Prostate-specific Antigen mRNA Research Articles

Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cancer (mCRPC) by integrating the VTX-1 CTC isolation system with the QIAGEN AdnaTest

BackgroundTherapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported.MethodsTo characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay.ResultsAR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA.ConclusionVTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.

Impact of Circulating Tumor Cell-Expressed Prostate-Specific Membrane Antigen and Prostate-Specific Antigen Transcripts in Different Stages of Prostate Cancer.

Prostate-specific membrane antigen (PSMA)-based images, which visually quantify PSMA expression, are used to determine prostate cancer micrometastases. This study evaluated whether a circulating tumor cell (CTC)-based transcript platform, including PSMA mRNA, could help identify potential prognostic markers in prostate cancer. We prospectively enrolled 21 healthy individuals and 247 patients with prostate cancer [localized prostate cancer (LPCa), n = 94; metastatic hormone-sensitive prostate cancer (mHSPC), n = 44; and metastatic castration-resistant prostate cancer (mCRPC), n = 109]. The mRNA expression of six transcripts [PSMA, prostate-specific antigen (PSA), AR, AR-V7, EpCAM, and KRT 19] from CTCs was measured, and their relationship with biochemical recurrence (BCR) in LPCa and mCRPC progression-free survival (PFS) rate in mHSPC was assessed. PSA-PFS and radiological-PFS were also calculated to identify potential biomarkers for predicting androgen receptor signaling inhibitor (ARSI) and taxane-based chemotherapy resistance in mCRPC. CTC detection rates were 75.5%, 95.3%, and 98.0% for LPCa, mHSPC, and mCRPC, respectively. In LPCa, PSMA [hazard ratio (HR), 3.35; P = 0.028) and PSA mRNA (HR, 1.42; P = 0.047] expressions were associated with BCR. Patients with mHSPC with high PSMA (HR, 4.26; P = 0.020) and PSA mRNA (HR, 3.52; P = 0.042) expressions showed significantly worse mCRPC-PFS rates than those with low expression. Increased PSA and PSMA mRNA expressions were significantly associated with shorter PSA-PFS and radiological PFS in mCPRC, indicating an association with drug resistance. PSMA and PSA mRNA expressions are associated with BCR in LPCa. In advanced prostate cancer, PSMA and PSA mRNA can also predict rapid progression from mHSPC to mCRPC and ARSI or taxane-based chemotherapy resistance.

Association of serum prostate-specific antigen (PSA) level and circulating tumor cell-based PSA mRNA in prostate cancer.

BackgroundProstate-specific antigen (PSA) is used for diagnosing prostate cancer, but does not reflect the characteristics of prostate cancer cells to allow assessment of cancer progression. PSA mRNA and circulating tumor cells (CTCs) could be potential biomarkers. However, the relationship between serum PSA levels and PSA mRNA in CTCs is unclear, and this study aimed to investigate this relationship.MethodsHealthy donors (HD, n = 9), and patients with local non-metastatic stage prostate cancer (n = 30), metastatic hormone–sensitive prostate cancer (mHSPC, n = 10), and metastatic castration–resistant prostate cancer (mCRPC, n = 75), were included. The expression of PSA mRNA in CTCs was measured by droplet digital PCR. Serum PSA (ng/mL) levels and PSA mRNA (copies/μL) in CTCs were then compared using Spearman correlation coefficients.ResultsPSA mRNA expression in CTCs was observed in 30% (9/30) of patients with localized cancer, 60.0% (6/10) among patients with mHSPC, 65.3% (49/75) among patients with mCRPC, and 0% among patients with HD, indicating that the detection rate of PSA mRNA increased with cancer stage. PSA mRNA expression in CTCs also increased from localized to metastatic stages. PSA mRNA levels rapidly increased in the mHSPC and mCRPC stages. Interestingly, PSA mRNA expression in CTCs was not correlated with serum PSA levels at the localized stage (R = 0.064, P = 0.512). However, there were significant correlations between serum PSA levels and PSA mRNA expression in mHSPC (R = 0.532, P = 0.041) and mCRPC (R = 0.566, P = 0.025). The number of CTCs isolated from mHSPC and mCRPC was not proportional to serum PSA and PSA mRNA levels.ConclusionCTC PSA mRNA has the potential to be used as a biomarker to complement serum PSA protein analysis or replace serum PSA in metastatic stages of prostate cancer.

Dual electrochemical genosensor for early diagnosis of prostate cancer through lncRNAs detection

The prostate specific antigen (PSA) test is the gold standard for the screening of prostate cancer (PCa), despite its limited clinical specificity. Long noncoding RNAs are released from the tumor tissue to the urine and show great potential for improving specificity in PCa diagnosis. This work reports on a sandwich-type hybridization assay to detect both the urinary biomarker prostate cancer antigen 3 (PCA3) and an endogenous control, the PSA mRNA. Multiple fluorescein-tagged hybridization assistant probes are used to promote the selective capture of this long noncoding RNA, and sensitivity by incorporating multiple redox enzymes per target molecule, after addition of antifluorescein Fab fragment-peroxidase conjugate. This strategy alleviates the problems associated with the low natural abundance of this marker, its large size, and complex secondary structure. The individual genosensors exhibit good sensitivity (2.48 ± 0.01 μA nM−1 and 6.4 ± 0.3 μA nM−1 for PCA3 and PSA, respectively), with wide linear ranges (from 25 pM to 10 nM for PCA3 and 1 nM for PSA), and detection limits in the low picomolar range (4.4 pM and 1.5 pM for PCA3 and PSA, respectively). This analytical performance is retained in the dual configuration without significant cross-talk, despite using the same enzyme label. The usefulness of this dual platform was demonstrated by analyzing RNA extracts from the prostate cancer cell line LNCaP and from urine samples of prostate cancer patients.

Anti-androgenic effect of astaxanthin in LNCaP cells is mediated through the aryl hydrocarbon-androgen receptors cross talk.

The aim of this study was to investigate the anti-androgenic effects of astaxanthin (AST) on human prostatic cancer cell growth, and its impact on androgen receptor (AR) signaling using prostate cancer (PCa) cell line LNCaP. LNCaP cells were treated with AST alone and in combination with CH223191 and flutamide (Flu) in the presence and absence of testosterone. MTT assay, cellular prostate-specific antigen (PSA) and dihydrotestosterone (DHT) production, mRNA levels of CYP1A1, PSA, Kallikrein-Related Peptidase 2 (KLK2), Transmembrane Serine Protease 2 (TMPRSS2), and AR genes were measured as endpoints. The expression of CYP1A1, PSA, KLK2, TMPRSS2, and AR mRNA levels was decreased which results in reducing the production of PSA and DHT in the presence of testosterone. Our data clearly demonstrate that AST has a potential ability to suppress the human prostate LNCaP cells growth at high concentrations. AST was able to repress the testosterone-induced transcription of AR-target genes. PRACTICAL APPLICATIONS: Astaxanthin is a natural compound with the most potent antioxidant activity among other antioxidants. In the current study, ASX suppressed the LNCaP cells at high concentrations. Furthermore, AST inhibited testosterone-induced transcriptional activation of androgen-related genes. AST induced the expression of CYP1A1, which is able to metabolize the steroid hormones. It seems that AST can act as AhR exogenous ligand by induction of CYP1A1, which results in testosterone metabolism and consequent suppression of AR genes. So that, AST could prevent the growth of testosterone-dependent PCa cells, downregulate downstream genes in testosterone pathways, and enhance the metabolism of testosterone via AhR pathway. Collectively, AST could be considered as a potential candidate for the treatment of PCa.

Estrogenic and antiandrogenic activities of methoxyflavans isolated from the fruit of Mauritia Flexuosa (Moriche Palm).

Moriche palm is consumed as both a fresh fruit and processed food in Peru and Brazil. Although its fruit contains phytoestrogens, the active compounds have not yet been identified. Therefore, we purified moriche palm extract (MPE) and identified compounds exhibiting estrogenic and antiandrogenic activities. Estrogenic activity was assessed by the estrogen-dependent growth of MCF-7 cells and increases in uterine weights in mice. Antiandrogenic activity was evaluated by 5α-reductase inhibitory activity and prostate-specific antigen (PSA) expression in LNCaP cells. In vivo antiestrogenic activity was also assessed based on testosterone-induced prostate growth in castrated mice. Four methoxyflavans were isolated from MPE and all, except for 7,4'-dihydroxy-5-methoxyflavan, promoted MCF-7 cell growth, indicating estrogenic activity. Uterine and ovary weights increased in mice orally administered MPE (400mg/kg) for 2weeks. Regarding antiandrogenic activity, among the four methoxyflavans isolated, 6,7,4'-trihydroxy-5-methoxyflavan (1 µg/ml) suppressed the mRNA and protein expression of PSA in LNCaP cells. Furthermore, prostate growth was suppressed in mice orally administered MPE (200mg/kg) for 2weeks. All methoxyflavans inhibited 5α-reductase activity with IC50 less than 10 µg/ml. Collectively, the present results demonstrated that orally administered MPE exhibited estrogenic and antiandrogenic activities. Methoxyflavans, particularly 6,7,4'-trihydroxy-5-methoxyflavan, appear to be the active compounds for these activities. PRACTICAL APPLICATIONS: The fruit of Mauritia flexuosa (moriche palm) has been used for beverages and processed foods. Although it is said to contain phytoestrogens, the active compounds have not yet been identified. In this study, we isolated and identified methoxyflavans exhibiting estrogenic and antiandrogenic activities. Among them, 6,7,4'-trihydroxy-5-methoxyflavan appeared to be the most effective compounds for these activities.

Prostatic aspirated cellular RNA analysis enables fast diagnosis and staging of prostate cancer

ObjectiveThe aim of this study is to investigate the potential application of prostatic aspirated cellular RNA analysis technique for fast diagnosing and staging prostate cancer. MethodsProstatic aspirated cells were obtained immediately after transrectal ultrasound (TRUS)-guided needle biopsy. Cellular RNA such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, prostate specific antigen (PSA) mRNA and prostate-specific RNA (PCA3) mRNA were analyzed by using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). PCA3 score was calculated as the ratio of PCA3 mRNA to PSA mRNA expression. Diagnostic performance of the fast-aspirated cellular RNA analysis technique for determining prostate cancer and metastatic status were evaluated by developing receiver operating characteristic curves (ROC), and the correlation between aspirated cellular RNA mRNA expressions and risk grouping was calculated, to investigate the underlying potential for PCa staging. ResultsPCA3 score was significantly higher in prostatic aspirated cells obtained from cancerous tissues than noncancerous tissues. The median aspirated cellular PCA3 score was higher in patients with PCa compared to BPH, and presenting an area under the ROC curve (AUC) of 0.87 (95%CI: 0.79–0.94) for PCa diagnosis. Multivariate regression analysis revealed that baseline median aspirated cellular PCA3 score (OR=9.316, 95%CI: 1.045–83.033, P<0.05) was an independent predictive factor for metastatic status in PCa patients. ConclusionThe ease of use and minimal complexity of fast prostatic aspirated cellular RNA analysis may be a valuable diagnostic technique, providing urgent diagnostic information for accurate PCa diagnosing and staging.

Dickkopf-1 Can Lead to Immune Evasion in Metastatic Castration-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets. Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort. Dickkopf-1 (DKK1) expression is increased in DNPC relative to prostate-specific antigen (PSA)-expressing mCRPC in the Stand Up to Cancer/Prostate Cancer Foundation discovery cohort (11.2 v 0.28 reads per kilobase per million mapped reads; q < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 v 0.99 fragments per kilobase of transcript per million mapped reads; P < .0001). DKK1 expression can be regulated by activated Wnt signaling in vitro and correlates with activating canonical Wnt signaling mutations and low PSA mRNA in mCRPC biopsies (P < .05). DKK1 hypomethylation was associated with increased DKK1 mRNA expression (Pearson r = -0.66; P < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells (P < .005) and lower numbers of activated NK cells (P < .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model. These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353).

Abstract 5378: A comparative analysis on the expression of PSA protein in serum and PSA mRNA in circulating tumor cells in prostate cancer

Abstract Introduction Unlike other cancers, prostate cancer has a distinguished biomarker for diagnosis and prognosis which is known as prostate specific antigen (PSA) protein in serum. In the early stage of prostate cancer, PSA protein in serum is highly expressed from primary tumor sites and thereby, the expression of PSA protein can be used in prognosis of prostate cancer during whole progression. However, due to low specificity of serum PSA protein, it raised concerns in over-diagnosis. Due to the advantages of noninvasive, real-time and pain-free for patients, circulating tumor cells (CTCs) is being researched as complementary alternative biomarkers for clinical and molecular analysis of cancer. Some studies reported that the levels of PSA protein and PSA mRNA in prostate cancer tissue are proportional to each other. Other studies showed that the expression level of PSA mRNA in peripheral blood and biochemical recurrence of PSA protein in serum are significant relationship. However, the relevance between PSA protein in serum and PSA mRNA in CTCs has not been clearly researched. In this study, we first report the relevance of the expression levels of PSA protein in serum and PSA mRNA in CTCs. Experimental method and analysis Prostate cancer patients were recruited from healthy donor (HD, n=5), local with non-metastatic stage (n=25), metastatic hormone sensitive prostate cancer (mHSPC, n=9) and metastatic castration-resistance prostate cancer (mCRPC, n=28). CTCs were isolated by high efficiency and purity using microfluidic technology based on lateral magnetophoresis, developed in our laboratory. Total mRNA was extracted from isolated CTC and the expression level of PSA mRNA in CTCs was measured by droplet digital PCR (ddPCR). Then, the expression levels of PSA protein (ng/ml) in serum and PSA mRNA (copies/µl) in CTCs were compared by statistical analysis. Results and discussion The expression of PSA mRNA in CTCs was observed in 40% of localized cancer, 55.56% of mHSPC, 89.29% of mCRPC patients and 0% of HD group. It indicates that the detection rate of PSA mRNA increases with stage of prostate cancer. The expression level of PSA mRNA in CTCs also increased from localized to metastatic stages. Interestingly, it increased very rapidly in mHSPC and mCRPC stages. In 7 of the blood samples of localized stage, the expression levels of PSA mRNA in CTCs were detected and they were not related to serum PSA protein. In mHSPC and mCRPC stage, the expression levels of PSA mRNA in CTCs were respectively detected in 5 and 25 samples, implicating that their expression levels are significantly related to the level of PSA protein in serum. Conclusions This result shows that the expression level of PSA mRNA in CTCs could be used as a new marker along with serum PSA protein in metastatic stages of prostate cancer, and that high-quality CTCs can be used as complementary alternative biomarkers for precision medicine in prostate cancer. Citation Format: Hyungseok Cho, Ki-Ho Han, Jae-Seung Chung. A comparative analysis on the expression of PSA protein in serum and PSA mRNA in circulating tumor cells in prostate cancer [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 5378.

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.

Evaluation of E2F3 and survivin expression in peripheral blood as potential diagnostic markers of prostate cancer

Abstract Background Prostate cancer (PC) incidence has risen globally. As there are no current independent biomarkers with high diagnostic efficiency to detect PC, this study was performed to investigate the relative gene expression levels of E2F3 and survivin in the whole blood of PC, benign prostate hyperplasia (BPH), and normal control individuals and to explore their diagnostic value. Material and methods Participants of the study were divided into three groups; normal control group (n=25), BPH patients (n=25), and PC patients (n=75). The E2F3 and survivin gene expression levels were assessed using real-time qPCR in addition to the measurement of free and total levels of prostate-specific antigen (PSA) using electrochemiluminescence assays. Results Survivin relative gene expression was over-expressed in PC and BPH patients compared to the normal control group, whereas, E2F3 did not differ significantly among the studied groups. Compared to PSA, E2F3 and survivin mRNA expression levels had lower diagnostic efficacy to differentiate PC from normal and BPH individuals with an area under curve (AUC) of 0.471 and 0.727, respectively. Further, survivin expression level was associated with increased the risk of PC. Conclusion Survivin and E2F3 relative expression levels in peripheral blood had low diagnostic performance to detect PC and individuals with high survivin expression levels may have higher risk to develop PC.

Effect of Paecilomyces tenuipes Extract on Testosterone-Induced Benign Prostatic Hyperplasia in Sprague-Dawley Rats.

Benign prostatic hyperplasia (BPH) is one of the major public health concerns, which has a high prevalence rate and causes significant decline in men’s quality of life. BPH is highly related to sexual hormone metabolism and aging. In particular, dihydrotestosterone (DHT), to which testosterone is modified by 5α-reductase (5AR), has a significant effect on BPH development. DHT binds to an androgen receptor (AR) and steroid receptor coactivator 1 (SRC-1); then, it induces the proliferation of a prostate cell and expression of prostate specific antigen (PSA). Paecilomyces tenuipes (P. tenuipes) is a mushroom that has been popularized by the artificial cultivation of fruiting bodies based on silkworms by researchers from the Republic of Korea. In a previous study, we identified the effect of PE on PSA mRNA expression in LNCaP cells. This suggests that PE may have an inhibitory effect on androgen signaling. Therefore, we confirmed the expression of androgen signaling-related factors, such as AR, SRC-1, and PSA in LNCaP. Furthermore, we confirmed the androgen signaling inhibitory effect of PE using the testosterone propionate (TP)-induced BPH rat model. A BPH rat model was established with a four-week treatment of daily subcutaneous injections of testosterone propionate (TP, 3 mg/kg) dissolved in corn oil after castration. The rats in the treatment group were orally gavaged P. tenuipes extract (PE), finasteride (Fi), or saw palmetto extract (Saw) with TP injection. DHT induced an increase in the expression levels of AR, SRC-1, and PSA proteins in LNCaP cells. On the contrary, the PE treatment reduced the expression levels. In vivo, the BPH group showed an increase in prostate size compared with the control group. The PE gavaged group showed a decrease in prostate size compared with the BPH group. In addition, the protein expressions of AR, 5AR2, and PSA were significantly lower in the PE gavaged group than BPH group in prostate tissue. These results suggest the beneficial effects of PE on BPH via the modulation of AR signaling pathway.

A pilot study of prostate-specific membrane antigen (PSMA) dynamics in men undergoing treatment for advanced prostate cancer.

Prostate-specific membrane antigen (PSMA) is a rational target for noninvasive detection of recurrent prostate cancer (PCa) and for therapy of metastatic castration-resistant prostate cancer (mCRPC) with PSMA-targeted agents. Here we conducted serial measurements of PSMA expression on circulating tumor cells (CTCs) to evaluate patterns of longitudinal PSMA dynamics over the course of multiple sequential therapies. A retrospective investigation of men with mCRPC undergoing evaluation at medical oncology clinics at our institution assessed the dynamics of PSMA expression in the context of different systemic treatments administered sequentially. Eligibility included patients who began systemic therapies with androgen receptor (AR)-directed agents or taxane agents for whom peripheral blood samples were tested for CTC mRNA of AR splice variant-7 (AR-V7), prostate-specific antigen (PSA), and PSMA (with >2 CTC + results) in a CLIA-accredited laboratory. From August 2015 to November 2017, we identified 96 eligible men. Fifteen had greater than or equal to 2 sequential therapies and evaluable CTC samples, greater than or equal to 1 expressing PSMA (PSMA+). Among the 15 patients included in this analysis, a total of 54 PSMA status evaluations were performed in the context of 48 therapies during a median follow-up of 18 months. At baseline, PSMA signal was detected ("positive") in 11 of 15 (73.3%) patients, while for 4 of 15 (26.7%) patients PSMA signal was undetectable ("negative"). In all but two patients, the baseline collection corresponded with a change in treatment. On the second assessment, PSMA increases were detected in all 4/4 (100%) PSMA-negative patients and 8 of 11 (72.7%) PSMA-positive patients. PSMA significantly decreased in a patient treated with 177 Lu-PSMA-617. Serum PSA declines were seen in 7 of 8 (88%) of the treatment periods where PSMA decreased. PSMA expression in CTCs is a dynamic marker. PSMA transcript declines appear to be associated with concurrent decreases in serum PSA. Sequential CTC sampling could provide a noninvasive response assessment to systemic treatment for mCRPC.

Cell-based evidence regarding the role of FSH in prostate cancer

IntroductionConversion of androgen-responsive prostate cancer (CaP) to castration-resistant CaP is associated with an acceleration of the disease that often requires treatment modalities other than androgen deprivation therapy only. Recently, follicle-stimulating hormone (FSH) has been shown to play a role in CaP growth, and clinical data showed that high serum concentration of FSH in chemically castrated CaP patients was associated with a shorter time of progression to castration-resistant CaP. In this study, we sought to investigate if FSH could have direct effects on CaP cells, possibly through the androgen receptor and androgen receptor regulated genes, such as prostate-specific antigen (PSA). Materials and methodsThe human CaP cell lines PC-3, LNCaP and C4-2, and nonmalignant PNT1A cells, were utilized to investigate the effects of FSH. qPCR, Western blotting analysis, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium assays were performed in order to analyze the FSH effects. ResultsThe FSH receptor was present in all cell lines except PNT1A. FSH significantly increased PSA mRNA (P < 0.01) and protein (P < 0.03) levels in C4-2 cells in a dose-dependent manner. In LNCaP cells, FSH also increased PSA protein level, although to a lesser extent than in C4-2 cells, and the expression was reduced by the antiandrogen enzalutamide. In PC-3 cells, FSH was shown to increase their proliferation (P < 0.03) and β-catenin expression. ConclusionThese findings demonstrate that FSH may have a direct effect in CaP in an androgen-depleted environment. However, further research is needed to understand the significance of direct FSH action in the maintenance of CaP growth at the different phases of transition from androgen dependence to androgen independence.

Serum PSA levels in patients with prostate cancer and other 33 different types of diseases.

The prostate-specific antigen (PSA), a glycoprotein, is a clinically used biomarker for screen and diagnosis of prostate cancer. Most of PSA in blood circulation is protein-bound, and only a small fraction is present as free PSA. In general, the serum total PSA levels are <4ng/mL in healthy controls and significantly increased in certain prostate cancer patients. Currently, <0.25 in the ratio of free PSA to total PSA (F/T value) is used as an indicator of prostate cancer. The mRNA of PSA is mainly expressed in prostate, and its protein product is a protease present in seminal plasma for the liquefaction of seminal coagulum in normal physiology. The F/T value as a prostate cancer biomarker has been questioned for its benefit over harm caused by its high false-positive rate of diagnosis. Furthermore, the molecular basis of how PSA is getting into blood circulation from seminal plasma is largely unknown. In current study, a total of 24,692 clinical lab test results of serum F/T values from healthy controls and patients with 34 different types of diseases including different types of cancers and nonneoplasm illnesses during the past 5 years were collected and analyzed statistically. Our data showed that even though the prostate cancer patients had the lowest median serum F/T values, both significantly increased and decreased serum F/T values were associated with different types of human diseases, such as acute cerebral infarction, coronary heart disease, uremia, and nephrotic syndrome. The possible molecular mechanisms of serum PSA as disease biomarkers are discussed.

Effects of inhibition of neural precursor cell-expressed developmentally down regulated 8 gene on biological behavior, and androgen receptor of human prostate cancer cells

Objective To explore the effect of lentivirus-mediated short hairpin RNA (shRNA) silencing neural precursor cell-expressed developmentally down regulated 8 (NEDD8) gene on androgen receptor (AR), prostate specific antigen (PSA), proliferation, apoptosis and invasion of human prostate cancer LNCap and VCaP cells. Methods LNCap and VCaP cells were transfected with lentivirus-mediated NEDD8-shRNA. The mRNA and protein expression of NEDD8, AR and PSA was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting, respectively. The proliferation, apoptosis and invasion abilities were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry and Transwell TM assay, respectively. Results After transfection with NEDD8-shRNA, NEDD8 mRNA expression in LnCaP and VCaP was (0.11±0.03) fold and (0.16±0.04) fold, respectively, as compared to the blank control group; AR mRNA expression was (0.52±0.04) fold and (0.42±0.05) fold; PSA mRNA expression was (0.49±0.04) fold and (0.59±0.06) fold. NEDD8, AR and PSA protein expression in LnCaP and VCaP was (11.26±2.65)% and (6.26±2.04)%, (30.36±4.35)% and (15.36±3.37)%, and (13.37±2.70)% and (11.36±2.67)%, respectively. As compared with the control groups, the mRNA and protein expression levels of NEDD8, AR and PSA were significantly inhibited after transfection with NEDD8-shRNA (P=0.000). As compared with the control groups, the proliferation of LnCaP and VCaP was significantly inhibited after transfection with NEDD8-shRNA. The apoptosis rate of LNCap and VCaP cells was (15.08±1.23)% and (20.37±1.76)% respectively, which was significantly enhanced as compared with the control groups (P=0.001, P=0.000). After transfection, the number of LnCaP and VCaP cells invading the Matrigel was (55.5±12.5) and (60.8±11.3) respectively, which was significantly reduced as compared with the control groups (P=0.000, P=0.004). Conclusion NEDD8 may downregulate AR at the transcriptional level. The lentivirus-mediated shRNA silencing NEDD8 gene could inhibit the proliferation and invasion, and promote apoptosis of LNCap and VCaP cells. Key words: Prostate cancer; Neural precursor cell-expressed developmentally down regulated factor 8; Androgen receptor; RNA interference

Abstract 2990: Sulforaphane enhances the anti-cancer efficacy of anti-androgens in prostate cancer cells (LNCaP and C4-2B) by increasing androgen receptor (AR) degradation

Abstract Introduction: Prostate Cancer (PC) cells utilize androgen for their growth. Therefore, androgen deprivation therapy (ADT) that blocks both systemic androgen production and androgen receptor (AR) signaling remains the mainstay of PC treatment. However, despite the efficacy of AR antagonists such as bicalutamide (BIC) and enzalutamide (ENZ), their sub-therapeutic efficacy in tumor microenvironments enable the selection and outgrowth of castration resistant PC (CRPC). Interestingly, CRPC cells continue to express AR and exploit the intratumoral androgen. Hence, alternate strategies to suppress AR expression in PC cells will be needed to enhance the efficacy of BIC and ENZ. The phytochemical, sulforaphane (SFN) is known to decrease AR protein levels. We hypothesize that co-exposure to SFN will increase the anti-cancer efficacy of BIC/ENZ. Methods: The AR expressing PC lines, both androgen-dependent (AD) LNCaP cells and androgen-independent CRPC cells (C4-2B), were used to investigate the effects of BIC or ENZ, alone and in combination with SFN. The following cellular parameters were evaluated: (a) cell proliferation by MTT-assay; (b) clonogenic ability by colony forming unit (CFU); (c) AR expression by immunoblot analysis; (d) subcellular AR localization by immunofluorescence microscropy (IFM); (e) AR and prostate specific antigen (PSA) gene expression by qRT-PCR; and (e) PSA protein secretion by ELISA. Studies were carried out under hormone deprived (charcoal-stripped FBS) conditions and/or in presence of AR agonist, R1881. Results: Co-exposure to SFN (10μM) significantly (p&amp;lt;0.05) enhanced the anti-proliferative effects of BIC (50μM) or ENZ (20μM) at 48 h post treatment, and suppressed R1881 stimulated PC growth, as well. At much lower concentrations, SFN (0.2μM) co-exposure enhanced the suppressive effects of BIC (0.5μM) or ENZ (0.2μM) on both the number and size of CFUs at 14 days. Western blot analysis revealed that SFN decreases AR protein levels in a time- and dose-dependent manner. IFM analysis indicated that SFN suppresses R1881-stimulated AR nuclear localization. Cycloheximide treatment suggested that SFN's effect on AR is primarily regulated at the translational level. Experiments with MG-132, a proteasomal inhibitor indicated that the SFN-induced AR degradation is partially regulated by the proteasomal pathway. Studies with qRT-PCR showed that SFN enhances the efficacy of BIC in suppressing PSA mRNA levels and decreased PSA protein secretion was also corroborated by ELISA. Conclusion: Sulforaphane decreases AR levels in both LNCaP and C4-2B cells to increase the anti-cancer efficacy of AR antagonists in a synergistic manner. This implies that adjuvant therapy with this safe phytochemical may be very promising. Citation Format: Namrata Khurana, Sudha Talwar, Partha Chandra, Asim B. Abdel-Mageed, Debasis Mondal, Suresh Sikka. Sulforaphane enhances the anti-cancer efficacy of anti-androgens in prostate cancer cells (LNCaP and C4-2B) by increasing androgen receptor (AR) degradation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2990.

Systems Pharmacology Modeling of Prostate-Specific Antigen in Patients With Prostate Cancer Treated With an Androgen Receptor Antagonist and Down-Regulator.

First‐in‐human (FIH) studies with AZD3514, a selective androgen receptor (AR) down‐regulator, showed decreases of >30% in the prostate‐specific antigen (PSA) in some patients. A modeling approach was adopted to understand these observations and define the optimum clinical use hypothesis for AZD3514 for clinical testing. Initial empirical modeling showed that only baseline PSA correlated significantly with this biological response, whereas drug concentration did not. To identify the mechanistic cause of this observation, a mechanism‐based model was first developed, which described the effects of AZD3514 on AR protein and PSA mRNA levels in LNCaP cells with and without dihydrotestosterone (DHT). Second, the mechanism‐based model was linked to a population pharmacokinetic (PK) model; PSA effects of clinical doses were subsequently simulated under different clinical conditions. This model was used to adjust the design of the ongoing clinical FIH study and direct the backup program.

The Effects of the Organic Flame‐Retardant 1,2‐Dibromo‐4‐(1,2‐dibromoethyl) Cyclohexane (TBECH) on Androgen Signaling in Human Prostate Cancer Cell Lines

The effects of the organic flame retardant 1,2-Dibromo-4-(1,2-dibromoethyl) cyclohexane (TBECH) on androgen receptor target gene expression were examined in the human LNCaP prostate cancer cell line. While γ-/δ-TBECH alone led to a significant increase in prostate-specific antigen (PSA) mRNA accumulation, both the α-/-TBECH and γ-/δ-TBECH mixtures repressed androgen-inducible PSA mRNA and protein accumulation in human LNCaP cells. Thus, we hypothesize that isomeric mixtures of TBECH may act as partial agonists of the androgen receptor.

Detection of AR-V7 mRNA in whole blood may not predict the effectiveness of novel endocrine drugs for castration-resistant prostate cancer.

A splice variant of androgen receptor (AR), AR-V7, lacks in androgen-binding portion and leads to aggressive cancer characteristics. Reverse transcription-polymerase chain reactions (PCRs) and subsequent nested PCRs for the amplification of AR-V7 and prostate-specific antigen (PSA) transcripts were done for whole blood of patients with prostate cancer and male controls. With primary reverse transcription PCRs, AR-V7 and PSA were detected in 4.5% and 4.7% of prostate cancer, respectively. With nested PCRs, AR-V7 messenger RNA (mRNA) was positive in 43.8% of castration-sensitive prostate cancer and 48.1% of castration-resistant prostate cancer (CRPC), while PSA mRNA was positive in 6.3% of castration-sensitive prostate cancer and 18.5% of CRPC. Whole-blood samples of controls showed AR-V7 mRNA expression by nested PCR. Based on multivariate analysis, expression of AR-V7 mRNA in whole blood was not significantly correlated with clinical parameters and PSA mRNA in blood, while univariate analysis showed a correlation between AR-V7 mRNA and metastasis at initial diagnosis. Detection of AR-V7 mRNA did not predict the reduction of serum PSA in patients with CRPC following abiraterone and enzalutamide administration. In conclusion, AR-V7 mRNA expression in normal hematopoietic cells may have annihilated the manifestation of aggressiveness of prostate cancer and the prediction of the effectiveness of abiraterone and enzalutamide by the assessment of AR-V7 mRNA in blood.