Hypoxia has been associated with malignant progression, metastasis and resistance to therapy. Hence, we studied expression of hypoxia–regulated genes in 100 prostate cancer (CaP) bulk tissues and 71 adjacent benign tissues. We found 24 transcripts significantly overexpressed (p≤0.02). Importantly, higher transcript levels of disc large (drosophila) homolog-associated protein 5 (DLGAP5)/discs large homolog 7 (DLG7)/hepatoma up-regulated protein (HURP), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with higher Gleason score and more advanced systemic progression. Since the products of HMMR and CCNB1 have been identified recently as molecular markers of CaP progression, we postulated that DLG7 has prognostic value too. To test this hypothesis, we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within six years of surgery) and controls (no progression within eight years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CI, 0.68 to 0.8). Overall, our data indicate that expression of DLG7, a hypoxia-controlled gene, holds prognostic potential in high-risk CaP; this also demonstrates that variation of oxygen tension may constitute a tool for identification of novel biomarkers for CaP.

Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents the main cause of cancer-related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by the resistance of advanced tumors. So far, the mechanisms governing tumor resistance to radiotherapy are not discussed in detail. Here, we demonstrate for the first time that the resistance of PCa to radiation therapy is attributed to elevated expression of Hepatoma Up-Regulated Protein (HURP). In PCa cells, the induction of HURP expression suppresses γ-irradiation-induced apoptosis. γ-irradiation-induced apoptosis of PCa cells is associated with expression of E2F1, p53, p21 proteins together with the phosphorylation of apoptosis signal-regulating kinase1 (ASK1), c-jun-N-terminal kinase (JNK) and Ataxia-telangiectasia mutated (ATM) and histone family member X (H2AX). Whereas, the induction of HURP expression is able to suppress γ-irradiation-induced effects on E2F1, p53, p21, ATM, ASK1, JNK and ATM, and H2AX. Also, inhibition of γ-irradiation-induced- cytochrome c release, cleavage of caspase-9, caspase-3, PARP, and reactive oxygen species (ROS) were noted in PCa cells induced for HURP expression. The observed radio-resistance of PCa is thought to be the consequence of HURP-mediated destabilization of p53 and ATM proteins that are essential for the modulation of γ-irradiation-induced apoptosis. Thus, based on our findings, PCa resistance to radiation therapy results from the deregulation of ASK1/ JNK; ATM/ H2AX; ATM/p53 and checkpoint kinase 2 (Chk2)/ E2F-1 in response to the elevated expression of HURP.

BackgroundUse of allogeneic cancer cells-based immunotherapy for treatment of established prostate cancer (PCa) has only been marginally effective. One reason for failure could stem from the mismatch of antigenic signatures of vaccine cells and cancer in situ. Hence, it is possible that vaccine cells expressed antigens differently than tumor cells in situ. We hypothesized that cells grown in vitro at low oxygen tension (pO2) provide a better antigen match to tumors in situ and could reveal a more relevant antigenic landscape than cells grown in atmospheric pO2.MethodsWe tested this hypothesis by comparing PCa cells propagated at pO2 = 2 kPa and 20 kPa. To identify potential tumor-associated antigens (TAAs), we prepared PCa cell lysates, resolved them by two-dimensional electrophoresis and immunoblotting using spontaneous antibodies from plasma derived from PCa patients and control subjects. Antibody-labeled spots were analyzed by MALDI-TOF mass spectrometry and validated by ELISA. We selected hypoxia-regulated HSP70 and hnRNP L and hypoxia-independent HSP60 and determined the frequency of plasma samples reacting with these molecules.ResultsFrequency of HSP60-reactive plasma was low in healthy controls [1.3 % (1/76)], while it was elevated in PCa patients [13.0 % (7/54); p < 0.05]. These data suggest a humoral immune response to HSP60 in PCa. Levels of autoantibodies to HSP70 did not differ from healthy controls [3.7 % (2/54)] in PCa patients [5.3 % (2/38)]. Similarly, hnRNP L autoantibodies did no differ between healthy controls [6.1 % (3/49)] and PCa patients [5.3 % (2/38)].ConclusionsOverall our results suggest the value of hypoxia as a modifier of the cellular and antigenic landscape of PCa cells. By modifying the immune reactivity of PCa cells in culture, manipulation of pO2 can be proposed as a new avenue for improving diagnosis, prognosis and immunotherapy for PCa.

2028 Background and Objectives: The HPA-1 polymorphism of αIIbβ3 arises from a Leu→Pro exchange at residue 33 of the β3 subunit resulting in HPA-1a (Leu33) or HPA-1b (Pro33). We have documented that patients with coronary artery disease who are carriers of HPA-1b (Pro33) experience their myocardial infarction 5.2 years earlier than HPA-1a/1a (Leu33) patients (J Thromb Haemost 2005; 3: 1522). Based on these observations, it has been postulated that HPA-1b (Pro33) is a prothrombotic variant of αIIbβ3. We have now generated a model overexpressing fluorescent proteins fused with αIIbβ3 in transfected HEK293 cells. Methods: :A yellow protein (YFP) and a cyan fluorescent protein (CFP) were cloned to the C-termini of the β3 and αIIb subunits prior to transfection of HEK293 cells, subsequently expressing the fusion proteins of both HPA-1 isoforms. Using flow cytometry, Western blotting and specific antibodies directed against αIIb or β3, we identified 12 HPA-1a and 11 HPA-1b positive clones. For further experiments only those cell lines expressing equal amounts of fluorescent fusion proteins, i.e. a 140 kD αIIb-CFP and a 113 kD β3-YFP, were used. Results: Functional integrity of both integrin variants and proper membrane insertion were documented by intact activation of transfected HEK293 cells through G protein-coupled receptors with organic acid (1-stearoyl-2-arachidonoyl-sn-glycerol) or direct phorbol 12-myristate 13-acetate-induced stimulation of protein kinase C and by specific binding of Alexa488 fibrinogen to αIIbβ3 in response to inside-out signaling. In the presencence of pertussis toxin or abciximab, activation or ligand binding of αIIbβ3 were completely (>98%) inhibited in both isoforms. Activation of αIIbβ3 stimulates the tyrosine kinase Src, constitutively associated with the the β-subunit of the integrin. To determine whether αIIbβ3-dependent outside-in signaling is responsible for a polymorphism-related modulation, we performed adhesion experiments under static conditions with fibrinogen (50 μg/ml) in the absence or presence of Mn2+ (0.5 mM). Specific activation of the phosphotyrosine motif (Src-pY418), as determined by Western blotting and quantified by densitometry (ratio of Src-pY418/total Src), was 15 + 1.5% higher in HPA-1b than HPA-1a cells in the presence of Mn2+ (n=6 independent experiments, p<0.01). To explore the molecular nature of this difference in terms of putative changes in the allostery of integrin αIIbβ3 with regard to the HPA-1 polymorphism, dynamic measurements were performed using fluorescence resonance energy transfer (FRET). The relative decrease in FRET signal, indicating spatial separation of the cytoplasmic tails of the α- and β-subunit as a consequence of integrin activation, was recorded every minute over 0.5 hrs in transfected HEK293 cells adherent onto fibrinogen. At every time point, the kinetic measurements revealed a significantly faster and more distict (> 5%) decrease in HPA-1b than in HPA-1a cells under static adhesion (p<0.009). Upon exposure of adherent HEK293 cells to increasing shear rates (stepwise elevation from 50 to 1600 sec-1 by doubling the initial shear rate every minute), the spatial separation of the integrin subunits occurred significantly faster and more distinct (> 10%) in HPA-1b (Pro33) than HPA-1a (Leu33) cells in response to shear (p<0.0014). Under the same conditions, the rate of HPA-1b cells still adherent onto immobilized fibrinogen was 80%, while the relative number of residual HPA-1a cells decreased to 20% upon exposure to 1600 sec-1 (p<0.0001). These displacement experiments suggest that the HPA-1b (Pro33) variant is more resistant to biomechanical stress than the HPA-1a (Leu33) isoform. Conclusions: Our findings suggest that the HPA-1 polymorphism can have a significant impact on the activation of αIIbβ3. This is evident from a higher outside-in signaling and a higher resistance to biomechanical stress upon exposure to increasing shear of HPA-1b (Pro33) in comparison with HPA-1a (Leu33) transfectants. The difference in spatial separation of the cytoplasmic tails of the integrin in response to activation, as demonstrated by FRET analyses under static and flow dynamic conditions, reflects allosteric changes that may contribute to the prothrombotic phenotype of the HPA-1b (Pro33) variant. Disclosures: No relevant conflicts of interest to declare.