A continuing problem in the treatment of prostate cancer (PCa) has been how best to deal with the significant number of patients who have either abnormally high prostate specific antigen (PSA) but negative biopsies or, alternatively, positive biopsies but low Gleason scores. An active surveillance protocol is most commonly followed in these cases but recent data indicates that, despite this cautious approach, up to 40% of these patients can eventually develop full-blown PCa. Thus, there is a strong need to develop more effective prevention strategies for this growing patient population. PAGE4 (prostate-associated gene 4) is a cancer testis antigen protein that has been shown to play an important role in PCa disease progression, suggesting that it may be a suitable target for chemo-preventative approaches. Expression of PAGE4 occurs specifically in the prostate. While its levels are very low in healthy adults, it is highly expressed in patients with frank (organ confined) PCa. Moreover, its expression increases in lesions affected by inflammation, indicating possible involvement in the stress response. Other stress-activated processes such as the JNK-1/c-Jun pathway also appear to play a role in the initiation of PCa. Our preliminary results link PAGE4 to this pathway. In particular, PAGE4 is a regulator of c-Jun transcriptional activity in vivo and interacts with c-Jun in vitro. We hypothesize that disrupting PAGE4/c-Jun interactions will provide a novel target for development of PCa chemo- preventative strategies. Our objectives are as follows: 1. Determine the PAGE4/c-Jun binding interface. The three-dimensional structure of the PAGE4/c-Jun complex will be characterized using NMR spectroscopy, identifying the contact residues involved at the binding interface in detail. The structural result will be used as the basis for validating the complex as a target for rational drug design in Aim 2. Our preliminary results indicate that such structural studies are feasible. 2. Validate the PAGE4/c-Jun interaction as a candidate for therapeutic development. The structural information obtained in Aim 1 will be used to generate residue-specific mutants of c-Jun that disrupt the binding interface. These mutants will be employed in vivo in a cell-based c- Jun transactivation assay to establish whether the PAGE4/c-Jun complex can be used as a drug target. Interfacial mutants with down-regulation of c-Jun transcriptional activity in a PCa cell line will validate the PAGE4/c-Jun complex as a therapeutic candidate. 3. Set up a high throughput in vitro assay to detect PAGE4/c-Jun binding. An in vitro assay for PAGE4/c-Jun binding will be developed using full-length PAGE4 and fluorescently labeled synthetic c-Jun peptides that correspond to the region necessary for interaction. The data obtained in Aims 1 and 2 will be used to identify candidate c-Jun peptides. The tightest binding c-Jun peptide obtained in Aim 3 will be used in a fluorescence-based assay to enable high throughput screening of compounds that might disrupt the PAGE4/c-Jun interaction. We do not anticipate compound screening within the R21 timeline, however.
A continuing problem in the treatment of prostate cancer (PCa) has been how best to deal with the significant number of patients who have either abnormally high prostate specific antigen (PSA) but negative biopsies or, alternatively, positive biopsies but low Gleason scores. Prostate-associated Gene 4 (PAGE4), a stress- response protein that interacts with the proto-oncogene c-Jun and is upregulated in PCa, is identified as a putative candidate for PCa chemoprevention in this patient population. The idea that PAGE4 interacts with c- Jun to regulate its transcriptional activity and that this activity may be disruped with small molecules is a completely novel concept that, if confirmed, will have a significant impact on treating and managing PCa.
