Background: While androgens have historically been considered to be the major sex steroid involved in the development, growth regulation, and pathobiology of the prostate, it is becoming increasingly evident that estrogens may also influence both normal and abnormal growth processes in the gland. We have found that the action of estrogen in the human prostate is mediated by a receptor known as estrogen receptor (ER)-2. This receptor may play a dual role in the gland. First, it appears to protect the gland from oxidative damages and inhibit aberrant growth. In this respect, it serves as a tumor suppressor. We made the novel observation that expression of ER-b is lost as prostate cancer progresses from low to high grade in the gland. However, the receptor may have a different function in metastatic prostate cancers. Both bone and lymph node metastases express high levels of this receptor. Hence, we have postulated that ER-b may promote survival of cancer cells in distance metastastic sites.

Hypothesis/Objectives: We have recently identified a non-genomic mechanism known as DNA methylation, which may play a role in the regulation of ER-b expression. We have data to support the hypothesis that the ER-b gene could be silence when clusters of CGs in the proximal end of the gene are modified by specific enzymes that add a bulky methyl group to the cytosine of the CG dinucleotide. This modification hinders interaction between transcriptional factors and the gene and therefore causes its silencing. In this application, we will use highly sensitive methodology to proofread the proximal region of the ER-b gene and look for methylated Cs in DNA extracted from normal tissues and low- and high-grade cancers. We will use laser capture microscopy to obtain pure population of cells so that we can study microscopic lesions one by one. These detailed and careful studies will reveal for us whether methylation of the CG clusters in the gene is responsible for its silencing. As a corollary goal, we will find out whether re-expression of the receptor could be traced to reversal of the methylation process.

Relevance and Innovation: Non-genomic mechanisms such as the one described above are believed to play important roles in prostate cancer initiation and progression. However, they have been largely understudied. Genes such as ER-b, which may have important regulatory roles in prostate cancer development and progression, warrant careful investigation. This proposal is of high relevance to prostate cancer research because it focuses beyond androgen and genetic changes. Once we build a solid knowledge base on non-genomic regulation of tumor suppressor or promoting genes, we could use a new class of therapeutic agents that targets methylation for prostate cancer prevention and treatment. We now know that DNA methylation could be reversed via a host of therapeutic agents such as 5-aza-2'deoxycytidine, which are nucleoside analogue inhibitors of DNMTs. These agents have been used clinically in the treatment of patients with chronic myeloid leukemia and in those with myelodysplastic syndrome. However, interests in the chemotherapeutic potentials of these drugs have been curbed by their hematopoietic toxicity, which has limited dose intensity. However, recently several experimental alternatives and a U.S. Food and Drug Administration-approved non-nucleoside inhibitor of DNMTs, procainamide, have held great promises. Looking into the future, once we understand how ER-b is regulated by DNA methylation, the use of these drugs may be an option for chemoprevention. Furthermore, the new class of methylation-sense-oligonucleotides that we propose to use in this proposal, if proven successful in re-methylating ER-b in matastatic PCa, could be developed into therapeutic agents for PCa treatment.