N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: implications for immunotherapy

Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in prostate cancer cells with levels proportional to tumor grade. The membrane association and correlation with disease stage portend a promising role for PSMA as an antigenic target for antibody-based therapies. Successful application of such modalities necessitates a detailed knowledge of the subcellular localization and trafficking of target antigen. In this study, we show that PSMA is expressed predominantly in the apical plasma membrane in epithelial cells of the prostate gland and in well-differentiated Madin-Darby canine kidney cells. We show that PSMA is targeted directly to the apical surface and that sorting into appropriate post-Golgi vesicles is dependent upon N-glycosylation of the protein. Integrity of the microtubule cytoskeleton is also essential for delivery and retention of PSMA at the apical plasma membrane domain, as destabilization of microtubules with nocodazole or commonly used chemotherapeutic Vinca alkaloids resulted in the basolateral expression of PSMA and increased the uptake of anti-PSMA antibody from the basolateral domain. These results may have important relevance to PSMA-based immunotherapy and imaging strategies, as prostate cancer cells can maintain a well-differentiated morphology even after metastasis to distal sites. In contrast to antigens on the basolateral surface, apical antigens are separated from the circulation by tight junctions that restrict transport of molecules across the epithelium. Thus, antigens expressed on the apical plasma membrane are not exposed to intravenously administered agents. The ability to reverse the polarity of PSMA from apical to basolateral could have significant implications for the use of PSMA as a therapeutic target.     

USP44 regulates centrosome positioning to prevent aneuploidy and suppress tumorigenesis

Most human tumors have abnormal numbers of chromosomes, a condition known as aneuploidy. The mitotic checkpoint is an important mechanism that prevents aneuploidy by restraining the activity of the anaphase-promoting complex (APC). The deubiquitinase USP44 was identified as a key regulator of APC activation; however, the physiological importance of USP44 and its impact on cancer biology are unknown. To clarify the role of USP44 in mitosis, we engineered a mouse lacking Usp44. We found that USP44 regulated the mitotic checkpoint and prevented chromosome lagging. Mice lacking Usp44 were prone to the development of spontaneous tumors, particularly in the lungs. Additionally, USP44 was frequently downregulated in human lung cancer, and low expression correlated with a poor prognosis. USP44 inhibited chromosome segregation errors independent of its role in the mitotic checkpoint by regulating centrosome separation, positioning, and mitotic spindle geometry. These functions required direct binding to the centriole protein centrin. Our data reveal a new role for the ubiquitin system in mitotic spindle regulation and underscore the importance of USP44 in the pathogenesis of human cancer.     

In vitro and in vivo responses of advanced prostate tumors to PSMA ADC, an auristatin-conjugated antibody to prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for therapy. PSMA ADC is an antibody-drug conjugate (ADC) that consists of a fully human anti-PSMA monoclonal antibody conjugated to monomethylauristatin E through a valine-citrulline linker. In this study, the antitumor activity of PSMA ADC was evaluated against a panel of prostate cancer cell lines in vitro and in a novel in vivo model of taxane-refractory human prostate cancer. In vitro cell killing was efficient for cells with abundant PSMA expression (>10(5) molecules/cell; IC(50) ≤ 0.022 nmol/L) and 1,000-fold less efficient for cells with undetectable PSMA (IC(50) > 30 nmol/L). Intermediate potency (IC(50) = 0.80 nmol/L) was observed for cells with approximately 10(4) molecules of PSMA per cell, indicating a threshold PSMA level for selective cell killing. Similar in vitro activity was observed against androgen-dependent and -independent cells that had abundant PSMA expression. In vitro activity of PSMA ADC was also dependent on internalization and proper N-glycosylation/folding of PSMA. In contrast, less potent and nonselective cytotoxic activity was observed for a control ADC, free monomethylauristatin E, and other microtubule inhibitors. PSMA ADC showed high in vivo activity in treating xenograft tumors that had progressed following an initial course of docetaxel therapy, including tumors that were large (>700 mm(3)) before treatment with PSMA ADC. This study defines determinants of antitumor activity of a novel ADC. The findings here support the clinical evaluation of this agent in advanced prostate cancer.     

前列腺癌组织中前列腺特异性膜抗原基因表达的临床意义

目的检测前列腺特异性膜抗原(PSMA)基因在前列腺癌组织中的表达,并探讨其意义。方法利用半定量(RT-PCR)法检测52例前列腺癌组织及35例前列腺增生组织中PSMA的表达。结果 (1)PSMA在前列腺癌及前列腺增生组织中的阳性表达率分别为84.6%(44/52)及68.6%(24/35),虽然前列腺癌组织中PSMA表达阳性率高于前列腺增生组织,但两者差异无统计学意义(P>0.05)。PSMA mRNA半定量结果在两组间存在差异,癌组织中PSMA的表达量明显高于增生组织中的表达,差异有统计学意义(P<0.05)。(2)PSMA基因表达量与肿瘤的分化程度有关,分化程度越低,表达量越高,高分化前列腺癌与低分化前列腺癌间差异有统计学意义(P<0.05)。结论 PSMA在前列腺癌中的表达与肿瘤的分化程度有关,且高于前列腺增生组织,提示PSMA可作为判断预后及分化程度的指标。     

Differing effects of microtubule depolymerizing and stabilizing chemotherapeutic agents on t-SNARE-mediated apical targeting of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a protein up-regulated in the vast majority of prostate cancers. Antibodies to PSMA have proved highly specific for prostate cancer cells, and the therapeutic potential of such antibodies is currently being assessed in clinical trials. We have previously shown that PSMA at the cell surface of polarized epithelial cells is predominantly expressed at the apical plasma membrane and that microtubule depolymerization abolishes apical PSMA targeting. In the current report, we implicate a functional role for a target membrane soluble N-ethylmaleimide-sensitive factor adaptor protein receptor, syntaxin 3, in the microtubule-dependent apical targeting of PSMA. PSMA and syntaxin 3 are similarly localized to the apical plasma membrane of the prostatic epithelium and Madin-Darby canine kidney cells. Introduction of a point mutation into syntaxin 3 abolishes its polarized distribution and causes PSMA to be targeted in a nonpolarized fashion. Additionally, treatment of polarized Madin-Darby canine kidney cells with vinblastine, a microtubule depolymerizing chemotherapeutic agent, causes both syntaxin 3 and PSMA to redistribute in a nonpolarized fashion. However, following treatment with the microtubule stabilizing chemotherapeutic agent Taxotere, both syntaxin 3 and PSMA continue to localize in a polarized manner at the apical plasma membrane. Thus, microtubule depolymerizing and stabilizing chemotherapeutic drugs might exact similar cytotoxic effects but have disparate effects on protein targeting. This phenomenon might have important clinical implication, especially related to antibody-mediated immunotherapy, and could potentially be exploited for therapeutic benefit.     

前列腺特异性膜抗原显像用于前列腺癌研究进展

前列腺特异性膜抗原(PSMA)于多数前列腺癌细胞及肿瘤新生血管内皮细胞内呈高表达，而PSMA显像为前列腺癌研究领域的热门方向。本文就PSMA显像用于前列腺癌进展进行综述。     

Disulfide-constrained peptides that bind to the extracellular portion of the prostate-specific membrane antigen

The prostate-specific membrane antigen (PSMA) is a well-characterized surface antigen, overexpressed in the most advanced, androgen-resistant human prostate cancer cells. We sought to exploit PSMA cell surface properties as a target for short peptides that will potentially guide protein-based therapeutics, such as viral vectors, to prostate cancer cells. Two separate phage display peptide strategies were applied, in parallel, to purified PSMA protein bound to two separate substrates. We reasoned that peptide sequences common to both substrate selections would be specific binders of PSMA. Additionally, the design allowed for stringent cross-selections, where phage populations from one selection condition could be applied to the alternative substrate. These strategies resulted in a series of phage displayed peptides able to bind to PSMA by ELISA and direct binding assays, both with purified protein and in prostate cancer cells. Cell binding is competitively inhibited by purified PSMA. The synthesized peptides are capable of enhancing PSMA carboxypeptidase enzymatic activity, suggesting protein folding stabilization. The discovery of these peptides provides the foundation for subsequent development of peptide targeted therapeutics against prostate cancer.     

Synthesis and in vitro evaluation of MR molecular imaging probes using J591 mAb‐conjugated SPIONs for specific detection of prostate cancer

Carcinoma of the prostate is the most frequent diagnosed malignant tumor in men and is the second leading cause of cancer‐related death in this group. The cure rate of prostate cancer is highly dependent on the stage of disease at the diagnosis and early detection is key to designing effective treatment strategies. The objective of the present study is to make a specific MR imaging probe for targeted imaging of cancer cells. We take advantage of the fact that many types of prostate cancer cells express high levels of prostate‐specific membrane antigen (PSMA) on their cell surface. The imaging strategy is to use superparamagnetic iron oxide nanoparticles (SPIONs), attached to an antibody (J591) that binds to the extracellular domain of PSMA, to specifically enhance the contrast of PSMA‐expressing prostate cancer cells. Conjugation of mAb J591 to commercial SPIONs was achieved using a heterobifunctional linker, sulfo‐SMCC. Two types of prostate cancer cell lines were chosen for experiments: LNCaP (PSMA+) and DU145 (PSMA?). MRI and cell uptake experiments demonstrated the high potential of the synthesized nanoprobe as a specific MRI contrast agent for detection of PSMA‐expressing prostate cancer cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Targeting prostate cancer: Prostate-specific membrane antigen based diagnosis and therapy

The high incidence rates of prostate cancer (PCa) raise demand for improved therapeutic strategies. Prostate tumors specifically express the prostate-specific membrane antigen (PSMA), a membrane-bound protease. As PSMA is highly overexpressed on malignant prostate tumor cells and as its expression rate correlates with the aggressiveness of the disease, this tumor-associated biomarker provides the possibility to develop new strategies for diagnostics and therapy of PCa. Major advances have been made in PSMA targeting, ranging from immunotherapeutic approaches to therapeutic small molecules. This review elaborates the diversity of PSMA targeting agents while focusing on the radioactively labeled tracers for diagnosis and endoradiotherapy. A variety of radionuclides have been shown to either enable precise diagnosis or efficiently treat the tumor with minimal effects to nontargeted organs. Most small molecules with affinity for PSMA are based on either a phosphonate or a urea-based binding motif. Based on these pharmacophores, major effort has been made to identify modifications to achieve ideal pharmacokinetics while retaining the specific targeting of the PSMA binding pocket. Several tracers have now shown excellent clinical usability in particular for molecular imaging and therapy as proven by the efficiency of theranostic approaches in current studies. The archetypal expression profile of PSMA may be exploited for the treatment with alpha emitters to break radioresistance and thus to bring the power of systemic therapy to higher levels.     

Changing the Goal Posts: Prostate-specific Membrane Antigen Targeted Theranostics in Prostate Cancer

ObjectiveProstate-specific membrane antigen (PSMA) theranostics is changing the face of prostate cancer diagnosis and therapy. PSMA, a transmembrane protein over-expressed in many prostate cancers, is a promising target for theranostics. Theranostics is the concept of small molecule proteins that are labelled to different radionuclides and can be used for either diagnosis or therapy, dependent on whether they are labelled with an imaging or therapy radionuclide. By directly targeting the cancer cells with imaging and then for therapy, this approach embodies the philosophy of precision medicine – right drug, right time, right dose. The question is how to best utilise these new imaging and therapy agents in clinical practice. This review will evaluate the importance of PSMA in prostate cancer, its role in diagnostic imaging, and its potential as a therapy of advanced prostate cancer.Data SourcesElectronic databases including MEDLINE, Scopus, professional websites were searched.ConclusionPSMA-directed theranostics has an expanding role in prostate cancer because of its utility as a sensitive diagnostic tool that can be coupled with efficacious and low-toxicity therapeutic options. Ongoing research is required to determine how to use this effective tool for best patient care.Implications for Nursing PracticePSMA theranostics is rapidly being incorporated into the routine care of men with prostate cancer. Understanding its strengths, its limitations, and where it may be valuable in clinical care is important in undertaking best patient practice.     

Nuclear pore protein NUP88 activates anaphase-promoting complex to promote aneuploidy

The nuclear pore complex protein NUP88 is frequently elevated in aggressive human cancers and correlates with reduced patient survival; however, it is unclear whether and how NUP88 overexpression drives tumorigenesis. Here, we show that mice overexpressing NUP88 are cancer prone and form intestinal tumors. To determine whether overexpression of NUP88 drives tumorigenesis, we engineered transgenic mice with doxycycline-inducible expression of Nup88. Surprisingly, NUP88 overexpression did not alter global nuclear transport, but was a potent inducer of aneuploidy and chromosomal instability. We determined that NUP88 and the nuclear transport factors NUP98 and RAE1 comprise a regulatory network that inhibits premitotic activity of the anaphase-promoting complex/cyclosome (APC/C). When overexpressed, NUP88 sequesters NUP98-RAE1 away from APC/C^CDH1, triggering proteolysis of polo-like kinase 1 (PLK1), a tumor suppressor and multitasking mitotic kinase. Premitotic destruction of PLK1 disrupts centrosome separation, causing mitotic spindle asymmetry, merotelic microtubule-kinetochore attachments, lagging chromosomes, and aneuploidy. These effects were replicated by PLK1 insufficiency, indicating that PLK1 is responsible for the mitotic defects associated with NUP88 overexpression. These findings demonstrate that the NUP88-NUP98-RAE1-APC/C^CDH1 axis contributes to aneuploidy and suggest that it may be deregulated in the initiating stages of a broad spectrum of human cancers.     

18F-前列腺特异性膜抗原(PSMA)-1007PET/CT参数评估前列腺癌骨转移

目的 观察¹⁸F-前列腺特异性膜抗原(PSMA)-1007 PET/CT参数评估前列腺癌(PCa)骨转移的价值。方法 根据有无骨转移将53例经病理确诊的PCa患者分为骨转移组(n=27)及无骨转移组(n=26)。于PET/CT三维图像上勾画PCa原发灶感兴趣容积，测量其最大标准摄取值(SUV_max)、前列腺肿瘤PSMA表达体积(PSMA-TVp)和前列腺肿瘤病灶PSMA表达总量(TL-PSMAp);比较组间PSMA表达参数差异，采用单因素logistic回归分析预测PCa骨转移的独立因素，绘制受试者工作特征曲线，分析PSMA表达参数评估骨转移的效能。结果 骨转移组PCa原发灶SUV_max、PSMA-TVp及TL-PSMAp均高于无骨转移组(P均<0.01)。原发灶SUV_max、PSMA-TVp及TL-PSMAp均为预测PCa骨转移的独立因素(OR=1.091、1.327、1.042,P均<0.05),其评估PCa骨转移的曲线下面积分别为0.734、0.908及0.929,PCa原发灶TL...     

Targeting PSMA with a Cu-64 Labeled Phosphoramidate Inhibitor for PET/CT Imaging of Variant PSMA-Expressing Xenografts in Mouse Models of Prostate Cancer

Purpose

Prostate-specific membrane antigen (PSMA) is highly up-regulated in prostate tumor cells, providing an ideal target for imaging applications of prostate cancer. CTT-1297 (IC₅₀?=?27 nM) is an irreversible phosphoramidate inhibitor of PSMA that has been conjugated to the CB-TE1K1P chelator for incorporation of Cu-64. The resulting positron emission tomography (PET) agent, [⁶⁴Cu]ABN-1, was evaluated for selective uptake both in vitro and in vivo in PSMA-positive cells of varying expression levels. The focus of this study was to assess the ability of [⁶⁴Cu]ABN-1 to detect and distinguish varying levels of PSMA in a panel of prostate tumor-bearing mouse models.

Procedures

CTT-1297 was conjugated to the CB-TE1K1P chelator using click chemistry and radiolabeled with Cu-64. Internalization and binding affinity of [⁶⁴Cu]ABN-1 was evaluated in the following cell lines having varying levels of PSMA expression: LNCaP late-passage?>?LNCaP early passage?≈?C4-2B?>?CWR22rv1 and PSMA-negative PC-3 cells. PET/X-ray computed tomography imaging was performed in NCr nude mice with subcutaneous tumors of the variant PSMA-expressing cell lines.

Results

[⁶⁴Cu]ABN-1 demonstrated excellent uptake in PSMA-positive cells in vitro, with ～80 % internalization at 4 h for each PSMA-positive cell line with uptake (fmol/mg) correlating to PSMA expression levels. The imaging data indicated significant tumor uptake in all models. The biodistribution for late-passage LNCaP (highest PSMA expression) demonstrated the highest specific uptake of [⁶⁴Cu]ABN-1 with tumor-to-muscle and tumor-to-blood ratios of 30?±?11 and 21?±?7, respectively, at 24 h post-injection. [⁶⁴Cu]ABN-1 cleared through all tissues except for PSMA-positive kidneys.

Conclusion

[⁶⁴Cu]ABN-1 demonstrated selective uptake in PSMA-positive cells and tumors, which correlated to the level of PSMA expression. The data reported herein suggest that [⁶⁴Cu]ABN-1 will selectively target and image variant PSMA expression and in the future will serve as a non-invasive method to follow the progression of prostate cancer in men.

     

前列腺特异性膜抗原PET/MRI在前列腺癌中的应用现状与前景

前列腺癌(prostate cancer,PCa)是常见的老年男性恶性肿瘤,并是其主要致死原因之一.前列腺特异性膜抗原(prostate specific membrane antigen,PSMA)是一种PCa组织特异性高表达的跨膜蛋白,这使得PSMA成为PCa良好的特异性分子影像靶点.目前,PSMA PET/CT在...     

Preclinical Dosimetry,Imaging, and Targeted Radionuclide Therapy Studies of Lu-177-Labeled Albumin-Binding,PSMA-Targeted CTT1403

Molecular Imaging and Biology - Prostate-specific membrane antigen (PSMA) continues to be the hallmark biomarker for prostate cancer as it is expressed on nearly all prostatic tumors. In addition,...     

分子影像指导下的一体化精准诊疗

近年来,国际临床医学领域排名Top10的热点前沿中,屡屡出现分子影像及其一体化精准诊疗,如靶向前列腺特异性膜抗原的前列腺癌精准诊断与治疗.分子影像指导下的一体化精准诊疗是一个飞速发展的领域:针对任何疾病的特征靶点,都有可能找到一种或多种分子探针,通过分子影像观察其在体内的表达及动态变化,用于疾病精准诊断,并一体化指导针...     

Expression and purification of prostate-specific membrane antigen in the baculovirus expression system and recognition by prostate-specific membrane antigen-specific T cells.

Antigen-specific immunotherapy of cancer depends on a consistent source of well-defined protein antigen. Production of recombinant protein offers the obvious solution to this problem but few comparisons of recombinant and native proteins in cellular immune assays have been reported. We report expression of a putative immunotherapy antigen, prostate-specific membrane antigen (PSMA), in insect cells using a baculovirus vector. T cells stimulated with recombinant PSMA or native PSMA derived from the LNCaP cell line recognized both native PSMA and recombinant, baculoviral PSMA. These data indicate that PSMA produced in Sf9 cells is immunologically cross-reactive with native PSMA and therefore suitable for immunotherapy as it is recognized by both cellular and humoral immune responses.     

Semiquantitative Parameters in PSMA-Targeted PET Imaging with [18F]DCFPyL: Intrapatient and Interpatient Variability of Normal Organ Uptake

Molecular Imaging and Biology - Prostate-specific membrane antigen (PSMA)-targeted positron emission tomography (PET) imaging has impacted the management of patients with prostate cancer (PCa) in...     

68Ga-PSMA-11与18F-PSMA-1007在前列腺癌中的应用

前列腺癌的发病率在全球男性恶性肿瘤中排名第2,其发病率及病死率呈快速上升趋势。前列腺特异性膜抗原（PSMA）PET/CT 在前列腺癌的检测方面具有高灵敏度和特异度,可用于原发性前列腺癌的定位与分期、复发性前列腺癌的监测。目前,以PSMA为靶点的显像剂主要是放射性核素⁶⁸Ga和¹⁸F的化合物。该文综述了临床常用的小分子抑制剂⁶⁸Ga-PSMA-11和¹⁸F-PSMA-1007的特点,以及其在前列腺癌显像中的优缺点,为临床诊断提供依据,并指导其在不同患者中的临床应用。     

Targeted Inhibition of Prostate Cancer Metastases with an RNA Aptamer to Prostate-specific Membrane Antigen

Cell-targeted therapies (smart drugs), which selectively control cancer cell progression with limited toxicity to normal cells, have been developed to effectively treat some cancers. However, many cancers such as metastatic prostate cancer (PC) have yet to be treated with current smart drug technology. Here, we describe the thorough preclinical characterization of an RNA aptamer (A9g) that functions as a smart drug for PC by inhibiting the enzymatic activity of prostate-specific membrane antigen (PSMA). Treatment of PC cells with A9g results in reduced cell migration/invasion in culture and metastatic disease in vivo. Importantly, A9g is safe in vivo and is not immunogenic in human cells. Pharmacokinetic and biodistribution studies in mice confirm target specificity and absence of non-specific on/off-target effects. In conclusion, these studies provide new and important insights into the role of PSMA in driving carcinogenesis and demonstrate critical endpoints for the translation of a novel RNA smart drug for advanced stage PC.