Estrogen regulates the growth of a proportion of breast cancers through an estrogen receptor (ER) mediated mechanism. Loss of the ER is associated with a loss of estrogen (E2) regulation and a resistance to antiestrogen therapy. Since no effective therapeutic intervention is available to treat the ER negative patient new initiatives are essential to develop a new treatment strategy. One approach would be either to reactivate the ER gene or to develop a targeted """"""""gene therapy"""""""" to re-introduce constitutive production of ER. Our strategic goal is to determine whether the ER could reassert control over the growth of receptor negative breast cancer cells. We are the first group to transfect a breast cancer cell line (MDA-MB-231) with sense and antisense cDNA for both mutant (valine replacing glycine at AA400) and wild type ER. The 8 different cloned cell lines, that constitutively produce ER, were selected by polycistronic production of mRNA for aminoglycoside phosphotransferase which confers resistance in media containing G418. The ER is functional and can activate vit ERETK-CAT however the wild type transfectant is approximately 20 times more sensitive that the mutant transfectant. The ER levels, determined by whole cell uptake of (3 HE-2) are in the range (300-520 fmole/mg protein) of MCF-7 breast cancer cells (500 fmole/mg protein). Interestingly enough E2 inhibits growth of all the transfectants in a concentration related manner. The inhibition is blocked by the pure antiestrogen ICI 164,384. In contrast an analog of the triphenylethylene 4 hydroxytamoxifen (40HT) is a partial agonist/antagonist in the wild type transfectants but an estrogen agonist in the mutant transfectants.
The aims of the proposal are to characterize the cell cycle kinetics and cell biology of our current and additional transfectants and to determine the effect of increasing the receptor number in other breast cancer cell lines. Colon cancer cell line HT-29 will be transfected with ER to broaden our finding to other target organs not previously regulated by estrogen. Additionally we have been intrigued by the alteration in pharmacology of 40HT with the mutant receptor; we will establish a model for antiestrogen drug resistance based on alterations in the ligand, receptor and hormone response element. There is evidence that the inhibitory effect of E2 on ER transfected receptor negative cells is universal so these studies will provide the first solid mechanistic evidence to pursue a new therapeutic approach to breast cancer. In the future the pharmacologist may not only alter the ligand requirements but also be able to target or reactive quiescent receptors as a new general therapeutic strategy for cancer.
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