SP04 and bicalutamide. The results indicated that SP04 increased ROS production and decreased androgen receptor expression dose-dependent manner. Additionally, we conducted a docking study to analyse the interaction patterns within the active site of the androgen receptor. ADMET analysis revealed that all the compounds exhibited favorable physicochemical properties and manageable toxicity profiles.

Selenium Nanoparticles Modulate Steroidogenesis-Related Genes and Improve Ovarian Functions via Regulating Androgen Receptors Expression in Polycystic Ovary Syndrome Rat Model.

Polycystic ovary syndrome (PCOS) occurs during the reproductive period in women and is characterized by reproductive, endocrine, and metabolic disorders. Androgen plays a decisive role in its pathogenesis due to the interaction between hyperandrogenism and insulin resistance, which might be improved by selenium nanoparticles (SeNPs). The present study aimed to clarify the effect of SeNPs on androgen synthesis and action in the PCOS model and the resulting effect on ovarian function. Fifty-five 7-week-old female albino rats (90-105g) were divided equally into five groups: control (C), fed a standard diet for 11weeks; high-fat diet (HFD) group, fed HFD for 11weeks; HFD and letrozole (L) (HFD + L), fed HFD for 11weeks and administrated orally with L, at a daily dose of 1mg/kg BW, for three weeks from the 7th to 9th week of the trial; HFD + L + 0.1SeNPs and HFD + L + 0.2SeNPs groups, treated the same as HFD + L group and orally gavaged SeNPs at daily doses of 0.1 and 0.2mg/kg BW, respectively, during the last 14day of the experiment. Daily determination of estrous cycle was performed, and at the end of the experimental period, BMI, serum glucose, insulin, HOMA-IR, lipid profile, sex hormones, TNF-α, IL6, oxidative stress biomarkers, ovarian mRNA expression of different proteins and enzymes involved in steroidogenesis, pathological examination, and immunohistochemical staining for androgen receptor (AR) were evaluated. Treatment of SeNPs restored estrous cyclicity, decreased BMI, and insulin resistance, improved dyslipidemia, reduced serum testosterone, and improved ovarian histopathology in PCOS rats. Furthermore, the anti-inflammatory and antioxidant impacts of SeNPs were remarkably noticed. Administration of SeNPs decreased androgen synthesis and expression of ovarian AR protein by decreasing the mRNA expression of STAR, Cyp11A1, Cyp17A1, and HSD17B3 and increasing the expression of Cyp19α1. Conclusively, SeNPs decreased androgen synthesis and blocked the vicious circle initiated by excessive androgen secretion via decreased AR expression. Thus, it may effectively treat PCOS cases by eliminating its reproductive, endocrine, and metabolic dysfunctions.

Cancer Stem Cells as the Key to Cancer: Special Emphasis on Prostate Cancer

Recent research into cancer stem cells has refined our knowledge of the origins, maintenance, and progression of cancer. The characteristics of tumor initiating cells and the stem-like properties of tumor side populations that appear to be responsible for tumor maintenance and metastasis have given insights into potential targets for the elimination of treatment-resistant and residual tumor cells. These insights have also provided inroads to understanding and preventing invasive and metastatic progression of cancer. In this review, we discuss recent advancements in understanding of tumor initiating cells and cancer stem cells and their implications on cancer pathobiology and treatment. The role of tumor initiating cell phenotypes on routes of metastasis and the use of stemness markers to guide prognosis and treatment are also discussed. Particular emphasis sections are included that focus on the role of stemness in the pathobiology and treatment of prostate cancer. Of particular interest is the correlation of stemness with decreased androgen receptor expression and resistance to anti-androgen therapy. The overview provided herein represents a primer for the understanding of current knowledge regarding cancer stem cells and their clinical implications in prostate and other cancer types.

The role of steroidogenic factor 1 (SF-1) in steroidogenic cell function of the testes and ovaries of mature mice.

The nuclear receptor steroidogenic factor 1 (SF-1) is essential for mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and depletion of SF-1 in steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility. Steroidogenic factor 1 (SF-1 or NR5A1) plays an essential role in the development of fetal gonads and regulates genes involved in steroid biosynthesis. Since SF-1 is expressed in multiple cell types in mouse gonads, we developed three novel conditional knockout (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (Nr5a1f/f) to identify its role in testes and ovaries of mature mice: Cytochrome P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f, Leydig and theca cell-specific), aromatase (Cyp19Cre/+;Nr5a1f/f, Sertoli and granulosa cell-specific), as well as a combination of both (Cyp17+Cyp19-Cre;Nr5a1f/f). Compared to control animals, Cyp19-Cre;Nr5a1f/f cKO males showed normal fertility and testicular function. The Cyp17Cre/+;Nr5a1f/f cKO males had smaller testis, with drastically reduced Leydig cell volumes and impaired steroidogenesis, though their reproductive performance remained comparable to controls. Some 50% of Cyp17Cre/++Cyp19Cre/+;Nr5a1f/f double-cKO (dKO) males were infertile, while the remaining 50% showed significantly reduced fertility. These dKO males also had smaller testis with degenerative seminiferous tubules, abnormal Leydig cell morphology and lower levels of intra-testicular testosterone. Abnormal Sertoli cell localization was noted in dKO testes, with increased Sox9, p27 and inhibin subunit ßb and decreased androgen receptor expression. Female mice from all genotypes showed normal reproductive capacity, though steroidogenic gene expression levels were significantly decreased in both Cyp17Cre/+;Nr5a1f/f cKO and dKO females. These results show the essential role of SF-1 in mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and that depletion SF-1 in all steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.

Suppression of androgen receptor signaling induces prostate cancer migration via activation of the CCL20-CCR6 axis.

The suppression of androgen receptor (AR) expression exacerbates the migration potential of prostate cancer. This study identified a previously unrecognized regulation of the AR-controlled pathway that promotes migration potential in prostate cancer cells. Prostate cancer cells that pass through a transwell membrane (mig cells) have a higher migration potential with a decreased AR expression than parental cells. In this study, we aimed to elucidate the mechanism of migration enhancement associated with the suppression of AR signaling. Expression of C-C motif ligand 20 (CCL20) is upregulated in mig cells, unlike in the parental cells. Knockdown of AR with small interfering RNA (siAR) in LNCaP and C4-2B cells increased CCL20 secretion and enhanced the migration of cancer cells. Mig cells, CCL20-treated cells, and siAR cells promoted cell migration with an enhancement of AKT phosphorylation and Snail expression, while the addition of a C-C chemokine receptor 6 (CCR6, the specific receptor of CCL20) inhibitor, anti-CCL20 antibody, and AKT inhibitor suppressed the activation of AKT and Snail. With 59 samples of prostate cancer tissue, CCL20 secretion was profuse in metastatic cases despite low AR expression levels. Snail expression was associated with the expression of CCL20 and CCR6. A xenograft study showed that the anti-CCL20 antibody significantly inhibited Snail expression, thereby suggesting a new therapeutic approach for castration-resistant prostate cancer with the inhibition of the axis between CCL20 and CCR6.

METTL3 boosts glycolysis and cardiac fibroblast proliferation by increasing AR methylation

Dysregulated glycolysis has been noted in several pathological processes characterized by supporting cell proliferation. Nonetheless, the role of glycolysis reprogramming is not well appreciated in cardiac fibrosis which is accompanied by increased fibroblasts proliferation. In this study, we investigated the cause and consequence of glycolysis reprogramming in cardiac fibrosis, using clinical samples, animal models, and cultured cells. Herein, we report that methyltransferase-like 3 (METTL3) facilitates glycolysis and cardiac fibroblasts proliferation, leading to cardiac fibrosis. The augmentation of glycolysis, an essential event during cardiac fibroblasts proliferation, is dependent on an increased expression of METTL3. A knockdown of METTL3 suppressed glycolysis, and inhibited cardiac fibroblast proliferation and cardiac fibrosis. Mechanistically, METTL3 epigenetically repressed androgen receptor (AR) expression in an m6A-YTHDF2- dependent manner, by targeting the specific AR m6A site. AR could interact with the glycolysis marker HIF-1α, and down-regulation of AR activates HIF-1α signaling, resulting in enhanced glycolysis and cardiac fibroblast proliferation. In contrast, the overexpression of AR significantly reduced the HIF-1α axis, decreased expression of glycolytic enzymes HK3, inhibited glycolysis, and repressed cardiac fibroblasts proliferation. Notably, increased METTL3 and YTHDF2 levels, decreased AR expression, increased HIF-1α and Postn expression and augmented glycolysis, and increased cardiac fibrosis were detected in human atrial fibrillation heart tissues. Our results found a novel mechanism by which METTL3-catalyzed m6A modification in cardiac fibrosis, wherein it facilitated glycolysis and cardiac fibroblasts proliferation by increasing AR methylation in an m6A-YTHDF2- dependent manner and provided new insights strategies to intervene cardiac fibrosis.

Abstract 2446: Low dose dimethyl sulfoxide (DMSO) downregulates the expression of androgen receptor (AR) and AR-variant 7 (AR-v7) in castration resistant prostate cancer (CRPC) cells

Abstract The highly aggressive prostate cancer (PC) cells utilize androgen receptor (AR) signaling to facilitate their growth and metastasis. Despite androgen deprivation therapy (ADT), recurrence of castration-resistant prostate cancer (CRPC) often occur due to constitutive AR signaling via both full-length AR (AR-FL) and AR splice variants, primarily AR-V7. Therefore, safe strategies to suppress both AR-FL & AR-V7 expression in CRPC cells will be of significant benefit. Several plant-derived organosulfur compounds, e.g. sulforaphane and diallyl-trisulfide, can potently suppress AR expression in CRPC cells, but are yet to be clinically approved. We investigated whether dimethyl sulfoxide (DMSO) an approved organosulfur compound often used as a solvent for pharmaceutical agents, can similarly suppress AR levels in the CRPC cell lines, C4-2B and 22Rv1. Exposure to low dose DMSO (0.1-1%) was not cytotoxic to these CRPC cells, and less than 20% cytotoxicity was seen with 2.5% DMSO even at 96 h post-exposure. Interestingly however, DMSO dose-dependently suppressed AR-FL in C4-2B cells and both AR-FL and AR-V7 in 22Rv1 cells within 24 h post-exposure. Exposure to DMSO also downregulated the expression of hetero-nuclear ribonucleoprotein H1 (hnRNPH1), which is known to regulate AR expression and splicing. Although DMSO exposure showed a dose- and time-dependent effect on reactive oxygen species (ROS) production by PC cells, the AR-suppressive effect of DMSO was not altered by the antioxidant n-acetyl cysteine (NAC). Furthermore, although DMSO did not affect cell viability at these clinically-achievable concentrations, a significant (p<0.01) decrease in the migratory ability of both CRPC cell lines was clearly evident. Our novel findings indicate that safe doses of DMSO may be used to decrease AR expression and metastatic ability in CRPC cells. Citation Format: Namrata Khurana, Hogyoung Kim, Talal Khan, Shohreh Kahhal, Amar Bukvic, Asim B. Abdel-Mageed, Suresh C. Sikka, Debasis Mondal. Low dose dimethyl sulfoxide (DMSO) downregulates the expression of androgen receptor (AR) and AR-variant 7 (AR-v7) in castration resistant prostate cancer (CRPC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2446.

Oncostatin M and its receptor in women with polycystic ovary syndrome and association with assisted reproductive technology outcomes

The role of adipokines in ovarian-related disorders such as polycystic ovary syndrome (PCOS) has been reported. However, the involvement of Oncostatin M (OSM), a recently identified adipokine, in ovarian function is unknown. Therefore, we investigated the association of the OSM signaling pathway with ovarian functions and PCOS pathogenesis. This case-control study enrolled 30 PCOS and 30 healthy women who underwent the intracytoplasmic sperm injection procedure. OSM and OSM receptor (OSMR) levels were evaluated in the follicular fluid (FF). Moreover, the expression of insulin receptor substrates (IRS1 and IRS2), OSM, OSMR, suppressor of cytokine signaling 3 (SOCS3), and androgen receptor (AR) genes were analyzed in the isolated cumulus cells (CCs). For the in-vitro experiment, the effect of recombinant OSM on the expression of related genes in isolated CCs was analyzed. Follicular concentrations of OSM and OSMR were significantly lower in PCOS (123.91±48.58 pg/ml and 0.93±0.35ng/ml, respectively) compared to control women (283.53±96.62pg/ml and 1.45±0.18ng/ml, respectively; p<0.001) and were positively correlated with the oocyte maturation (r=0.611 and r=0.611, respectively) and fertilization (r=0.592 and r=0.627, respectively) rates in the PCOS group. Furthermore, the SOCS3 expression was upregulated about eight times in PCOS patients compared to the controls (p<0.05). The treatment of cells with recombinant OSM significantly increased SOCS3, OSMR, IRS-1, and -2 expression and decreased AR expression. The decreased levels of OSM and its receptor in PCOS patients, possibly mediated by SOCS3, could negatively affect oocyte maturation and fertilization rates.

Chronic exposure to aquacultural stressors affects pituitary-testis axis in the Mozambique tilapia Oreochromis mossambicus.

Reproduction in fish is modulated by several factors that include environmental and endocrine components. The aim of this study was to elucidate the effect of aquacultural stressors along the pituitary-testis axis in a continuously breeding cichlid fish Oreochromis mossambicus. The fish (35.05 ± 2.20g) were divided into three groups (n = 10 in each group, n = 5 in each replicate), namely initial controls (euthanized on the day of initiation of experiment), time-matched controls (kept undisturbed), and stressed fish, which were subjected to different kinds of randomized aquacultural stressors such as handling, chasing, frequent netting, and low water levels, daily for a period of 21days. Although the gonadosomatic index and the mean numbers of spermatogonia-A and spermatogonia-B did not differ significantly among different experimental groups, significant decrease was observed in the mean numbers of primary spermatocytes, secondary spermatocytes, early spermatids, and late spermatids in fish exposed to stressors compared to those of initial controls and time-matched controls. While the diameter of the seminiferous lobule was significantly lower, the size of the lumen and the serum levels of cortisol were significantly increased in stressed fish compared with initial controls and time-matched controls. Furthermore, weak androgen receptor immunoreactivity was observed in the Sertoli cells of the testis in contrast to the strongly immunoreactive androgen receptors in initial controls and time-matched controls. Concomitant with this, there was a significant decrease in the percent area and the intensity of luteinizing hormone (LH) immunoreactive content in the proximal pars distalis (PPD) region of the pituitary gland in stressed fish compared with initial controls and time-matched controls. Overall, these results suggest that exposure to chronic aquacultural stressors causes suppression of LH synthesis in the pituitary gland concomitant with decreased androgen receptor expression and blockade of recruitment of germline cells at the meiosis stage. This inhibition appears to be mediated through the hypothalamic-pituitary-interrenal axis in the tilapia O. mossambicus.

RBP4 regulates androgen receptor expression and steroid synthesis in Sertoli cells from Bactrian camels.

Retinol-binding protein (RBP4) plays an important role in the transport and metabolism of retinol. In addition, RBP4 contributes to testicular homeostasis, including maintenance of spermatogenesis and synthesis of androgens that mediate their physiological functions through the androgen receptor. RBP4 in Sertoli cells regulates testosterone and dihydrotestosterone synthesis and secretion, although the mechanisms have yet to be revealed. In this study, we examined the expression and function of RBP4 in Sertoli cells isolated from Bactrian camels. qRT-PCR analysis of various Bactrian camel tissues revealed high expression of RBP4 in the testis and epididymis. To examine RBP4 function, Sertoli cells isolated from testes were transfected with an RBP4 overexpression plasmid or RBP4-targeting siRNA. RBP4 overexpression resulted in significant inhibition of transcription and translation of the steroidogenic enzymes 3βHSD and SRD5A1 concomitant with a significant decrease in androgen receptor expression and dihydrotestosterone secretion. Conversely, RBP4 knockdown significantly increased the expression of 3βHSD, SRD5A1 and androgen receptor and enhanced the secretion of dihydrotestosterone and testosterone. These data reveal a novel role for RBP4 in regulating steroid synthesis in Sertoli cells from Bactrian camels.

Novel Strategy of Proxalutamide for the Treatment of Prostate Cancer through Coordinated Blockade of Lipogenesis and Androgen Receptor Axis.

Objective: Prostate cancer (PCa) is the most common malignant tumor diagnosed in men in developed countries. In developing countries, the PCa morbidity and mortality rates are also increasing rapidly. Since androgen receptor (AR) is a key driver and plays a critical role in the regulation of PCa development, AR-targeted agents provide a key component of current therapy regimens. However, even new-generation AR antagonists are prone to drug resistance, and there is currently no effective strategy for overcoming advanced PCa aggressiveness, including drug-resistance progression. The aim of this study was to evaluate the potential efficacy and novel therapy strategy of proxalutamide (a newly developed AR antagonist) in PCa. Methods: Four PCa cell lines with various biological heterogeneities were utilized in this study, namely, androgen-sensitive/-insensitive with/without AR expression. Proliferation, migration and apoptosis assays in PCa cells were used to evaluate the effective therapeutic activity of proxalutamide. The changes in lipid droplet accumulation and lipidomic profiles were analyzed to determine the influence of proxalutamide on lipogenesis in PCa cells. The molecular basis of the effects of proxalutamide on lipogenesis and the AR axis was then further investigated. Results: Proxalutamide significantly inhibited the proliferation and migration of PCa cells, and its inhibitory effect was superior to that of enzalutamide (Enz, second-generation AR antagonist). Proxalutamide induced the caspase-dependent apoptosis of PCa cells. Proxalutamide significantly diminished the level of lipid droplets in PCa cells, changed the lipid profile of PCa cells and reduced the content of most lipids (especially triglycerides) in PCa cells. Proxalutamide attenuated de novo lipogenesis by inhibiting the expression of ATP citrate lyase (ACL), acetyl CoA carboxylase (ACC), fatty acid synthase (FASN) and sterol regulatory element-binding protein-1 (SREBP-1). Moreover, proxalutamide also decreased AR expression in PCa cells, and its inhibitory effect on lipogenesis did not depend on its ability to down-regulate AR expression. However, Enz had no effect on AR expression, lipid accumulation or lipid de novo synthesis in PCa cells. Conclusions: By co-targeting the AR axis and endogenous adipogenesis, a novel and promising strategy was established for proxalutamide to combat the progress of PCa. The unique effect of proxalutamide on the metabolic reprogramming of PCa provides a potential solution to overcome the resistance of current AR-targeted therapy, which will help to effectively prolong its clinical service life.

Immunohistochemical examination of androgen receptor and estrogen receptor alpha expressions in obstructive and non-obstructive azoospermia

ABSTRACT In this study, the expression of the androgen receptor (AR) and estrogen receptor alpha (ERα) in testicular tissue of male patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) were evaluated by immunohistochemistry. NOA (n = 23) and OA (n = 21) groups were created according to clinical and laboratory archival records. Testicular sperm extraction tissue sections were evaluated according to Johnsen’s tubular biopsy scoring (JTBS) method. ERα and AR immunostaining results were evaluated semiquantitatively. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and estradiol were analyzed. Serum FSH and LH concentrations were greater, and testosterone concentrations were lower than the normal values in the NOA group, whereas the OA group revealed normal hormonal values. Serum estradiol concentrations in groups were in the normal range. JTBSs were significantly lower in the NOA group. Decreased AR expression and increased ERα expression were observed in the NOA group compared to the OA group. This suggests that ERα and AR are expressed in Sertoli cells, Leydig cells, and myoid cells and are required for normal testicular function. Decreased expression of the AR and increased expression of ERα in the testis may negatively affect spermatogenesis. Abbreviations: AR: androgen receptor; ER: estrogen receptor; ERα: estrogen receptor alpha; FSH: follicle-stimulating hormone; JTBS: Johnsen’s tubular biopsy scoring; LH: luteinizing hormone; NOA: non-obstructive azoospermia; OA: obstructive azoospermia; TESE: testicular sperm extraction

Histone acetyltransferase 1 upregulates androgen receptor expression to modulate CRPC cell resistance to enzalutamide.

Castration‐resistant prostate cancer (CRPC) is the latest stage of PCa, and there is almost no effective treatment available for the patients with CRPC when next‐generation androgen deprivation therapy drugs, such as enzalutamide (ENZ), fail. The androgen receptor (AR) plays key roles in PCa and CRPC progression and drug resistance. Histone acetyltransferase 1 (HAT1) has recently been reported to be highly expressed in some tumors, such as lung carcinoma. However, what relationship between the AR and HAT1, and whether or how HAT1 plays roles in CRPC progression and drug resistance remain elusive. In the present study, we found that HAT1 is highly expressed in PCa cells, and the overexpression of HAT1 is linked with CRPC cell proliferation. Moreover, the HAT1 expression is positively correlated with the expression of AR, including both AR‐FL (full‐length) and AR‐V7 (variant 7), which is mainly mediated by a bromodomain containing protein 4 (BRD4) ‐mediated pathway. Furthermore, knockdown of HAT1 can re‐sensitize the response of CRPC cells to ENZ treatment in cells and mouse models. In addition, ascorbate was observed to decrease AR expression through downregulation of HAT1 expression. Collectively, our findings reveal a novel AR signaling regulation pathway in PCa and CRPC and suggest that HAT1 serves as a critical oncoprotein and an ideal target for the treatment of ENZ resistance in CRPC patients.

Host versus cell-dependent effects of β-arrestin 1 expression in prostate tumorigenesis.

Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease has been attributed to androgen independence, resulting in a lack of response to androgen deprivation therapy and sustained activation of other growth pathways. The scaffold proteins β-arrestin 1 and 2 (βarr1 and βarr2), which are known to mediate G protein-coupled receptor desensitization and internalization, were also shown to modulate prostate tumorigenesis. βarr1 is significantly overexpressed (>4-fold) in PCa cells relative to βarr2. In this study, we investigated the effect of βarr1 overexpression in PCa development and progression using the mouse and human PCa cell xenografts, and autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) models deficient in β-arrestin depletion of βarr1 in TRAMP mice (TRAMP/βarr1-/-) increased PCa growth and decreased overall survival relative to control TRAMP or TRAMP/βarr2-/- animals. Prostate tissues from TRAMP/βarr1-/- tumors displayed an increase in androgen receptor (AR) expression, whereas overexpression of βarr1 in TRAMP-C1 (TRAMP-C1-βarr1-GFP) which derived from TRAMP decreased AR expression, cell proliferation and tumor growth in nude mice xenografts, relative to control TRAMP-C1-GFP. Knockdown of βarr1 expression in human MDA PCa 2b cells (MDA PCa 2b-βarr1-/-) also decreased AR expression cell proliferation and tumor growth relative to control (MDA PCa 2b-Sham) cells. Interestingly, both TRAMP-C1-βarr1-GFP and MDA PCa 2b-βarr1-/- xenografts showed a decrease in AKT phosphorylation but an increase in MAPK activation. Altogether, the data indicate that the effect of βarr1 in modulating AR signaling to regulate PCa aggressiveness is cell and host autonomous.

Depletion of UXT, a novel TSG101 interaction protein, leads to enhanced CEP55 attenuation through lysosome degradation

The expression level of CEP55, a centrosome and midbody-associated protein is pivotal for cell cytokinesis and is significantly correlated with tumor stage. Our previous study demonstrated that ectopic expression of TSG101 can decrease androgen receptor expression level through the lysosome degradation pathway. Here, we further extended the investigation of TSG101 in modulating protein levels through lysosomes, and identified ubiquitously expressed transcript (UXT) to be a novel TSG101 interaction partner associated with TSG101-containing cytoplasmic vesicles. We also demonstrated that CEP55 can be recruited to TSG101 cytoplasmic vesicles resulting in downregulation of CEP55 through lysosome degradation. Moreover, UXT depletion promoted TSG101 vesicle-lysosome association and elevated autophagic carrier flux to enhance CEP55 degradation upon TSG101 overexpression. In summary, we identified a novel CEP55 regulation pathway mediated by TSG101 overexpression via lysosome degradation and revealed that UXT plays a role in the late endosome/autophagosome-lysosome fusion event, engaging in TSG101-mediated lysosome degradation.

Nucleolin represses transcription of the androgen receptor gene through a G-quadruplex.

The androgen receptor (AR) is a major driver of prostate cancer development and progression. Men who develop advanced prostate cancer often have long-term cancer control when treated with androgen-deprivation therapies (ADT). Still, their disease inevitably becomes resistant to ADT and progresses to castration-resistant prostate cancer (CRPC). ADT involves potent competitive AR antagonists and androgen synthesis inhibitors. Resistance to these types of treatments emerges, primarily through the maintenance of AR signaling by ligand-independent activation mechanisms. There is a need to find better ways to block AR to overcome CRPC. In the findings reported here, we demonstrate that the nuclear scaffold protein, nucleolin (NCL), suppresses the expression of AR. NCL binds to a G-rich region in the AR promoter that forms a G-quadruplex (G4) structure. Binding of NCL to this G4-element is required for NCL to suppress AR expression, specifically in AR-expressing tumor cells. Compounds that stabilize G4 structures require NCL to associate with the G4-element of the AR promoter in order to decrease AR expression. A newly discovered G4 compound that suppresses AR expression demonstrates selective killing of AR-expressing tumor cells, including CRPC lines. Our findings raise the significant possibility that G4-stabilizing drugs can be used to increase NCL transcriptional repressor activity to block AR expression in prostate cancer. Our studies contribute to a clearer understanding of the mechanisms that control AR expression, which could be exploited to overcome CRPC.

Host- versus cell-dependent effects of β-Arrestin 1 expression in prostate tumorigenesis

Abstract Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease is androgen independent resulting in lack of response to androgen deprivation therapy. The scaffold proteins β-arrestin 1 and 2 (βarr1 and βarr2) which are known to mediate G protein-coupled receptors (GPCRs) desensitization and internalization, were also shown to modulate prostate tumorigenesis. βarr1 is overexpressed (~4 fold) in PCa cells relative to βarr2. In this study, we investigate the effect of β-arrestins expression in prostate tumorigenesis using in-vivo xenografts and autochthonous transgenic adenocarcinoma mouse prostate (TRAMP) model deficient in β-arrestins expression. Depletion of βarr1 in TRAMP mice (TRAMP/βarr1−/−) increased PCa growth and decreased overall survival, relative to TRAMP and TRAMP/βarr2−/− animals. Tumors from TRAMP/βarr1−/− mice displayed increase androgen receptor (AR), but decrease prostate specific antigen (PSA) expression. In contrast, depletion of βarr1 in MDA PCa 2b cells (MDA PCa 2b-βarr1−/−) decreased cell proliferation, wound closure and anchorage independent cell growth compared to wild type MDA PCa 2b and MDA PCa 2b-βarr2−/− cells. Nude mice xenografts of MDA PCa 2b-βarr1−/− cells also showed significant decrease in tumor growth. Interestingly, tumor lysates from MDA PCa 2b-βarr1−/− xenografts exhibited decrease AR and increase PSA expression, relative to control tumors. Altogether, the data indicate that βarr1 modulates AR/PSA signaling to regulate prostate cancer aggressiveness. However, the effect of βarr1 is context-sensitive depending on its expression in the host relative to the tumor cells.

Association between Lymph Node Status and Expression Levels of Androgen Receptor, miR-185, miR-205, and miR-21 in Breast Cancer Subtypes

Breast cancer is the most commonly diagnosed cancer among women. Difficulties in treating breast cancer are associated with the occurrence of metastases at early stages of disease, leading to its further progression. Recent studies have shown that changes in androgen receptor (AR) and microRNAs' expressions are associated with mammary gland carcinogenesis, in particular, with the formation of metastases. Thus, to identify novel metastatic markers, we evaluated the expression levels of AR; miR-185 and miR-205, both of which have been confirmed to target AR; and miR-21, transcription of which is regulated by AR, in breast cancer samples (n = 89). Here, we show that the molecular subtypes of breast cancer differ in the expression profiles of AR and AR-associated microRNAs. In addition, the expression of AR and these microRNAs may depend on the expression of PR, ER, and HER2 receptors. Our results show that the possibility of using AR and microRNAs as markers depends on the tumor subtype: a decrease in AR expression may be the marker for the presence of lymph node metastases in patients with HER2-positive subtypes of breast cancer, and disturbance of miR-205, miR-185, and miR-21 expressions may be the marker in patients with a luminal B HER2-positive subtype. Cases with metastases in this type of breast cancer are characterized by a higher level of miR-205 and a lower level of miR-185 and miR-21 in tumor tissues compared to nonmetastatic cases. A decrease in the miR-185 level is also associated with lymph node metastasis in luminal B HER2-negative breast cancer. Thus, the expression levels of AR, miR-185, miR-205, and miR-21 can serve as markers to predict cancer spread to the lymph node in luminal B- and HER2-positive subtypes of breast cancer.

Insight into the molecular sex dimorphism of ischaemic stroke in rat cerebral cortex: Focus on neuroglobin, sex steroids and autophagy.

Including sex is of paramount importance in preclinical and clinical stroke researches, and molecular studies dealing in depth with sex differences in stroke pathophysiology are needed. To gain insight into the molecular sex dimorphism of ischaemic stroke in rat cerebral cortex, male and female adult rats were subjected to transient middle cerebral artery occlusion. The expression of neuroglobin (Ngb) and other functionally related molecules involved in sex steroid signalling (oestrogen and androgen receptors), steroidogenesis (StAR, TSPO and aromatase) and autophagic activity (LC3B-II/LC3B-I ratio, UCP2 and HIF-1α) was assessed in the ipsilateral ischaemic and contralateral non-ischaemic hemispheres. An increased expression of Ngb was detected in the injured female cerebral cortex. In contrast, increased expression of oestrogen receptor α, GPER, StAR, TSPO and UCP2, and decreased androgen receptor expression were detected in the injured male cortex. In both sexes, the ischaemic insult induced an upregulation of LC3B-II/-I ratio, indicative of increased autophagy. Therefore, the cerebral cortex activates both sex-specific and common molecular responses with neuroprotective potential after ischaemia-reperfusion, which globally results in similar stroke outcome in both sexes. Nonetheless, these different potential molecular targets should be taken into account when neuroprotective drugs aiming to reduce brain damage in ischaemic stroke are investigated.

Abstract P5-05-06: Umbelliferon derivatives for androgen receptor positive triple negative breast cancer

Abstract Purpose: Triple negative breast cancer (TNBC), which lacks hormone receptors and HER-2 over expression, has a poor prognosis due to the absence of targeted therapeutics. Gene expression profiling identified different subtypes of TNBC, several of which express androgen receptor (AR). AR inhibitors could provide a much-needed, less toxic, therapeutic intervention for patients with AR+ TNBC. However, clinical trials have shown modest activity of these agents in patients with metastatic AR+ TNBC to date. Conversion from AR antagonist to agonist is a well-recognized mechanism of resistance for AR inhibitors in the treatment of prostate cancer. To that end, we synthesized novel 7-substituted umbelliferon derivatives (UMBDs) with a distinct scaffold modified from available AR antagonists, designed to avoid such conversion. We have previously demonstrated that one of these agents, UMBD# 3 (compound 7a), demonstrates sub-micro molar inhibitory activity in a human prostate cancer with greater than 30 to 50 fold potency over the anti-androgens bicalutamide (Bica) and enzalutamide (Enza). In our current study, we tested the efficacy of UMBD# 3 in an AR+ TNBC cells that are sensitive or have acquired resistance to Bica. Methods: Bica-resistant cell lines were generated via multiple passaging of wild type (WT) MDA-MB-231 cells in media containing varying concentrations of Bica. Cell viability assays were conducted over a 72-hour period post seeding of AR+TNBC cell lines with AR antagonists Bica, Enza or UMBD#3. IC50 for respective drugs was determined via linear regression on normalized control values. Real time PCR and western blots were used to determine changes of AR at mRNA and protein level respectively, in response to the different agents. In order to isolate potential mechanistic pathway, we evaluated two proteins that are highly expressed in breast cancer, namely Poly (ADP-ribose) polymerase-1 (PARP-1) and Cadherin-11 (CAD-11) and assessed their expression in AR+ TNBC cells treated with UMBD#3. Results: The IC 50 for UMBD#3 was 6 and 4-fold lower than for Bica or Enza, respectively, in the WT AR+ TNBC cells (Bica, Enza and UMBD#3 IC50 were 136 ± 12.19, 91±12 and 20±7 µM respectively). The IC-50 for Bica in the Bica-resistant AR+ TNBC cells was 729 ± 42 µM, 6-fold higher than the WT cells. The IC50 for UMBD#3 in the Bica-resistant cells was 23.4±10 µM, similar to the WT cells. UMBD#3 caused a decrease in AR expression in the Wt and Bica-resistant calls. Expression of CAD-11 and PARP-1 were decreased in AR+ TNBC cells treated with UMBD#3 both at mRNA level and protein level. Conclusion: We show for the first time in vitro the anti-proliferative effect of a novel AR antagonist, UMBD#3, in WT and resistant AR+ TNBC. UMBD#3 appears to act via downregulation of AR with resultant decreased expression PARP and CAD-11. Given the apparent inherent resistance of AR+ TNBC to standard anti-androgens, further evaluation of this agent is warranted. Citation Format: Saswati Bhattacharya, Pam Westmark, Noah Siegel, Yuanqi Cai, Sahar Kandil, Andrew D Westwell, Christopher McGuigan, Ruth O'Regan. Umbelliferon derivatives for androgen receptor positive triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-06.