The ubiquitin-proteasome system in prostate cancer and its transition to castration resistance.

Prostate cancer is the most common carcinoma in the male population. In its initial stage, the disease is androgen-dependent and responds therapeutically to androgen deprivation treatment but it usually progresses after a few years to an androgen-independent phase that is refractory to hormonal manipulations. The proteasome is a multi-unit protease system that regulates the abundance and function of a significant number of cell proteins, and its inhibition results in cancer cell growth inhibition and apoptosis and is already exploited in the clinic with the use of proteasome inhibitor bortezomib in multiple myeloma. In order to be recognized by the proteasome, a target protein needs to be linked to a chain of the small protein ubiquitin. In this paper, we review the role of ubiquitin-proteasome system (UPS) in androgen receptor-dependent transcription as well as in the castration resistant stage of the disease, and we discuss therapeutic opportunities that UPS inhibition offers in prostate cancer.

The role of ubiquitin-proteasome system in glioma survival and growth.

High-grade gliomas represent a group of aggressive brain tumors with poor prognosis due to an inherent capacity of persistent cell growth and survival. The ubiquitin-proteasome system (UPS) is an intracellular machinery responsible for protein turnover. Emerging evidence implicates various proteins targeted for degradation by the UPS in key survival and proliferation signaling pathways of these tumors. In this review, we discuss the involvement of UPS in the regulation of several mediators and effectors of these pathways in malignant gliomas.

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma.

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.

Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

A recyclable assay to analyze the NH(2)-terminal trimming of antigenic peptide precursors.

The proteasome plays an essential role in the production of MHC class I-restricted antigenic peptides. Recent results have indicated that several peptidases, including tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, could act downstream of the proteasome by trimming NH(2)-terminal extensions of antigenic peptide precursors liberated by the proteasome. In this study, we have developed a solid-phase peptidase assay that allowed us to efficiently purify and immobilize proteasome, tripeptidyl peptidase II, and puromycin-sensitive aminopeptidase. Whereas the first peptidase was active against small fluorogenic peptides, the latter two could also digest antigenic peptide precursors and could be used repeatedly with different precursors. Using three distinct antigenic peptide precursors, we found that tripeptidyl peptidase II never cleaved within the antigenic peptide sequence, suggesting that, aside from its proteolytic activities, it may also play a role in protecting antigenic peptides from complete hydrolysis in the cytosol. This method should be valuable for high throughput screenings of substrate specificity and potential inhibitors.

Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing.

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.

FANCM regulates DNA chain elongation and is stabilized by S-phase checkpoint signalling.

FANCM binds and remodels replication fork structures in vitro. We report that in vivo, FANCM controls DNA chain elongation in an ATPase-dependent manner. In the presence of replication inhibitors that do not damage DNA, FANCM counteracts fork movement, possibly by remodelling fork structures. Conversely, through damaged DNA, FANCM promotes replication and recovers stalled forks. Hence, the impact of FANCM on fork progression depends on the underlying hindrance. We further report that signalling through the checkpoint effector kinase Chk1 prevents FANCM from degradation by the proteasome after exposure to DNA damage. FANCM also acts in a feedback loop to stabilize Chk1. We propose that FANCM is a ringmaster in the response to replication stress by physically altering replication fork structures and by providing a tight link to S-phase checkpoint signalling.

Light-induced degradation of phyA is promoted by transfer of the photoreceptor into the nucleus.

Higher plants possess multiple members of the phytochrome family of red, far-red light sensors to modulate plant growth and development according to competition from neighbors. The phytochrome family is composed of the light-labile phyA and several light-stable members (phyB-phyE in Arabidopsis). phyA accumulates to high levels in etiolated seedlings and is essential for young seedling establishment under a dense canopy. In photosynthetically active seedlings high levels of phyA counteract the shade avoidance response. phyA levels are maintained low in light-grown plants by a combination of light-dependent repression of PHYA transcription and light-induced proteasome-mediated degradation of the activated photoreceptor. Light-activated phyA is transported from the cytoplasm where it resides in darkness to the nucleus where it is needed for most phytochrome-induced responses. Here we show that phyA is degraded by a proteasome-dependent mechanism both in the cytoplasm and the nucleus. However, phyA degradation is significantly slower in the cytoplasm than in the nucleus. In the nucleus phyA is degraded in a proteasome-dependent mechanism even in its inactive Pr (red light absorbing) form, preventing the accumulation of high levels of nuclear phyA in darkness. Thus, light-induced degradation of phyA is in part controlled by a light-regulated import into the nucleus where the turnover is faster. Although most phyA responses require nuclear phyA it might be useful to maintain phyA in the cytoplasm in its inactive form to allow accumulation of high levels of the light sensor in etiolated seedlings.

Processing of tumor-associated antigen by the proteasomes of dendritic cells controls in vivo T-cell responses.

Dendritic cells are unique in their capacity to process antigens and prime naive CD8(+) T cells. Contrary to most cells, which express the standard proteasomes, dendritic cells express immunoproteasomes constitutively. The melanoma-associated protein Melan-A(MART1) contains an HLA-A2-restricted peptide that is poorly processed by melanoma cells expressing immunoproteasomes in vitro. Here, we show that the expression of Melan-A in dendritic cells fails to elicit T-cell responses in vitro and in vivo because it is not processed by the proteasomes of dendritic cells. In contrast, dendritic cells lacking immunoproteasomes induce strong anti-Melan-A T-cell responses in vitro and in vivo. These results suggest that the inefficient processing of self-antigens, such as Melan-A, by the immunoproteasomes of professional antigen-presenting cells prevents the induction of antitumor T-cell responses in vivo.

Oxidized LDL modulates apoptosis of regulatory T cells in patients with ESRD.

Increased levels of oxidized low-density lipoproteins (oxLDL) contribute to the increased risk for atherosclerosis, which persists even after adjusting for traditional risk factors, among patients with ESRD. Regulatory T cells (CD4+/CD25+ Tregs), which down-regulate T cell responses to foreign and self-antigens, are protective in murine atherogenesis, but whether similar immunoregulation occurs in humans with ESRD is unknown. Because cellular defense systems against oxLDL involve proteolytic degradation, the authors investigated the role of oxLDL on proteasome activity of CD4+/CD25+ Tregs in patients with ESRD. CD4+/CD25+ Tregs isolated from uremic patients' peripheral blood, especially that of chronically hemodialyzed patients, failed to suppress cell proliferation, exhibited cell-cycle arrest, and entered apoptosis by altering proteasome activity. Treating CD4+/CD25+ Tregs with oxLDL or uremic serum ex vivo decreased the number and suppressive capacity of CD4+/CD25+ Tregs. In vitro, oxLDL promoted the accumulation of p27Kip1, the cyclin-dependent kinase inhibitor responsible for G1 cell cycle arrest, and increased apoptosis in a time- and concentration-dependent manner. In summary, proteasome inhibition by oxLDL leads to cell cycle arrest and apoptosis, dramatically affecting the number and suppressive capacity of CD4+/CD25+ Tregs in chronically hemodialyzed patients. This response may contribute to the immune dysfunction, microinflammation, and atherogenesis observed in patients with ESRD.

High frequency of functionally active Melan-a-specific T cells in a patient with progressive immunoproteasome-deficient melanoma.

Tumor-reactive T cells play an important role in cancer immunosurveillance. Applying the multimer technology, we report here an unexpected high frequency of Melan-A-specific CTLs in a melanoma patient with progressive lymph node metastases, consisting of 18 and 12.8% of total peripheral blood and tumor-infiltrating CD8+ T cells, respectively. Melan-A-specific CTLs revealed a high cytolytic activity against allogeneic Melan-A-expressing target cells but failed to kill the autologous tumor cells. Loading of the tumor cells with Melan-A peptide reversed the resistance to killing, suggesting impaired function of the MHC class I antigen processing and presentation pathway. Mutations of the coding region of the HLA-A2 binding Melan-A26-35 peptide or down-regulation of the MHC class I heavy chain, the antigenic peptide TAP, and tapasin could be excluded. However, PCR and immunohistochemical analysis revealed a deficiency of the immunoproteasomes low molecular weight protein 2 and low molecular weight protein 7 in the primary tumor cells, which affects the quantity and quality of generated T-cell epitopes and might explain the resistance to killing. This is supported by our data, demonstrating that the resistance to killing can be partially reversed by pre-exposure of the tumor cells to IFN-gamma, which is known to induce the immunoproteasomes. Overall, this is the first report of an extremely high frequency of tumor-specific CTLs that exhibit competent T-cell-effector functions but fail to lyse the autologous tumor cells. Immunotherapeutic approaches should not only focus on the induction of a robust antitumor immune response, but should also have to target tumor immune escape mechanisms.