The expression of prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA), two well characterized marker proteins, remains highly active in the hormone refractory stage of prostate cancer. In this study, an artificial chimeric enhancer (PSES) composed of two modified regulatory elements controlling the expression of PSA and PSMA genes was tested for its promoter activity and tissue specificity using the reporter system. As a result, this novel PSES promoter remained silent in PSA- and PSMA-negative prostate and non-prostate cancer cell lines, but mediated high levels of luciferase in PSA- and PSMA-expressing prostate cancer cell lines in the presence and absence of androgen. To determine whether PSES could be used for in vivo gene therapy of prostate cancer, a recombinant adenovirus, Ad-PSES-luc, was constructed. Luciferase activity in prostate cancer cell lines mediated by Ad-PSES-luc was 400- to 1000-fold higher than in several other non-prostate cell lines, suggesting the high tissue-specificity of the PSES promoter in an adenoviral vector. Finally, recombinant virus Ad-PSES-luc was injected into mice to evaluate the tissue-discriminatory promoter activity in an experimental animal. Unlike Ad-CMV-luc, the luciferase activity from systemic injection of Ad-PSES-luc was fairly low in all major organs. However, when injected into prostate, Ad-PSES-luc drove high luciferase activity almost exclusively in prostate and not in other tissues. Our results demonstrated the potential use of PSES for the treatment of androgen-independent prostate cancer patients.

PSES is a chimeric enhancer containing enhancer elements from prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) genes that are prevalently expressed in androgen-independent prostate cancers. PSES shows strong activity equivalent to cytomegalovirus (CMV) promoter, specifically in PSA/PSMA-positive prostate cancer cells, the major cell types in prostate cancer in the absence of androgen. We developed a recombinant adenovirus (AdE4P-SESE1a) by placing adenoviral E1a and E4 genes under the control of the bidirectional enhancer PSES and enhanced green fluorescent protein gene for the purpose of intratumoral virus tracking under the control of CMV promoter. Because of PSES being very weak in nonprostatic cells, including HEK293 and HER911 that are frequently used to produce recombinant adenovirus, AdE4PSESE1a can only be produced in the HER911E4 cell line which expresses both E1 and E4 genes. AdE4PSESE1a showed similar viral replication and tumor cell killing activities to wild-type adenovirus in PSA/PSMApositive prostate cancer cells. The viral replication and tumor cell killing activities were dramatically attenuated in PSA/ PSMA-negative cells. To test whether AdE4PSESE1a could be used to target prostate tumors in vivo, CWR22rv s.c. tumors were induced in nude mice and treated with AdE4PSESE1a via intratumoral and tail vein injection. Compared to tumors treated with control virus, the growth of CWR22rv tumors was dramatically inhibited by AdE4PSESE1a via tail vein injection or intratumoral injection. These data show that adenoviral replication can be tightly controlled in a novel fashion by controlling adenoviral E1a and E4 genes simultaneously with a single enhancer. (Cancer Res 2005; 65(5): 1941-51)

No abstract

Prostate cancer is the second most commonly diagnosed cancer in men and accounts for significant mortality and morbidity in the United States. Initially androgen-dependent, prostate cancer ultimately becomes androgen-independent, which makes the disease extremely difficult to cure. In this study, we examined the use of conditionally replication-competent adenovirus for the treatment of hormone-independent prostate cancer. We utilized PSME, an enhancer element for prostate-specific PSMA expression, to control viral E1A protein expression and achieve exclusive virus replication in prostate. Western blotting confirmed that PSME mediated high E1A protein expression in PSMA-positive, androgen-independent prostate cancer cells (C4-2 and CWR22rv), but was much less active in PSMA-negative cancer cells (PC-3 and A549). Consistent with E1A protein expression, the recombinant adenovirus Ad5-PSME-E1a replicated in C4-2 and CWR22rv almost as efficiently as wild type with low levels of androgen, but its replication was significantly attenuated in PSMA-negative cells. In the in vitro killing assay, Ad5-PSME-E1a lysed all C4-2 and CWR22rv cells 5 days after infection, with minimal effect on PSMA-negative cells. In addition, injections of 1.7 x 10(8) plaque-forming units in a CWR22rv xenograft model in nude mice induced significant tumor growth delay, with a substantial necrotic area. These studies suggest that PSME-driven replication-competent adenovirus may be a new therapeutic modality for prostate cancer patients after hormone ablation therapy.