-edited) The goal of this National Action Plan on Breast Cancer Innovative Small Grant Program application is to identify a new gene which may be very important in the etiology and susceptibility of women to breast cancer. Also, this gene may have a critical role in the progression of estrogen dependent (ER positive) tumors to autonomy (ER negative). During the course of studies funded by US ARMY Breast Cancer Program grant, the investigator discovered that the well described plasma glycoprotein, sex-hormone-binding globulin (SHBG), is an inhibitor of estrogen-dependent breast cancer cell growth in serum-free defined culture, and that physiologic concentrations of estrogens reverse the inhibition to restore optimum growth. The role for SHBG is entirely new. Prior to this work, it was known only that SHBG was the plasma transport protein for sex hormones and that sex hormone target tissues had high affinity binding sites for SHBG. The innovation of this work is not only the discovery of a new SHBG regulatory function, but also the contribution of a new framework for study of negative regulation of breast tissue. The loss or impairment of this control might well be expected to increase susceptibility to breast cancer. The applicant plans to isolate the SHBG receptor by methods that include (i) lectin affinity chromatography (ii) chemical crosslink of a fluorescence labeled SHBG peptide to the receptors, (iii) biotinylation of SHBG followed by binding to the receptors and isolation of the SBBG-receptor complexes with avidin-Sepharose, (iv) immunoprecipitation of SHBG-receptor complexes with antibody against SHBG, and (v) affinity chromatography using SHBG-Sepharose. The receptor preparations will be further purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electroelution, the receptor will be trypsin treated, the fragments separated by reverse phase high pressure liquid chromatography and their amino acid sequences established by microsequencing methods. Oligonucleotide probes and antibodies will be generated from these data for cDNA cloning from human breast cancer cell libraries. The receptor cDNA will be sequenced to deduce the full amino acid sequence. Using the cDNA as probe, the investigator plans to seek a genomic clone. Finally, using 125I-SHBG 32P-labeled cDNA and serum- free tissue culture methods, the investigator will see if ER negative cells have lost SHBG control due to the absence of receptor or because a defective receptor is expressed. With the genetic tools developed in this program, the applicant plans in the future to initiate a larger collaborative program with investigators studying genetic susceptibility to breast cancer and to develop diagnostic methods to better differentiate between estrogen responsive and autonomous tumors.
