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.

Genomic Correlates of Prostate-Specific Membrane Antigen Expression and Response to 177Lu-PSMA-617: A Retrospective Multicenter Cohort Study.

PURPOSE: While 177Lu-PSMA-617 (LuPSMA) is an effective therapy for many patients with metastatic castration-resistant prostate cancer (mCRPC), biomarkers associated with outcomes are not well defined. We hypothesized that prostate cancer mutational profile may associate with clinical activity of LuPSMA. We devised a study to evaluate associations between mCRPC mutational profile with LuPSMA clinical outcomes. METHODS: This was a multicenter retrospective analysis of patients with mCRPC with next-generation sequencing (NGS) who received LuPSMA. PSA50 response (ie, ≥50% decline in prostate-specific antigen [PSA]) rate, PSA progression free survival (PSA PFS), and overall survival (OS) were compared between genetically defined subgroups. RESULTS: One hundred twenty-six patients with NGS results who received at least one cycle of LuPSMA were identified. The median age was 73 (IQR, 68-78) years, 124 (98.4%) received ≥1 prior androgen receptor-signaling inhibitor, and 121 (96%) received ≥1 taxane-based chemotherapy regimen. Fifty-eight (46%) patients with a DNA damage repair gene mutation (DNA damage response group) and 59 (46.8%) with a mutation in TP53, RB1, or PTEN tumor suppressor genes (TSG group) were identified. After adjusting for relevant confounders, the presence of ≥1 TSG mutation was associated with shorter PSA PFS (hazard ratio [HR], 1.93 [95% CI, 1.05 to 3.54]; P = .034) and OS (HR, 2.65 [95% CI, 1.15 to 6.11]; P = .023). There was improved OS favoring the DNA damage response group (HR, 0.37 [95% CI, 0.14 to 0.97]; P = .044) on multivariable analysis. Univariate analysis of patients with ATM mutations had significantly higher rates of PSA50 response, PSA PFS, and OS. CONCLUSION: Outcomes on LuPSMA varied on the basis of mutational profile. Prospective studies to define the clinical activity of LuPSMA in predefined genomic subgroups are justified.

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

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

Parallel Metabolomics and Lipidomics of a PSMA/GCPII Deficient Mouse Model Reveal Alteration of NAAG Levels and Brain Lipid Composition.

Glutamate carboxypeptidase II (GCPII, also known as PSMA or FOLH1) is responsible for the cleavage of N-acetyl-aspartyl-glutamate (NAAG) to N-acetyl-aspartate and glutamate in the central nervous system and facilitates the intestinal absorption of folate by processing dietary folyl-poly-γ-glutamate in the small intestine. The physiological function of GCPII in other organs like kidneys is still not known. GCPII inhibitors are neuroprotective in various conditions (e.g., ischemic brain injury) in vivo; however, their utilization as potential drug candidates has not been investigated in regard to not yet known GCPII activities. To explore the GCPII role and possible side effects of GCPII inhibitors, we performed parallel metabolomic and lipidomic analysis of the cerebrospinal fluid (CSF), urine, plasma, and brain tissue of mice with varying degrees of GCPII deficiency (fully deficient in Folh1, -/-; one allele deficient in Folh1, +/-; and wild type, +/+). Multivariate analysis of metabolites showed no significant differences between wild-type and GCPII-deficient mice (except for NAAG), although changes were observed between the sex and age. NAAG levels were statistically significantly increased in the CSF, urine, and plasma of GCPII-deficient mice. However, no difference in NAAG concentrations was found in the whole brain lysate likely because GCPII, as an extracellular enzyme, can affect only extracellular and not intracellular NAAG concentrations. Regarding the lipidome, the most pronounced genotype-linked changes were found in the brain tissue. In brains of GCPII-deficient mice, we observed statistically significant enrichment in phosphatidylcholine-based lipids and reduction of sphingolipids and phosphatidylethanolamine plasmalogens. We hypothesize that the alteration of the NAA-NAAG axis by absent GCPII activity affected myelin composition. In summary, the absence of GCPII and thus similarly its inhibition do not have detrimental effects on metabolism, with just minor changes in the brain lipidome.

A Novel Role of Prostate-Specific Membrane Antigen in Telomere Stability in Prostate Cancer Cells.

Prostate-specific membrane antigen (PSMA) expression increases with prostate cancer grade and progression; however, the role of PSMA in prostate cancer progression remains poorly understood. Telomere stability is essential for the survival and genome stability of cancer cells. We found massive telomere DNA damage in PSMA-negative prostate cancer cells (PC-3 and DU145) compared with PSMA-positive prostate cancer (LNCaP) cells. The ectopic expression of PSMA suppressed telomere DNA damage in PC3 cells. PSMA inhibitor, 2-PMPA, and PSMA knockdown induced telomere DNA damage in PSMA-positive LNCaP cells but not in PSMA-negative PC-3 cells, suggesting that PSMA plays a critical role in telomere stability in prostate cancer cells. In addition, we observed that inhibition of PSMA or inhibition of glutamate receptor, which mediates PSMA-dependent activation of AKT, suppressed AKT phosphorylation, and caused telomere DNA damage. Furthermore, 2-PMPA-induced telomere DNA damage in LNCaP cells was associated with telomere aberrations, such as telomere-telomere fusions, sister-chromatid telomere fusions, and telomere breakages. AKT is reported to promote cell growth by stabilizing telomere association with telomere-binding proteins TRF1 and TPP1. We observed that TRF1 and TPP1 transfection of LNCaP cells attenuated the inhibitory effect of 2-PMPA on cell growth and telomere DNA damage. Together, these observations indicate that PSMA role in maintaining telomere stability in prostate cancer cells is mediated by AKT. Thus, these studies reveal an important role of PSMA in maintaining telomere stability that can promote cell survival and, thereby, prostate cancer progression. IMPLICATIONS: Role of PSMA in telomere stability suggests a strong correlation between PSMA expression and prostate cancer progression.

Deletion of glutamate carboxypeptidase II (GCPII), but not GCPIII, provided long-term benefits in mice with traumatic brain injury.

MAIN PROBLEM: N-acetylaspartylglutamate (NAAG) has neuroprotective effects in traumatic brain injury (TBI) by activating metabotropic glutamate receptor 3 (mGluR3) and reducing glutamate release. Glutamate carboxypeptidase II (GCPII) is the primary enzyme responsible for the hydrolysis of NAAG. It remains unclear whether glutamate carboxypeptidase III (GCPIII), a homolog of GCPII, can partially compensate for GCPII's function. METHODS: GCPII-/- , GCPIII-/- , and GCPII/III-/- mice were generated using CRISPR/Cas9 technology. Mice brain injury model was established through moderate controlled cortical impact (CCI). The relationship between GCPII and GCPIII was explored by analyzing injury response signals in the hippocampus and cortex of mice with different genotypes at the acute (1 day) and subacute (7 day) phase after TBI. RESULTS: In this study, we found that deletion of GCPII reduced glutamate production, excitotoxicity, and neuronal damage and improved cognitive function, but GCPIII deletion had no significant neuroprotective effect. Additionally, there was no significant difference in the neuroprotective effect between the combination of GCPII and GCPIII deletion and GCPII deletion alone. CONCLUSION: These results suggest that GCPII inhibition may be a therapeutic option for TBI, and that GCPIII may not act as a complementary enzyme to GCPII in this context.

[Prokaryotic expression of prostate-specific membrane antigen (PSMA) nanoantibody gene and screening of natural phage nanoantibody library].

Objective To screen nanobodies against prostate specific membrane antigen (PSMA). Methods Based on the naive phage display library, three rounds of screening were performed targeting the PSMA antigen, and positive clones were identified by ELISA and sequencing was performed. The positive cloned gene sequence was inserted into the pET28a prokaryotic expression vector and transformed into E.coli BL21. The expression of the recombinant protein was induced by IPTG and purified using Ni column, with the purified product verified by SDS-PAGE. Results Four PSMA nanobodies VHH1, VHH2, VHH3 and VHH4 were obtained by screening. The VHH1 failed to obtain protein expression, while the VHH2, VHH3 and VHH4 proteins were expressed. The purity of anti-PSMA nanobodies showed high and relative molecular mass (Mr) of about 17 000. Conclusion The sequence of anti-PSMA nanobody was successfully obtained by screening the naive phage nanobody library and were subjected to prokaryotic expression and purified.

Prostate-Specific Membrane Antigen Expression and Response to DNA Damaging Agents in Prostate Cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA) targeting therapies such as Lutetium-177 (177Lu)-PSMA-617 are affecting outcomes from metastatic castration-resistant prostate cancer (mCRPC). However, a significant subset of patients have prostate cancer cells lacking PSMA expression, raising concerns about treatment resistance attributable at least in part to heterogeneous PSMA expression. We have previously demonstrated an association between high PSMA expression and DNA damage repair defects in mCRPC biopsies and therefore hypothesized that DNA damage upregulates PSMA expression. EXPERIMENTAL DESIGN: To test this relationship between PSMA and DNA damage we conducted a screen of 147 anticancer agents (NCI/NIH FDA-approved anticancer "Oncology Set") and treated tumor cells with repeated ionizing irradiation. RESULTS: The topoisomerase-2 inhibitors, daunorubicin and mitoxantrone, were identified from the screen to upregulate PSMA protein expression in castration-resistant LNCaP95 cells; this result was validated in vitro in LNCaP, LNCaP95, and 22Rv1 cell lines and in vivo using an mCRPC patient-derived xenograft model CP286 identified to have heterogeneous PSMA expression. As double-strand DNA break induction by topoisomerase-2 inhibitors upregulated PSMA, we next studied the impact of ionizing radiation on PSMA expression; this also upregulated PSMA protein expression in a dose-dependent fashion. CONCLUSIONS: The results presented herein are the first, to our knowledge, to demonstrate that PSMA is upregulated in response to double-strand DNA damage by anticancer treatment. These data support the study of rational combinations that maximize the antitumor activity of PSMA-targeted therapeutic strategies by upregulating PSMA.

Impact of Androgen Receptor Activity on Prostate-Specific Membrane Antigen Expression in Prostate Cancer Cells.

Prostate-specific membrane antigen (PSMA) is an essential molecular regulator of prostate cancer (PCa) progression coded by the FOLH1 gene. The PSMA protein has become an important factor in metastatic PCa diagnosis and radioligand therapy. However, low PSMA expression is suggested to be a resistance mechanism to PSMA-based imaging and therapy. Clinical studies revealed that androgen receptor (AR) inhibition increases PSMA expression. The mechanism has not yet been elucidated. Therefore, this study investigated the effect of activation and inhibition of androgen signaling on PSMA expression levels in vitro and compared these findings with PSMA levels in PCa patients receiving systemic therapy. To this end, LAPC4, LNCaP, and C4-2 PCa cells were treated with various concentrations of the synthetic androgen R1881 and antiandrogens. Changes in FOLH1 mRNA were determined using qPCR. Open access databases were used for ChIP-Seq and tissue expression analysis. Changes in PSMA protein were determined using western blot. For PSMA staining in patients' specimens, immunohistochemistry (IHC) was performed. Results revealed that treatment with the synthetic androgen R1881 led to decreased FOLH1 mRNA and PSMA protein. This effect was partially reversed by antiandrogen treatment. However, AR ChIP-Seq analysis revealed no canonical AR binding sites in the regulatory elements of the FOLH1 gene. IHC analysis indicated that androgen deprivation only resulted in increased PSMA expression in patients with low PSMA levels. The data demonstrate that AR activation and inhibition affects PSMA protein levels via a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression rates may be mandatory to increase PSMA by androgen deprivation.

Expression of glutamate carboxypeptidase II in the glial tumor recurrence evaluated in vivo using radionuclide imaging.

Glutamate carboxypeptidase II (GCP), also known as prostate specific membrane antigen (PSMA) has been found to be expressed in glioma vasculature in in-vitro studies. GCP expression can be traced with the use of [68Ga]Ga-PSMA-11 PET/CT used routinely for prostate cancer imaging. The aim of this paper was to analyze GCP expression in the recurrent glial tumors in vivo. 34 patients (pts.) aged 44.5 ± 10.3 years with suspicion of recurrence of histologically confirmed glioma grade III (6 pts.) and grade IV (28 pts.) were included in the study. All patients underwent contrast-enhanced MR and [68Ga]Ga-PSMA-11 PET/CT. No radiopharmaceutical-related adverse events were noted. PET/CT was positive in all the areas suspected for recurrence at MR in all the patients. The recurrence was confirmed by histopathological examinations or follow-up imaging in all cases. The images showed a very low background activity of the normal brain. Median maximal standard uptake value (SUVmax) of the tumors was 6.5 (range 0.9-15.6) and mean standard uptake value (SUVmean) was 3.5 (range 0.9-7.5). Target-to-background (TBR) ratios varied between 15 and 1400 with a median of 152. Target-to-liver background ratios (TLR) ranged from 0.2 to 2.6, the median TLR was 1.3. No significant difference of the measured parameters was found between the subgroups according to the glioma grade. High GCP expression in the recurrent glioma was demonstrated in-vivo with the use of [68Ga]Ga-PSMA-11 PET/CT. As the treatment options in recurrent glioma are limited, this observation may open new therapeutic perspectives with the use of radiolabeled agents targeting the GCP.

Predicting Effects of Site-Directed Mutagenesis on Enzyme Kinetics by QM/MM and QM Calculations: A Case of Glutamate Carboxypeptidase II.

Quantum and molecular mechanics (QM/MM) and QM-only (cluster model) modeling techniques represent the two workhorses in mechanistic understanding of enzyme catalysis. One of the stringent tests for QM/MM and/or QM approaches is to provide quantitative answers to real-world biochemical questions, such as the effect of single-point mutations on enzyme kinetics. This translates into predicting the relative activation energies to 1-2 kcal·mol-1 accuracy; such predictions can be used for the rational design of novel enzyme variants with desired/improved characteristics. Herein, we employ glutamate carboxypeptidase II (GCPII), a dizinc metallopeptidase, also known as the prostate specific membrane antigen, as a model system. The structure and activity of this major cancer antigen have been thoroughly studied, both experimentally and computationally, which makes it an ideal model system for method development. Its reaction mechanism is quite well understood: the reaction coordinate comprises a "tetrahedral intermediate" and two transition states and experimental activation Gibbs free energy of ∼17.5 kcal·mol-1 can be inferred for the known kcat ≈ 1 s-1. We correlate experimental kinetic data (including the E424H variant, newly characterized in this work) for various GCPII mutants (kcat = 8.6 × 10-5 s-1 to 2.7 s-1) with the energy profiles calculated by QM/MM and QM-only (cluster model) approaches. We show that the near-quantitative agreement between the experimental values and the calculated activation energies (ΔH⧧) can be obtained and recommend the combination of the two protocols: QM/MM optimized structures and cluster model (QM) energetics. The trend in relative activation energies is mostly independent of the QM method (DFT functional) used. Last but not least, a satisfactory correlation between experimental and theoretical data allows us to provide qualitative and fairly simple explanations of the observed kinetic effects which are thus based on a rigorous footing.

Prostate-specific membrane antigen modulates the progression of prostate cancer by regulating the synthesis of arginine and proline and the expression of androgen receptors and Fos proto-oncogenes.

The expression of prostate-specific membrane antigen (PSMA) is strikingly upregulated during oncogenesis and prostate cancer (PCa) progression, but the functions of this antigen in PCa remain unclear. Here, we constructed PSMA-knockdown LNCaP and 22rv1 cell lines and performed metabonomic and transcriptomic analyses to determine the effects of PSMA on PCa metabolism and transcription. The metabolism of arginine and proline was detected using specific kits. The mRNA and protein expression levels of the identified differentially expressed genes were quantified by RT-qPCR and Western blotting. The proliferation of each cell line was evaluated through CCK-8, EdU and colony formation assays. The migration and invasion abilities of each cell line were detected using wound healing and transwell assays, respectively. PSMA knockdown led to metabolic disorder and abnormal transcription in PCa and resulted in inhibition of the proliferation and metastasis of PCa cells in vitro and in vivo. The depletion of PSMA also promoted the biosynthesis of arginine and proline, inhibited the expression of AR and PSA, and induced the expression of c-Fos and FosB. PSMA plays an important role in the metabolism, proliferation and metastasis of human PCa and may be a promising therapeutic target.

Prostate-Specific Membrane Antigen (PSMA) Theranostics for Treatment of Oligometastatic Prostate Cancer.

Theranostics, a combination of therapy and diagnostics, is a field of personalized medicine involving the use of the same or similar radiopharmaceutical agents for the diagnosis and treatment of patients. Prostate-specific membrane antigen (PSMA) is a promising theranostic target for the treatment of prostate cancers. Diagnostic PSMA radiopharmaceuticals are currently used for staging and diagnosis of prostate cancers, and imaging can predict response to therapeutic PSMA radiopharmaceuticals. While mainly used in the setting of metastatic, castrate-resistant disease, clinical trials are investigating the use of PSMA-based therapy at earlier stages, including in hormone-sensitive or hormone-naïve prostate cancers, and in oligometastatic prostate cancers. This review explores the use of PSMA as a theranostic target and investigates the potential use of PSMA in earlier stage disease, including hormone-sensitive metastatic prostate cancer, and oligometastatic prostate cancer.

Deep Membrane Proteome Profiling Reveals Overexpression of Prostate-Specific Membrane Antigen (PSMA) in High-Risk Human Paraganglioma and Pheochromocytoma, Suggesting New Theranostic Opportunity.

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors arising from chromaffin cells of adrenal medulla or sympathetic or parasympathetic paraganglia, respectively. To identify new therapeutic targets, we performed a detailed membrane-focused proteomic analysis of five human paraganglioma (PGL) samples. Using the Pitchfork strategy, which combines specific enrichments of glycopeptides, hydrophobic transmembrane segments, and non-glycosylated extra-membrane peptides, we identified over 1800 integral membrane proteins (IMPs). We found 45 "tumor enriched" proteins, i.e., proteins identified in all five PGLs but not found in control chromaffin tissue. Among them, 18 IMPs were predicted to be localized on the cell surface, a preferred drug targeting site, including prostate-specific membrane antigen (PSMA), a well-established target for nuclear imaging and therapy of advanced prostate cancer. Using specific antibodies, we verified PSMA expression in 22 well-characterized human PPGL samples. Compared to control chromaffin tissue, PSMA was markedly overexpressed in high-risk PPGLs belonging to the established Cluster 1, which is characterized by worse clinical outcomes, pseudohypoxia, multiplicity, recurrence, and metastasis, specifically including SDHB, VHL, and EPAS1 mutations. Using immunohistochemistry, we localized PSMA expression to tumor vasculature. Our study provides the first direct evidence of PSMA overexpression in PPGLs which could translate to therapeutic and diagnostic applications of anti-PSMA radio-conjugates in high-risk PPGLs.

Two Sides of Quantum-Based Modeling of Enzyme-Catalyzed Reactions: Mechanistic and Electronic Structure Aspects of the Hydrolysis by Glutamate Carboxypeptidase.

We report the results of a computational study of the hydrolysis reaction mechanism of N-acetyl-l-aspartyl-l-glutamate (NAAG) catalyzed by glutamate carboxypeptidase II. Analysis of both mechanistic and electronic structure aspects of this multistep reaction is in the focus of this work. In these simulations, model systems are constructed using the relevant crystal structure of the mutated inactive enzyme. After selection of reaction coordinates, the Gibbs energy profiles of elementary steps of the reaction are computed using molecular dynamics simulations with ab initio type QM/MM potentials (QM/MM MD). Energies and forces in the large QM subsystem are estimated in the DFT(PBE0-D3/6-31G**) approximation. The established mechanism includes four elementary steps with the activation energy barriers not exceeding 7 kcal/mol. The models explain the role of point mutations in the enzyme observed in the experimental kinetic studies; namely, the Tyr552Ile substitution disturbs the "oxyanion hole", and the Glu424Gln replacement increases the distance of the nucleophilic attack. Both issues diminish the substrate activation in the enzyme active site. To quantify the substrate activation, we apply the QTAIM-based approaches and the NBO analysis of dynamic features of the corresponding enzyme-substrate complexes. Analysis of the 2D Laplacian of electron density maps allows one to define structures with the electron density deconcentration on the substrate carbon atom, i.e., at the electrophilic site of reactants. The similar electronic structure element in the NBO approach is a lone vacancy on the carbonyl carbon atom in the reactive species. The electronic structure patterns revealed in the NBO and QTAIM-based analyses consistently clarify the reactivity issues in this system.

Promoter Methylation of PRKCB, ADAMTS12, and NAALAD2 Is Specific to Prostate Cancer and Predicts Biochemical Disease Recurrence.

The molecular diversity of prostate cancer (PCa) has been demonstrated by recent genome-wide studies, proposing a significant number of different molecular markers. However, only a few of them have been transferred into clinical practice so far. The present study aimed to identify and validate novel DNA methylation biomarkers for PCa diagnosis and prognosis. Microarray-based methylome data of well-characterized cancerous and noncancerous prostate tissue (NPT) pairs was used for the initial screening. Ten protein-coding genes were selected for validation in a set of 151 PCa, 51 NPT, as well as 17 benign prostatic hyperplasia samples. The Prostate Cancer Dataset (PRAD) of The Cancer Genome Atlas (TCGA) was utilized for independent validation of our findings. Methylation frequencies of ADAMTS12, CCDC181, FILIP1L, NAALAD2, PRKCB, and ZMIZ1 were up to 91% in our study. PCa specific methylation of ADAMTS12, CCDC181, NAALAD2, and PRKCB was demonstrated by qualitative and quantitative means (all p < 0.05). In agreement with PRAD, promoter methylation of these four genes was associated with the transcript down-regulation in the Lithuanian cohort (all p < 0.05). Methylation of ADAMTS12, NAALAD2, and PRKCB was independently predictive for biochemical disease recurrence, while NAALAD2 and PRKCB increased the prognostic power of multivariate models (all p < 0.01). The present study identified methylation of ADAMTS12, NAALAD2, and PRKCB as novel diagnostic and prognostic PCa biomarkers that might guide treatment decisions in clinical practice.

Synthesis of KUE-siRNA Conjugates for Prostate Cancer Cell-Targeted Gene Silencing.

The delivery of siRNAs to selectively target cells poses a great challenge in RNAi-based cancer therapy. The lack of suitable cell-targeting methods seriously restricts the advance in delivering siRNAs to extrahepatic tissues. Based on prostate-specific membrane antigen (PSMA)-targeting ligands, we have synthesized a series of lysine-urea-glutamate (KUE)-siRNA conjugates and verified their effective cell uptake and gene silencing properties in prostate cancers. The results indicated that the KUE-siRNA conjugates could selectively enter PSMA+ LNCaP cells, eventually down-regulating STAT3 expression. Based on post-synthesis modification and receptor-mediated endocytosis, this strategy of constructing ligand-siRNA conjugates might provide a general method of siRNA delivery for cell-targeted gene silencing.

Impact of short-term Dutasteride treatment on prostate-specific membrane antigen expression in a mouse xenograft model.

BACKGROUND: Dutasteride has been shown to increase expression of the prostate-specific membrane antigen (PSMA) in prostate cancer cells in previous in vitro studies. This 5-alpha-reductase inhibitor is commonly used for the treatment of symptomatic benign prostatic enlargement. The modulation of PSMA expression might affect PSMA-based prostate cancer imaging and therapy. AIM: The purpose of this work was to further analyze concentration-dependent effects of Dutasteride on PSMA expression in a mouse xenograft model. METHODS AND RESULTS: Four groups of mice bearing LNCaP xenografts were treated for 14 days with daily intraperitoneal injections of either vehicle control or different concentrations of Dutasteride (0.1, 1, 10 mg/kg). Total expression of PSMA, androgen receptor (AR), and caspase-3 protein was analyzed using immunoblotting (WES). In addition, PSMA, cleaved caspase-3 and Ki-67 expression was assessed and quantified by immunohistochemistry. Tumor size was measured by caliper on day 7 and 14, tumor weight was assessed following tissue harvesting. The mean PSMA protein expression in mice increased significantly after treatment with 1 mg/kg (10-fold) or 10 mg/kg (sixfold) of Dutasteride compared to vehicle control. The mean fluorescence intensity significantly increased by daily injections of 0.1 mg/kg Dutasteride (1.6-fold) as well as 1 and 10 mg/kg Dutasteride (twofold). While the reduction in tumor volume following treatment with high concentrations of 10 mg/kg Dutasteride was nonsignificant, no changes in AR, caspase-3, cleaved caspase-3, and Ki-67 expression were observed. CONCLUSION: Short-term Dutasteride treatments with concentrations of 1 and 10 mg/kg significantly increase the total PSMA protein expression in a mouse LNCaP xenograft model. PSMA fluorescence intensity increases significantly even using lower daily concentrations of 0.1 mg/kg Dutasteride. Further investigations are needed to elucidate the impact of Dutasteride treatment on PSMA expression in patients.

Synthesis and evaluation of tumor-homing peptides for targeting prostate cancer.

High toxicity caused by chemotherapeutic drugs and the acquisition of drug resistance by cancer cells are the major drawbacks in cancer therapy. A promising approach to overcome the posed barriers is conjugating tumor-homing peptides to drugs or nanocarriers. Such high-affinity peptides can specifically target surface markers overexpressed by cancer cells, ensuring a rapid and cancer-specific uptake of the drugs. Since prostate-specific membrane antigen (PSMA) is overexpressed by aggressive prostate cancer cells, targeting this surface protein with peptide conjugates can lead to the development of effective strategies against prostate cancer. In this study, we aimed to determine which PSMA-binding peptide among peptides 563, 562 and 9-mer, show the highest selectivity towards PSMA using 22Rv1 prostate cancer cells, a cell line with moderate PSMA levels. Tumor-homing peptides were synthesized by fluorenylmethoxycarbonyl-based solid-phase peptide synthesis (Fmoc-SPPS) strategy, and evaluated for their prostate cancer cell-specific targeting efficiencies by flow cytometry. Our results showed that the PSMA-binding capacity of peptide 563 was superior to those of 562, 9-mer, and 5-mer; therefore, can be utilized as a potent-targeting agent not only in the treatment of high PSMA positive but also moderate PSMA positive prostate cancer tumors.

Antihormone treatment differentially regulates PSA secretion, PSMA expression and 68Ga-PSMA uptake in LNCaP cells.

BACKGROUND: In recent years, a variety of innovative therapeutics for castration-resistant prostate cancer have been developed, including novel anti-androgenic drugs, such as abiraterone or VPC-13566. Therapeutic monitoring of these pharmaceuticals is performed either by measuring PSA levels in serum or by imaging. PET using PSMA ligands labeled with Fluor-18 or Gallium-68 is the most sensitive and specific imaging modality for detection of metastases in advanced prostate cancer. To date, it remains unclear how PSMA expression is modulated by anti-hormonal treatment and how it correlates with PSA secretion. METHODS: We analyzed modulation of PSMA-mRNA and protein expression, 68Ga-PSMA uptake and regulation of PSA secretion by abiraterone or VPC-13566 in LNCaP cells in vitro. RESULTS: We found that abiraterone and VPC-13566 upregulate PSMA protein and mRNA expression but block PSA secretion in LNCaP cells. Both anti-androgens also enhanced 68Ga-PSMA uptake normalized by the number of cells, whereas abiraterone and VPC-13566 reduced 68Ga-PSMA uptake in total LNCaP monolayers treated due to cell death. CONCLUSION: Our data indicate that PSA secretion and PSMA expression are differentially regulated upon anti-androgen treatment. This finding might be important for the interpretation of 68Ga-PSMA PET images in monitoring therapies with abiraterone and VPC-13566 in prostate cancer patients, but needs to be validated in vivo.