This application builds on data generated under the auspices of a prior OIG award. Estramustine remains one of the most significant drugs for the combination treatment of hormone refractory prostate cancer. Because of the recent success of estramustine in ovarian cancer, we will expand translational studies to consider this disease. Our past work has defined a dual mechanism of action for the drug, anti-microtubule and estrogenic. We now wish to characterize two genes which may significantly influence the efficacy of the drug. The first gene is a novel ABC transporter, ABC2, which we have shown to be over-expressed in a gene amplified region of chromosome 9q in estramustine resistant cells and serves to sequester the drug into peroxisomes, removed from its primary site of action (microtubules). Peroxisomes are integral into the metabolism of a number of endogenous steroids and are also involved in metabolic detoxification of exogenous drugs. The increase of ABC2 is linked with enhanced peroxisome formation and we wish to determine if this is a general property of drug (and hormone) resistant cells. The second novel gene and its product which will be studied is prostatin, a protein found at high levels in the ventral prostate of rodents. The equivalent human gene(s) have not been described and we will make use of our preliminary data and reagents to carry out a complete characterization of prostatin. Our motivation for this goal is the belief that prostatin will have affinity for estramustine and contribute to the prostate specific effects of the drug. Thus, we wish to clone and characterize the cDNAs for both ABC2 and prostatin and compare and contrast their cellular roles in influencing drug and hormone response.
The specific aims document a series of molecular and protein techniques which will determine the relationship of ABC2 to other transporters, measure binding affinities with drugs, consolidate data showing its peroxisome localization, determine the role of estramustine as peroxisome proliferator, provide biophysical chemical characterization of recombinant protein and assess the consequence of ABC2 gene deletion in the mouse. For prostatin, primary sequence and structure will be compared with other non-receptor ligand binding proteins, subcellular localization will be assessed by confocal fluorescent microscopy, the influence of transfection on drug response will be measured, recombinant protein will be subjected to kinetic drug affinity measurements and biophysical chemical analysis. With the requisite tools generated in the early phases of the project, we will seek to assess the importance of both ABC2 and prostatin as markers/predictors of disease stage and response in clinical prostate and ovarian cancer.
