The overall objective of these efforts is to understand resistance to antiestrogen (AES) therapy by estrogen receptor (ERc)-positive breast cancers. In particular, these efforts focus on the relationships between elevated antiestrogen binding sites (AESBS) activity, the resulting restriction of AES from ERc (referred to as AES appropriation) and AES resistance. These efforts utilize normal tissues with low and high AESBS activity (uterus vs. liver), as well as AES-sensitive MCF-7 breast cancer cells with low AESBS activity and AES-resistant LY-2 cells with high AESBS activity. First, different treatment agents will be used to understand how factors that control AESBS activity affect AES appropriation by examining induction, negative regulation, changes in molecular capacity, affinity, cooperativity and catalytic function. Size exclusion HPLC will be used to simultaneously assess AES binding to both AESBS and ERc. TLC will evaluate AES appropriation by determining the degree to which AES associates with ERc in the presence of [3H]-estradiol. SDS-PAGE will be used to examine the capacity of the AESBS holomer in terms of subunit activity. The potential for reversing resistance due to AES appropriation will be examined by evaluating the effect on LY-2 cell proliferation of several agents that reduce AESBS binding without interfering with ERc binding. Second, these same techniques will be used to determine the contribution of AESBS structure to binding activity by characterizing individual subunit activity, subunit stoichiometry, the relationship of ligand occupancy to subunit assembly, the protease-mapped structure of AESBS subunits and the involvement of components that do not bind ligand in the AESBS holomer in order to see if differences exist between AESBS from low and high activity sources. Third, AESBS activity will be characterized in human breast tumors in order to correlate AESBS activity to tamoxifen resistance, and comparisons will be made to other prognostic indicators including: steroid receptor activity, DNA-ploidy, S-phase fraction, HER-2/neu oncogene expression and pathological classification. These analyses will include specimens from ERc-positive tumors not treated with tamoxifen and ERc- negative tumors so that recurrence and survival in general can be isolated. Flow cytometry will be used in ploidy/S-phase analyses, while HER-2/neu oncogene expression will be determined by immunohistochemistry. Efforts proposed here are based on a straightforward mechanism of therapeutic incapacitation through AES appropriation mediated by AESBS. This mechanism allows estrogen sensitivity to persist and suggests a strategy for reversing resistance by moderating AESBS activity so that AES can act through the ERc. This work is significant because it will develop an understanding of AESBS activity and structure, lead to the reversal of resistance and define the role of AESBS in human breast cancer.
| Pavlik, E J; Nelson, K; Srinivasan, S et al. (1992) Resistance to tamoxifen with persisting sensitivity to estrogen: possible mediation by excessive antiestrogen binding site activity. Cancer Res 52:4106-12 |
