The homodimer of prostate-specific membrane antigen is a functional target for cancer therapy

Prostate-specific membrane antigen (PSMA) is a type 2 integral membrane glycoprotein that serves as an attractive target for cancer immunotherapy by virtue of its abundant and restricted expression on the surface of prostate carcinomas and the neovasculature of most other solid tumors. However, relatively little is known about the molecular structure of this target. Here, we report that PSMA is expressed on tumor cells as a noncovalent homodimer. A truncated PSMA protein, lacking transmembrane and cytoplasmic domains, also formed homodimers, indicating that the extracellular domain is sufficient for dimerization. PSMA dimers but not monomers displayed a native conformation and possessed high-level carboxypeptidase activity. A unique dimer-specific epitope was identified by using one of a panel of novel mAbs. When used to immunize animals, dimer but not monomer elicited antibodies that efficiently recognized PSMA-expressing tumor cells. These findings on PSMA structure and biology may have important implications for active and passive immunotherapy of prostate and other cancers.     

Noninvasive measurement of androgen receptor signaling with a positron-emitting radiopharmaceutical that targets prostate-specific membrane antigen

Despite encouraging clinical results with next generation drugs (MDV3100 and abiraterone) that inhibit androgen receptor (AR) signaling in patients with castration-resistant prostate cancer (CRPC), responses are variable and short-lived. There is an urgent need to understand the basis of resistance to optimize their future use. We reasoned that a radiopharmaceutical that measures intratumoral changes in AR signaling could substantially improve our understanding of AR pathway directed therapies. Expanding on previous observations, we first show that prostate-specific membrane antigen (PSMA) is repressed by androgen treatment in multiple models of AR-positive prostate cancer in an AR-dependent manner. Conversely, antiandrogens up-regulate PSMA expression. These expression changes, including increased PSMA expression in response to treatment with the antiandrogen MDV3100, can be quantitatively measured in vivo in human prostate cancer xenograft models through PET imaging with a fully humanized, radiolabeled antibody to PSMA, (64)Cu-J591. Collectively, these results establish that relative changes in PSMA expression levels can be quantitatively measured using a human-ready imaging reagent and could serve as a biomarker of AR signaling to noninvasively evaluate AR activity in patients with CRPC.     

Prostatitis and serum prostate-specific antigen

Prostatitis is an inflammatory condition of the prostate and has been divided into four categories according to the National Institutes of Health classification. This article reviews the various types of prostatitis and their effect on serum prostate-specific antigen levels. Various proposed mechanisms of this elevation include leakage of prostatespecific antigen (PSA) into the blood stream, hypervascularity, and altered vascular permeability secondary to inflammation. Acute prostatitis can lead to an increase in PSA, which usually returns to normal levels with appropriate antibiotics within 1 to 3 months. Patients with chronic prostatitis have a less well-defined decrease in PSA after an antibiotic course. Whether a course of antibiotics prior to biopsy increases the yield has not been well established. Asymptomatic inflammation of the prostate has been recognized to be an important confounding factor in patients with an elevated PSA. Inflammation has been proposed to be a precursor of prostate adenocarcinoma.     

Ewing's-sarcoma-associated HBA-71 tumor antigen represents a new differentiation marker of human thymocytes

Summary The monoclonal HBA-71 antibody recognizes a new human tumor-associated antigen of Ewing's sarcoma and peripheral neuroectodermal tumors, which is also expressed in some normal tissues, including thymus, islets of Langerhans, ependyme, adenohypophysis, Sertoli/Leydig and granulosa cells. Besides a tumor-specific reciprocal chromosomal translocation t (11:22), the expression of the HBA-71 antigen is the only marker which can be used for reliable differential diagnosis of these rare malignancies of childhood and adolescence among other small round cell tumors. The HBA-71 antigen is further characterized here by ultrastructural, functional and cell-matrix interaction studies. In immunohistochemical staining the HBA-71 reacted with the cell surface of human cortical thymocytes. The HBA-71 antigen was also found to be localized at the cell-surface glycocalyx of tumor cells using immunogold staining and electron microscopy. A panel of additional monoclonal antibodies with reactivity patterns similar to those of the HBA-71 antibody was obtained by immunization of mice with ES cell lines and boostering with thymocytes. The HBA-71 antibody triggers proliferation of thymocytes and to a lesser extent also stimulates peripheral mononuclear blood cells. Antibody-induced thymocyte cultures exhibit the phenotype of immature, CD3_low thymocytes with uniform and stable expression of the HBA-71 antigen. In contrast to the thymocytes the HBA-71 antibody has an inhibitory effect on the continuous growth of the HBA-71⁺ tumor cell lines. The HBA-71 antigen may be involved in the regulation to growth of the positive normal and malignant tissues. Positive modulation of the antigen expression was induced in Ewing's sarcoma cell lines in response to insulin, insulin-like growth factor I (IGF-I) and by interaction of the cells with the extracellular matrixAbbreviations used ES Ewing's sarcoma - PNET peripheral neuroectodermal tumors - FITC fluorescein-isothiocyanate - PMBC peripheral mononuclear blood cells Supported by Bürgermeisterfond der Stadt Wien, grant no. 703     

Association of prostate-specific membrane antigen with caveolin-1 and its caveolae-dependent internalization in microvascular endothelial cells: implications for targeting to tumor vasculature

Prostate-specific membrane antigen (PSMA) is a transmembrane protein with a highly restricted profile of expression. Expression is primarily limited to secretory cells of the prostatic epithelium, with elevated levels observed in prostate cancer. As an integral membrane protein correlated with prostate cancer, PSMA offers a potentially valuable target for immunotherapy. PSMA is also detected in the neovasculature of a variety of solid tumors but not in the endothelial cells of preexisting blood vessels. Although the significance of PSMA expression in these cells remains elusive, this pattern of expression implies that PSMA may perform a functional role in angiogenesis and may offer a therapeutic target for the treatment of a broad spectrum of solid tumors. In this study, we have expressed PSMA in human microvascular endothelial cells and demonstrate that PSMA binds to caveolin-1 and undergoes internalization via a caveolae-dependent mechanism. The association between PSMA and caveolae in endothelial cells may provide important insight into PSMA function and ways to best exploit this protein for therapeutic benefit.     

Crystal structure of the dimeric extracellular domain of human carbonic anhydrase XII, a bitopic membrane protein overexpressed in certain cancer tumor cells

Overexpression of the zinc enzyme carbonic anhydrase (CA; EC ) XII is observed in certain human cancers. This bitopic membrane protein contains an N-terminal extracellular catalytic domain, a membrane-spanning alpha-helix, and a small intracellular C-terminal domain. We have determined the three-dimensional structure of the extracellular catalytic domain of human CA XII by x-ray crystallographic methods at 1.55-A resolution. The structure reveals a prototypical CA fold; however, two CA XII domains associate to form an isologous dimer, an observation that is confirmed by studies of the enzyme in solution. The identification of signature GXXXG and GXXXS motifs in the transmembrane sequence that facilitate helix-helix association is additionally consistent with dimeric architecture. The dimer interface is situated so that the active site clefts of each monomer are clearly exposed on one face of the dimer, and the C termini are located together on the opposite face of the dimer to facilitate membrane interaction. The amino acid composition of the active-site cleft closely resembles that of the other CA isozymes in the immediate vicinity of the catalytic zinc ion, but differs in the region of the nearby alpha-helical "130's segment." The structure of the CA XII-acetazolamide complex is also reported at 1.50-A resolution, and prospects for the design of CA XII-specific inhibitors of possible chemotherapeutic value are discussed.     

Insulin-like growth factor I is not a useful marker of prostate cancer in men with elevated levels of prostate-specific antigen

High serum levels of insulin-like growth factor I (IGF-I) and low levels of IGF-binding protein-3 (IGFBP-3) have been shown to correlate with increased prostate cancer risk. To evaluate this, IGF-I, IGFBP-3, and prostate-specific antigen (PSA) were measured in serum from 665 consecutive men (179 with prostate cancer), aged 55-67 yr, with elevated serum prostate-specific antigen (PSA; > or = 4 microg/L) in a screening trial. Men in the highest quartile of IGF-I levels had an odds ratio (OR) for prostate cancer of 0.50 [95% confidence interval (CI) 0.26-0.97] when adjusting for serum IGFBP-3. IGFBP-3 itself was not significantly associated with prostate cancer risk (OR, 1.24; 95% CI, 0.68-2.24). Prostate volume was larger in men without than in those with prostate cancer (P < 0.001), and after adjustment for prostate volume, the negative association between serum IGF-I and prostate cancer risk was no longer significant (OR, 0.57; 95% CI, 0.28-1.16). In screen-positive men with elevated serum PSA, serum IGF-I is not a useful diagnostic test for prostate cancer, but it may be associated with benign prostatic hyperplasia and enlargement.     

A retrospective and prospective overview of prostate-specific antigen

Since the identification of prostate-specific antigen (PSA), continued technological advances have provided highly sensitive assays for its quantification. Given its lack of disease specificity, and its recent detection at low levels in an increasing number of non-prostatic tissues, PSA is far from being the perfect “tumour” marker (biological marker). However, the positive predictive value of PSA for assessing cancer risk makes PSA the most useful “tumour” marker for monitoring progression and response to treatment among patients with prostate cancer. Earlier detection through screening for elevated levels of PSA, while controversial, has been proposed as a way to decrease prostate cancer mortality. Haematogenous identification of PSA mRNA may provide stage-related prognostic information, and the use of ultrasensitive assays for PSA may permit earlier identification of residual or recurrent cancer, following treatment and the initiation of adjuvant therapy. Various PSA-related concepts, including the ratio of “free” PSA and complexes of PSA with the protease inhibitor, α₁-antichymotrypsin, to total PSA, have been proposed and placed within diagnostic and management algorithms. Elevations of PSA in other irregularities of the prostate, notably in benign prostatic hyperplasia, and the increasing frequency and number of non-prostatic tissues, including those in women, expressing PSA, have implications for future immunoassays for PSA and strategies for immunotherapy using PSA-based monoclonal antibodies or vaccines, as well as for the molecular basis for its anomalous expression and physiological function(s).     

Practitioner-level determinants of inappropriate prostate-specific antigen screening

BACKGROUND: None of the major clinical practice guidelines recommend that prostate-specific antigen (PSA) screening be routinely performed in asymptomatic men older than 75 years or younger than 40 years. We investigated the practitioner-level determinants of inappropriate PSA screening in 7 Veterans Health Administration (VHA) hospitals. METHODS: Data on PSA test use from 1997 to 2004 were obtained from VHA databases for 181 139 male patients and the 4823 health care providers who ordered their tests. Patients were excluded from the study population if they underwent PSA testing for nonscreening reasons, as indicated by prostate cancer-specific medications, diagnoses, and procedures. Inappropriate PSA test use was defined as PSA screening in patients older than 75 years or younger than 40 years. Univariate and multivariate Poisson regressions were performed. RESULTS: The mean +/- SD percentage of inappropriate tests by health care provider was 19.3% +/- 15.0%, with 18.4% +/- 14.9% in patients older than 75 years and 0.8% +/- 3.0% in patients younger than 40 years. Practitioners who were urology specialists, male, infrequent PSA test orderers, and affiliated with specific hospitals had significantly higher levels of inappropriate PSA screening. Compared with attending physicians, nurses and physician assistants had significantly lower levels of inappropriate screening. Under multivariate modeling, infrequent PSA test ordering and hospital affiliation retained statistical significance. The percentage of inappropriate PSA screening increased significantly with the age of male health care providers (P<.001). CONCLUSIONS: This study elucidates several important provider-level determinants of PSA screening misuse and substantiates that PSA screening is frequently performed counter to evidence-based guidelines. Further work is needed to determine the degree to which "prostatempathy" contributes to PSA misuse by older male providers.     

Effect of finasteride, a 5 alpha-reductase inhibitor on prostate tissue androgens and prostate-specific antigen.

Whole tissue dihydrotestosterone (DHT) and testosterone (T) concentrations have been measured after finasteride, a 5 alpha-reductase inhibitor, and this was compared to other methods of androgen inhibition. In 10 patients treated for 1 week with finasteride, whole tissue DHT decreased to a mean of 0.302 ng/g. This value was significantly less than DHT values in control patients (mean, 4.5 ng/g) and patients treated with either surgical castration (mean, 1.14 ng/g), megestrol (160 mg/day) plus diethylstilbestrol (0.1 mg), or megestrol plus ketoconazole (1200 mg/day; mean, 0.609). Tissue T levels were significantly increased in finasteride-treated patients (1.18 ng/g) compared to a mean of 0.171 ng/g in controls and 0.105 ng/g in megestrol-treated patients. Part of the prostate tissue obtained at surgery was mechanically separated into epithelium and stroma. The epithelial cells were homogenized, the supernate was assayed for prostate-specific antigen (PSA), an androgen-dependent protein, and the pellet was assayed for DHT and DNA. Epithelial cell PSA and DHT values significantly decreased in finasteride- and megestrol- plus estrogen-treated patients compared to controls. However, the slope and position of the regression lines for DHT vs. PSA in patients treated with megestrol plus low dose estrogen (r = 0.79) differed significantly (P less than 0.05) from the regression line relating epithelial DHT to PSA in patients treated with finasteride (r = 0.82). Since the regression slope for finasteride described a greater increase in PSA per unit change in DHT compared to the slope for megestrol plus estrogen, which lowers both DHT and T, finasteride, despite its drastic lowering of DHT, may have a modest residual androgenic effect related to its effect on tissue T.     

From the Cover: Suppression of human prostate tumor growth by a unique prostate-specific monoclonal antibody F77 targeting a glycolipid marker

In our effort to find diagnostic markers and to develop therapeutic approaches for prostate cancer, we have identified an mAb that is capable of binding to a cell surface antigen specifically expressed on both androgen-dependent and androgen-independent prostate cancer cells. Immunohistological studies revealed that this mAb, called F77, stained 112 of 116 primary and 29 of 34 metastatic human prostate cancer specimens. Although the mAb F77 alone directly promotes prostate cancer cell death, it also mediates complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. In addition, mAb F77 can significantly inhibit androgen-independent PC3 and Du145 tumor growth in nude mice. Antigen characterization revealed that mAb F77 recognizes a very small molecular species with glycolipid properties. F77 antigen is concentrated in the lipid-raft microdomains, which serve as platforms for the assembly of associating protein complexes. Thus, the present study indicates that mAb F77 defines a unique prostate cancer marker and shows promising potential for diagnosis and treatment of prostate cancer, especially for androgen-independent metastatic prostate cancer.     

Human malignancy-associated nucleolar antigen as a marker for tumor cells in patients with acute leukemia

Tumor burden in adult patients with acute leukemia is assessed using the percentage of blast cells in the bone marrow or blood. It is clear, however, that not all blast cells are leukemic cells, especially during rapid marrow regeneration. Similarly, some leukemia cell lines have been shown to differentiate in vitro, and the same process also occurs in vivo. Therefore, the leukemic burden may be due to more differentiated cells as well as to blast cells. The purpose of this study was to investigate whether the human malignancy-associated nucleolar antigen (HMNA) could be used as a marker for leukemic cells and to examine its potential as a diagnostic tool. The proportion of HMNA-positive cells in the bone marrow of patients with acute leukemia was determined by indirect immunofluorescence with antibodies to HMNA and was compared with the differential counts routinely made in the clinic laboratory. The percentages of HMNA-positive cells among the nucleated cells in the marrow of 72 patients with clinical evidence of leukemia were significantly higher (range 9%-98%, median 83%) than those observed for nonleukemic individuals (range less than 0.05%-2.5%, median 1%) or for fractions of marrow cells from normal volunteers enriched for normal early progenitor cells (less than or equal to 2%). Patients with leukemia in remission had a lower percentage of HMNA- positive cells (range 0%-83%, median 3%). The percentage of HMNA- positive cells increased as patients approached relapse. Although the percentage of HMNA-positive cells was related to the percentage of blast cells in the bone marrow of the patients with leukemia, some partially differentiated cells were also HMNA-positive in some specimens, and some blastic cells were HMNA-negative in other specimens. These studies indicate the potential usefulness of HMNA as a marker for leukemic cells.     

The interaction among protein C inhibitor, prostate-specific antigen, and the semenogelin system

The kallikrein-like serine protease, prostate-specific antigen (PSA), is mixed in human seminal plasma with its protein substrates semenogelin (Sg) -I, Sg-II, and protein C inhibitor (PCI), which are produced in seminal vesicles. In the seminal plasma, PSA degrades Sg-I, and Sg-II, which are major components in insoluble coagula, and PCI inhibits PSA by forming a PSA-PCI complex. Digestion of seminal coagula with PSA releases PCI and PSA-PCI complex from the coagula into a soluble phase, suggesting the presence of active PCI within the coagula. PCI forms a ternary complex with PSA and Sg-II in the seminal plasma. The binding of Sg-II to PSA and PCI is influenced by pH, ionic strength, heparin, negatively charged dextran sulfate, divalent cations, and particularly by Zn 2 +. These observations suggest that binding of PCI to Sg in seminal vesicles regulates the PSA-catalyzed degradation of Sg in seminal plasma; the complex formation among PCI, PSA, and Sg is modulated by several factors in seminal plasma.     

Prostate-specific antigen and prostate-specific antigen derivatives as predictors of benign prostatic hyperplasia progression

Benign prostatic hyperplasia (BPH) is the most common urologic affliction in aging men, leading to adverse clinical outcomes in a significant proportion of the population. Serum prostate-specific antigen (PSA) has been established as a marker for prostate cancer for the past two decades but more recently has been recognized as an equally important marker of BPH presence and progression. Over this time, the discovery and study of multiple isoforms of PSA has led to even more sensitive and specific methods to differentiate BPH from prostate cancer. Herein we review the expression, processing, and biochemistry of PSA and its derivatives and discuss the potential of these isoforms, both individually and in combination, to serve as determinants of BPH severity and progression.     

Using the free-to-total prostate-specific antigen ratio to detect prostate cancer in men with nonspecific elevations of prostate-specific antigen levels

BACKGROUND: Prostate-specific antigen (PSA) levels between 4.0 to 10.0 ng/ml have poor specificity in prostate cancer screening, leading to unnecessary biopsies. OBJECTIVE: To determine whether the free-to-total PSA ratio (F/T PSA) improved the diagnostic accuracy of these nonspecific PSA levels. MEASUREMENTS AND MAIN RESULTS: Medline was searched from 1986 to 1997. Additional studies were identified from article bibliographies and by searching urology journals. Two investigators independently identified English-language studies providing F/T PSA ratio test-operating characteristics data on ≧10 cancer patients with PSA values between 2.0 and 10.0 ng/ml. Twenty-one of 90 retrieved studies met selection criteria. Two investigators independently extracted data on methodology and diagnostic performance. Investigator-selected cut points for the optimal F/T PSA ratio had a median likelihood ratio of 1.76 (interquartile range, 1.40 to 2.11) for a positive test and 0.27 (0.20 to 0.40) for a negative test. Assuming a 25% pretest probability of cancer, the posttest probabilities were 37% following a positive test and 8% following a negative test. The summary receiver operating characteristic curve showed that maintaining test sensitivity above 90% was associated with false positive rates of 60% to 90%. Methodologic problems limited the validity and generalizability of the literature. CONCLUSIONS: A negative test reduced the posttest probability of cancer to approximately 10%. However, patients may find that this probability is not low enough to avoid undergoing prostate biopsy. The optimal F/T PSA ratio cut point and precise estimates for test specificity still need to be determined. This work was presented in part at the Society of General Internal Medicine annual meeting, Washington, DC, May 1997. This work was supported by the VA Medical Center, Albuquerque, NM.