Prostate-specific Membrane Antigen Gene 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.

Exploring the role of combined external beam radiotherapy and targeted radioligand therapy with [177Lu]Lu-PSMA-617 for prostate cancer - from bench to bedside.

Management of prostate cancer (PC) might be improved by combining external beam radiotherapy (EBRT) and prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) with lutetium-177 (177Lu)-labeled PSMA inhibitors. We hypothesized a higher efficacy of the combination due to augmentation of the radiation dose to the tumor and interactions of EBRT with PSMA expression potentially increasing radiopharmaceutical uptake. Therefore, this study analyzed the influence of radiation on PSMA expression levels in vitro. The results were translated to evaluate the efficacy of the combination of photon EBRT and [177Lu]Lu-PSMA-617 in a murine PC xenograft model. Finally, a clinical case report on a combined elective field EBRT with RLT dose escalation illustrates a proof-of-concept. Methods: PSMA gene and protein expression were assessed in human PSMA-overexpressing LNCaP cells after irradiation using reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry and On-Cell Western assays. In the in vivo therapy study, LNCaP tumor-bearing BALB/c nu/nu mice were irradiated once with 2 Gy X-ray EBRT and injected with 40 MBq [177Lu]Lu-PSMA-617 after 4 h or received single or no treatment (n = 10 each). Tumor-absorbed doses by [177Lu]Lu-PSMA-617 were calculated according to the Medical Internal Radiation Dosimetry (MIRD) formalism after deriving time-activity curves using a gamma probe. An exemplified patient case is demonstrated where fractionated EBRT (54 Gy to prostate; 45 Gy to pelvic lymphatics) and three cycles of [177Lu]Lu-PSMA-617 (3.4-6.0 GBq per cycle) were sequentially combined under concurrent androgen deprivation for treating locally advanced PC. Results: At 4 h following irradiation with 2-8 Gy, LNCaP cells displayed a PSMA protein upregulation by around 18% relative to non-irradiated cells, and a stronger upregulation on mRNA level (up to 2.6-fold). This effect was reversed by 24 h when PSMA protein levels were downregulated by up to 22%. Mice treated with the combination therapy showed significantly improved outcomes regarding tumor control and median survival (p < 0.0001) as compared to single or no treatment. Relative to monotherapy with PSMA-RLT or EBRT, the tumor doubling time was prolonged 1.7- or 2.7-fold and the median survival was extended by 24% or 60% with the combination, respectively. Additionally, tumors treated with EBRT exhibited a 14% higher uptake of the radiopharmaceutical as evident from the calculated tumor-absorbed dose, albeit with high variability in the data. Concerning the patient case, the tri-modality treatment was well tolerated and the patient responded with a long-lasting complete biochemical remission for five years following end of PSMA-RLT. The patient then developed a biochemical relapse with oligo-recurrent disease on follow-up imaging. Conclusion: The present preclinical and clinical data demonstrate that the combination of EBRT with dose escalation by PSMA-RLT improves tumor control and potentially prolongs survival. This may pave the way for further clinical investigations of this approach to explore the curative potential of the combination therapy.

PD04-04 MOLECULAR CORRELATES WITH PSMA EXPRESSION IN PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023PD04-04 MOLECULAR CORRELATES WITH PSMA EXPRESSION IN PROSTATE CANCER Adam Weiner, Eric Li, JJ Zhang, Elai Davicioni, Edward Schaeffer, Ashley Ross, Luca Valle, Paul Boutros, Jeremie Calais, Amar Kishan, and Robert Reiter Adam WeinerAdam Weiner More articles by this author , Eric LiEric Li More articles by this author , JJ ZhangJJ Zhang More articles by this author , Elai DavicioniElai Davicioni More articles by this author , Edward SchaefferEdward Schaeffer More articles by this author , Ashley RossAshley Ross More articles by this author , Luca ValleLuca Valle More articles by this author , Paul BoutrosPaul Boutros More articles by this author , Jeremie CalaisJeremie Calais More articles by this author , Amar KishanAmar Kishan More articles by this author , and Robert ReiterRobert Reiter More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Not all prostate cancer (PCa) is detected by prostate-specific membrane antigen (PSMA)-based imaging or responsive to PSMA-based radiopharmaceuticals. We sought to define the molecular processes associated with PSMA expression. METHODS: Using multiple datasets (n=5 Homo sapiens; n=2 mouse xenograft models; n=1 cell line), we correlated PSMA gene expression (FOLH1) with biological processes and treatments. RESULTS: In an exploratory analysis of a prospective cohort of 7,000 tumors from radical prostatectomy, tumors with high PSMA RNA expression (one standard deviation above mean) tended to demonstrate more androgen receptor activity, high genomic risk scores (Decipher), more markers of homologous recombination deficiency, less immune content, lower predicted response to radiotherapy (“PORTOS”), and enrichment for P13K/AKT/mTOR pathways (Figure 1). In a validation cohort, adenocarcinoma tumors (n=49) expressed more PSMA than benign tissue (n=12; Wilcoxon p=0.020) and castration resistant tumors (n=27; p=0.015). An additional cohort showed adenocarcinoma (n=110) expressed more PSMA than neuroendocrine tumors (n=20; p=4.4e-6). In a mouse xenograft model (n=1) of high-grade adenocarcinoma, PSMA expression declined as the model underwent conversion to castration resistance and subsequent neuroendocrine transformation following castration. From both mouse and human data, treatment with androgen deprivation was associated with an initial increase in PSMA expression. From TCGA (n=491), tumors with high PSMA expression also tended to have a higher mutational burden and fewer tumor infiltrating lymphocytes in a subset with immunohistochemistry data (n=330). To assess the association between PSMA and P13K/AKT/mTOR, we used data from a cell line exposed to the MAPK inhibitor trametinib which upregulates P13K/AKT/mTOR pathways. VCaP cells treated with both enzalutamide and trametinib expressed two-fold more PSMA compared to those treated with the individual medications or neither. CONCLUSIONS: PCa with high PSMA expression is characterized by relevant differences in biological processes. If corroborated with PSMA-PET avidity, these results could optimize precision PCa care by informing adjunctive molecular testing or treatment paradigms for low or high PSMA expressing tumors. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e143 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Adam Weiner More articles by this author Eric Li More articles by this author JJ Zhang More articles by this author Elai Davicioni More articles by this author Edward Schaeffer More articles by this author Ashley Ross More articles by this author Luca Valle More articles by this author Paul Boutros More articles by this author Jeremie Calais More articles by this author Amar Kishan More articles by this author Robert Reiter More articles by this author Expand All Advertisement PDF downloadLoading ...

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.

Investigating PSMA differential expression in canine uroepithelial carcinomas to aid disease-based stratification and guide therapeutic selection

BackgroundIn male dogs, uroepithelial cancers include invasive urothelial carcinoma (iUC) and prostate carcinoma (PCA). The inability to distinguish iUC involving the prostate from PCA results in indiscriminate clinical management strategies that could be suboptimal as first-line chemotherapy for iUC (cisplatin) and PCA (docetaxel) differ in people. Prostate specific membrane antigen (PSMA) is a transmembrane protein, and its overexpression has been identified in human prostate carcinoma and neovasculature associated with solid tumor growth. This study investigates whether differential PSMA expression exists between presumptive canine iUC and PCA among cell lines and archived patient samples, which might allow for improved accuracy in disease-based stratification and optimal chemotherapy selection. Additionally, in vitro sensitivities of reported canine iUC and PCA cell lines to uroepithelial directed chemotherapeutic agents were characterized.ResultsNormalized PSMA gene and protein expressions were not significantly different between 5 iUC and 4 PCA cell lines. PSMA protein expression was uniformly observed in uroepithelial cancers regardless of anatomic origin from archived patient samples, further confirming that PSMA cannot differentiate iUC from PCA. In vitro sensitivity of cell lines to uroepithelial directed chemotherapeutics revealed that vinblastine exerted the broadest cytotoxic activity.ConclusionsDifferential expression of PSMA was not identified between canine iUC and PCA cell lines or archived patient samples, and PSMA alone cannot be used for disease stratification. Nonetheless given its conserved overexpression, PSMA may be a targetable surface marker for both canine iUC and PCA. Lastly, in uroepithelial carcinomas, vinblastine might exert the broadest anticancer activity regardless of cellular origin.

Clinical and molecular features of low prostate-specific membrane antigen (PSMA) expression in patients (pts) with metastatic castration resistant prostate cancer (mCRPC).

167 Background: Low PSMA uptake on positron-emission tomography is seen in up to 30% of mCRPC pts and represents a clinically distinct subgroup with adverse outcomes. We assessed transcriptional and clinical features associated with low PSMA ( FOLH1) gene expression in mCRPC. Methods: A retrospective analysis of mCRPC biopsy samples with RNA-seq data was undertaken. Normalized FOLH1 expression was compared across histologic subtypes and sites of disease. We assessed the association between FOLH1 expression, selected androgen receptor (AR) target genes, master regulators of neuroendocrine differentiation, and previously validated AR activity and treatment-associated small cell neuroendocrine carcinoma (t-SCNC) transcriptional signature scores using Pearson correlations. Associations between FOLH1 and both PSA50 response to subsequent AR-targeted therapy and overall survival (OS) were examined by logistic regression and Cox proportional hazard models, respectively. Results: Samples from 97 pts were identified, of which 18% harbored t-SCNC histology. 45% of pts had visceral metastases at the time of biopsy, and 41% received subsequent AR-targeted therapy. Median FOLH1 expression was lower in pts with visceral metastases vs no visceral metastases (14.7 vs 15.6, p = 0.02) but was not significantly different across t-SCNC vs adenocarcinoma biopsies (14.3 vs 15.4, p = 0.13). FOLH1 expression was positively correlated with AR transcriptional activity and AR target genes, and negatively correlated with master regulators of neuroendocrine differentiation and t-SCNC transcriptional signature scores (Table). Low FOLH1 expression did not predict PSA50 response to subsequent AR-targeted therapy (OR 0.97, p = 0.8), but was associated with shorter OS on univariate analysis (HR 1.09, 95% CI 1.02-1.16, p=0.01). A post-hoc analysis revealed a trend towards decreased median OS in pts with FOLH1 expression &lt;12 (7.5 vs 17.1 months, log-rank p = 0.06). Conclusions: In this retrospective analysis of mCRPC pts, low FOLH1 expression was associated with transcriptional features of t-SCNC, decreased AR activity, and shorter OS. These findings are hypothesis-generating and prospective validation is needed.[Table: see text]

Prostate-specific Membrane Antigen and Fluciclovine Transporter Genes are Associated with Variable Clinical Features and Molecular Subtypes of Primary Prostate Cancer

18F-Fluciclovine-based positron emission tomography (PET) imaging is recommended in the USA for biochemical recurrence (BCR) after prostate cancer treatment. However, prostate-specific membrane antigen (PSMA)-based PET imaging is more common worldwide, supported by international guidelines, and is now approved by the Food and Drug Administration in the USA for initial staging of primary prostate cancer. Little is known about the molecular profiles of lesions detected by PSMA-targeted PET/computed tomography (CT) versus 18F-fluciclovine PET/CT. We examined the expression of PSMA (FOLH1) and the fluciclovine transporter genes LAT1–4 and ASCT1/2 in a combined cohort of more than 18 000 radical prostatectomy specimens and their associations with clinical outcomes. Expression of PSMA and all but one fluciclovine transporter gene was higher in prostate cancer than in benign tissue. PSMA expression was associated with Gleason score (GS) ≥8 and lymph node involvement (LNI), and had a positive linear correlation with Decipher risk score. By contrast, expression of the fluciclovine transporters LAT2, LAT3, and ASCT2 was negatively associated with GS ≥ 8, LNI, and high Decipher score. The top decile of PSMA expression was associated with poorest metastasis-free survival (MFS), while the bottom deciles of LAT3 and ASCT2 expression were associated with poorest MFS. Patient summaryWe measured the expression of genes that encode the targets for two different radiotracers in PET (positron emission tomography) scans of the prostate. We found that PSMA gene expression (PSMA-based tracer) is associated with worse clinical outcomes, while expression of ASCT2, LAT2, and LAT3 genes (fluciclovine tracer) is associated with better outcomes.

Characterization of PSMA and 18F-fluciclovine transporter gene expression in localized prostate cancer.

295 Background: While 18F-fluciclovine PET/CT is approved in the US and recommended by the NCCN, prostate-specific membrane antigen (PSMA) PET/CT is more common in Europe/Australia and recommended by the EAU. Less is known about the biology of lesions detected by either modality. 18F-fluciclovine PET relies on radiotracer uptake by amino acid transporters LAT1-4 and ASCT1-2. PSMA PET is dependent on surface expression of PSMA. We compared relative expression of PSMA and fluciclovine transporter genes in radical prostatectomy (RP) samples to determine their distribution across subtypes and correlation with outcomes. Methods: Gene expression data of 19,102 RP samples were analyzed using the Affymetrix Human Exon 1.0 ST microarray. 1,135 patients had long term follow up. Associations between expression of PSMA and fluciclovine transporter genes (LAT1-4 and ASCT1-2) and pathologic variables, molecular subtypes, and clinical outcomes were conducted. Results: All fluciclovine transporter genes (LAT 1-4, ASCT1-2) were expressed at lower levels than PSMA (p &lt;0.0001). PSMA expression was positively correlated with genomic risk score and pathologic Gleason score (p&lt;0.0001), but LAT2-3 and ASCT2 were inversely correlated with genomic risk in primary tumors (p&lt;0.0001) and less expressed in GS 9-10 tumors (p&lt;0.0001). PSMA expression was associated with worse metastasis-free survival (MFS) (HR 1.45, p=0.001) and lymph node involvement (HR 2.14, p&lt;0.0001). Expression of LAT2, LAT3, ASCT2 expression was associated with better MFS (HR 0.85, 0.63, 0.74, p&lt;0.0001-0.04). After multivariable adjustment, PSMA expression remained independently prognostic of poorer MFS (HR 1.3, p=0.028). Luminal B subtype was notable for PSMA overexpression; Luminal A was enriched in ASCT2 and LAT2 (p&lt;0.0001). PSMA expression did not correlate with ERG fusion prostate cancers, but LAT2, ASCT1, and ASCT2 were overexpressed in ERG fusion negative tumors (p&lt;0.0001). Conclusions: PSMA expression is associated with more aggressive disease and poorer clinical outcomes than fluciclovine transporter genes in localized prostate cancer. Molecular subtypes of prostate cancer vary in PSMA and fluciclovine transporter gene expression.

Differential expression of PSMA and 18F-fluciclovine transporter genes in metastatic castrate-resistant and treatment-emergent small cell/neuroendocrine prostate cancer.

24 Background: 18F-fluciclovine (Axumin) PET/CT imaging is recommended by the NCCN in the setting of biochemical recurrence, while prostate-specific membrane antigen (PSMA) PET/CT is preferred by the EAU. The utility of these methods in the post-androgen deprivation therapy (ADT) setting however, is less defined. Our objective was to compare relative gene expression of the molecular targets of these imaging modalities— fluciclovine transporter genes (LAT1-4, ASCT1-2) and PSMA—in metastatic castrate resistant prostate cancer (mCRPC) and treatment-emergent small cell/neuroendocrine prostate cancer (t-SCNC). Methods: Genome-wide expression profiles of five mCRPC cohorts (Aggarwal, Grasso, Kumar, Beltran, Robinson, et al) were used to characterize relative expression of fluciclovine transporter (LAT1-4, ASC1-2) and PSMA (FOLH1) genes. 3 cohorts (Kumar, Beltran, Aggarwal) were enriched with t-SCNC tumors. The GSE35988 cohort included primary tumors and mCRPC. RNA expression profiling methods were consistent within cohorts. Results: 518 mCRPC specimens were included. In the GSE35988 cohort, PSMA expression was downregulated in mCRPC when compared to primary localized tumors (p=0.01). PSMA expression was further depressed in t-SCNC when compared with mCRPC (p&lt;0.001). Of the fluciclovine transporter genes, LAT1 and LAT4 were overexpressed in mCRPC when compared to primary tumors, while ASC2 was less expressed (p&lt;0.001). LAT1 was further overexpressed in t-SCNC when compared to mCRPC, while LAT2 was less expressed (p&lt;0.001). PSMA expression was negatively correlated with LAT1 (p&lt;0.001) but positively correlated with LAT2 (p=0.006). Other fluciclovine transporters were not correlated. Conclusions: Expression of PSMA and a subset of fluciclovine transporter genes are inversely correlated in mCRPC and t-SCNC. These findings suggest that fluciclovine-based imaging may play a role in castrate resistant states. Clinical comparison between PSMA- and fluciclovine-based imaging modalities in mCRPC and t-SCNC is warranted.

Differential Expression of Glucose Transporters and Hexokinases in Prostate Cancer with a Neuroendocrine Gene Signature: A Mechanistic Perspective for 18F-FDG Imaging of PSMA-Suppressed Tumors.

Although the incidence of de novo neuroendocrine prostate cancer (PC) is rare, recent data suggest that low expression of prostate-specific membrane antigen (PSMA) is associated with a spectrum of neuroendocrine hallmarks and androgen receptor (AR) suppression in PC. Previous clinical reports indicate that PCs with a phenotype similar to neuroendocrine tumors can be more amenable to imaging by 18F-FDG than by PSMA-targeting radioligands. In this study, we evaluated the association between neuroendocrine gene signature and 18F-FDG uptake-associated genes including glucose transporters (GLUTs) and hexokinases, with the goal of providing a genomic signature to explain the reported 18F-FDG avidity of PSMA-suppressed tumors. Methods: Data-mining approaches, cell lines, and patient-derived xenograft models were used to study the levels of 14 members of the SLC2A family (encoding GLUT proteins), 4 members of the hexokinase family (genes HK1-HK3 and GCK), and PSMA (FOLH1 gene) after AR inhibition and in correlation with neuroendocrine hallmarks. Also, we characterize a neuroendocrine-like PC (NELPC) subset among a cohort of primary and metastatic PC samples with no neuroendocrine histopathology. We measured glucose uptake in a neuroendocrine-induced in vitro model and a zebrafish model by nonradioactive imaging of glucose uptake using a fluorescent glucose bioprobe, GB2-Cy3. Results: This work demonstrated that a neuroendocrine gene signature associates with differential expression of genes encoding GLUT and hexokinase proteins. In NELPC, elevated expression of GCK (encoding glucokinase protein) and decreased expression of SLC2A12 correlated with earlier biochemical recurrence. In tumors treated with AR inhibitors, high expression of GCK and low expression of SLC2A12 correlated with neuroendocrine histopathology and PSMA gene suppression. GLUT12 suppression and upregulation of glucokinase were observed in neuroendocrine-induced PC cell lines and patient-derived xenograft models. A higher glucose uptake was confirmed in low-PSMA tumors using a GB2-Cy3 probe in a zebrafish model. Conclusion: A neuroendocrine gene signature in neuroendocrine PC and NELPC associates with a distinct transcriptional profile of GLUTs and hexokinases. PSMA suppression correlates with GLUT12 suppression and glucokinase upregulation. Alteration of 18F-FDG uptake-associated genes correlated positively with higher glucose uptake in AR- and PSMA-suppressed tumors. Zebrafish xenograft tumor models are an accurate and efficient preclinical method for monitoring nonradioactive glucose uptake.

Neuroendocrine differentiation of prostate cancer leads to PSMA suppression.

Prostate-specific membrane antigen (PSMA) is overexpressed in most prostate adenocarcinoma (AdPC) cells and acts as a target for molecular imaging. However, some case reports indicate that PSMA-targeted imaging could be ineffectual for delineation of neuroendocrine (NE) prostate cancer (NEPC) lesions due to the suppression of the PSMA gene (FOLH1). These same reports suggest that targeting somatostatin receptor type 2 (SSTR2) could be an alternative diagnostic target for NEPC patients. This study evaluates the correlation between expression of FOLH1, NEPC marker genes and SSTR2. We evaluated the transcript abundance for FOLH1 and SSTR2 genes as well as NE markers across 909 tumors. A significant suppression of FOLH1 in NEPC patient samples and AdPC samples with high expression of NE marker genes was observed. We also investigated protein alterations of PSMA and SSTR2 in an NE-induced cell line derived by hormone depletion and lineage plasticity by loss of p53. PSMA is suppressed following NE induction and cellular plasticity in p53-deficient NEPC model. The PSMA-suppressed cells have more colony formation ability and resistance to enzalutamide treatment. Conversely, SSTR2 was only elevated following hormone depletion. In 18 NEPC patient-derived xenograft (PDX) models we find a significant suppression of FOLH1 and amplification of SSTR2 expression. Due to the observed FOLH1-supressed signature of NEPC, this study cautions on the reliability of using PMSA as a target for molecular imaging of NEPC. The observed elevation of SSTR2 in NEPC supports the possible ability of SSTR2-targeted imaging for follow-up imaging of low PSMA patients and monitoring for NEPC development.

Antitumor effect of dendritic cells transfected with prostate-specific membrane antigen recombinant adenovirus on prostate cancer: An in vitro study.

The aim of the present study was to construct a dendritic cell (DC) vaccine transfected with a prostate-specific membrane antigen (PSMA) recombinant adenovirus and to observe the ability of the recombinant DCs in eliciting a PSMA-directed T‑cell response to prostate cancer (PCa) in vitro. Replication‑defective adenoviral vectors, were constructed using the Adxsi system. The virus titer was measured by TCID50 assay, and the expression of the PSMA gene was identified by polymerase chain reaction (PCR) generation of peripheral blood mononuclear cell (PBMC) derived DCs in vitro. In addition, a PE‑7AAD apoptosis and necrosis kit was applied to detect the survival of DCs at different MOI values to determine the optimal MOI. Morphological changes of DC were observed under a fluorescence microscope, expression of the PSMA protein was detected by western blotting 48 h after transfection, the expression of DC markers prior to and following transfection was detected by direct immunofluorescence, and the interleukin (IL)-12 concentration in the culture supernatant of the three groups was measured by ELISA. The antitumor effect of DC-stimulated cytotoxic T lymphocytes (CTLs) on different PCa cell lines was analyzed using a Cell Counting Kit‑8 assay. The optimal MOI value was determined to be 200. The PSMA protein was expressed in DCs, and the recombinant adenovirus did not impact the maturation and morphological changes of the DCs. The expression levels of the co-stimulatory molecules, CD80, CD83, CD86 and HLA‑DR, were significantly higher in the Ad‑PSMA‑DC group than in the other two groups (P<0.05). The concentration of IL‑12 in the supernatant of the Ad‑PSMA‑DC group (79.51±1.60 pg) was comparable with that of the Ad‑GFP‑DC group (not significantly different), and the two were significantly higher than the non‑transfection group (P<0.05). The optimal effector to target (E:T) ratio was determined to be 40:1. However, at the same E:T ratio, the cytotoxic activity of PSMA‑DC‑T against the LNCap cells was markedly stronger than its activity against the other target cells (DU145 and 22RV; P<0.05); furthermore, the the cytotoxic activity of PSMA-DC-T against the LNCap cells was significantly higher than the anti‑LNCap effect of DC‑T cells in other groups (P<0.05). In vitro experiments indicated that mature DCs transfected with Ad‑PSMA secreted high concentrations of IL‑12 and elicited potent antitumor immune responses targeting PSMA‑expressing cells by stimulating the cytotoxic activity of CTLs.

PGC and PSMA in prostate cancer diagnosis: tissue analysis from biopsy samples

The discovery of new diagnostic tools for the diagnosis of prostate cancer (PCa) has become an important field of research. In this study, we analyzed the diagnostic value of the expression of the pepsinogen C (PGC) and prostate-specific membrane antigen (PSMA) genes in tissue samples obtained from prostate biopsies. This study was comprised of 51 consecutive patients who underwent transrectal ultrasound (TRUS)-guided prostate biopsies between January 2010 and March 2010. The biopsies were performed with 12 cores, and an additional core was randomly retrieved from the peripheral zone from each patient for study purposes. The expression of the PGC and PSMA genes was analyzed from the cDNA from the samples via the qRT-PCR technology. The expression patterns of patients with PCa were compared with those of patients without a PCa diagnosis. PSMA was overexpressed in only 43.4% of PCa cases, and PGC was overexpressed in 72.7% of cases. The median expression of PSMA was 1.5 times (0.1 to 43.9) and the median PGC expression was 8.7 times (0.1 to 50.0) the expression observed in prostatic tissue from TRUS-guided biopsies of normal patients. Analysis of patients with high-risk PCa indicated that PGC was overexpressed in 71.4% of cases (with a median expression of 10.6 times), and PSMA was overexpressed in only 35.7% of cases (with a median expression of 4.5 times). Among patients with low-risk PCa, PGC was also overexpressed in 71.4% of cases (with a median expression of 5.9 times), and PSMA was overexpressed in only 42.8% of cases (with a median expression of 2.5 times). PGC gene expression is significantly higher in prostatic tissue in men affected by PCa when compared to normal prostates. Further analyses are necessary to confirm our results.

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.

The Proinflammatory Cytokine, IL-6, and its Interference with bFGF Signaling and PSMA in Prostate Cancer Cells

The aim of the present work was to study the expression of the proinflammatory cytokine, interleukin-6 (IL-6), mediated by bFGF signaling and its possible crosstalk with prostate-specific membrane antigen (PSMA) in LNCaP and PC3-PSMA prostate cancer cell lines. PC3 cells stably transfected with PSMA gene were used for restoring PSMA expression. LNCaP and PC3-PSMA cells were exposed to 10ng/mL of basic fibroblast growth factor (bFGF). IL-6 production was measured by ELISA assay, and levels of PSMA expression were assessed by flow cytometry. AKT, ERK1/2, and p38 phosphorylation were detected by Western blot. bFGF enhances IL-6 production in LNCaP and PC3-PSMA prostate cancer cells. The effect of bFGF on stimulating IL-6 secretion was greater in LNCaP than in PC3-PSMA cells. In the presence of bFGF, PSMA expression was activated after 4days of treatment in LNCaP and PC3-PSMA cells. This activation was not maintained after long term of treatment in both metastatic cell lines. Solely MAPKs pathways (ERK1/2 and p38) were activated after bFGF stimulation in both metastatic cell lines, whereas AKT did not show any activation. The interference of the proinflammatory cytokine, IL-6, with bFGF signaling and PSMA, should be of high clinical relevance in the treatment of metastatic prostate cancer. In developing novel therapeutic modalities targeting IL-6, significant attention should be given to PSMA and its inactivation to fight against prostate cancer.

Immune response against prostate cancer cells induced by recombinant adenovirus encoding truncated prostate specific membrane antigen gene and 4-1BBL gene modified dendritic cells in vitro

Objective To study the cytotoxic T lymphocyte response induced by dendritic cells (DCs) transduced with recombinant adenovirus vector bearing truncated human prostate specific membrane antigen (tPSMA) gene and mouse 4-1BB ligand (m4-1BBL) gene against prostate cancer cells in vitro.Methods tPSMA cDNAs and m4-1BBL cDNAs were cloned into pDC316 vector,which was then transfected into HEK 293 cells with adenovirus backbone vector pBHGlox_E1,3Cre to obtain adenovirus expression vector Ad-tPSMA-IRES-m4-1BBL.tPSMA and m4-1BBL proteins were detected by using Western blotting.DCs generated from C57BL/6 mice were transduced with Ad-tPSMA-IRES-m4-1BBL,and the phenotype of DCs was analysed by flow cytometry; Proliferation of T cells stimulated by Ad-tPSMA-IRES-m4-1BBL-transduced DCs in allogeneic mixed lymphocyte reactions and cytotoxic cytotoxic T lymphocyte (CTL) activity induced by Ad-tPSMA-IRES-m4-1BBL-transduced DCs were detected by cell counting kit-8 (CCK-8) assay.Results Recombinant adenovirus Ad-tPSMA-IRES-m4-1BBL was constructed,and tPSMA gene and m4-1BBL gene were expressed correctly.pDC316-tPSMA-IRES-m4-1BBL-transduced DCs upregulated the expression of major histocompatibility complex (MHC) Ⅱ [(87.1 ± 4.7) ％],CD80 [(1.6 ± 5.4) ％] and CD86 [(80.1 ± 2.8) ％].Proliferation rate of T cells stimulated by pDC316-tPSMA-IRES-m4-1BBL-transduced DCs (A450 =0.61 ± 0.02) was significantly higher than that in DCs control group (0.38 ±0.13) and Ad-eGFP-transfected DCs group (0.42 ±0.18) (P ＜0.05).pDC316-tPSMA-IRES-m4-1BBL-transduced DCs induced obvious CTL specific cytotoxicity [(53.5 ± 0.1)％] against tPSMA-expressing prostate cancer cells in vitro.Conclusion DCs vaccines modified by recombinant adenovirus pDC316-tPSMA-IRES-m4-1BBL can induce effective CTL specific cytotoxicity against tPSMA-expressing prostate cancer cells. Key words: Prostate cancer; Dendritic cells; Recombinant adenovirus

Abstract 354: Monitor androgen blockade therapy with functional androgen receptor reporting system

Abstract Hormonal manipulation remains the first line treatment for advanced prostate cancer. It includes surgical and medical means of androgen deprivation and androgen receptor (AR) blockade therapies. However, the majority of patients will develop castration resistant prostate cancer, often with metastasis that accounts for the lethality of this disease. Therefore, a reliable imaging modality capable of active surveillance of tumor recurrence and/or new incidence of metastasis would greatly benefit the timing and decision-making in adopting second line therapies. To this end, we recently constructed a prostate specific yet androgen independent promoter system, namely PSES-TSTA (Prostate Specific Enhancer Sequence coupled with Two-Step Transcriptional Amplification). We used this system to drive PET and optical imaging reporters and successfully visualized subcutaneous tumor and intra-tibial metastasis in castrated mice. The androgen independency of PSES-TSTA, which secured viability of this imaging approach when only castrated level of androgen is available, is attributed to the composition of the chimeric PSES sequence - an androgen inducible element derived from the prostate specific antigen (PSA) gene promoter and an androgen suppressible element from the prostate specific membrane antigen gene enhancer (PSME). Another challenge faced by patients after hormonal ablation therapy is the need for effective second-line therapies. Interestingly, a growing body of evidence points out that even at castration resistant stage, AR remains active and most prostate cancer still rely on AR signaling axis for proliferation and survival. Accordingly, much effort has been devoted to the development of second generation anti-androgen agents. Therefore, it would be greatly beneficial to establish a practical and reliable reporting system that reflects the functional status of the androgen-AR axis so that the efficacy of such new drugs can be monitored at the molecular level. Inspired by previous results, we separated the two components of PSES and generated two imaging reporters: PSA-driven Firefly Luciferase (PSA-FL) and PSME-driven Renilla Luciferase (PSME-RL). Based on their respective response to androgen signaling, we anticipated that the PSA-FL signal will decrease while the PSME-RL signal will increase in the face of an effective anti-androgen therapy. Indeed, we observed such pattern of reporter gene expression in multiple prostate cancer cell lines. Importantly, this system is able to differentiate the potency and efficacy between the first generation and the more potent second generation AR antagonists. Moreover, the simultaneous application of the PSME- and PSA-driven reporter system is advantageous over the single PSA promoter-based reporter in that it rules out the effect of tumor mass reduction on the readout because both up- and down-regulation of gene expression are being assessed and imaged. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 354. doi:1538-7445.AM2012-354

A phase I dose escalation trial of vaccine replicon particles (VRP) expressing prostate-specific membrane antigen (PSMA) in patients (pts) with castration-resistant prostate cancer (CRPC): Preliminary results.

140 Background: Prostate-specific membrane antigen (PSMA), a type II transmembrane protein overexpressed in prostate cells as they emerge into the androgen-independent state, remains a target to which vaccines with novel adjuvants or naked DNA has been directed. The PSMA-Viral Replicon Package (VRP) is a propagation-defective vector system from an attentuated strain of an alpha virus (VEE) encoding PSMA for use as a vaccine in prostate cancer. The PSMA gene is encoded by a subgenomic messenger RNA (mRNA), leading to high level expression of PSMA protein and presentation to the immune system.. We report preliminary results of a first in human trial using PSMA VRP to elicit immune responses targeting PSMA. Methods: Up to 18 pts with chemotherapy naïve CRPC metastatic to bone or soft tissue were permitted with 12 pts enrolled with ECOG status of 0 or 1. PSMA VRP was administered SC doses of either 0.9×107 IU or 0.36×108 IU for a total of 5 doses given on weeks 1, 4, 7, 10 and 18. Adverse events, PSA, circulating tumor cells, and clinical disease progression were assessed. Immune assessment was performed on patients' sera by ELISA and flow cytometry of serum binding to PSMA expressing tumor cells (3T3) and cellular (IFN-g EliSpot) components. Patients underwent imaging with bone and CAT scans every 3 months. Results: Three pts received 0.9×107 IU and 9 received 0.36×108 IU. Of these, 8 pts received all 5 vaccines. Four pts were taken off study for disease progression. One pt at the 0.36×108 continues on study and will be receiving his seventh vaccine. The demographic featuresof the 12 subjects enrolled were similar (mean age 69±11 yrs). No significant adverse events were noted; no aberrant laboratory abnormalities were associated with treatment. Weak humoral responses were observed in 1 patient in the 0.9×107 IU group and in 3 treated at 0.36×108. Conclusions: In this first-in-man evaluation, vaccination with PSMA VRP appeared to be well-tolerated but does not appear to show impact on disease progression. Further studies are ongoing to assess immune responses and identify potential pathways for enhancement of the PSMA VRP approach. [Table: see text]

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 and its truncated form PSM′

Prostate specific membrane antigen (PSMA) is a type II transmembrane protein overexpressed in prostate cancer as well as in the neovasculature of several non-prostatic solid tumors. In addition to full-length PSMA, several splice variants exist in prostatic tissue. Notably, the N-terminally truncated PSMA variant, termed PSM', is prevalent in healthy prostate, and the ratio of PSMA/PSM' mRNA has been shown to correlate with cancer progression. The widely accepted hypothesis is that the PSM' protein is a translation product arising from the alternatively spliced PSM' mRNA. Differential ultracentrifugation, cell surface biotinylation, Western blotting, and enzyme activity measurement were used to study the origin and localization of the PSMA/PSM' variants in prostatic (LNCaP; lymph-node carcinoma of the prostate) and non-prostatic (HEK293) cell lines. These experiments were further complemented by analysis of the N-glycosylation patterns of the PSMA/PSM' proteins and by site-directed mutagenesis. We identified PSM' protein expression in both the LNCaP cell line and a non-cancerous HEK293 human cell line transfected with a plasmid encoding full-length PSMA. Differential centrifugation revealed that PSM' is localized predominantly to the cytosol of both these cell lines and is proteolytically active. Furthermore, the PSM' protein is N-glycosylated by a mixture of high-mannose and complex type oligosaccharides and therefore trafficked beyond the cis-Golgi compartment. Our data suggest that the PSM' protein is likely not generated by alternative splicing of the PSMA gene but by different mechanism, probably via an endoproteolytic cleavage of the full-length PSMA.