Recombinant adenovirus mediated prostate-specific enzyme pro-drug gene therapy regulated by prostate-specific membrane antigen (PSMA) enhancer/promoter

Gene directed enzyme pro-drug therapy (GDEPT) is one of the adjuvant therapeutic regimens for advanced prostate adenocarcinoma, and this research intended to explore how to apply targeting therapy of prostate adenocarcinoma under the mediation of a promoter/enhancer of prostate-specific membrane antigen (PSMA(EP)) as a specific regulatory element. Recombinant adenoviruses (Ad-PSMA(E-P)-enhanced green fluorescent protein [EGFP], Ad-CMV-EGFP, Ad-PSMA(E-P)-CD, and Ad-CMV-CD) were constructed and could express cytosine deaminase (CD) or the EGFP reporter gene driven by a PSMA(EP) or cytomegalovirus (CMV) promoter. LNCaP, CL-1, MCF-7, and A549 were infected with CD-produced recombinant adenoviruses and treated with pro-drug 5-fluorocytosine (5-FC) in vivo and vitro; then, the growth inhibition of the cells and the cell cycle variation were assessed by an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay and flow cytometry. Growth suppression of the xenograft tumor was also adopted to evaluate the efficiency of the suicide system. Morphologic changes after treatment in vivo were assessed with hematoxylin and eosin staining. In the 4 examined cancer cell lines, PSMA-positive prostate cancer cells LNCap and CL-1 were exclusively sensitive to the Ad-PSMA(E-P)-CD/5-FC system. The S phase of cell cycle arrest was thought to be involved in the cytotoxicity of 5-fluorouracil (5-FU) converted from 5-FC by CD. CL-1 implanted Athymic BALB/c mice showed growth inhibition of tumors when they were treated with the Ad-PSMA(E-P)-CD/5-FC system without systemic conversion toxicity. The PSMA-based, CD-produced adenovirus, deserving further investigation in the future, might be a good candidate for targeting gene therapy of prostate adenocarcinoma.     

前列腺特异性膜抗原启动子增强子调控重组质粒的构建及鉴定

目的探讨前列腺特异性膜抗原(PSMA)启动子和增强子调控目的基因表达的前列腺细胞特异性，了解增强子增强转录效率的能力。方法采用PCR方法从前列腺癌细胞DNA中分别扩增PSMA启动子和增强子序列，先后克隆到含有报告基因绿色荧光蛋白(GFP)的质粒载体，脂质体介导基因转染不同癌细胞并观察GFP在不同细胞的表达情况。结果成功构建质粒pEGFP—PS—MAPro和pEGFP-PSMAEP，转染结果显示PSMA表达阳性的LNCaP细胞中存在GFP的有效表达，且pEGFP—PSMAEP的调控转录能力较pEGFP—PSMAPro强20倍。结论PSMA启动子增强子调控GFP表达的重组质粒的构建证明该调控序列具有前列腺细胞特异性，增强子能够明显增强启动子的转录效率，增加了目的基因的表达水平。PSMA启动子和增强子的共调控可以保证基因表达的强度和细胞特异性，为前列腺癌基因靶向性治疗研究提供实验依据。     

Imaging with prostate-specific membrane antigen (PSMA) in prostate cancer

Radioimmunoscintigraphy using a radio-labelled antibody to prostate-specific membrane antigen (PSMA) has growing applications as a means of tissue-specific imaging based on functional characteristics and complements traditional staging investigations. Clinical applications in men with carcinoma of the prostate are being refined, and this study reports outcomes with this technique in our practice. Prostatic immunoscintigraphy scans were performed with In-111 CYT 356 in 49 men with carcinoma of the prostate, obtaining sequential images in two and three dimensions at 10 min, 24 and 48 h. Of the 49 men, 36 had clinically localized cancer, 10 had recurrent disease after radical radiotherapy or radical prostatectomy and three had rising PSA after primary endocrine treatment. Scan findings are discussed in the context of clinical management. Of the 36 men with clinically localized cancer, seven had increased uptake in regional and distant lymph nodes. Of these seven, three were treated with hormone manipulation, two by radical prostatectomy and two by radical radiotherapy. Among 10 patients who had recurrence after radical treatment of the primary tumour, scans showed local recurrence alone in four, and six had regional or distant metastases. Three patients treated with primary hormone manipulation had scans for rising PSA, and of these one had a positive regional node and two had distant soft tissue and bone metastases. In conclusion, prostatic radio-immunoscintigraphy scans highlight tissues involved by prostate cancer, including the prostate, lymph nodes, soft tissues and bone metastases as well as pelvic recurrence. Results may contribute to the clinical management of individual patients, although histological confirmation may be appropriate when considering alternative treatment. Prostate Cancer and Prostatic Diseases (2000) 3, 47-52     

The clinical role of prostate-specific membrane antigen (PSMA)

Prostate cancer remains the most common cancer type in men in the United States. Efforts are increasing to evaluate and to discover diagnostic and therapeutic markers for prostate cancer patients. One of these, prostate-specific membrane antigen (PSMA), is a transmembrane protein highly expressed in all types of prostatic tissue, especially cancer. The radio-immunoconjugate form of the anti-PSMA monoclonal antibody (mAb) 7E11, known as the ProstaScint scan, is currently being used to diagnose prostate cancer metastasis and recurrence. Early promising results from various Phase I and II trials have utilized PSMA as a therapeutic target. Recently, PSMA expression in endothelial cells of tumor-associated neovasculature has been described. PSMA's possible role in malignant angiogenesis newly expands the realm of its possible beneficial uses, especially as new anti-PSMA mAbs continue to be developed and refined.     

Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer

BACKGROUND: Prostate-specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases. In addition, unlike prostate-specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer. Recently, both the promoter and the enhancer driving prostate-specific expression of the PSMA gene were cloned. We describe here our analysis of the PSMA enhancer for the most active region(s) and present a way of using the enhancer in combination with the E. coli cytosine deaminase gene for suicide-driven gene therapy that converts the nontoxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic drug 5-fluorouracil (5-FU) in prostate cancer cells. METHODS: Deletion constructs of the full-length PSMA enhancer were subcloned into a luciferase reporter vector containing either the PSMA or SV-40 promoter. The most active portion of the enhancer was then determined via luciferase activity in the C4-2 cell line. We then replaced the luciferase gene with the E. coli cytosine deaminase gene in the subclone that showed the most luciferase activity. The specificity of this technique was examined in vitro, using the prostate cancer cell line LNCaP, its androgen-independent derivative C4-2, and a number of nonprostatic cell lines. The toxicity of 5-FC and 5-FU on transiently transfected cell lines was then compared. RESULTS: The enhancer region originally isolated from the PSMA gene was approximately 2 kb. Deletion constructs revealed that at least two distinct regions seem to contribute to expression of the gene in prostate cancer cells, and therefore the best construct for prostate-specific expression was determined to be 1, 648 bp long. The IC(50) of 5-FC was similar in all cell lines tested (>10 mM). However, transfection with the 1648 nt PSMA enhancer and the PSMA promoter to drive the cytosine deaminase gene enhanced toxicity in a dose-dependent manner more than 50-fold, while cells that did not express the PSMA gene were not significantly sensitized by transfection. CONCLUSIONS: Suicide gene therapy using the PSMA enhancer may be of benefit to patients who have undergone androgen ablation therapy and are suffering a relapse of disease.     

Salvage lymph node dissection for prostate-specific membrane antigen (PSMA) positron emission tomography (PET)-identified oligometastatic disease

IntroductionThe availability of prostate-specific membrane antigen (PSMA) positron emission tomography (PET)/computed tomography (CT) imaging, particularly in the setting of rising prostate-specific antigen (PSA) after definitive treatment, has led to oligometastatic prostate cancer being increasingly identified. Despite the enthusiasm surrounding treating oligometastatic disease, it has been relatively understudied. We sought to review our salvage lymphadenectomy experience in the PSMA PET/CT era.MethodsWe retrospectively reviewed patients undergoing lymphadenectomy following curative-intent primary therapy with rising PSA who had undergone a PSMA PET/CT identifying oligometastatic disease (defined as ≤5 PSMA-avid lesions) between January 2016 and April 2020. The primary endpoint was complete response, defined as achieving a PSA <0.2 ng/ml without concomitant androgen deprivation therapy (ADT).ResultsTwenty-two patients were included. Primary curative therapy included radical prostatectomy (86.4%) and brachytherapy (13.6%). Median PSA at salvage surgery was 1.72 ng/ml. Pelvic lymph node dissection was the most performed procedure (72.7%). Median node yield was 10.5, with a median of 1.5 positive nodes on pathology. Eight patients (36.4%) achieved PSA <0.2, with six (27.3%) remaining with PSA <0.2 after a median followup of 23.1 months. Nine (40.9%) had an initial PSA decline, but nadired ≥0.2, and in five (22.7%) the PSA rose immediately after surgery. Overall, ADT was started in seven patients (31.8%) at a median of 10.1 months post-salvage surgery.ConclusionsIn our series of salvage dissection for PSMA-PET-detected nodal oligometastases, approximately a third achieved PSA <0.2; yet, it was only durable in 27%. Prospective trials of salvage nodal radiation are ongoing, however, more prospective trials of salvage node dissection are needed.     

Prostate-specific membrane antigen (PSMA) enzyme activity is elevated in prostate cancer cells

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a glutamate carboxypeptidase that cleaves terminal carboxy glutamates from both the neuronal dipeptide N-acetylaspartylglutamate (NAAG) and gamma-linked folate polyglutamate. The prostate enzyme has activity in both the membrane and cytosolic fractions termed PSMA and PSMA', respectively. METHODS: Using a NAAG hydrolytic radioenzymatic assay, we quantitated the enzymatic activity of PSMA and PSMA' in normal, benign prostatic hyperplasia (BPH), and prostate cancer (PC) tissues from radical prostatectomies. PSMA enzyme activity was evaluated in each tissue type and expressed per milligram protein and epithelial cell content. RESULTS: PSMA and PSMA' enzyme activities were significantly elevated in prostate cancer when compared to normal prostate tissue and BPH. Ratios of PSMA to PSMA' were also decreased in BPH as compared to cancerous and normal tissue. CONCLUSIONS: Prostate carcinogenesis is associated with an elevation in PSMA and PSMA' enzyme activity. In contrast, no such enhancement in PSMA activity is observed with benign neoplastic changes in BPH. Thus, the enhancement observed in prostate cancer is not simply related to a generalized prostatic hyperplasia, but is specific to its malignancy.     

The emerging role of prostate-specific membrane antigen(PSMA)PET-CT in patients with high-risk prostate cancer:moving the bar in high-risk prostate cancer

T he proPSMA trial is a Phase Ⅲmulticentric trial that examined the role of gallium-68-prostate-specific membrane antigen(PSMA)-positron emission tomography/com...     

Improved effects of a double suicide gene system on prostate cancer cells by targeted regulation of prostate-specific membrane antigen promoter and enhancer

Objective:   To explore the specific killing effect on prostate cancer cells of a dual cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) expression plasmid system controlled by the prostate-specific membrane antigen (PSMA) promoter and enhancer.
Methods:   The CD gene was used to construct the recombinant plasmid prostate-specific membrane antigen_{promoter/enhancer}-CD (pPSMA_E/P-CD). The specific regulatory function of the pPSMA_E/P promoter was demonstrated by detection of enhanced green fluorescent protein (EGFP) expression in the LNCaP cell line. Survival of cells transfected with different plasmids and treated with 5-fluorocytosine (5-FC) was measured by microculture tetrazolium assay. Cell cycle changes were measured by flow cytometry.
Results:   Target-specific expression of PSMA_E/P was observed in the prostate cancer cell line. Cytotoxicity of 5-FC was greater against LNCaP cells transfected with pPSMA_E/P-CD and UPRT and pPSMA_E/P-CD than control groups. Percentages of cells in S phase were 37.5% (LNCaP) and 30.6% (5-FC treatment) in the un-transfected groups, whereas they were 23.9% and 12.4% in the double and single suicide gene groups, respectively.
Conclusions:   Our findings confirm the cytotoxic efficacy of the pPSMA_E/P-CD + 5-FC and pPSMA_E/P-CD and UPRT + 5-FC suicide gene systems. The CD and UPRT gene system quickly and directly converted 5-FC into 5-FU, and then into toxic metabolites. The CD and UPRT double suicide gene system was more effective in inducing tumor cell apoptosis with 5-FC than the single suicide gene system. Thus, this construct can specifically target prostate cancer cells and might have a role in gene therapy against prostate cancer.     

The prognostic value of lymph node staging with prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT) and extended pelvic lymph node dissection in node-positive patients with prostate cancer

Objectives

To investigate whether patients with suspected pelvic lymph node metastases (molecular imaging [mi] N1) on staging prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT) had a different oncological outcome compared to those in whom the PSMA PET/CT did not reveal any pelvic lymph node metastases (miN0).

Patients and Methods

All patients with pelvic lymph node metastatic (pN1) disease after robot-assisted radical prostatectomy (RARP) and extended pelvic lymph node dissection (ePLND) between January 2017 and December 2020 were included. To assess predictors of biochemical progression of disease after RARP, a multivariable Cox regression analysis was performed, including number of tumour-positive lymph nodes, diameter of the largest nodal metastasis, and extranodal extension.

Results

In total, 145 patients were diagnosed with pN1 disease after ePLND. The median biochemical progression-free survival in patients with miN0 on PSMA PET/CT was 13.7 months, compared to 7.9 months in patients with miN1 disease (P = 0.006). On multivariable Cox regression analysis, both number of tumour-positive lymph nodes (>2 vs 1–2: hazard ratio [HR] 1.97; P = 0.005) and diameter of the largest nodal metastasis (HR 1.12; P < 0.001) were significant independent predictors of biochemical progression of disease.

Conclusion

Patients in whom pelvic lymph node metastases were suspected on preoperative PSMA imaging (miN1), patients diagnosed with >2 tumour-positive lymph nodes, and patients with a larger diameter of the largest nodal metastasis had a significantly increased risk of biochemical disease progression after surgery.     

Focal positive prostate-specific membrane antigen (PSMA) expression in ganglionic tissues associated with prostate neurovascular bundle: Implications for novel intraoperative PSMA-based fluorescent imaging techniques

ObjectiveProstate specific membrane antigen (PSMA) is primarily expressed in glandular prostatic tissue and is frequently utilized to detect primary or metastatic prostatic adenocarcinoma (CaP). A purported novel application of PSMA detection is the intraoperative real-time identification of CaP using radioimmunoscintigraphy to define the extension of the surgical resection. Considering that PSMA expression has been reported in other tissues, we evaluated its immunoexpression in prostatic neurovascular bundle elements to assess the convenience and safety of the aforementioned procedure.Materials and methodsTwenty consecutive specimens of radical prostatectomy (RP) were retrieved from our surgical pathology archives. PSMA immunoexpression (Clone 3E6, DAKO) was assessed in a representative section from each specimen containing neurovascular bundle elements.ResultsPSMA expression was documented in 20/20 of examined CaP slides. Most cases exhibited an apical/cytoplasmic or cytoplasmic with membranous accentuation pattern of staining. Focal weak to moderate cytoplasmic staining was detected in associated ganglionic tissue in 3/15 of the examined RP. In all cases, staining was cytoplasmic, less extensive, and weaker than the pattern observed in CaP. None of the peripheral nerve sheath cells or lymphovascular components of the examined neurovascular bundles were positive for PSMA.ConclusionsWe found focal positive PSMA expression in the ganglionic cells of the prostatic neurovascular bundle. Our results suggest that the radioimmunoscintigraphic detection of radiolabeled PSMA antibodies might not be entirely specific for prostatic cells; this observation must be taken into account should an intraoperative PSMA-based fluorescent imaging technique be used to define the extension of the surgical procedure.