Prostate-specific Membrane 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.

18F-DCFPyL (PSMA) PET as a radiotherapy response assessment tool in metastatic prostate cancer.

Prostate Specific Membrane Antigen (PSMA) - positron emission tomography (PET) guides metastasis-directed radiotherapy (MDRT) in prostate cancer (PrCa). However, its value as a treatment response assessment tool after MDRT remains unclear. Importantly, there is limited understanding of the potential of radiotherapy (RT) to alter PSMA gene (folate hydrolase 1; FOLH1) expression. We reviewed a series of 11 men with oligo-metastatic PrCa (25 metastasis sites) treated with MDRT before re-staging with 18F-DCFPyL (PSMA) PET upon secondary recurrence. Acute effects of RT on PSMA protein and mRNA levels were examined with qPCR and immunoblotting in human wild-type androgen-sensitive (LNCap), castrate-resistant (22RV1) and castrate-resistant neuroendocrine (PC3 and DU145) PrCa cell lines. Xenograft tumors were analyzed with immunohistochemistry. Further, we examined PSMA expression in untreated and irradiated radio-resistant (RR) 22RV1 (22RV1-RR) and DU145 (DU145-RR) cells and xenografts selected for survival after high-dose RT. The majority of MDRT-treated lesions showed lack of PSMA-PET/CT avidity, suggesting treatment response even after low biological effective dose (BED) MDRT. We observed similar high degree of heterogeneity of PSMA expression in both human specimens and in xenograft tumors. PSMA was highly expressed in LNCap and 22RV1 cells and tumors but not in the neuroendocrine PC3 and DU145 models. Single fraction RT caused detectable reduction in PSMA protein but not in mRNA levels in LNCap cells and did not significantly alter PSMA protein or mRNA levels in tissue culture or xenografts of the other cell lines. However, radio-resistant 22RV1-RR cells and tumors demonstrated marked decrease of PSMA transcript and protein expression over their parental counterparts. PSMA-PET may be a promising tool to assess RT response in oligo-metastatic PrCa. However, future systematic investigation of this concept should recognize the high degree of heterogeneity of PSMA expression within prostate tumors and the risk for loss of PSMA expression in tumor surviving curative courses of RT.

Molecular analysis of circulating tumor cells of metastatic castration-resistant Prostate Cancer Patients receiving 177Lu-PSMA-617 Radioligand Therapy.

Rationale: Lu-177-PSMA-617 radioligand therapy (RLT) is currently under approval for treatment of metastatic castration resistant prostate cancer (mCRPC) patients with late stage disease. However, previous studies demonstrated both heterogeneity of prostate specific membrane antigen (PSMA) expression, as well as response to PSMA treatment among mCRPC patients. Thus, there is an unmet need for identifying predictive parametres prior or under PSMA-RLT treatment. We therefore aimed to correlate several clinical and molecular parameters with response to PSMA treatment in a cohort of mCRPC patients undergoing PSMA RLT followed by a detailed analysis of promising candidates.Methods: Nineteen patients, median age 68.8 years (range: 56.9 - 83.3) with mCRPC were included in this study. We performed baseline analysis of clinical parameters based on PSMA PET/CT, (metabolic tumor volume (MTV), total tumor volume (TTV)), serum PSA, ALP, LDH and gene expression analysis of circulating tumor cells (expression of AR full length (AR-FL), AR splice variant 7 (AR-V7), PSA and PSMA) as well as common markers for neuroendocrine differentiation (NED).Results: Patients presented with bone, lymph node, and visceral metastases (89%, 68%, and 21%, respectively). All patients were pretreated with docetaxel, either abiraterone or enzalutamide, or both. Biochemical response in terms of PSA decline ≥50 or ≥30% was observed in 42% and 63%, respectively. There were significant correlations between PSA and PSMA mRNA expression, as well as tumor volumes (both MTV and TTV), AR-FL and AR-V7 mRNA expression. However, there was no correlation with response to PSMA treatment. Furthermore, none of these parameters was significantly correlated with baseline serum PSA values. Common NED markers were shown to be specifically high expressed and revealed impact on OS independent from AR-V7 gene expression.Conclusion: We demonstrate that AR-FL and its splice variant AR-V7 might serve as prognostic biomarkers displaying high tumor burden in mCRPC patient prior to PSMA-RLT. Contrary, PSMA, which has been discussed as a biomarker for PSMA targeted treatment, does not display strong prognostic ability - at least on the mRNA level. Surprisingly, none of these parameters correlates to response to PSMA treatment. In contrast, commom NED markers such as SYP and ENO2 as well as FOXA1 expression level seem to predict OS, but not PFS, more reliably. We admit that a limitation of our study is the focus on mRNA expression of potential biomarkers only. Further investigations analyzing the potential role of protein expression of these markers are therefore warranted.

PSMA heterogeneity and DNA repair defects in prostate cancer.

5002 Background: Prostate-specific membrane antigen (PSMA) is a promising target for theranostics in metastatic castration resistant prostate cancer (mCRPC). Methods: Membranous PSMA (mPSMA) expression was immunohistochemically evaluated in castration sensitive (CSPC) (n = 38) and mCRPC (n = 60) tissue biopsies, and associations with molecular aberrations (next-generation sequencing; NGS) and clinical outcome were determined. Results: mPSMA expression was significantly higher (p = 0.005) in mCRPC biopsies (median H-score [interquartile range]; 55.0 [2.8-117.5]) compared to CSPC biopsies (17.5 [0.0-60.0]). Furthermore, patients with higher mPSMA expression ( > median H-score) at diagnosis had higher Gleason Grade (p = 0.04) and shorter OS (p = 0.006). Critically, 42% (16/38) of CSPC biopsies and 27% (16/60) of mCRPC biopsies were completely negative for mPSMA expression. In addition, CSPC and mCRPC biopsies expressing mPSMA demonstrated marked intra-tumor heterogeneity in expression levels, commonly exhibiting areas without detectable PSMA (CSPC – 100%; mCRPC – 84%), while heterogeneous mPSMA expression between metastases from the same patient was also observed. Subsequent genomic analysis showed that mCRPC patients with deleterious DNA damage repair (DDR) aberrations have higher (p = 0.016) mPSMA expression (87.5 [25.0-247.5]) than those without these (20 [0.3-98.8]). Furthermore, 9 of the 11 patients (82%) responding to PARP inhibition had a mPSMA H-Score above the median. The association between mPSMA expression and DDR aberrations was validated in an independent cohort with known DDR aberrations. Tumors with DDR aberrations had significantly higher mPSMA (ATM 212.5 [136.3-300] p = 0.005; BRCA2 300 [165-300] p = < 0.001) than unselected mCRPC biopsies (55.0 [2.75-117.5]). Finally, analyses of 122 mCRPC biopsy transcriptomes confirmed a negative correlation between PSMA and BRCA2 mRNA expression (p = 1.5x10-5). Conclusions: mPSMA expression in CSPC and mCRPC exhibits marked intra- and inter-patient heterogeneity, limiting the clinical utility of PSMA-targeted theranostics. We show for the first time that DDR gene aberrations associate with high mPSMA expression and may serve as predictive biomarkers for PSMA-targeted therapies.

Sox7 negatively regulates prostate-specific membrane antigen (PSMA) expression through PSMA-enhancer.

PSMA expression in the prostate epithelium is controlled by a cis-element, PSMA enhancer (PSME). PSME contains multiple binding sites for Sox proteins, and in this study, we identified Sox7 protein as a negative regulator of PSMA expression through its interaction with PSME. The statistical correlation between Sox7 and PSMA mRNA expression was evaluated using five prostate cancer studies from cBioportal. In vitro and in vivo interaction between Sox7 and PSME was evaluated by chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and luciferase reporter assay. Synthetic oligonucleotides were generated to define the sites in PSME that interact with Sox7 protein. Sox7 mutants were generated to identify the region of this protein required to regulate PSMA expression. Sox7 was also stably expressed in LNCaP/C4-2 and 22Rv1 cells to validate the regulation of PSMA expression by Sox7 in vivo. Sox7 mRNA expression negatively correlated with PSMA/FOLH1 and PSMAL/FOLH1B mRNA expression in Broad/Cornell, TCGA and MSKCC studies, but not in two studies containing only metastatic prostate tumors. PC-3 cells mostly expressed the 48.5 KDa isoform 2 of Sox7, and the depletion of this isoform did not restore PSMA expression. Ectopic expression of canonical, wild-type Sox7 in C4-2 and 22Rv1 cells suppressed PSMA protein expression. ChIP assay revealed that canonical Sox7 protein preferentially interacts with PSME in vivo, and EMSA identified the SOX box sites #2 and #4 in PSME as required for its interaction. Sox7 was capable of directly binding to PSME and suppressed PSME-mediated transcription. The NLS regions of Sox7, but not its β-catenin interacting motif, are essential for this suppressing activity. Furthermore, restoration of wild-type Sox7 expression but not Sox7-NLS mutant in Sox7-null prostate cancer cell lines suppressed PSMA expression. The inactivation of canonical Sox7 is responsible for the upregulated expression of PSMA in non-metastatic prostate cancer.

Alterations in androgen deprivation enhanced prostate-specific membrane antigen (PSMA) expression in prostate cancer cells as a target for diagnostics and therapy.

BackgroundProstate-specific membrane antigen (PSMA) is a promising target for diagnostics and therapy of prostate carcinoma (PCa). Based on the hypothesis that PSMA expression can be modulated by variations in androgen deprivation therapy (ADT), we investigated the binding of a PSMA-directed radiopharmaceutical in vitro in order to get an insight of the interactions between altered premedication and PSMA expression before repetitive PSMA-directed PET/CT for therapy response and targeted therapy implementation.MethodsThe human castration-resistant PCa cell line VCaP (CRPC) was treated with either 1 nmol/L testosterone (T) over 20 passages yielding the androgen-sensitive cell line (revCRPC) or with 5 μmol/L abiraterone acetate (AA) generating the abiraterone-tolerant subtype CRPCAA. In these cell lines, T and AA were varied by either supply or withdrawal of T and AA. PSMA expression of the three cell culture models was detected by Western blot and immunohistochemical staining. For quantitative measurement of tracer uptake, 0.3 nmol/L 68Ga-labelled PSMA-HBED-CC peptide (100–300 kBq/ml) was added to different treated parallel cultures (n = 9 each). Time-dependent uptake per 106 cells of each culture was calculated and evaluated. PSMA mRNA expression was investigated by qPCR.ResultsPSMA expression increased dependently on intensified ADT in all three basic cell lines. 68Ga-PSMA-HBED-CC uptake almost doubled during 3 h in all cell lines (p < 0.01). Compared to the basic cells, pre-incubation with abiraterone for 48 h resulted in a significant increased uptake in CRPC (p < 0.001). In revCRPC, 48-h AA pre-incubation resulted in an eightfold higher uptake after 3 h (p < 0.001). Additional withdrawal of external testosterone increased the uptake up to tenfold (p < 0.01). The increase of PSMA expression upon ADT and AA treatments was confirmed by qPCR and Western blot data. Furthermore, in CRPCAA, 48-h AA withdrawal increased the uptake up to fivefold (p < 0.01).ConclusionsThe investigated three PCa cell culture subtypes represent a serial preclinical model of androgen deprivation therapy as a proxy for clinical situations with differing basal PSMA expression. The uptake of PSMA-binding tracers could be stimulated by therapeutic effective short-term variation in premedication in all stages of ADT response. These complex interactions have to be considered in the interpretation of diagnostic imaging using PSMA ligands as well as in the optimal timing of PSMA-based therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-015-0145-8) contains supplementary material, which is available to authorized users.

Prostate-specific membrane antigen as a marker of pancreatic cancer cells

The aim of this study was to identify the expression of prostate-specific membrane antigen (PSMA) and analyze the correlation between PSMA with clinical characteristics in patients with pancreatic cancer. The expression of PSMA protein and mRNA was detected by immunohistochemistry and real-time quantitative polymerase chain reaction in pancreatic cancer tissues, pancreatic intraepithelial neoplasia or normal pancreatic tissues, respectively. And clinical characteristics and prognosis of patients were investigated. PSMA was expressed in pancreatic cancer cells, both in protein and mRNA levels. Moreover, the PSMA levels were associated with the prognosis of patients with pancreatic ductal adenocarcinoma. The overall survival time of pancreatic cancer patients with high expression of PSMA was significantly shorter than that of the low ones. Moreover, the PSMA levels were correlated with clinicopathological features including the histological grade and pathological tumor-node-metastasis stage. PSMA is involved in the carcinogenesis of pancreatic cancer, and it might serve as a potential therapeutic target for pancreatic cancer.

A novel regulation of PSMA and PSA expression by Q640X AR in 22Rv1 and LNCaP prostate cancer cells

We have investigated the expression of prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) transcripts in androgen-dependent (LNCaP) and androgen-independent (22Rv1) prostate cancer cell lines. We also enquired whether Q640X CTE-truncated androgen receptor (AR) has an impact on transcription of mRNA for PSMA and PSA in transfected androgen-sensitive prostate cancer LNCaP cells. Wild type LNCaP, 22Rv1 prostate cancer cells, prostate stromal cells (PrSC) and LNCaP cells transfected with p-Q640X AR, p-WT AR or p-C3 empty plasmids were studied. The expression of PSMA and PSA were detected by real-time PCR after transfection for 4 and 7 days. Expression of mRNAs for PSA was sixfold greater than PSMA in wild type LNCaP cells. In contrast, the wild type androgen refractory 22Rv1 cell line reacted almost exactly the opposite way reverse to LNCaP cells, since the transcription of mRNA for PSMA almost twofold greater than PSA. Non-transfected human PrSC responded similarly to PSMA mRNA and PSA mRNA was not detected in these cells. Q640X AR transfected LNCaP cells downregulated the expression of PSMA and PSA genes after 7 days. Our results demonstrate that Q640X mutated AR may have an important regulatory role in mediating the PSMA and PSA genes expression during the progression of prostate cancer from androgen-dependence to androgen-independence. Understanding their functional properties and mechanisms by which ARs involved in regulation of PSMA and PSA expression will allow the identification of new target therapies for the treatment of hormone-resistant prostate cancer.

Quantitative RT-PCR analysis of PSA and prostate-specific membrane antigen mRNA to detect circulating tumor cells improves recurrence-free survival nomogram prediction after radical prostatectomy

Circulating tumor cell (CTC) analysis is a potential new biomarker in prostate cancer. We hypothesize that quantitative detection of CTCs in patients pre- and post-radical prostatectomy (RP) using quantitative TaqMan® fluorogenic RT-PCR will improve the accuracy of the Kattan nomogram to predict the probability of recurrence-free survival (RFS) post-RP. Ninty-two patients who underwent RP between 2004 and 2009 had venous blood samples taken pre- (Day - 1) and post-operatively (Day + 7). We performed quantitative Taqman® RT-PCR to detect circulating prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) mRNA. We calculated both the logarithmic ratio of Day + 7/Day - 1 for PSA (PSAr) and PSMA (PSMAr) expression (log(Day+7/Day-1) ) and the Kattan nomogram predicted probability of disease recurrence for each patient. We then analyzed how the AUC-ROC analysis for the Kattan nomogram prediction alone (K) compared to the addition of the PSAr and PSMAr in predicting 5-year RFS. The mean age (years), PSA (ng/ml), and follow-up (mo) was 65.1, 9.13, and 72, respectively. The AUCs for K, PSAr + K, and PSMAr + K were 0.752 (95%CI 0.620-0.860), 0.830 (95%CI 0.740-0.911), and 0.837 (95%CI 0.613-0.923), respectively (P = 0.03). The Kattan 5-year PSA RFS was 75%. The actual 5-year PSA RFS survival rate was 77%. Data from modern quantitative RT-PCR to detect circulating prostate-derived PSA and PSM mRNA pre- and post-RP improves the accuracy of the Kattan nomogram to predict biochemical recurrence.

Role of TMPRSS2-ERG gene fusion in negative regulation of PSMA expression.

Prostate specific membrane antigen (PSMA) is overexpressed in prostatic adenocarcinoma (CaP), and its expression is negatively regulated by androgen stimulation. However, it is still unclear which factors are involved in this downregulation. TMPRSS2-ERG fusion is the most common known gene rearrangement in prostate carcinoma. Androgen stimulation can increase expression of the TMPRSS2-ERG fusion in fusion positive prostate cancer cells. The purpose of this investigation is to determine whether PSMA expression can be regulated by the TMPRSS2-ERG gene fusion. We employed two PSMA positive cell lines: VCaP cells, which harbor TMPRSS2-ERG fusion, and LNCaP cells, which lack the fusion. After 24 hours of androgen treatment, TMPRSS2-ERG mRNA level was increased in VCaP cells. PSMA mRNA level was dramatically decreased in VCaP cells, while it only has moderate change in LNCaP cells. Treatment with the androgen antagonist flutamide partially restored PSMA expression in androgen-treated VCaP cells. Knocking down ERG by siRNA in VCaP cells enhances PSMA expression both in the presence and absence of synthetic androgen R1881. Overexpressing TMPRSS2-ERG fusions in LNCaP cells downregulated PSMA both in the presence or absence of R1881, while overexpressing wild type ERG did not. Using PSMA-based luciferase reporter assays, we found TMPRSS2-ERG fusion can inhibit PSMA activity at the transcriptional level. Our data indicated that downregulation of PSMA in androgen-treated VCaP cells appears partially mediated by TMPRSS2-ERG gene fusion.

Study on micrometastases in pelvic lymph nodes detected by real-time reverse transcriptase polymerase chain reaction with clinically localized prostate cancer before radical prostatectomy

To clarify the significance of micrometastases in pelvic lymph nodes in patients with neoadjuvant hormonal therapy (NHT) before radical prostatectomy (RP). Twenty-one patients with clinically localized prostate cancer who received NHT between August 2007 and March 2010 were observed. The patients were clarified into four groups: pathological examination was positive (group A), real-time PCR examination targeting prostate specific antigen (PSA) mRNA and prostate specific membrane antigen (PSMA) mRNA were positive (group B), pathological examination and real-time PCR examination targeting PSA mRNA and PSMA mRNA were both negative (group C), and the control group (group D). After a standard bipedal lymphangiography the films were reviewed carefully by an experienced radiologist. If positive lymph nodes were seen or suspected, a thin-walled 22 gauge needle were directed transabdominally under fluoroscopic control into the area of question and an aspirate was obtained. The expression of PSA and PSMA in aspirate were assessed by a fully quantitative real-time PCR. The specimens were regarded in which either PSA mRNA or PSMA mRNA were positive as showing the "presence of micrometastasis". Lymph node specimens were also stained immunohistochemically with an antibody PSA after RP. Pathological examination detected lymph node metastases from 3 cases, and real-time PCR further identified lymph node micrometastases from 14 cases with no pathological evidence of nodal involvement. The expression level of PSA mRNA and PSMA mRNA were statistically significant in patients with histological confirmed lymph node metastases and micrometastases detected by real-time PCR despite the lack of histological evidence, and the expression level of PSA mRNA and PSMA mRNA in aspirate were higher than the lymph node between the group A and group B. Although residual foci of atrophic prostate cancer cells in resected lymph nodes after NHT can be difficult to diagnose by pathological examination, the present results show the usefulness of quantitative real-time PCR targeting PSA and PSMA mRNA for detected micrometastatic tumour foci in pelvic lymph nodes from fine needle aspiration biopsy of lymph nodes before RP.

230 INHIBITION OF PROSTATE SPECIFIC MEMBRANE ANTIGEN GENE-EXPRESSION IS MEDIATED BY TMPRSS2:ERG GENE FUSION IN VCAP PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2010230 INHIBITION OF PROSTATE SPECIFIC MEMBRANE ANTIGEN GENE-EXPRESSION IS MEDIATED BY TMPRSS2:ERG GENE FUSION IN VCAP PROSTATE CANCER CELLS Pravin Rao, Lihong Yin, and Warren Heston Pravin RaoPravin Rao Lakewood, OH More articles by this author , Lihong YinLihong Yin Cleveland, OH More articles by this author , and Warren HestonWarren Heston Cleveland, OH More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.288AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate specific membrane antigen (PSMA) is a type II transmembrane glycoprotein that is over-expressed in prostatic adenocarcinoma (CaP). Levels of PSMA expression have been correlated with aggressive disease and risk of early recurrence after radical prostatectomy (RP). Studies have shown that a significant fraction of CaP's harbor a signature gene fusion between the androgen-responsive TMPRSS2 gene and transcription factors in the ETS family – most commonly ERG. The TMPRSS2-ERG fusion is likely the cause for over-expression of ERG in CaP. PSMA, unlike ERG, is negatively regulated by androgen. Elucidation of the relationship between TMPRSS2 gene fusions and PSMA expression could reveal mechanisms to alter PSMA expression and disease course. The purpose of this investigation was to determine whether PSMA gene expression could be regulated by the TMPRSS2-ERG fusion in prostate cancer cells. METHODS VCaP cells (which harbor the TMPRSS2:ERG fusion) and LNCaP cells (which do not) were incubated with vehicle or the androgen-receptor antagonist flutamide prior to treatment with synthetic androgen R1881. In addition, transfection of siRNA against human ERG was performed to create ERG knockdown in VCaP cells. Reverse transcription (RT) PCR as well as real-time RT PCR were used to detect and quantify transcription of ERG, TMPRSS2:ERG, PSMA, and androgen receptor (AR) genes. Using the comparative threshold cycles method, the quantification of these transcripts was performed, and expression was compared in various androgen-treated and untreated VCaP and LNCaP cells. RESULTS After 24-hour androgen treatment, ERG and TMPRSS2-ERG mRNA level were increased in VCaP cells and unchanged in LNCaP cells. PSMA and androgen receptor (AR) mRNA level were dramatically decreased in VCaP cells and modestly decreased in LNCaP cells. Treatment with the androgen antagonist flutamide partially restored PSMA and AR expression in androgen-treated VCaP cells. Knocking down ERG by siRNA in VCaP cells enhances PSMA mRNA expression both in the presence and absence of R1881, while it has minimal change of AR mRNA in VCaP-ERG knock down cells. CONCLUSIONS Down-regulation of PSMA in androgen-treated VCaP cells appears partially mediated by the TMPRSS2:ERG gene fusion. Since PSMA has been associated with aggressive disease and early biochemical failure after RP, further elucidation of the relationship between TMPRSS2 gene fusions and PSMA could reveal potential novel targets for treatment of prostate cancer. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e90 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pravin Rao Lakewood, OH More articles by this author Lihong Yin Cleveland, OH More articles by this author Warren Heston Cleveland, OH More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Prostate Specific Membrane Antigen mRNA in Blood as a Potential Predictor of Biochemical Recurrence after Radical Prostatectomy

We investigated whether the detection of prostate specific membrane antigen (PSMA) in blood preoperatively has predictive value for biochemical recurrence (BCR) after radical prostatectomy in patients with prostate cancer. All 134 patients scheduled to receive radical prostatectomy for prostate cancer were prospectively enrolled. The authors used nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect PSMA mRNA-bearing cells in peripheral blood, and analyzed the ability of PSMA mRNA positivity to predict BCR after surgery. PSMA-mRNA was detected in 24 (17.9%) patients by RT-PCR. Over a median follow-up of 20 months (range, 3 to 46 months), BCR developed in 15 patients (11.2%) and median time to BCR was 7 months (range, 3 to 25 months). Kaplan-Meier analysis revealed a significant difference between those positive or negative for PSMA in terms of recurrence-free actuarial probability (log rank P=0.0039). Multivariate analysis showed that positivity for PSMA mRNA (HR: 3.697, 95% CI 1.285-10.634, P=0.015) and a biopsy Gleason score of ≥7 (HR: 4.500, 95% CI 1.419-14.274, P=0.011) were independent preoperative predictors of BCR. The presence of PSMA mRNA in peripheral blood can be used to predict BCR after radical prostatectomy.

Gene expression of prostate-specific membrance antigen

Objective The aim of this research is to clone cDNA of human prostate-specific membrane antigen(PSMA),construct pcDNA3-PSMA eukaryotic expression vector, and then identify the expression of PSMA protein. We hope this experiment will give a good basis on the research of the prostate therapy based on DC vaccine. Methods Total RNA was extracted from prostate cancer tissues. A pair of primers were designed according to M99487 in Genebank and obtain the PSMA cDNA fragment by reverse transcription-polymerase chain reaction. The PCR products were then cloned into empty plasmid pcDNA3.Further sequence analysis was carried out and the recombinant plasmids PcDNA3.0-PSMA were transfected into PC-3 cells. In G418 selective medium(400μg/ml),two positive clones expressing PSMA mRNA, named D4 and B3 respectively, were identified by RT-PCR. Using rabbit anti-human PSMA monoclonal antibody, we identified the location of expressed PSMA protein by indirect immunofluorescence analysis. To verified the molecular weight of expressed PSMA protein, western blotting analysis of total cell extracts was carried out using chemiluminescence western blotting kit. Results Sequence analysis confirmed the homogeneity between cloned PSMA cDNA and M99487 was 99.7%.After transfection. two cell clones expressing PSMA mRNA were identified by RT-PCR.The indirect immunofluorescence assay confirmed that expressed PSMA protein was located on membrane and had good immunoreactivity. Western blotting analysis showed the molecular weight of expressed PSMA was 100kD.as was PSMA protein from LNCaP cells. Conclusion We have obtained the PSMA cDNA from prostate cancer tissues. The PcDNA3.0-PSMA eukaryotic expression vector was constructed and the target PSMA protein which had immunoreactivity expressed on cell membrane. The molecular weight of expressed PSMA was 100kD,the right molecular weight as we expected. Our research layed foundation for treatment of prostate neoplasms by DC vaccines transfect with PSMA. Key words: Prostate; Antigeson

Commentary on Improved sensitivity for detecting micrometastases in pelvic lymph nodes by real-time reverse transcriptase polymerase chain reaction (RT-PCR) compared with conventional RT-PCR in patients with clinically localized prostate cancer undergoing radical prostatectomy: Terakawa T, Miyake H, Kurahashi T, Furukawa J, Takenaka A, Fujisawa M, Division of Urology, Kobe University Graduate School of Medicine, Kobe, Japan

To compare the usefulness between real-time reverse transcriptase polymerase chain reaction (RT-PCR) with that of conventional RT-PCR for detecting micrometastases in pelvic lymph nodes (PLN) dissected during radical prostatectomy (RP) for prostate cancer. In all, 120 patients with clinically localized prostate cancer who underwent RP and pelvic lymphadenectomy were included. Expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) in 2,215 PLNs obtained from these 120 patients were assessed by fully quantitative real-time RT-PCR as well as conventional RT-PCR. Specimens in which either PSA or PSMA mRNA was positive were regarded as showing the “presence of micrometastasis.” Pathologic examinations detected tumor cells in 29 PLNs from 11 patients, while real-time RT-PCR and conventional RT-PCR further identified micrometastasis in 143 and 81 PLNs from 32 and 19 patients, respectively, with no pathologic evidence of nodal involvement; that is, the sensitivity of real-time RT-PCR for detecting micrometastases was significantly higher than that of conventional RT-PCR. In this series, biochemical recurrence occurred in 32 patients, and in both assays, there were significant differences in biochemical recurrence-free survival between patients with and with no micrometastases. However, despite the significant association of micrometastases detected by both assays with biochemical recurrence on univariate analysis, the presence of micrometastases detected by real-time RT-PCR but not that detected by conventional RT-PCR appeared to be useful as an independent predictor on multivariate analysis. Although micrometastatic tumor foci in PLNs that were missed by routine pathologic examination could be diagnosed by both real-time RT-PCR and conventional RT-PCR assays, it would be strongly recommended to use real-time RT-PCR to detect micrometastases considering its high sensitivity and the close association between the outcome of this assay and the probability of biochemical recurrence.

Improved sensitivity for detecting micrometastases in pelvic lymph nodes by real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) compared with conventional RT‐PCR in patients with clinically localized prostate cancer undergoing radical prostatectomy

To compare the usefulness between real-time reverse transcriptase polymerase chain reaction (RT-PCR) with that of conventional RT-PCR for detecting micrometastases in pelvic lymph nodes (PLN) dissected during radical prostatectomy (RP) for prostate cancer. In all, 120 patients with clinically localized prostate cancer who underwent RP and pelvic lymphadenectomy were included. Expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) in 2215 PLNs obtained from these 120 patients were assessed by fully quantitative real-time RT-PCR and as well as conventional RT-PCR. Specimens, in which either PSA or PSMA mRNA was positive, were regarded as showing the 'presence of micrometastasis'. Pathological examinations detected tumour cells in 29 PLNs from 11 patients, while real-time RT-PCR and conventional RT-PCR further identified micrometastasis in 143 and 81 PLNs from 32 and 19 patients, respectively, with no pathological evidence of nodal involvement; that is, the sensitivity of real-time RT-PCR for detecting micrometastases was significantly higher than that of conventional RT-PCR. In this series, biochemical recurrence occurred in 32 patients, and in both assays, there were significant differences in biochemical recurrence-free survival between patients with and with no micrometastases. However, despite the significant association of micrometastases detected by both assays with biochemical recurrence on univariate analysis, the presence of micrometastases detected by real-time RT-PCR but not that detected by conventional RT-PCR appeared to be useful as an independent predictor on multivariate analysis. Although micrometastatic tumour foci in PLNs that were missed by routine pathological examination could be diagnosed by both real-time RT-PCR and conventional RT-PCR assays, it would be strongly recommended to use real-time RT-PCR to detect micrometastases considering its high sensitivity and the close association between the outcome of this assay and the probability of biochemical recurrence.

Phase I trial of a PSMA DNA vaccine for patients(pts) with good risk renal cell carcinoma(RCC)

16073 Background: Prostate Specific Membrane Antigen (PSMA) is a self antigen first identified as a marker for epithelial cells in the prostate but is expressed on the vascular endothelium of metastatic RCC. PSMA mRNA transcripts and protein expression have been seen in the endothelium of tumor-associated neovasculature of non-metastatic malignancies. No PSMA mRNA or protein expression has been found in vascular endothelial cells of corresponding benign tissues. The clinical importance of PSMA expression is supported by the observation that PSMA RNA detected by RT-PCR in the circulation of RCC pts correlates with disease progression. A phase I trial was designed using a xenogeneic murine versus human PSMA DNA vaccine to evaluate safety in pts with RCC. Eighteen pts with clear cell RCC status post nephrectomy, cytokine therapy, or post metastastectomy with high risk of recurrent disease were enrolled. Good risk pts were defined as having a KPS of >80%, prior nephrectomy, normal calcium and hemoglobin levels. Methods: Six pts each were assigned to dose levels (500, 1500 and 3000μg) and were randomized to receive 3 murine or 3 human PSMA DNA vaccines im every 3 weeks. After the third vaccine, pts were crossed-over to the other species for an additional 3 vaccines. If pts remained disease-free or had clinically responding disease, booster immunizations were administered bimonthly for up to 4 additional booster vaccinations at weeks 24, 32, 40 and 48. Results: Ten of 15 treated pts were evaluable. The vaccine was well-tolerated. One pt from the 500μg dose level who received the murine vaccine first, and 2 pts from the 1500μg dose and 1 pt from the 3000μg dose who received the human PSMA DNA vaccine first, remain on study at 167, 114, 125 and 68 weeks, respectively. FACS analysis of pts’ sera against 3T3 fibroblasts genetically transduced to secrete PSMA showed no reactivity at the 500μg dose level; 1 of 2 pts at 1500μg and 1 pt at the 3000μg dose showed reactivity against PSMA, respectively. Minimal activity was seen by ELISA. Conclusions: The vaccine was safe at all dose levels without autoimmunity. The 2 pts at the 1500μg dose level have stable lung lesions. Studies are ongoing to examine other parameters of immune responsiveness. Supported by the Byrne Foundation No significant financial relationships to disclose.

Prostate-Specific Membrane Antigen Expression Is a Potential Prognostic Marker in Endometrial Adenocarcinoma

The aim of this study was to determine the role of prostate-specific membrane antigen (PSMA) as a prognostic marker in endometrial adenocarcinoma (EAC) and to explore whether its down-regulation could be due to epigenetic mechanism. First, we examined the expression and the prognostic value of PSMA by semiquantitative reverse transcription-PCR and immunohistochemistry in EAC tissue samples. Second, to explore the role of CpG methylation in down-regulation PSMA in EAC, we evaluated PSMA CpG island methylation using methylation-specific PCR in cells lines and in a subset of patients' samples. Furthermore, association of the status of tumor methylation to the clinical and histologic variables was also evaluated. Higher PSMA mRNA levels were associated with stage I (P = 0.046) and PSMA protein intensity by immunohistochemistry (P = 0.032). In multivariate analysis, loss of PSMA expression was associated with a worse disease-free survival (P = 0.02). PSMA was methylated in prostate cell lines (DU145 and PC3) and endometrial cell lines. In addition, PSMA was methylated in 5 of 18 samples (all 5 had low PSMA mRNA value). There was a significant association between PSMA methylation and loss of protein expression by immunohistochemistry and PSMA-RNA level with P value of 0.036 and 0.011, respectively. In addition, there was an association between PSMA methylation and tumor size (P = 0.025). In summary, (a) PSMA is underexpressed in advanced stage EAC, (b) loss of PSMA expression can be considered as a prognostic marker in patients with EAC, and (c) loss of PSMA expression in a subset of EAC cases could be due to epigenetic silencing.

High expression of PSM‐E correlated with tumor grade in prostate cancer: A new alternatively spliced variant of prostate‐specific membrane antigen

Prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer (PCa) has been targeted for therapy and diagnosis of PCa. In the current study, PSMA cDNA was cloned from PCa tissue by RT-PCR. After sequencing, a new spliced variant of PSMA (PSM-E) was discovered and its specificity in PCa was evaluated. PSM-E and PSMA mRNA were measured in LNCaP, PC-3 and prostate or nonprostatic malignancies. Following transfection of PC-3 with PSM-E cDNA in the pcDNA3.0 vector, PSM-E expression was measured by immunofluorescence and Western-blot. PSM-E and PSMA mRNA levels were quantified by a real-time PCR assay in normal prostate (n = 7), benign prostatic hyperplasia (BPH) (n = 22) and PCa (n = 41). The correlation between their levels and tumor grade was analyzed. PSM-E cDNA is identical to PSMA except for a 97-nucleotide region and a 93-nucleotide region. PSM-E and PSMA mRNA were detected in PCa and LNCaP, not in PC-3; PSMA could be detected in some nonprostatic tumors whereas PSM-E not. The expression of PSM-E protein was detected in transfected cells. Significant difference of PSM-E mRNA levels was observed among normal prostate, BPH and PCa (P < 0.001), and PSM-E levels increased with increasing Gleason score (r = 0.514, P < 0.001). PSMA mRNA levels were higher in BPH and PCa than in normal prostate (P < 0.001), but no difference between BPH and PCa, no significant correlation was observed between PSMA levels and Gleason score (r = 0.229, P = 0.057). PSM-E may be a potential prognostic indicator for PCa progression and may be a new target antigen for therapy of PCa.