University of Virginia Health System

Women with estrogen receptor positive breast cancers frequently respond initially to inhibition of estrogen secretion or action but later relapse. Secondary therapies designed to further lower estrogen levels induce additional remissions but progression to a hormone refractory state invariable follows. The mechanisms mediating these secondary responses and later progression are incompletely understood. Based upon our prior studies, we postulate that tumor cells can adapt to conditions of estrogen deprivation by developing enhanced sensitivity to estrogens. Using a model system of long term estradiol withdrawal, we have directly demonstrated the ability of cells to develop hypersensitivity to estradiol both in vitro and in vivo. Our studies indicated that hypersensitivity is not mediated at the level of estrogen receptor transcription. Rather, adaptation to estradiol deprivation results in overproduction of growth factor signaling pathways which interact with receptor mediated events to enhance estradiol sensitivity. Cross talk between separate estrogen signaling and growth factor signaling pathways occurs at both upstream and downstream levels. These interactions affect not only cell proliferation but also cell death. We plan to further examine these adaptive mechanisms and then exploit them to develop novel new therapeutic approaches to the treatment of breast cancer. These novel approaches involve combined simultaneous blockade of growth factor and estrogen pathways as well as stimulation of apoptosis with pulse estradiol administration. To study upstream events, we have identified four specific aims. Specific Aim 1 is to demonstrate the upregulation of tyrosine kinase mediated receptor functionality in Long Term Estradiol Deprived (LTED) versus wild type cells. To accomplish this, we will utilize multi-array DNA methodology as well as specific anti-receptor antibodies and tyrosine kinase inhibitors to block DNA synthesis. Specific Aim 2 is to demonstrate the separate roles of activated RAS on cell proliferation and on cell death. This specific aim tests the hypothesis that RAS stimulates MAP kinase which enhances cell proliferation and also PI-3K which prevents cell death. We will examine the effect of MAP kinase and PI-3-kinase inhibitors separately and in combination on DNA synthesis and on apoptosis to demonstrate separate effects on these two mechanistic events. Specific Aim 3 tests the innovative hypothesis that estrogens can actively stimulate apoptosis under appropriate conditions. We will provide in vitro evidence of hormonal stimulation of apoptosis by blocking PI-3-kianse in the presence of estradiol stimulation of c-myc and MAP kinase. Under these circumstances, we will examine the rate of apoptosis occurring in response to estradiol given in the presence of the PI-3-kinase inhibitors and MAP kinase inhibitors alone and in combination. We will also examine the effect of the RAS inhibitor FTS on both cell proliferation and cell death. Specific aim 4 is to further test this hypothesis under in vivo conditions by use of estradiol and an inhibitor of PI-3-kinase activity. Regarding downstream events, our hypothesis is that an adaptive process occurs in which growth factor and estradiol mediated events cooperatively interact at a point of intersection in the cell cycle to mediate hypersensitivity. At this intersection point, c-myc, a direct estrogen-mediated early response gene, and products of the growth factor stimulated RAS pathway cooperate to regulate key cell cycle proteins. We postulate that this interaction results in suppression of the cyclin inhibitor p-27, bypass of the need for phosphorylation of the Rb protein, enhanced activation of E2F transcription factors, and an increase in cyclin E kinase activity. To test this hypothesis, we will pursue four specific aims. Specific Aim 1 will evaluate the role of E2F transcription factors and the cyclins in the process of adaptive hypersensitivity. Specific Aim 2 will seek to demonstrate down regulation of cyclin inhibitors and resultant effects on E2F levels and cell proliferation. We postulate that adaptation to estrogen deprivation involves down regulation of inhibitors of cyclin dependent kinase activation. We will measure the levels of the two classes of inhibitors, the INK 4 a-d group as well as the Cip-1/Waf-1/Kip1/Kip2 class (p-21, p-27, p-57). To directly assess cause and effect relationships, we will use stable transfectants to determine if p-27 activation decreases sensitivity to estradiol in LTED deprived cells. Specific Aim 3 will evaluate the role of growth factor pathways on E2F production. We will use strategies both for induction of hypersensitivity in wild type cells and for reversion of hypersensitive cells back to normal sensitivity. Inhibitors of MAP kinase and RAS plasma membrane binding will be used to interdict the effects of activated RAS in hypersensitive cells and to cause reversion of hypersensitivity. Induction of hypersensitivity in wild type cells will involve TGF alpha administration as well as use of tetracycline regulatable, stable transfection constructs to cause over-expression of MAP kinase. Specific Aim 4 will determine whether LTED deprivation induces anti-apoptotic regulatory mechanisms.

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