Advances in Prostate Cancer Immunotherapies

Prostate cancer is a major cause of mortality in men in the Western world.Although treatment of early stage prostate cancer with radiation therapy or prostatectomy is efficient in most cases, some patients develop a fatal hormone-refractory disease. Treatments in this case are limited to aggressive chemotherapies, which can reduce serum prostate-specific antigen (PSA) levels in some patients. Taxane- and platinum-compound-based chemotherapies produce a survival benefit of only a few months. Therefore, it is crucial to develop novel, well tolerated treatment strategies. Over the past years, immunotherapy of hormone-refractory prostate cancer has been studied in numerous clinical trials. The fact that the prostate is a non-essential organ makes prostate cancer an excellent target for immunotherapy. Administration of antibodies targeting the human epidermal growth factor receptor-2 or the prostate-specific membrane antigen led to stabilisation of PSA levels in several patients. Vaccination of prostate cancer patients with irradiated allogeneic prostate cell lines has demonstrated that whole cell-based vaccines can significantly attenuate increases in PSA. Two different recombinant viral expression vectors have been applied in prostate cancer treatment: poxvirus and adenovirus vectors. Both vaccines have the advantages of using a natural method to induce immune responses and achieving high levels of transgene expression. Vaccinia viruses in combination with recombinant fowlpox or canarypox virus have been used to express recombinant PSA. Several studies demonstrated that this approach is safe and can lead to stabilisation of PSA values. A very promising approach in prostate cancer immunotherapy is vaccination of patients with dendritic cells. Thereby, peptides, recombinant proteins, tumour lysates or messenger RNA have been used to deliver antigens to autologous dendritic cells. Loading of dendritic cells with up to five different peptides derived from multiple proteins expressed in prostate cancer demonstrated that cytotoxic T-cell responses could be elicited in prostate cancer patients. Sipuleucel-T (APC8015), an immunotherapy product consisting of antigen-presenting cells, loaded ex vivo with a recombinant fusion protein consisting of prostatic acid phosphatase linked to granulocyte-macrophage colony-stimulating factor, demonstrated in a phase III, placebo-controlled trial n improvement in median a time to disease progression. The improvement in overall survival was 4.5 months for sipuleucel-T-treated patients compared with the placebo group. Although there is a minor increase in overall survival of metastatic prostate cancer patients with some approaches, more effective therapeutic strategies need to be developed

The unique functions of tissue-specific proteasomes

The 26S proteasome is the main protease in eukaryotes. Proteolysis occurs within the cylindrical 20S proteasome that is constitutively expressed in most tissues. However, three tissue-specific versions of the 20S proteasome have been discovered to date. The immunoproteasome is optimized to process antigens and it directs the differentiation of T helper (Th) cells. The thymoproteasome is selectively expressed in cortical epithelial cells of the thymus where it plays an essential role in the positive selection of T lymphocytes. Finally, the spermatoproteasome is found in the testes where it is required during spermatogenesis. Here, we outline how tissue-specific proteasomes adapt to functional needs in their respective tissues and how their selective inhibition may be used to interfere with autoimmune diseases and cancer

No essential role for tripeptidyl peptidase II for the processing of LCMV-derived T cell epitopes

The proteasome is critically involved in the production of MHC class I-restricted T cell epitopes. Approximately 20% of all peptides generated by the proteasome are too large for direct presentation by MHC class I molecules. Reits et al. (Immunity 2004. 20:495–506) suggested that a major portion of proteasomal products are larger than 15 amino acids and require further degradation by the tripeptidyl peptidase II (TPPII) before becoming ligands of MHC class I molecules. Using the well-characterized lymphocytic choriomeningitis virus (LCMV) model, the role of TPPII in the processing of several LCMV-derived T cell epitopes was investigated. In contrast to Reits' proposal, TPPII inhibition and TPPII overexpression experiments revealed that five out of six LCMV-derived CD8+ T cell epitopes were not affected by inhibition of TPPII, while one epitope (GP276) was slightly reduced upon TPPII overexpression. Additionally, we demonstrated that the processing of two epitopes derived from ovalbumin and murine cytomegalovirus were not altered by TPPII inhibition. We propose that TPPII is not generally required for the production of MHC class I peptides, but the presentation of some peptides can be negatively affected by TPPII

Prostaglandin E2 inhibits IL-23 and IL-12 production by human monocytes through down-regulation of their common p40 subunit

The heterodimeric cytokine IL-23 is important for the maintenance of Th17 cells, which are pivotal mediators of autoimmune diseases like rheumatoid arthritis, colitis, and multiple sclerosis. Prostaglandin E2 (PGE2) is a soluble regulator of inflammation that has both pro- and anti-inflammatory properties. PGE2 has been shown to elevate the IL-23 production by dendritic cells (DC). Monocytes are also producers of IL-23 but the effect of PGE2 on IL-23 production by human monocytes has hardly been investigated. We show here that PGE2 blocks the production of IL-23 by LPS-stimulated monocytes in an IL-10 and IL-1 independent manner. This effect was due to the down-regulation of the p40 subunit of IL-23 on mRNA and protein level. The p40 subunit is shared by IL-12 and, consistently, PGE2 also lowered the IL-12 production by monocytes. These effects of PGE2 were cAMP-dependent since the cAMP enhancer forskolin strongly reduced IL-23 and IL-12 production by monocytes. Taken together, PGE2 acts in an anti-inflammatory manner by lowering IL-23 production by monocytes while it has the opposite effect in DC. Our data may help to reconcile controversial point of views on the pro- and anti-inflammatory nature of PGE2 by making a strong case for a cell type-dependent function

Investigations into the auto-FAT10ylation of the bispecific E2 conjugating enzyme UBA6-specific E2 enzyme 1

The cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) targets its substrates for degradation by the proteasome. FAT10 is conjugated to its substrates via the bispecific, ubiquitin-activating and FAT10-activating enzyme UBA6, the likewise bispecific conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By MS analysis, we found that USE1 undergoes self-FAT10ylation in cis, mainly at Lys323. Mutation of Lys323 to an arginine did not abolish auto-FAT10ylation of USE1, but every other lysine could instead be modified with FAT10. Similarly to bulk FAT10 substrates, FAT10ylation of USE1 accelerated its proteasomal degradation. Interestingly, the USE1–FAT10 conjugate continued to be an active E2 enzyme, because both FAT10 and ubiquitin could still be thioester-linked to the USE1–FAT10 conjugate. We therefore suggest that the major function of USE1 auto-FAT10ylation is to serve as a negative feedback mechanism to limit the conjugation of FAT10 upon its cytokine-mediated induction by reducing the amount of USE1 through proteasomal degradation of the USE1–FAT10 conjugate

PLGA microspheres for improved antigen delivery to dendritic cells as cellular vaccines

Dendritic cells (DC) are currently employed as cellular vaccines in clinical trials of tumor immunotherapy. In most trials, peptide epitopes derived from tumor antigens are being exogenously loaded onto human DC for binding to MHC class I molecules. While this is a convenient method, it suffers from the drawback that the persistence of class I/peptide complexes on the cell surface is in the order of a few hours. This drawback limits the success of vaccination. We have investigated biodegradable poly(D,L-lactide-co-glycolide) microspheres (PLGA-MS) as delivery tools for antigen loading of human monocyte-derived DC (hMoDC). Immature hMoDC readily take up PLGA-MS and present epitopes from encapsulated proteins or peptides both on MHC class I and class II. Interestingly, antigen presentation by hMoDC was markedly prolonged when hMoDC were charged with PLGA-MS-encapsulated as opposed to soluble antigens. The properties of hMoDC with respect to migration, cytokine secretion, survival and allostimulation were not adversely affected by the uptake of PLGA-MS. In this article, we will review the properties of PLGA-MS as an adjuvant and summarize recent data on their potential for antigen delivery to dendritic cells

FAT10ylation as a signal for proteasomal degradation

The Nobel prize has been awarded for the discovery of ubiquitin as a transferable signal for the degradation of proteins by the 26S proteasome. While isopeptide linkage of a protein with a single ubiquitin does not serve as a degradation signal for the proteasome, poly-ubiquitylation via several different lysine residues within ubiquitin leads to efficient proteasomal degradation. Ubiquitin-like modifiers have not been shown to directly mediate proteasomal degradation except for the cytokine inducible modifier HLA-F adjacent transcript 10 (FAT10), which consists of two ubiquitin-like domains. FAT10 ends with a free diglycine motif at its C-terminus which is required for isopeptide linkage to hundreds of different substrates. In contrast to ubiquitin, a single FAT10 suffices to bind to the 26S proteasome and to efficiently mediate proteasomal degradation in a ubiquitin-independent manner. Here we review the data on ubiquitin-independent degradation by FAT10, on how FAT10 is conjugated to its substrates, how FAT10 binds to the 26S proteasome, and how the ubiquitin-like (UBL)-ubiquitin-associated (UBA) protein NUB1L accelerates FAT10 mediated proteolysis. Finally, with a glimpse on recently identified substrates, we will discuss the currently emerging knowledge about the biological functions of FAT10. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System

The Proteasome, a New Target for Cancer Therapy

The proteasome is a multicatalytic protease and the principal non-lysosomal proteolytic system in all eukaryotic cells. It plays a central role in virtually all regulatory pathways as for instance cell-cycle regulation, differentiation, and apoptosis. The proteasome degrades regulatory proteins and their inhibitors and, thus is an interesting target for therapeutic drugs. Inhibitors of the proteasome are small molecules that function by stabilizing various proteins, including cell-cycle regulators, tumor suppressors and growth factors. Because proteasome inhibition blocks cellular proliferation and induces apoptosis, these agents have been tested as anticancer drugs in tumor models and have shown impressive potential. In addition, treatment with proteasome inhibitors can sensitize cells to other cancer treatments like radio- or chemotherapy. This review introduces the ubiquitin-proteasome pathway and outlines the recent progress in the development of proteasome inhibition as a treatment option for clinical cancer therapy.publishe

The immunoproteasome subunit LMP7 is required in the murine thymus for filling up a hole in the T cell repertoire

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible variant of the proteasome which has been implicated in regulating inflammatory responses and antigen presentation. In the thymus, medullary thymic epithelial cells (mTECs) and cortical thymic epithelial cells (cTECs) do express different proteasome subunits exerting chymotrypsin-like activities suggesting distinct functions in thymic T cell selection. Employing the lymphocytic choriomeningitis virus (LCMV) infection model, we could show that the immunoproteasome subunit LMP7 was absolutely required for the generation of LCMV GP118-125 -specific T cells although the class I mediated presentation of GP118-125 was not dependent on LMP7. Using bone marrow chimeras and adoptive transfer of LMP7-deficient CD8(+) T cells into RAG1-deficient mice we show that LMP7-deficient mice lacked GP118-125 -specific T cell precursors and that LMP7 was required in radioresistant cells - most likely thymic epithelial cells - to enable their selection. Since LMP7 is strongly expressed in negatively selecting mTECs but barely in positively selecting cTECs our data suggest that LMP7 was required to avoid excessive negative selection of GP118-125 -specific T cell precursors. Taken together, this study demonstrates that the immunoproteasome is a crucial factor for filling up holes within the cytotoxic T cell repertoire.publishe

The ubiquitin-like protein FAT10 forms covalent conjugates and induces apoptosis

FAT10 is a ubiquitin-like protein that is encoded in the major histocompatibility complex class I locus and is synergistically inducible with interferon-gamma and tumor necrosis factor alpha. The molecule consists of two ubiquitin-like domains in tandem arrangement and bears a conserved diglycine motif at its carboxyl terminus commonly used in ubiquitin-like proteins for isopeptide linkage to conjugated proteins. We investigated the function of FAT10 by expressing murine FAT10 in a hemagglutinin-tagged wild type form as well as a diglycine-deficient mutant form in mouse fibroblasts in a tetracycline-repressible manner. FAT10 expression did not affect major histocompatibility complex class I cell surface expression or antigen presentation. However, we found that wild type but not mutant FAT10 caused apoptosis within 24 h of induction in a caspase-dependent manner as indicated by annexin V cell surface staining and DNA fragmentation. Wild type FAT10, but not its diglycine mutant, was covalently conjugated to thus far unidentified proteins, indicating that specific FAT10 activating and conjugating enzymes must be operative in unstimulated fibroblasts. Because FAT10 expression causes apoptosis and is inducible with tumor necrosis factor alpha, it may be functionally involved in the programmed cell death mediated by this cytokine