Prostate-specific Antigen Secretion Research Articles

Prostate-specific antigen: An unfamiliar protein in the human salivary glands.

The presence of prostate-specific antigen (PSA) in saliva and salivary glands has been reported. Nevertheless, its release pathway in these glands remains to be elucidated. Here, we showed PSA subcellular distribution focusing on its plausible route in human salivary parenchyma. Sections of parotid and submandibular glands were subjected to the immunohistochemical demonstration of PSA by the streptavidin-biotin method revealed by alkaline phosphatase. Moreover, ultrathin sections were collected on nickel grids and processed for immunocytochemical analysis, to visualize the intracellular distribution pattern of PSA through the observation by transmission electron microscopy. By immunohistochemistry, in both parotid and submandibular glands PSA expression was detected in serous secretory acini and striated ducts. By immunocytochemistry, immunoreactivity was retrieved in the cytoplasmic compartment of acinar and ductal cells, often associated with small cytoplasmic vesicles. PSA labeling appeared also on rough endoplasmic reticulum and in the acini's lumen. A negligible PSA labeling appeared in most of the secretory granules of both glands. Our findings clearly support that human parotid and submandibular glands are involved in PSA secretion. Moreover, based on the immunoreactivity pattern, its release in oral cavity would probably occur by minor regulated secretory or constitutive-like secretory pathways.

Microfluidic-based prostate cancer model for investigating the secretion of prostate-specific antigen and microRNAs in vitro

The study of prostate cancer in vitro relies on established cell lines that lack important physiological characteristics, such as proper polarization and expression of relevant biomarkers. Microphysiological systems (MPS) can replicate cancer microenvironments and lead to cellular phenotypic changes that better represent organ physiology in vitro. In this study, we developed an MPS model comprising conventional prostate cancer cells to evaluate their activity under dynamic culture conditions. Androgen-sensitive (LNCaP) and androgen-insensitive (PC3) cells were grown in conventional and 3D cultures, both static and dynamic. Cell morphology, the secretion of prostate-specific antigen, and the expression of key prostate markers and microRNAs were analyzed. LNCaP formed spheroids in 3D and MPS cultures, with morphological changes supported by the upregulation of cytokeratins and adhesion proteins. LNCaP also maintained a constant prostate-specific antigen secretion in MPS. PC3 cells did not develop complex structures in 3D and MPS cultures. PSA expression at the gene level was downregulated in LNCaP-MPS and considerably upregulated in PC3-MPS. MicroRNA expression was altered by the 3D static and dynamic culture, both intra- and extracellularly. MicroRNAs associated with prostate cancer progression were mostly upregulated in LNCaP-MPS. Overall dynamic cell culture substantially altered the morphology and expression of LNCaP cells, arguably augmenting their prostate cancer phenotype. This novel approach demonstrates that microRNA expression in prostate cancer cells is sensitive to external stimuli and that MPS can effectively promote important physiological changes in conventional prostate cancer models.

Abstract 3904: Simvastatin intensifies anti-androgen efficacy against treatment-resistant prostate cancer cells

Abstract Androgen receptor (AR) signaling inhibitors (ARSIs) bicalutamide (Bic) and enzalutamide (Enza) are used in the treatment of castration resistant prostate cancer (CRPC). Prostate cancer (PCa) cells eventually develop treatment resistance against these drugs causing clinical challenges. The mechanisms behind treatment resistance are complex and only partially known. Androgen signaling is linked to lipid and cholesterol metabolism, and they likely play a role in the development of treatment resistance. Cholesterol producing mevalonate pathway can be inhibited by statins, but it is unknown whether statin treatment could enhance effects of ARSIs. We studied combined effects of simvastatin (Sim) with or without Bic or Enza in VCaP originated PCa cell lines resistant to these ARSIs. Unique treatment resistant cell lines were created with long-term cultures of VCaP cells. The cells were kept under anti-androgen influence until the cells were growing normally despite the ARSIs. We analyzed changes in cell growth, RNA expression, and relevant AR signaling and cholesterol metabolism protein expression. Prostate-specific antigen (PSA) secretion was measured as a marker of AR signaling activity. In Bic or Enza resistant cell lines, combination of 2.5 or 5 μM simvastatin + 10 μM Bic/Enza decreased cell growth more than Sim alone, i.e., ARSIs demonstrated efficacy in combination therapy against ARSI resistant cells. In Bic and Enza resistant cells, combination treatment increased the expression of 60 and 26 and decreased the expression of 33 and 48 genes, respectively, compared with simvastatin alone. Altogether, 16 genes expression changed similarly in both cell lines in response to combination therapy. In both cell lines, over 70% of all gene changes were seen on AR-regulated genes. In Bic resistant cells, Sim decreased PSA levels with or without Bic. On the contrary, in Enza resistant cells PSA levels were increased with combination of Sim and Enza. We showed that combined treatment with ARSI and Sim inhibited cell growth more than Sim alone in ARSI resistant cell lines. This suggests that inhibition of the mevalonate pathway could enhance androgen signaling inhibition and potentially circumvent ARSI resistance mechanisms. This phenomenon is supported by epidemiological studies as patients treated with anti-androgens responded better during simultaneous statin treatment. Gene expression differed in combination treatment compared to Sim alone. Future studies are needed on whether interventions on lipid and cholesterol metabolism could enhance treatment of CRPC. Knowledge about the precise mechanisms behind the treatment responses and resistance might lead to new therapeutic applications. Citation Format: Aino Siltari, Olga Korhonen, Paavo Raittinen, Merja Bläuer, Heimo Syvälä, Teuvo L. Tammela, Teemu J. Murtola. Simvastatin intensifies anti-androgen efficacy against treatment-resistant prostate cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3904.

Vitamin D, a Regulator of Androgen Levels, Is Not Correlated to PSA Serum Levels in a Cohort of the Middle Italy Region Participating to a Prostate Cancer Screening Campaign

Prostate cancer (PCa) is the most common non-cutaneous malignancy in men worldwide, and it represents the fifth leading cause of death. It has long been recognized that dietary habits can impact prostate health and improve the benefits of traditional medical care. The activity of novel agents on prostate health is routinely assessed by measuring changes in serum prostate-specific antigen (PSA) levels. Recent studies hypothesized that vitamin D supplementation reduces circulating androgen levels and PSA secretion, inhibits cell growth of the hormone-sensitive PCa cell lines, counteracts neoangiogenesis and improves apoptosis. However, the results are conflicting and inconsistent. Furthermore, the use of vitamin D in PCa treatments has not achieved consistently positive results to date. In order to assess the existence of a correlation between the PSA and 25(OH)vitamin D levels as widely hypothesized in the literature, we analyzed the serum PSA and 25(OH)vitamin D concentration on a cohort of one hundred patients joining a PCa screening campaign. Additionally, we performed medical and pharmacological anamnesis and analyzed lifestyle, as sport practice and eating habits, by administering a questionnaire on family history. Although several studies suggested a protective role of vitamin D in PCa onset prevention and progression, our preliminary results revealed a clear absence of correlation between the serum vitamin D and PSA concentration levels, suggesting that vitamin D has no impact on PCa risk. Further investigations enrolling a huge number of patients are needed with particular attention to vitamin D supplementation, calcium intake, solar radiation that influences vitamin D metabolism and other potential indicators of health to confirm the absence of correlation observed in our study.

A low prostate specific antigen predicts a worse outcome in high but not in low/intermediate-grade prostate cancer

ObjectiveThe relationship between prostate-specific antigen (PSA) and prostate cancer (PCa) grade was traditionally thought to be linear but recent reports suggest this is not true in high-grade cancers. We aimed to compare the association between PSA and PCa-specific mortality (PCSM) in clinically localised low/intermediate and high-grade PCa. Subjects/patients and methodsRetrospective cohort study using the National Prostate Cancer Audit database in England of men treated with external beam radiotherapy (EBRT), EBRT and brachytherapy boost (EBRT + BT), radical prostatectomy or no radical local treatment between 2014 and 2018. Multivariable competing-risk regression was used to examine the association between PSA, Gleason, and PCSM. Multivariable restricted cubic spline regression was used to explore the non-linear associations of PSA and PCSM. Results102,089 men were included, of whom 71,138 had low/intermediate-grade and 22,425 had high-grade PCa. In high-grade, 4-year PCSM was higher with PSA ≤5 than PSA 5.1–10 for men treated with EBRT (hazard ratio 1.96 (95% confidence interval 1.15–3.34) or no radical local treatment (hazard ratio 1.99 (95% confidence interval 1.33–2.98). Restricted cubic spline regression showed that PSA and PCSM have a non-linear association in high-grade but a linear association in low/intermediate-grade PCa. ConclusionThe low-PSA/high-grade combination in M0 PCa treated with EBRT has a higher PCSM than those with high-grade and intermediate PSA levels. In high-grade disease, the PSA association was non-linear; by contrast, low/intermediate-grade had a linear relationship. This confirms a more aggressive biology in low PSA secreting high-grade PCa and a worse outcome following treatment.

Chromogranin A: a useful biomarker in castration-resistant prostate cancer

PurposeThe natural history of prostate cancer (PC) almost always evolves to castration-resistant prostate cancer (CRPC) status, sometimes comprising pure or mixed neuroendocrine prostate cancers (NEPC) differentiation. In CRPC, monitoring using only prostate-specific antigen (PSA) is not optimal since neuroendocrine differentiated cells do not secrete PSA. Thus, monitoring with PSA and chromogranin A (CgA) may be useful. This review aims to evaluate evidence for the usefulness of CgA assessments during the monitoring of prostate cancer.MethodThis review was based on three recent meta-analysis concerning CgA and prostate cancer. Further data were obtained from PubMed and Embase databases by searches using keywords, including chromogranin A and prostate cancer.ResultsCgA levels remain largely unchanged during the early PC evolution. The development of NEPC is characterised by lower PSA secretion and increased CgA secretion. Data supporting the prognostic value of high CgA baseline levels for survival are contrasting and scarce. However, increasing CgA levels early during treatment of metastatic (m)CRPC suggests resistance to treatment and predicts shorter survival, particularly in men with high baseline levels of CgA levels. In men with mCRPC, the first-line chemotherapy may be more appropriate than other agents when baseline CgA levels are high. Also, increasing CgA levels during treatment may indicate disease progression and may warrant a change of therapy.ConclusionCgA monitoring at baseline and regularly during mCRPC management may be useful for monitoring disease evolution. An increased CgA baseline levels and increasing CgA levels may assist physicians with choosing and modifying therapy.

The Relationship Between PSA and Total Testosterone Levels in Men With Prostate Cancer

Prostate-specific antigen (PSA) secretion is a testosterone (T) dependent process. Published data suggest that a low T level is an independent predictor of higher-grade prostate cancer (PC). To evaluate the relationship between T and PSA in patients with PC. All men diagnosed with PC with a recorded pre-treatment total T level measurement were included in this analysis. We analyzed demographic, clinical, and pathological data. Patients were stratified according to pretreatment PSA levels: <2 ng/mL, 2-4 ng/mL, >4 ng/mL. Low T was defined as total T < 10.4 nmol/L (300 ng/dL), very low T < 6.9 nmol/L (200 ng/dL). T levels by PSA groups according to the PC pathology. In this retrospective study, mean patient age was 61 years among 646 men. The distribution by PSA group was: 8% (<2), 17% (2-4), and 76% (>4). The mean T level across the entire cohort was 13 nmol/L (374 ng/dL). Overall, 30% had a T level < 10.4 nmol/L (300 ng/dL). The mean total T level by PSA group was: <2 ng/mL, 7 nmol/L (206 ng/dL); 2-4 ng/mL, 13 nmol/L (362 ng/dL); >4 ng/mL, 14 nmol/L (393 ng/dL), P < .001. PSA <4 ng/mL was a significant predictor of low T in men with PC GS ≥8. PSA <2 ng/mL was a significant predictor of very low T independent of the PC pathology. These findings suggest that clinicians should consider measuring T levels when a patient diagnosed with PC GS ≥8 and PSA level <4 ng/mL, and for each patient with PSA level <2 ng/mL independent of the PC pathology. Our study has several strengths including (i) inclusion of a large population of men, (ii) use of a database which is audited and reviewed for accuracy annually, and (iii) use of an accurate T assay (LCMS). Nonetheless, there are limitations: (i) the subjects of the study are from a single institution, and (ii) we did not measure free T levels. In men with PC with GS ≥8, PSA level <4 ng/mL predicts low T. PSA <2 ng/mL predicts very low T independent of the PC pathology. Flores JM, Bernie HL, Miranda E, et al. The Relationship Between PSA and Total Testosterone Levels in Men With Prostate Cancer. J Sex Med 2022;19:471-478.

Tissue microarray profiling and integrative proteomics indicate the modulatory potential of Maytenus royleanus in inhibition of overexpressed TPD52 in prostate cancers

Maytenus roylanus (MEM) is a plant with anti-proliferative effects against prostate cancer. We aimed to explore the mechanism of action of MEM in prostate cancer (PCa) by employing an in vitro global proteome approach to get useful information of various signaling pathways and effected genes to define the mechanism of MEM action in prostate cancer. We conducted a global proteome analysis of CWR22Rv1after treatment with methanolic extract of MEM. The result of the proteomic profiling of in vitro PCa cells demonstrated the reduction in tumor protein D52 (TPD52) expression after treatment with methanolic extract of MEM. Down-regulation of TPD52 expression at mRNA level was observed by MEM treatment in CWR22Rν1 and C4-2 cells in a dose-dependent fashion probably by cleavage of Caspase 3 and PARP, or by modulation of cyclin-dependent kinases in CWR22Rν1 and C4-2 cells. The progressive character of the TRAMP model demonstrates a chance to evaluate the potential of chemo-preventive agents for both initial and late stages of prostate cancer development, and induction in TPD52 protein expression with development as well as the progression of prostate cancer was observed in the TRAMP model. Analyses of the tissue microarray collection of 25 specimens confirmed the clinical significance of our findings identifying TPD52 as a potential marker for PCa progression. We determined that knockdown of TPD52 (CWR22Rν1 cells), a considerable downregulation was seen at the protein level. Downregulation of TPD52 inhibited the migration and invasive behavior of prostate cancer cells as observed. Moreover, we observed that the siRNA-TPD52 transfection of CWR22Rν1 cells resulted in tumor growth inhibition with a marked reduction in the secretion of prostate-specific antigen (PSA) in the serum. Intraperitoneal injection of MEM considerably slowed tumor growth in athymic mice, inhibited TPD52 expression, and caused a marked reduction in PSA levels of serum as demonstrated by immunoblot screening and immune-histochemical staining. This report illustrates a molecular overview of pathological processes in PCa, indicating possible new disease biomarkers and therapeutic targets.

In Vitro Assessment of Diet-Derived Phytochemicals on PSA Secretion

Prostate cancer (PCa) is increasingly a global public health burden. The prostate specific antigen (PSA) is the major biomarker for diagnosing and managing PCa. Due to the importance of PSA as a biomarker and as a downstream product of the androgen receptor (AR), it was imperative to expose LNCaP cells to selective diet-derived phytochemicals and investigate the effect on PSA protein secretion. The phytochemicals: Curcumin, 3,3’-Diindolylmethane (DIM) and Epigallcatechin-3-gallate (EGCG) were utilised singly and in paired combinations as in vitro treatments. The ELISA technique was utilised to assess secreted PSA levels. Results obtained indicated that DIM posed inhibitory effects to PSA production and secretion. Uniquely, DIM lowered PSA levels significantly when compared to curcumin and EGCG. This finding corroborates with previous reports where DIM was observed to inhibit AR protein expression. It is suggestive that DIM targets the androgen receptor and its downstream pathway that produces PSA. In conclusion, DIM may hold overwhelming potential chemoprevention and management of PCa, and can be harnessed for enhanced clinical applications.

Effects of Curcumin Combined With the 5-alpha Reductase Inhibitor Dutasteride on LNCaP Prostate Cancer Cells.

Curcumin is a natural compound of turmeric, which inhibits prostate cancer cell proliferation. This study examined whether treatment of LNCaP prostate cancer cells with the combination of curcumin and dutasteride, a 5-alpha reductase inhibitor, affect proliferation and the amount of testosterone and dihydrotestosterone. LNCaP Cells were incubated with curcumin or the combination of curcumin and dutasteride and cell proliferation was measured at 72 h. LC-MS/MS was used to determine testosterone and dihydrotestosterone concentrations in prostate cancer cells. Curcumin combined with dutasteride suppressed proliferation and affected apoptosis of LNCaP cells. The combination of curcumin and dutasteride also reduced the amount of testosterone and dihydrotestosterone in LNCaP cells. The secretion of prostate-specific antigen was inhibited by the combination treatment in a dose-dependent manner. Treatment with the combination of curcumin and dutasteride may interfere with the intra-tumoral androgen activity.

Abstract 2693: Detection of PSA secretion from single prostate cancer cells with and without drug stimulation

Abstract Treatment options available for cancer patients are increasing rapidly. Biomarkers to predict which therapies will be effective are however lacking, resulting in the frequent administration of ineffective therapies. Emergence of therapy resistance and both inter-patient and intra-patient heterogeneity play an important role in the differences in response to treatment in prostate cancer (PC). Therefore, research into the nature of the heterogeneity and its significance will aid in developing an improved individualized treatment strategy. Here we use a microwell technology that can enable the testing of drug effectivity and heterogeneity of tumor cells. Several techniques exist to study secreted proteins, including enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISpot), intracellular staining, and cytokine capture. The common immunoassays that directly measure secreted proteins on a solid support (ELISA and ELISpot) fail to correlate the captured analytes with the producing cell and cannot do this repeatedly on the same cell. We used the microwell technology to sort viable single cells derived from the prostate cancer cell lines LnCAP and VcCAP. The Prostate Specific Antigen (PSA) produced by LnCAP and VcAP cells is measured with or without drug stimulation (r1881, Enzalutamide or Abiraterone). To measure PSA secretion the microwell is connected to a membrane coated with antibodies against PSA. We demonstrate that PSA secreted by cells can be measured and we quantified the amounts for &amp;gt;1200 single PC cells. Before cells were subjected to drug stimulation PSA secretion was measured. Results indicate a heterogeneous PSA secretion profile for both cell lines; the average LnCAP secretion patterns were 4,7 (sd=3,3), 11,3 (sd=5,9) and 2,4 (sd=1,1) pg/cell for respectively, non-stimulated, r1881 androgen steroid and Enzalutamide respectively. LnCAPs stimulated with Abiraterone secreted 4,7 (sd=1,9) pg/cell, indicating that enzalutamide has a higher inhibitory effect on PSA secretion than abiraterone. VcAPs secreted 3,8 (sd=1,8), 6,5 (sd=4,1) and 0,9 (sd=0,7) pg/cell for non-stimulated, r1881 androgen steroid and enzalutamide stimulated cells respectively. Abiraterone stimulated cells secreted 2,0 (sd=1,5) pg/cell. The results further suggest that a small percentage of LnCAP of the cells (&amp;lt;1%) did not respond to Enzalutamide treatment indicating resistance to the drug. We expect that this approach can enable to study and predict the efficacy of the clinical response to drug and ultimately improve the success rate of treatment of PC patients. This research is part of the HTSM research programme and was partly funded by the NWO, The Netherlands Organization for Scientific Research (Utrecht, The Netherlands) and is part of Project McSPRinter under project number 15327. Citation Format: Fikri Abali, Narghes Baghi, Arjan Tibbe, Leon Terstappen. Detection of PSA secretion from single prostate cancer cells with and without drug stimulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2693.

PSA secretion and cell proliferation are affected by NCoR1 silencing in prostate cancer cells

The androgen receptor (AR) plays an important role in normal prostate gland development as well as in prostatic neoplasms. Transcriptional regulation by AR is modulated by its interaction with co-activators or co-repressors, such as NCoR1 (nuclear receptor co-repressor 1), which is involved in the reduction of AR activity over the target gene transcription. To identify the role of NCoR1 in the prostate cancer androgen independence in a cell line model, we aimed to evaluate the effects of silencing NCoR1 on prostate-specific antigen ( PSA) gene expression, the proliferative response and PSA secretion on the supernatant of C4-2B and LNCaP cells that were submitted to small interfering RNAs (siRNAs) transfection and to treatments with different androgen dosages. In LNCaP and C4-2B cells with no dihydrotestosterone (DHT) treatment, a decrease of PSA mRNA expression was observed 48 hours and 72 hours after gene silencing in the siNCoR group when compared to the control and siNC groups. LNCaP and C4-2B cells showed a biphasic pattern in response to dihydrotestosterone treatment in transfected groups (siNCoR and siNC) as well as in the control condition (no transfection). The secretion of PSA in cell supernatant of LNCaP and C4-2B cells was higher in the siNCoR group, and, regarding the hormonal treatment, higher in the 10 -8 M DHT group. A reduction in the levels of NCoR1 seems to have a double influence on the activity of AR in PCa cells. These results suggest that NCoR can act as AR a co-repressor depending upon hormonal stimulation.

Abstract P6-05-06: Significance of prostate specific antigen as a predictor of androgen receptor activity in breast cancer

Abstract BACKGROUND: Preclinical data suggest that androgen receptor (AR) activity plays a role in breast cancer (BC). Clinical trials testing AR-targeted therapies in BC have shown clinical benefit and additional trials are currently underway. However, a method is needed to predict which patients may benefit the most. Prostate specific antigen (PSA) is the secreted product of the androgen-responsive gene kallikrein related peptidase 3 (KLK3). Serum PSA (sPSA) is an established indicator of tumor burden in prostate cancer. Recently, we found that sPSA can be detected in BC patients and is positively correlated with serum testosterone levels, AR expression in the primary tumor, and aromatase inhibitor resistance (Hanamura, et al. SABCS 2018). HYPOTHESIS: Serum PSA may reflect AR activity in BC and have utility as a predictive marker for identifying patients that would benefit from AR-targeted therapy. OBJECTIVE: To establish that PSA is regulated by AR in BC, we examined the relationship between AR and PSA in BC cell lines, BC patient derived xenograft (PDX), and publically-available datasets of patient gene expression profile. METHODS: Nine BC cell lines representing three clinical subtypes (ERα+, HER2+ and TNBC) were analyzed for response to dihydrotestosterone (DHT) and the AR antagonist enzalutamide (Enza) by crystal violet assay and further analyzed by western blot for PSA. Next, PSA was measured in conditioned media from BT474 BC cells by chemiluminescence immunoassay. Finally, HCI-009, an AR+ TNBC PDX model, grown in NOD-scid gamma mice were given vehicle control versus DHT and were used to identify AR regulated genes by RNA-sequencing, followed by verification at the protein level by IHC. Genes increased in HCI-009 from mice given DHT treatment as compared to vehicle were defined as “PDX androgen-responsive gene set” and imported into gene set enrichment analysis (GSEA). To test whether PSA reflects AR activity, two publically available datasets, The Cancer Genome Atlas (TCGA) (n=1100) and the population-based Multi center Sweden Cancerome Analysis Network (n=405), were mined for correlation between KLK3 andAR or ESR1mRNA (for comparison), and with GSEA with regard to known androgen- or estrogen-responsive gene sets. RESULTS: Although the proliferative response to DHT varied among cell lines, Enza decreased proliferation of the majority of AR+ BC lines regardless of the proliferative response to DHT. Western blot analysis showed PSA to be regulated in an AR-dependent manner (induced by DHT and suppressed by Enza). In BT474 cells, PSA secretion into the media was DHT-responsive and suppressed by Enza. Gene expression analysis of the HCI-009 PDX model showed KLK3 as one of most DHT responsive genes. IHC analysis showed PSA and other AR regulated proteins are significantly higher in tumors from mice given DHT as compared to control and that tumor growth was increased in these mice compared to control mice. In the TCGA and Sweden cohort, a stronger positive correlation was found between KLK3 and ARmRNA expression compared to KLK3 and ESR1 mRNA. An androgen-responsive gene set was significantly enriched in patients with a high KLK3, but the estrogen-responsive gene set was enriched in patients with a low KLK3. The PDX androgen-responsive gene set was also significantly enriched in patients with a high KLK3. CONCLUSIONS: Results of in vitro studies suggest that PSA reflects AR activity and the responsiveness of BC cells to AR antagonism. In comprehensive analysis of gene expression profiling, KLK3 was positively correlated with AR activity, but negatively correlated with ER activity. Together with our previous findings, these data suggest the need for further studies to determine whether sPSA or other AR-regulated secreted proteins are useful as a clinical predictive marker of response to AR targeted therapies. Citation Format: Toru Hanamura, Jessica L Christenson, Kathleen I O’Neill, Emmanuel Rosas, Nicole S Spoelstra, Michelle M Williams, Jennifer K Richer. Significance of prostate specific antigen as a predictor of androgen receptor activity in 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 P6-05-06.

Purple rice extract inhibits testosterone-induced rat prostatic hyperplasia and growth of human prostate cancer cell line by reduction of androgen receptor activation.

The preventive effects of purple rice crude ethanolic extract (PRE) were firstly investigated on testosterone-induced benign prostatic hyperplasia (BPH) in castrated rats. As compared to vehicle-treated rats, lower prostate weights were found in the BPH rats that received PRE 1g/kg bw. In addition, the PRE treatment down-regulated the androgen receptor (AR) expression in the dorsolateral prostate of those rats. In human prostate cancer cell line, LNCaP, PRE could reduce the cell growth, down-regulate the expression of AR and suppress prostate-specific antigen (PSA) secretion. Moreover, PRE also inhibited an activity of 5α-reductase from rat liver microsomes and the mutagenicity of Salmonella Typhimurium induced by standard mutagen. These results demonstrate that PRE altered testosterone-induced BPH in rats and retarded prostate cancer cell growth by modulating AR expression. It is therefore recommended that further investigation is undertaken into the chemopreventive potential of PRE in androgen-AR mediated diseases. PRACTICAL APPLICATIONS: This study revealed the mechanisms of purple rice extract on testosterone-induced rat benign prostatic hyperplasia. Such information, purple rice components show promise as an effective chemopreventive agent for prostatic hyperplasia prevention by alternating the influence of testosterone through its receptor. Thus, purple rice might be developed as food supplement for reduction of prostatic hyperplasia or cancer in elder men.

Serum Prostate-Specific Antigen Level in Women With Polycystic Ovary Syndrome: A Systematic Review and Meta-analysis.

We undertook a comprehensive search of all published original articles evaluating the diagnostic value of prostate-specific antigen (PSA) for the detection of polycystic ovary syndrome (PCOS). Finally, 25 studies with a total of 1865 participants (1104 cases and 761 controls) met the inclusion criteria. Mean serum PSA levels were significantly higher in PCOS women compared to healthy women [Standardized mean difference (SMD)=1.73 ng/ml, 95% CI=1.22 to 2.25, p<0.001]. Only 5 studies reported accuracy indicators and were included in the bivariate random-effects model; with a total of 509 samples that included 332 (65.22%) confirmed PCOS women. The sensitivity of serum PSA for the diagnosis of PCOS varied between 66.3 and 90.6%, and, the specificity ranged from 62.5-86.7%. Meta-analysis revealed a pooled sensitivity of 72.3% (95% CI 67.1-77.00%) and specificity of 74.6% (95% CI 67.5-80.8%) with an area under the summary ROC curve equal to 0.82 (95% CI 0.75-0.83). The summary estimates were 2.92 (95% CI 1.98-4.31) for positive LR and 0.37 (95% CI 0.26-0.51) for negative LR. The pooled estimate of diagnostic odds ratio (DOR) of serum PSA was 9.01 (95% CI 4.35-18.64). Considering the methodological weaknesses existed in studies, lack of sufficient data regarding the accuracy indicators, and the complexity of the exact pathophysiology of PSA secretion in women, the precise position of PSA, as a diagnostic test for the detection of PCOS, in clinical practice is undetermined, and further studies are still needed.

The Role of Alcohol-Induced Golgi Fragmentation for Androgen Receptor Signaling in Prostate Cancer.

Multiple epidemiologic observations and meta-analysis clearly indicate the link between alcohol abuse and the incidence and progression of prostate cancer; however, the mechanism remains enigmatic. Recently, it was found that ethanol (EtOH) induces disorganization of the Golgi complex caused by impaired function of the largest Golgi matrix protein, giantin (GOLGB1), which, in turn, alters the Golgi docking of resident Golgi proteins. Here, it is determined that in normal prostate cells, histone deacetylase 6 (HDAC6), the known regulator of androgen receptor (AR) signaling, localizes in the cytoplasm and nucleus, while its kinase, glycogen synthase kinase β (GSK3β), primarily resides in the Golgi. Progression of prostate cancer is accompanied by Golgi scattering, translocation of GSK3β from the Golgi to the cytoplasm, and the cytoplasmic shift in HDAC6 localization. Alcohol dehydrogenase-generated metabolites induces Golgi disorganization in androgen-responsive LNCaP and 22Rv1 cells, facilitates tumor growth in a mouse xenograft model and activates anchorage-independent proliferation, migration, and cell adhesion. EtOH-treated cells demonstrate reduced giantin and subsequent cytoplasmic GSK3β; this phenomenon was validated in giantin-depleted cells. Redistribution of GSK3β to the cytoplasm results in phosphorylation of HDAC6 and its retention in the cytoplasm, which, in turn, stimulates deacetylation of HSP90, AR import into the nucleus, and secretion of prostate-specific antigen (PSA). Finally, the relationship between Golgi morphology, HDAC6 cytoplasmic content, and clinicopathologic features was assessed in human prostate cancer patient specimens with and without a history of alcohol dependence. IMPLICATIONS: This study demonstrates the importance of alcohol-induced Golgi fragmentation in the activation of AR-mediated proliferation.

Ex vivo treatment of prostate tumor tissue recapitulates in vivo therapy response.

BackgroundIn vitro models of prostate cancer (PCa) are not always reliable to evaluate anticancer treatment efficacy. This limitation may be overcome by using viable tumor slice material. Here we report on the establishment of an optimized ex vivo method to culture tissue slices from patient‐derived xenografts (PDX) of prostate cancer (PCa), to assess responses to PCa treatments.MethodsThree PDX models were used that are characterized by different androgen receptor (AR) expression and different homology directed DNA repair capacities, due to a breast cancer associated two (BRCA2) wild‐type or mutated status. Tumors were removed from mice, sliced using a vibratome and cultured for a maximum of 6 days. To test the sensitivity to androgen antagonist, tumor slices from the AR‐expressing and AR‐negative PDX tumors were treated with the anti‐androgen enzalutamide. For sensitivity to DNA repair intervention, tumors slices from BRCA2 wild‐type and mutated PDXs were treated with the poly (ADP‐ribose) polymerase‐1 inhibitor olaparib. Treatment response in these tumor slices was determined by measuring slice morphology, cell proliferation, apoptosis, AR expression level, and secretion of prostate specific antigen (PSA).ResultsWe compared various culture conditions (support materials, growth media, and use of a 3D smooth rocking platform) to define the optimal condition to maintain tissue viability and proliferative capacity up to least 6 days. Under optimized conditions, enzalutamide treatment significantly decreased proliferation, increased apoptosis, and reduced AR‐expression and PSA secretion of AR‐expressing tumor slices compared to AR‐negative slices, that did not respond to the intervention. Olaparib treatment significantly increased cell death in BRCA2 mutated tumors slices as compared to slices from BRCA2 wild type tumors.ConclusionsEx vivo treatment of PCa PDX tumor slices with enzalutamide and olaparib recapitulates responses previously observed in vivo. The faithful retention of tissue structure and function in this ex vivo model offers an ideal opportunity for treatment efficacy screening, thereby reducing costs and numbers of experimental animals.

The Activity of Urolithin A and M4 Valerolactone, Colonic Microbiota Metabolites of Polyphenols, in a Prostate Cancer In Vitro Model.

The gut microbiota-derived metabolites of ellagitannins and green tea catechins, urolithin A (uroA) and 5-(3',4',5'-trihydroxyphenyl)-γ-valerolactone (M4), respectively, are among the main compounds absorbed into human system after ingestion of these polyphenols. The aim of this study was to establish the effects of M4, uroA, and their combinations on LNCaP cells, an androgen dependent prostate cancer in vitro model.. The LNCaP cells were incubated with increasing concentrations of tested metabolites. The cell proliferation was determined by measurement of DNA-bisbenzimide H 33 258 complexes fluorescence. The isobolographic analysis was used to establish the type of interaction between metabolites. The apoptosis, androgen receptor (AR) localization, and phosphorylation of Akt kinase were measured by flow cytometry. Prostate-specific antigen (PSA) secretion was determined by ELISA. M4 showed modest antiproliferative activity in LNCaP cells (IC50 = 117 µM; CI: 81 - 154). UroA decreased proliferation (IC50 = 32.7 µM; CI: 24.3 - 41.1) and induced apoptosis of LNCaP cells. The mixture of M4 with uroA had synergistic antiproliferative effect. Moreover, M4 potentiated inhibition of PSA secretion and enhanced retention of AR in cytoplasm caused by uroA. Interestingly, uroA increased levels of pSer473 Akt in LNCaP cells. These results show that colonic metabolites may contribute to chemoprevention of prostate cancer by varied polyphenol-rich diet or composite polyphenol preparations.

PO-442 Ex vivo prostate cancer culture system recapitulates in vivo response to androgen antagonist

IntroductionIn vitro models of prostate cancer (PCa) do not always accurately represent the different stages of PCa nor reliably predict treatment efficacy. Therefore, we have developed an experimental platform in which we cultured tumour slices from patient-derived prostate cancer xenografts (PDX) and tested whether ex vivo PCa culture can recapitulate the in vivo response to the androgen antagonist enzalutamide.Material and methodsIn this ex vivo PDX culture system we used tumours from two PCa models in mice, one androgen receptor (AR) expressing model and one AR-negative model. Tumours were removed from the mice and sliced using a vibratome. We characterised the tumour slices by analysing morphology, cell proliferation, apoptosis, expression levels of AR and excretion of prostate specific antigen (PSA). To test the sensitivity to androgen antagonist, tumour slices from the AD and AI models were treated with enzalutamide.Results and discussionsWe optimised support materials, growth medium and use of a 3D smooth rocking platform to increase oxygen and nutrient exchange. Under optimal condition, the tissue slice cultures maintained morphology, proliferative capacity and viability for at least six days. AR expression was largely maintained and tumour slices continued to secrete PSA in AR expressing tumour slices and responded to enzalutamide with a significant decrease of S-phase cells, increase of apoptotic cells, dramatic reduction of AR expression and inhibition of PSA secretion. As expected, the AR-negative model did not respond to the AR antagonist.ConclusionThis thorough characterisation of PCa PDX tumour slices in an ex vivo culture system showed that the response to enzalutamide recapitulates that observed in vivo. The faithful retention of tissue structure and function in this ex vivo model offers an ideal opportunity for drug screenings, thereby reducing costs and numbers of experimental animals.

Evaluation of SHetA2 Analogs as Anti‐Cancer Agents on Human Prostate Cancer Cells

BackgroundSHetA2 is a flexible heteroarotinoid compound that has shown potential anticancer effects on many cancer cell lines as well as animal models of cancer. However, due to its high lipophilicity, poor target specificity and low bioavailability, it may not be an ideal drug candidate. To overcome these limitations, over twenty thiochroman analogs of SHetA2 were synthesized and screened for their growth inhibitory effects in three different human prostate cancer cells. The screening yielded three analogs, SL‐01‐18, SL‐01‐30 and SL‐01‐39 that showed growth inhibitory effects comparable to SHetA2. All three analogs were more potent in inhibiting the growth of androgen receptor (AR) positive cell line, LNCaP, than the AR negative cell lines, DU145 and PC3. Here, we further evaluated the mechanism of action of these three analogs in LNCaP cell line.ObjectiveThe aim of the study was to evaluate the mechanism of action of the three SHetA2 analogs and compare their effects to SHetA2 in androgen receptor positive cell line, LNCaP.MethodsApoTox‐Glo assay was performed on LNCaP cells after 24 hours exposure to SHetA2 and its three analogs to determine the cell viability, cytotoxicity and caspase 3/7 activity. Bromodeoxyuridine (BrdU) incorporation assay was performed to measure the proportion of LNCaP cells in S‐phase of the cell cycle after drug treatments. Western blot analysis was performed on LNCaP cells treated with the SHetA2 analogs to analyze the effect on the expression of androgen receptor (AR) protein. Finally, prostate specific antigen (PSA)‐ELISA assay was performed to determine PSA concentration on LNCaP supernatants upon treatment with SHetA2 and its analogs.ResultsApoTox‐Glo assay results showed decreased cell viability, increased caspase activity and very little cytotoxicity in LNCaP cells treated with SHetA2 and its analogs. BrdU incorporation assay in LNCaP cells revealed a significant reduction in the proportion of cells in the S‐phase of the cell cycle with all four drug treatments, Finally, Western blot analysis revealed time and concentration‐dependent decrease in AR expression in treated cells while PSA‐ELISA assay indicated significant decrease in PSA secretion from LNCaP cells treated with SHetA2 and its analogs for 72 hours.ConclusionSHetA2 and its thiochroman analogs, SL‐01‐18, SL‐01‐30 and SL‐01‐39 inhibited cell growth of AR positive LNCaP cells while not being cytotoxic to these cells. In addition to stalling the cells from entering the S‐phase of the cell cycle, these agents also raised the likelihood of the cells to undergo apoptosis by activating the executioner caspase 3/7. A decrease in secreted PSA levels as well as concentration‐dependent decrease in AR protein expression suggests that the SHetA2 analogs may elicit their action by interfering with androgen receptor signaling. However, understanding how exactly are the analogs affecting androgen receptor signaling, inducing cell cycle arrest and/or causing programmed cell death requires further investigation.Support or Funding InformationTouro CA COP‐MS Program for funding this research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.