Tissue-specificity of prostate specific antigens: Comparative analysis of transcript levels in prostate and non-prostatic tissues
Introduction
With 28,900 deaths in 2003, prostate cancer (PCa) remains to be the second leading cause of cancer-related deaths among men in the United States [1]. Primary local treatment of PCa relies on radical prostatectomy and radiotherapy. Despite optimisation of these therapies about 30% of patients suffer from recurrent disease.
The screening for serum levels of PSA has greatly facilitated detection of PCa at early stages when the tumor has an encouragingly high cure rate [2]. But nevertheless, there is still no curative treatment available for patients who suffer from minimal residual disease or for those who have metastases at the time of diagnosis. Although PCa begins as an androgen-dependent tumor, the beneficial effects of androgen deprivation are often temporary and the development of therapy-resistant PCa that is essentially incurable seems to be almost inevitable during later stages [3]. Whereas the 10-year disease-specific survival of patients with localised PCa is up to 90% depending on the Gleason score, therapy-resistant disease will lead to the death of about 75% of the patients within 2–5 years [4]. This dismal prognosis has triggered the search of novel therapeutic approaches like immunotherapy based on T cells or tumor specific antibodies [5].
Induction of antitumor responses by tumor-specific vaccines and restoration of tumor-specific immunity after resection of progressively growing solid tumors in experimental cancer models, have documented the ability of the immune system to defend against tumors. Consequently, immunotherapeutic approaches are currently explored in numerous clinical trials [6], [7], [8].
Treatment with anti-tumor antibodies was the first accepted cancer immunotherapy [9]. The majority of these antibodies are directed against membrane molecules expressed on tumor cells. This can lead to antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) or, in the case of antibody-conjugates, enable improvement of the therapeutic index for a particular cytotoxic agent (chemotherapeutic, toxin, or radionuclide) by targeting that agent to the tumor while simultaneously reducing systemic exposure [10].
T cell-based immunotherapies involve either vaccines aimed at activating tumor reactive T cells in vivo or adoptive transfer of T cells stimulated ex vivo [11]. Such therapies can be targeted against any protein selectively made by a tumor cell. T cells may not only kill tumors through direct cytotoxicity, but can also recruit a vast array of effector cells, like NK cells, macrophages, and neutrophils, which in turn kill tumor cells by production of reactive oxygen species, granzymes, and death receptor ligands [12]. At present, targeting cytotoxic T lymphocytes (CTLs) at PCa is a preferred strategy in experimental immunotherapy.
Effective T cell-based therapy of PCa critically depends on the identification of appropriate target antigens. To be used for the therapeutic activation of CTLs, the antigens have to fulfil two qualifications. First, when activating patient's CTLs against tumor antigens, induction of strong autoimmune reactions against vital organs is a major concern. To reduce the probability of such harmful side effects, a careful selection of the target antigens based on tissue restricted expression is required. In the case of PCa, these antigens might also be expressed in normal prostate tissue, and can therefore be classified as tissue-specific antigens (TSAs). Second, for an increased cytotoxic efficiency, the target antigens must be expressed on the tumor cells at a sufficient concentration.
In addition to well-established prostate TSAs like PSA [13], prostatic acid phosphatase (PAP) [14] and prostate specific membrane antigen (PSMA) [15], a number of novel prostate specific proteins have recently been described (reviewed in [16]). Nevertheless, currently there is no study available that compares the tissue restriction and the expression levels of these TSAs. In this study, we determine by quantitative real-time PCR the expression levels and tissue-specificities of candidate prostate antigens, which might be helpful when choosing TSAs for immunotherapy of PCa.
A number of antigens that had been published as prostate-associated gene products were not included in this study since they have already been described as being either not specific for prostate tissues, down-regulated in PCa or seem to represent non-coding RNAs.
Section snippets
Prostate cDNA synthesis and cloning of PCR products
The cDNA synthesis was performed using 1 μg of normal prostate total RNA (BD Biosciences, Clontech, Palo Alto, USA) and random hexamer primers (Advantage™ RT-for-PCR Kit; BD Biosciences, Clontech) according to the manufacturers protocol. PCR was carried out by using 3 μL of the cDNA as template. Amplification was performed using Titanium Taq polymerase (BD Biosciences, Clontech) according to the manufacturers protocol. The specific primers (MWG-BIOTECH AG, Ebersberg, Germany) for each prostate
Selection of prostate tissue-specific antigens
The TSA candidates selected to be included in this study had to be well characterised prostate TSAs. In addition they had to meet at least one of the following criteria: (a) being used in clinical studies, (b) containing an immunogenic T cell epitope or (c) being up-regulated in PCa.
Specificity and level of expression were determined for the following prostate TSAs (Table 1): PSA, prostate stem cell antigen (PSCA) [17], prostate specific G-protein coupled receptor (PSGR) [18], [19], PAP,
Discussion
An important concept of tumor immunotherapy, either T cell-based or antibody-mediated, is that a potential target antigen has to be specifically overexpressed by the target cells. Even a protein expressed in other normal tissues may serve as an immunotherapy target if the normal cells that express it are expendable [23], like normal prostate cells in the case of PCa. As the list of prostate TSAs grows, it might be important to quantify their expression and to discuss the complexity of an
Acknowledgement
We thank Sandra Schwind for the excellent technical assistance. This study was supported by the Deutscher Akademischer Austausch Dienst (to A. Cunha), by the Wihelm-Vaillant-Stiftung, Maistrasse 11, 80377 Muenchen (to B. Weigle) and by the BMBF (NBL3, grant 9980011/18862) (to M. Bachmann).
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Both authors contributed equally to this work.