PSA Production Research Articles

Role of the sigma factor AlgU in regulating growth, virulence, motility, exopolysaccharide production, and environmental stress adaptation of Pseudomonas syringae pv. actinidiae QSY6

The extracytoplasmic function (ECF) sigma factor AlgU is involved in the regulation of various virulence-related pathways in Pseudomonas syringae, especially alginate biosynthesis and motility, and the role of AlgU differs among P. syringae pathovars. However, to date, the mechanism of its regulation in virulence of P. syringae pv. actinidiae (Psa) is still unclear. ECF sigma factors are a class of alternative sigma factors that typically function with anti-sigma factors as part of cell-surface signaling systems. Under non-inducing conditions, AlgU remains inhibited by anti-sigma factors such as MucA and MucB. To investigate the function of AlgU in Psa, mutant strains lacking algU or lacking algU with mucA and mucB genes, as well as complementary and overexpression strains of algU were generated, respectively. The results showed that AlgU was highly conserved among P. syringae pathovars and positively regulated growth rate, pathogenicity, and resistance to osmotic and oxidative stress of Psa QSY6. While AlgU did not affect the motility and exopolysaccharide production of Psa, its abundant expression enhanced the swimming ability of QSY6 and reduced its production of extracellular polysaccharides. Furthermore, AlgU regulates a number of virulence-related factors, including the Hrp system, the type VI secretion system, and flagellar synthesis. Specifically, AlgU induced the expression of hrpL and hrpRS in vivo, and repressed the transcription of hrpL and tssC in vitro, while promoting the expression of hrpS, fliC, and tssJ. This study contributes to a better understanding of the mechanisms of virulence regulation of AlgU in Psa.

Testosterone (T) challenge in men post-radical prostatectomy (RP) with profoundly low testosterone

ABSTRACT Introduction Prostate specific antigen is the primary marker of prostate cancer recurrence. PSA secretion is T dependent with T levels below saturation point associated with sub-optimal PSA production. Men with an undetectable PSA level after RP with very low T levels might have an artificially low PSA level. Objective We aimed to evaluate the effects of raising T level in such men. Methods Study population consisted of (i) men post-RP (ii) with undetectable PSA level (iii) total T levels <200 ng/dl (two levels, LCMS) who (iv) underwent in-office intramuscular injection of 100mg of T cypionate (v) had a PSA level checked 5 days later, and if undetectable every month after that, 3 months later then every 6 months and (vi) follow-up of at least 24 months on T therapy. Results 22 men have been challenged thus far. Mean age 62±18 years. Mean preoperative PSA 6.2. Mean post-RP TT value 140± 35 ng/dL 2.2. Median Gleason sum 7. 23% had GS ≥8. Mean post-RP, pre-challenge T value 140± 35 ng/dL. Mean time post-RP to challenge was 7± 9 months. Mean TT value 5 days after IMT challenge 640± 220 ng/dL, all with TT levels above 400. 5/22 has a PSA elevation, 2 immediately after IMT challenge (PSA values 0.07, 0.08). All other men started T therapy immediately and had PSA elevation at 9, 14 and 22 months after commencing T therapy (PSA values 0.08, 0.12, 0.11). All patients with PSA elevation had continued rise in PSA and had detectable disease on imaging. All men with PSA recurrence had either GS 7 with unfavorable pathology (2) or GS ≥8 (3). Conclusions This T challenge approach permits the early detection of prostate cancer recurrence permitting early intervention. About 10% of such men had a PSA rise immediately after T challenge. Disclosure Work supported by industry: no.

Testosterone (T) Challenge in Men Post-Radical Prostatectomy (RP) with Profoundly Low Testosterone

ABSTRACT Introduction Prostate specific antigen is the primary marker of prostate cancer recurrence. PSA secretion is T dependent with T levels below saturation point associated with sub-optimal PSA production. Men with an undetectable PSA level after RP with very low T levels might have an artificially low PSA level. Objective We aimed to evaluate the effects of raising T level in such men. Methods Study population consisted of (i) men post-RP (ii) with undetectable PSA level (iii) total T levels <200 ng/dl (two levels, LCMS) who (iv) underwent in-office intramuscular injection of 100mg of T cypionate (v) had a PSA level checked 5 days later, and if undetectable every month after that, 3 months later then every 6 months and (vi) follow-up of at least 24 months on T therapy. Results 22 men have been challenged thus far. Mean age 62±18 years. Mean preoperative PSA 6.2. Mean post-RP TT value 140± 35 ng/dL 2.2. Median Gleason sum 7. 23% had GS ≥8. Mean post-RP, pre-challenge T value 140± 35 ng/dL. Mean time post-RP to challenge was 7± 9 months. Mean TT value 5 days after IMT challenge 640± 220 ng/dL, all with TT levels above 400. 5/22 has a PSA elevation, 2 immediately after IMT challenge (PSA values 0.07, 0.08). All other men started T therapy immediately and had PSA elevation at 9, 14 and 22 months after commencing T therapy (PSA values 0.08, 0.12, 0.11). All patients with PSA elevation had continued rise in PSA and had detectable disease on imaging. All men with PSA recurrence had either GS 7 with unfavorable pathology (2) or GS ≥8 (3). Conclusions This T challenge approach permits the early detection of prostate cancer recurrence permitting early intervention. About 10% of such men had a PSA rise immediately after T challenge. Disclosure No

Modeled Early Longitudinal PSA Kinetics Prognostic Value in Rising PSA Prostate Cancer Patients after Local Therapy Treated with ADT +/- Docetaxel.

Simple SummaryIn prostate cancer patients with rising prostate-specific antigens (PSAs) after primary local therapy, and at high risk of metastatic disease based on a high Gleason score or ISUP grades 4–5 and/or short PSA doubling time, androgen-deprivation therapy (ADT) was shown to be effective with or without salvage radiotherapy. In the present paper, the analysis of a phase III trial dataset comparing ADT +/− docetaxel demonstrates that longitudinal PSA kinetics during the first 100 days of treatment, assessed using mathematical modeling, is associated with patient prognosis. Indeed, multivariate analyses showed that the modeled PSA production rate constant KPROD, and the elimination rate constant KELIM, exhibited strong and independent prognostic values regarding PSA progression-free survival (PSA-PFS) and overall survival (OS), respectively. As it was shown with the longitudinal CA-125 kinetic in ovarian cancers, the modeled longitudinal PSA kinetics during the 100 days of treatment may represent a novel prognostic factor in patients with metastatic prostate cancers.Background: In metastatic prostate cancer (PCa) patients, androgen-deprivation therapy (ADT) combined with chemotherapy or next-generation androgen receptor targeted agents is a new standard treatment. The objective of the present study is to assess longitudinal PSA kinetics during treatment using mathematical modeling, to identify the modeled PSA kinetic parameters able to exhibit early prognostic/predictive values. Methods: Phase III clinical trial dataset (NCT00764166) comparing ADT +/− docetaxel in 250 locally treated patients for PCa with rising PSA levels, who were at high risk of metastatic disease was assessed. A kinetic-pharmacodynamic (K-PD) model was used to fit PSA kinetics during the first 100 treatment days, to estimate the modeled PSA production rate K (KPROD) and elimination constant rate K (KELIM). The prognostic value of these parameters, considered as categorized (favorable vs. unfavorable) covariates regarding PSA progression-free survival (PSA-PFS) and overall survival (OS), was assessed using univariate/multivariate analyses. Results: Data from 177/250 patients was assessed. KELIM exhibited a significant prognostic value regarding PSA-PFS and KPROD regarding OS (univariate analysis). In the PSA-PFS final multivariate model, KELIM and the primary therapy type were significant. The OS multivariate model integrated both KPROD and baseline PSA doubling-time. Conclusion: In this first study assessing the modeled PSA kinetics prognostic value in PCa patients treated with systemic treatments, KELIM and KPROD exhibited respective prognostic values regarding PSA-PFS and OS.

Abiraterone switches castration-resistant prostate cancer dependency from adrenal androgens towards androgen receptor variants and glucocorticoid receptor signalling.

IntroductionCastration‐resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signalling, which is largely driven by conversion of adrenal androgen precursors lasting after castration. Abiraterone, an inhibitor of the steroidogenic enzyme CYP17A1, has been demonstrated to reduce adrenal androgen synthesis and prolong CRPC patient survival. To study mechanisms of resistance to castration and abiraterone, we created coculture models using human prostate and adrenal tumours.Materials and MethodsCastration‐naïve and CRPC clones of VCaP were incubated with steroid substrates or cocultured with human adrenal cells (H295R) and treated with abiraterone or the antiandrogen enzalutamide. Male mice bearing VCaP xenografts with and without concurrent H295R xenografts were castrated and treated with placebo or abiraterone. Response was assessed by tumour growth and PSA release. Plasma and tumour steroid levels were assessed by LC/MS‐MS. Quantitative polymerase chain reaction determined steroidogenic enzyme, nuclear receptor and AR target gene expression.ResultsIn vitro, adrenal androgens induced castration‐naïve and CRPC cell growth, while precursors steroids for de novo synthesis did not. In a coculture system, abiraterone blocked H295R‐induced growth of VCaP cells. In vivo, H295R promoted castration‐resistant VCaP growth. Abiraterone only inhibited VCaP growth or PSA production in the presence of H295R. Plasma steroid levels demonstrated CYP17A1 inhibition by abiraterone, whilst CRPC tumour tissue steroid levels showed no evidence of de novo intratumoural androgen production. Castration‐resistant and abiraterone‐resistant VCaP tumours had increased levels of AR, AR variants and glucocorticoid receptor (GR) resulting in equal AR target gene expression levels compared to noncastrate tumours.ConclusionsIn our model, ligand‐dependent AR‐regulated regrowth of CRPC was predominantly supported via adrenal androgen precursor production while there was no evidence for intratumoural androgen synthesis. Abiraterone‐resistant tumours relied on AR overexpression, expression of ligand‐independent AR variants and GR signalling.

Measurement of the Drug Sensitivity of Single Prostate Cancer Cells.

Simple SummaryCells communicate mainly through the secretion of proteins. Impaired protein secretion can indicate the development of disease. Cancer cell heterogeneity and acquired resistance to therapy are, however, reducing the effectiveness of cancer treatments. As cancer cells change during the course of the disease, sampling of cancer cells at the time of treatment is needed in order to determine which drugs will be effective. This paper describes a method for measuring secreted prostate specific antigen (PSA) protein from thousands of prostate cancer (PCa) cells. Furthermore, we show that the PSA secretion of individual cells in microwells can be stimulated or inhibited with drugs. To this end, we believe that this method could accelerate the development of new drugs, improve our understanding of resistance to therapy, and, ultimately, improve personalized cancer therapy.The treatment of cancer faces a serious challenge as cancer cells within patients are heterogeneous and frequently resistant to therapeutic drugs. Here, we introduce a technology enabling the assessment of single cancer cells exposed to different drugs. PCa cells were individually sorted in self-seeding microwells, cultured for 24 h, and then exposed to several drugs to induce (R1881) or inhibit (Enzalutamide/Abiraterone) the secretion of a protein (PSA). Cell viability and PSA secretion of each individual prostate cell were monitored over a 3-day period. The PSA protein secreted by each cell was captured on a PVDF membrane through a pore in the bottom of each well. The basal PSA secretion was found to be 6.1 ± 4.5 and 3.7 ± 1.9 pg/cell/day for LNCaP and VCaP, respectively. After exposure to R1881, the PSA secretion increased by ~90% on average and was not altered for ~10% of the cells. PSA production decreased in the majority of cells after exposure to enzalutamide and abiraterone.

Lidocaine-pressure sensitive adhesive patches from STR-5L block rubber: Preparations, in vitro characterizations, and stability studies

This study aimed to formulate the lidocaine-pressure sensitive adhesive (PSA) patches by melt blending technique using STR-5L block rubber as the main polymer, hydroxyethyl cellulose (HEC) as a tackifier, and paraffinic oil as a softener. STR-5L with 80 Mooney viscosity was masticated for 15 min using two rolls mill machines, and then HEC, paraffinic oil and lidocaine were added, respectively. Lidocaine-PSA patches were constructed after 75 min of mixing and rolled out with a gap of 0.1 mm. Their physicochemical properties, in vitro drug release and permeation, and stability were evaluated. Lidocaine base (LB) and lidocaine hydrochloride salt (LH) were used as the models of hydrophobic and hydrophilic drugs, respectively. The results were found that STR-5L block rubber with HEC and paraffinic oil blends could be used as a PSA matrix for both lidocaine base and salt delivery. Moreover, either LB or LH powder could be mixed during the PSA production process. Lidocaine-PSA patches exhibited higher hydrophilic properties when compared with the plain rubber patch due to the hydrophilic property of HEC. LB showed the molecular dispersion in the PSA patch but LH showed the crystalline character in the PSA patch when observed by differential scanning calorimeter and X-ray diffractometer. The drug release of both LB and LH from PSA patches were not significantly different. However, the LB permeation through pig skins was better than LH's one due to its hydrophobic characteristic. The most suitable condition to keep lidocaine-PSA patches in this study was at 4 °C of storage temperature.

Development of 2-(5,6,7-Trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide as a Potent, Selective, and Orally Available Inhibitor of Human Androgen Receptor Targeting Its Binding Function-3 for the Treatment of Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer (CRPC). Targeting the androgen receptor (AR) Binding Function-3 (BF3) site offers a promising option to treat CRPC. However, BF3 inhibitors have been limited by poor potency or inadequate metabolic stability. Through extensive medicinal chemistry, molecular modeling, and biochemistry, we identified 2-(5,6,7-trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide (VPC-13789), a potent AR BF3 antagonist with markedly improved pharmacokinetic properties. We demonstrate that VPC-13789 suppresses AR-mediated transcription, chromatin binding, and recruitment of coregulatory proteins. This novel AR antagonist selectively reduces the growth of both androgen-dependent and enzalutamide-resistant PCa cell lines. Having demonstrated in vitro efficacy, we developed an orally bioavailable prodrug that reduced PSA production and tumor volume in animal models of CRPC with no observed toxicity. VPC-13789 is a potent, selective, and orally bioavailable antiandrogen with a distinct mode of action that has a potential as novel CRPC therapeutics.

MP36-01 TESTOSTERONE (T) CHALLENGE IN MEN POST-RADICAL PROSTATECTOMY (RP) WITH PROFOUNDLY LOW TESTOSTERONE

You have accessJournal of UrologySexual Function/Dysfunction: Medical, Hormonal & Non-surgical Therapy II (MP36)1 Sep 2021MP36-01 TESTOSTERONE (T) CHALLENGE IN MEN POST-RADICAL PROSTATECTOMY (RP) WITH PROFOUNDLY LOW TESTOSTERONE John P. Mulhall, Serkan Deveci, Jose Flores, Kazuhito Matsushita, Jose Torremade, and Carolyn A. Salter John P. MulhallJohn P. Mulhall More articles by this author , Serkan DeveciSerkan Deveci More articles by this author , Jose FloresJose Flores More articles by this author , Kazuhito MatsushitaKazuhito Matsushita More articles by this author , Jose TorremadeJose Torremade More articles by this author , and Carolyn A. SalterCarolyn A. Salter More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002045.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate specific antigen is the primary marker of prostate cancer recurrence. PSA secretion is T dependent with T levels below saturation point associated with sub-optimal PSA production. Men with an undetectable PSA level after RP with very low T levels might have an artificially low PSA level. We aimed to evaluate the effects of raising T level in such men. METHODS: Study population consisted of (i) men post-RP (ii) with undetectable PSA level (iii) total T levels <200 ng/dl (two levels, LCMS) who (iv) underwent in-office intramuscular injection of 100mg of T cypionate (v) had a PSA level checked 5 days later, and if undetectable every month after that, 3 months later then every 6 months and (vi) follow-up of at least 24 months on T therapy. RESULTS: 22 men have been challenged thus far. Mean age 62±18 years. Mean preoperative PSA 6.2. Mean post-RP TT value 140±35 ng/dL 2.2. Median Gleason sum 7. 23% had GS ≥8. Mean post-RP, pre-challenge T value 140± 35 ng/dL. Mean time post-RP to challenge was 7±9 months. Mean TT value 5 days after IMT challenge 640±220 ng/dL, all with TT levels above 400. 5/22 has a PSA elevation, 2 immediately after IMT challenge (PSA values 0.07, 0.08). All other men started T therapy immediately and had PSA elevation at 9, 14 and 22 months after commencing T therapy (PSA values 0.08, 0.12, 0.11). All patients with PSA elevation had continued rise in PSA and had detectable disease on imaging. All men with PSA recurrence had either GS 7 with unfavorable pathology (2) or GS ≥8 (3). CONCLUSIONS: This T challenge approach permits the early detection of prostate cancer recurrence permitting early intervention. About 10% of such men had a PSA rise immediately after T challenge. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e636-e636 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information John P. Mulhall More articles by this author Serkan Deveci More articles by this author Jose Flores More articles by this author Kazuhito Matsushita More articles by this author Jose Torremade More articles by this author Carolyn A. Salter More articles by this author Expand All Advertisement Loading ...

Identification and Characterization of a Trillin Rhamnosyltransferase From Dioscorea zingiberensis.

Dioscorea zingiberensis accumulates abundant steroidal saponins, such as dioscin, which is the principal bioactive ingredient displaying a wide range of pharmacological activities. Diosgenin is the aglycone of dioscin, and recently, genes encoding cytochrome P450 enzymes in the late steps of diosgenin biosynthesis have been isolated. Diosgenin was successfully synthesized in the cholesterol-producing yeasts. From diosgenin to dioscin, one glucose and two rhamnose groups need to be added. Although genes encoding UDP-glucosyltransferases converting diosgenin to trillin were isolated, genes encoding UDP-rhamnosyltransferases involved in dioscin biosynthesis remain unknown. In this study, we isolated the cDNA encoding the trillin rhamnosyltransferase (designated DzGT1) from D. zingiberensis. Heterologous expression of DzGT1 in Escherichia coli cells showed that the gene product exhibits an enzyme activity that glycosylates the trillin to form prosapogenin A of dioscin (PSA). The transcript level of DzGT1 is in accord with PSA accumulation in different organs of D. zingiberensis. Integration of the biochemical, metabolic, and transcriptional data supported the function of DzGT1 in dioscin biosynthesis. The identification and characterization of DzGT1 will help understand the metabolism of steroidal saponins in D. zingiberensis and provide candidate UDP-rhamnosyltransferase for efficient production of PSA, dioscin, and relevant steroidal saponins in microbial hosts.

Abstract B017: Role of CYP3A5 in modulating androgen receptor signaling and its relevance in African American prostate cancer patients

Abstract Introduction: African-American men (AA) often present with aggressive castration-resistant prostate cancer (CRPC), due to highly active androgen receptor (AR). AR is a ligand-activated transcription factor that promotes expression of genes responsible for cell proliferation and growth. Previously, we have shown that CYP3A5 promotes AR nuclear translocation and activation leading to increased prostate cancer (PC) growth. 73% of AAs carrying wild-type CYP3A5 (*1/*1) express full-length functional CYP3A5, whereas 95% of Caucasians carry mutant (*3/*3) variant producing truncated nonfunctional protein. Difference in CYP3A5 expression leads to highly active AR and aggressive disease in AAs. Additionally, most of the PC patients are prescribed concomitant medications to manage age-related comorbidities. Many of these drugs are known modulators of CYP3A5; modulation of CYP3A5 may alter efficacy of ADT in these patients. Our work is focused on characterizing the effect of CYP3A5 modulating drugs on AR signaling in AAs. Methods: q-RT-PCR based profiler assay was used to study effect of CYP3A5 modulation on AR-regulated genes using cDNA from nontarget (NT) and CYP3A5 siRNA treated cells. Confocal microscopy and cell fractionation assays were performed to evaluate and confirm the effect of CYP3A5 modulating drugs on AR nuclear translocation. Genes shortlisted from q-PCR based profiler assays were analyzed for change of expression in response to CYP3A5 modulating drugs. MDAPCA2b (AA origin, *1/*3) and LNCaP (Caucasian, *3/*3) cell lines were used for above experiments. Results: CYP3A5 siRNA pool treatment downregulates AR regulated genes identified using q-PCR based profiler assay, performed with cDNA from CYP3A5 siRNA pool and NT treated MDAPCA2b cells. These downregulated genes include SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1 and SPDEF with p-value of ≤0.005. These genes are known to regulate AR nuclear translocation, cell cycle progression, and cell growth. CYP3A5 siRNA treated MDACPA2b/ LNCaP cells showed decreased AR nuclear translocation and PSA production. Commonly prescribed drugs that are either CYP inhibitors (amiodarone, ritonavir) or inducers (phenytoin, rifampicin) were tested for their ability to alter AR signaling. The results show that the CYP inducers promoted AR nuclear migration and downstream signaling whereas CYP3A5 inhibitors blocked it. Further, CYP3A5 siRNA treated MDAPCa2b cells do not show any increase in AR nuclear migration with phenytoin treatment (CYP3A inducer), confirming that the activation of AR activity is specific to changes in CYP3A5 activity. Conclusions: Concomitantly prescribed CYP3A5 modulating drugs can alter downstream AR signaling, cell growth, and ADT efficacy in men, more so in AAs expressing wild-type CYP3A5. We propose to further characterize drug-induced CYP3A5 modulation of AR signaling to develop a guideline for physicians coprescribing CYP3A5 modulating drugs to treat comorbidities in elderly patients undergoing ADT. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr, Ranjana Mitra. Role of CYP3A5 in modulating androgen receptor signaling and its relevance in African American prostate cancer patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B017.

Abstract 2026: Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer

Abstract Well-differentiated prostate cancer (PCa) cells are generally androgen/androgen receptor (AR)-dependent, i.e. AR signaling regulates cell cycle and differentiation. Loss of AR signaling after androgen deprivation therapy (ADT) triggers AR-independent outgrowth, generating poorly differentiated uncontrollable PCa cells. To prevent development and progression of castration-resistant prostate cancer (CRPC), we hypothesize that preservation of AR signaling after ADT is an important target. Tumor stroma surrounding PCa cells is enriched in fibroblasts secreting AR-activating factors, e.g. EGF, IGF1, and IL-6. Thus, we investigated the role of fibroblasts in AR activation of human prostate cancer LNCaP sublines differing in androgen sensitivity under androgen deprivation. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Original fibroblasts pcPrFs (-M5, -M6, -M7) were isolated from needle biopsy samples of patients with PCa. The PCa cells alone or PCa cells plus pcPrF-M5 were grafted beneath the renal capsule of male athymic nude mice, and then mice were castrated on 14 days post transplantation. Tumor volume of E9 cells + pcPrF-M5 became diminished post castration but that of F10 and AIDL cells + pcPrF-M5 was gradually increased even post castration. In E9 cells + pcPrF-M5 tumors, serum PSA became detected on day 21 post castration. Interestingly, serum PSA was gradually increased even in F10 cells inoculated alone. In AIDL tumors with or without pcPrF-M5, serum PSA was not detected because of mutated AR in AIDL cells. Cell growth of E9 and AIDL cells was directly increased by treating with EGF, whereas that in F10 cells was not affected. Phosphorylation of AKT and ERK1/2 in E9 and AIDL cells was also significantly increased by treating with EGF, whereas that in F10 cells was not affected. In the condition of co-cultures with fibroblasts, PSA production was directly increased in E9 cells but not in F10 and AIDL cells. Protein expression of AR splice variant 7 (AR-V7) was only detected in F10 cells. The reduced AR-dependency of PCa cells is an important clinical development because of its association with the cell’ progression to CRPC. Here our results showed that fibroblasts secreting AR-activating factors preserved AR signaling in E9 cells after ADT, indicating that this type of PCa cells can be controlled by ADT. In contrast, we suggest that loss of fibroblasts-dependent AR activation in F10 and AIDL cells may be responsible for development and progression of CRPC. Citation Format: Kenichiro Ishii, Izumi Matsuoka, Takeshi Sasaki, Manabu Kato, Kohei Nishikawa, Hideki Kanda, Yoshifumi Hirokawa, Kazuhiro Iguchi, Kiminobu Arima, Masatoshi Watanabe, Yoshiki Sugimura. Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2026.

Harnessing Androgen Receptor Pathway Activation for Targeted Alpha Particle Radioimmunotherapy of Breast Cancer

The impact of androgen receptor (AR) activity in breast cancer biology is unclear. We characterized and tested a novel therapy to an AR-governed target in breast cancer.Experimental Design: We evaluated the expression of prototypical AR gene products human kallikrein 2 (hK2) and PSA in breast cancer models. We screened 13 well-characterized breast cancer cell lines for hK2 and PSA production upon in vitro hormone stimulation by testosterone [dihydrotestosterone (DHT)]. AR-positive lines were further evaluated by exposure to estrogen (17β-Estradiol) and the synthetic progestin D-Norgestrel. We then evaluated an anti-hK2-targeted radiotherapy platform (hu11B6), labeled with alpha (α)-particle emitting Actinium-225, to specifically treat AR-expressing breast cancer xenografts under hormone stimulation. D-Norgestrel and DHT activated the AR pathway, while 17β-Estradiol did not. Competitive binding for AR protein showed similar affinity between DHT and D-Norgestrel, indicating direct AR-ligand interaction. In vivo production of hK2 was sufficient to achieve site-specific delivery of therapeutic radionuclide to tumor tissue at >20-fold over background muscle uptake; effecting long-term local tumor control. [225Ac]hu11B6 targeted radiotherapy was potentiated by DHT and by D-Norgestrel in murine xenograft models of breast cancer. AR activity in breast cancer correlates with kallikrein-related peptidase-2 and can be activated by D-Norgestrel, a common contraceptive, and AR induction can be harnessed for hK2-targeted breast cancer α-emitter radiotherapy.

Abstract B001: CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance

Abstract Purpose: Androgen receptor (AR) plays a major role in promoting growth and progression of prostate cancer and thus has been successfully targeted therapeutically. Therapeutic resistance to androgen deprivation therapy (ADT) accounts for much of the morbidity and mortality attributable to prostate cancer. Therapeutic resistance is typically accompanied by insufficient blockade of AR activation, whose initial step involves translocation of AR to the nucleus. In recent studies we demonstrated that CYP3A5 facilitates the nuclear translocation of AR, promoting prostate cancer growth. This work focuses on understanding the role of CYP3A5 in regulating AR nuclear translocation and therapeutic resistance. Method: To understand how CYP3A5 effects AR downstream signaling, we used RT2 profiler PCR arrays to help identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy and cell fractionation experiments were used to further confirm the effect of CYP3A5 modulation of AR signaling. As concomitant CYP3A5 inducer/inhibitor medications can affect intratumoral CYP3A5 activity and thus may modify AR downstream signaling, we utilized cell fractionation and confocal microscopy to analyze the effect of these medications on AR nuclear translocation. Further, to decipher the role of CYP3A5 in the AR translocation process we performed a series of co-immunoprecipitation (co-IP) experiments with CYP3A5 and components of HSP90-AR complex. We used extracts of LNCaP transfected with a CYP3A5-Flag-tagged construct for IP, with either Flag-agarose or HSP90 and AR antibodies with A/G beads. Finally, to decipher CYP3A5’s role in therapeutic resistance we analyzed cDNA after CYP3A5 siRNA treatment using drug resistance RT2 profiler PCR array. Results: The q-PCR based profiler assay revealed a set of nine AR regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005. MYC and PIK3R3 are known to be involved in regulating cell growth by either affecting cell cycle progression and/or apoptosis. PSA, known to aid in the cell growth, is regulated by SPDEF, a transcription factor. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. Concomitantly prescribed CYP3A inhibitors amiodarone and ritonavir blocked AR nuclear migration and decreased PSA expression whereas inducers phenytoin and rifampicin promoted increased AR nuclear migration and increased PSA production, supporting our hypothesis. Co-immunoprecipitation experiments revealed that CYP3A5 is a part of the HSP90-AR complex. Both Flag-immunoprecipitation and indirect immunoprecipitation revealed that CYP3A5 associates with the HSP90-AR complex. The CYP3A5 siRNA also downregulated 11 genes (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB, and HPRT1) known to confer drug resistance, with a p value of ≤0.005. The role of TOP2A, MYC, and BRCA is well known in prostate cancer drug resistance; thus, their downregulation with CYP3A5 siRNA treatment may predict better outcome. Conclusion: Our results indicate that CYP3A5 is a part of the HSP90-AR complex and positively regulates AR nuclear translocation promoting AR downstream signaling and therapeutic resistance. Therefore, targeting CYP3A5 to block AR nuclear translocation may be a therapeutic adjunctive approach in advanced prostate cancer. As CYP3A5 siRNA also downregulates drug-resistance genes, targeting CYP3A5 may also help overcome therapeutic resistance. As many concomitant medications can alter CYP3A5 activity, thus they can modify AR signaling. We hypothesize that AR directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors. These observations may play a significant role in African Americans who express high level of wild-type CYP3A5 and often have aggressive prostate cancer. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B001.

Abstract B48: Effect of concomitant CYP3A5-modulating drugs on androgen-receptor signaling in African American men undergoing androgen deprivation therapy

Abstract Introduction: In a recent study we have established that CYP3A5 augments nuclear translocation of androgen receptor (AR) and promotes prostate cancer (PC) growth. CYP3A isoforms are responsible for metabolizing almost 50% of the commonly prescribed drugs, many of which are CYP3A5 inhibitors and inducers. PC patients are elderly with comorbidities and are often prescribed concomitant medications while undergoing androgen-deprivation therapy (ADT), the most preferred treatment for PC. CYP3A5 is polymorphic, and African Americans (AA, 73%) express higher levels of CYP3A5 due to presence of wild-type CYP3A5 (*1/*1) compared to Caucasians (95%) expressing the mutant (*3/*3) producing only 13% of the active full-length CYP3A5 protein. Due to the expression of wild-type CYP3A5 in AA, concomitant medications modulating CYP3A5 activity can drastically alter AR activity and ADT efficacy in these patients. Our current work is focused on deciphering the mechanism of how commonly used CYP3A5-modulating medications can alter AR signaling in AA carrying the wild-type CYP3A5. Method: We genotyped the androgen-responsive prostate cancer cell lines to determine their CYP3A5 status using TaqMan® Drug Metabolism assay. We used cDNA from cells treated with either nontarget or CYP3A5 siRNA in RT2 profiler PCR arrays to identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy was used to study AR nuclear localization after exposure to commonly used CYP3A5 inducer/inhibitor drugs. Cell fractionation experiments were used to further confirm the effect of these drugs on AR signaling. Luciferase-based reporter assay was used to test effect of CYP3A5-modulating drugs on AR activity. Results: Our genotyping assay revealed that MDA PCa 2b (African American) carries *1/*3 CYP3A5 gene compared to LNCaP (Caucasian) carrying *3/*3 genotype. To compare the differences due to CYP3A5 genotype on AR signaling, we selected MDAPCa2b and LNCaP cells for further studies. Consistent with earlier observations with LNCaP, MDA PCa 2b cell lines also showed decreased nuclear translocation of AR with CYP3A5 siRNA in both confocal and cell fractionation experiments. We also observed blocking of AR nuclear localization with azamulin, a CYP3A-specific inhibitor in both cell lines. The q-PCR-based profiler assay revealed a set of nine AR-regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005 in MDA PCa 2b cells. MYC is known to be involved in cell cycle progression and apoptosis whereas PIK3R3 codes for the regulatory subunit of PI3 kinase regulating cell growth. SPDEF codes for a transcription factor promoting PSA known to aid in the cell growth. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. We tested some commonly prescribed drugs that are either CYP inhibitors (amiodarone, ritonavir) or inducers (phenytoin, rifampicin) and evaluated their ability to alter AR signaling. The results revealed that the CYP inducers promoted increased AR nuclear migration and increased PSA production whereas CYP3A5 inhibitors blocked AR nuclear migration, decreasing PSA expression in both cell lines. A luminescence-based reporter assay using androgen response elements also supported that these CYP3A inducer and inhibitor drugs alter AR signaling. Conclusion: Our results indicate that concomitant medications that can alter CYP3A5 activity can modify AR signaling and PC growth. These results suggest that these CYP-modulating drugs can alter efficacy of ADT severely in AA men predominantly expressing wild-type CYP3A5 (*1/*1). We hypothesize that AR-directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors in patients undergoing ADT. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. Effect of concomitant CYP3A5-modulating drugs on androgen-receptor signaling in African American men undergoing androgen deprivation therapy [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B48.

Dipotassium phosphate improves the molecular weight stability of polysialic acid in Escherichia coli K235 culture broth

This work elucidated the intrinsic mechanism underlying the influence of K2HPO4 on PSA production and molecular weight (MW) stability. Among the different potassium salts mixed with K2HPO4 in the initial medium, those with buffering capacity were favorable for PSA production. In the bioreactor culture with pH control, adding an appropriate concentration of K2HPO4 could enhance PSA production. A dual-phase pH control strategy with ammonia water and KOH could also increase the yield and maintain the MW stability of PSA. Zeta potential test, UV/circular dichroism spectra, and transmission electric microscopy were utilized to explore the configuration of K2HPO4–PSA complex. The results from this study can serve a good basis for the industrial-scale production of PSA with stable MW.

Tissue-scale, personalized modeling and simulation of prostate cancer growth

Recently, mathematical modeling and simulation of diseases and their treatments have enabled the prediction of clinical outcomes and the design of optimal therapies on a personalized (i.e., patient-specific) basis. This new trend in medical research has been termed "predictive medicine." Prostate cancer (PCa) is a major health problem and an ideal candidate to explore tissue-scale, personalized modeling of cancer growth for two main reasons: First, it is a small organ, and, second, tumor growth can be estimated by measuring serum prostate-specific antigen (PSA, a PCa biomarker in blood), which may enable in vivo validation. In this paper, we present a simple continuous model that reproduces the growth patterns of PCa. We use the phase-field method to account for the transformation of healthy cells to cancer cells and use diffusion-reaction equations to compute nutrient consumption and PSA production. To accurately and efficiently compute tumor growth, our simulations leverage isogeometric analysis (IGA). Our model is shown to reproduce a known shape instability from a spheroidal pattern to fingered growth. Results of our computations indicate that such shift is a tumor response to escape starvation, hypoxia, and, eventually, necrosis. Thus, branching enables the tumor to minimize the distance from inner cells to external nutrients, contributing to cancer survival and further development. We have also used our model to perform tissue-scale, personalized simulation of a PCa patient, based on prostatic anatomy extracted from computed tomography images. This simulation shows tumor progression similar to that seen in clinical practice.

PYK2 via S6K1 regulates the function of androgen receptors and the growth of prostate cancer cells.

Androgen receptor (AR) is a steroid hormone receptor that functions as a transcription factor for regulating cell growth and survival. Aberrant AR function becomes a risk factor for promoting the progression of prostate cancer (PCa). In this study, we examined the roles of proline-rich tyrosine kinase 2 (PYK2) and ribosomal S6 kinase 1 (S6K1) in regulating AR expression and activity and growth properties in PCa cells. Compared with normal prostate tissues, PCa tumors exhibited high levels of PYK2 and S6K1 expression. Furthermore, the expression levels of PYK2 and S6K1 were significantly correlated with nuclear AR expression in PCa tissues. We further found the association between PYK2, S6K1, and AR in their protein expression and phosphorylation levels among normal prostate PZ-HPV-7 cells and prostate cancer LNCaP and 22Rv1 cells. Overexpression of the wild-type PYK2 in PZ-HPV-7 and LNCaP cells promoted AR and S6K1 expression and phosphorylation as well as enhanced cell growth. In contrast, expression of the mutated PYK2 or knockdown of PYK2 expression in LNCaP or 22Rv1 cells caused reduced expression or phosphorylation of AR and S6K1 as well as retarded cell growth. Under an androgen-deprived condition, PYK2-promoted AR expression and phosphorylation and PSA production in LNCaP cells can be abolished by knocking down S6K1 expression. In summary, our data suggested that PYK2 via S6K1 activation modulated AR function and growth properties in PCa cells. Thus, PYK2 and S6K1 may potentially serve as therapeutic targets for PCa treatment.

Abstract C97: Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling

Abstract Introduction Castration resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signalling, driven by adrenal precursors and potentially de novo steroid synthesis in other organ tissues including prostate. Abiraterone, an inhibitor of the steroidogenic enzyme CYP17A1 and the AR has been demonstrated to prolong survival of CRPC patients. In this study, we created a co-culture model using human prostate and adrenal tumours to study abiraterone resistance. Materials and Methods Human androgen-dependent PC (VCaP) and CPRC clones were cultured with substrates for de novo androgen synthesis or with adrenal androgens, or cultured with human adrenal cells (H295R) and treated with either the CYP17A1 inhibitor abiraterone or the antiandrogen MDV3100. Male mice bearing VCaP tumours and human adrenal H295R xenografts were castrated and treated with placebo or abiraterone. Tumour response was assessed by tumour growth, PSA release, steroid quantitation by (LC/MS-MS), immunohistochemistry and mRNA expression analysis of steroidogenic enzymes and nuclear receptors. Results In vitro, physiological levels of adrenal androgen precursors DHEA and androstenedione induced cell growth in parental and CRPC VCaP sub clones, whereas precursor steroids pregnenolone and progesterone for de novo synthesis did not. In a co-culture model, abiraterone blocked H295R-induced growth of VCaP cells. Likewise, in vivo, H295R tumours stimulated castration-resistant VCaP growth. This stimulative effect was inhibited by abiraterone, reducing - but not fully blocking - growth and PSA production. In the absence of H295R tissue, VCaP xenografts grew slow but became castration resistant nonetheless. In contrast to the observed effects on VCaP growing in castrate animals bearing H295R tumours, abiraterone was unable to inhibit the slow VCaP growth and low PSA production in castrate mice without H295R xenografts. LC/MS-MS analysis of plasma and tumour tissue could not confirm increased de novo production of androgens. Castrate and abiraterone-resistant VCaP tumours were characterised by increased levels of AR, AR variants and glucocorticoid receptor (GR) expression, resulting in equal AR target gene expression levels. Conclusions Our data indicate that AR ligand dependent regrowth of CRPC is predominantly supported via adrenal steroid production. Abiraterone resistant disease of VCaP relies on AR overexpression, expression of ligand independent AR variants and GR signalling. Citation Format: Jan Matthijs Moll, Johannes Hofland, Wilma Teubel, Corrina M.A. de Ridder, Anne E. Taylor, Ralph Graeser, Wiebke Arlt, Guido W. Jenster, Wytske M. van Weerden. Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C97.

URINARY RETENTION EFFECT IN THE INCREASED OF PSA EXPRESSION WITHIN PROSTATIC TISSUE

Objective: To determine whether the increased of PSA expression within prostatic tissue and subsequent systemic blood circulation in acute urinary retention cases of nonmalignant origin were caused by acute inflammation on the prostatic gland. Following this inflammation, PSA willincrease, producing byacinar epitelial cells in the gland and continue to capillary vessels before entering the blood circulation. Material &amp; method: Thirty male Rattus Norvegicus were randomly allocated into 3 groups. A control group underwent urethral manipulation, treatment-1 group and -2 group underwent proximal urethral ligation. Prostatectomy was performed after 24 hours in the control and treatment-1 group. Ligation was removed after 24 hours in treatment-2 group, and prostatectomy performed after 4x24 hours. Each prostate specimen was examined for PSA expression by immunohistochemistry methods in the prostatic gland. Statistical analysis of study data was analyzed by descriptive statistics and performed ANOVA with level of significance α = 0.01.Results: Study results showed an increase PSA expression significantly after urinary retention and returned to normal values four days later after relief of retention.Conclusion: Urinary retention caused acute inflammation on the prostatic gland and increased PSA expression within prostatic tissue. Entry of PSA into stroma and subsequent systemic blood circulation occur through significant increase in PSA production by acinar epithelial cells.