Robin Fuchs-Young

Raloxifene is a Tissue‐Selective Agonist/Antagonist That Functions through the Estrogen Receptor

Raloxifene (LY 139481), previously called keoxifene, is a benzothiophene derivative that binds to the estrogen receptor (ER) with high affinity (kd = 0.54-0.11 nM).’ This compound has tissue-specific activity, acting as an antiestrogen in breast and uterus, but functioning as an estrogen agonist in bone and on lipid metabolism. The molecular mechanisms of this tissue specificity are still under investigation, but preliminary evidence indicates that raloxifene-ER complexes associate with unique response elements, thus indicating that multiple transcriptional pathways may mediate these e f f e ~ t s . ~ ’ ~

Inhibition of estrogen-stimulated growth of uterine leiomyomas by selective estrogen receptor modulators

Uterine leiomyoma is the most frequent gynecologic neoplasm in women. By using a panel of cell lines derived from spontaneous Eker rat leiomyomas, we examined the estrogen‐responsive phenotype of these tumor cells. Leiomyoma‐derived ELT cell lines proliferated in response to estrogen, and estrogen‐induced cell proliferation could be inhibited by the estrogen antagonist ICI 182780 and the selective estrogen‐receptor modulators (SERMs) raloxifene and tamoxifen. In addition to inhibiting cell growth, these antagonists also inhibited estrogen‐induced increases in progesterone‐receptor expression. These data indicate that SERMs such as raloxifene and tamoxifen act as estrogen antagonists in uterine myometrial cells and suggest that this class of compounds may be effective for treatment of this important gynecologic neoplasm.

Altered Hormonal Responsiveness of Proliferation and Apoptosis During Myometrial Maturation and the Development of Uterine Leiomyomas in the Rat

Abstract Uterine leiomyomas are responsive to the ovarian steroids, estrogen and progesterone; however, a mechanistic understanding of the role of these hormones in the development of this common gynecologic lesion remains to be elucidated. We have used the Eker rat uterine leiomyoma model to investigate how ovarian hormones regulate or promote the growth of these tumors. Proliferative and apoptotic rates were quantitated in normal uterine tissues and leiomyomas in response to endogenous ovarian steroids. In 2- to 4-mo-old animals, cell proliferation in the normal uterus corresponded with high serum levels of steroid hormones during the estrous cycle, and apoptosis occurred in the rat uterus in all cell types following sharp, cyclical declines in serum hormone levels. It is interesting that the responsiveness of uterine mesenchymal cells changed between 4 and 6 mo of age, with significant decreases in both proliferative and apoptotic rates observed in myometrial and stromal cells of cycling animals. Leiomyomas displayed much higher levels of proliferation than did age-matched myometrium; however, their apoptotic index was significantly decreased in comparison with normal myometrium. This disregulation between proliferative and apoptotic responses, which were tightly regulated during ovarian cycling in the normal myometrium, may contribute to the disruption of tissue homeostasis and underlie neoplastic growth of these tumors.

Transcriptional regulation of estrogen receptor-alpha by p53 in human breast cancer cells.

Estrogen receptor alpha (ER) and p53 are critical prognostic indicators in breast cancer. Loss of functional p53 is correlated with poor prognosis, ER negativity, and resistance to antiestrogen treatment. Previously, we found that p53 genotype was correlated with ER expression and response to tamoxifen in mammary tumors arising in mouse mammary tumor virus-Wnt-1 transgenic mice. These results lead us to hypothesize that p53 may regulate ER expression. To test this, MCF-7 cells were treated with doxorubicin or ionizing radiation, both of which stimulated a 5-fold increase in p53 expression. ER expression was also increased 4-fold over a 24-h time frame. In cells treated with small interfering RNA (siRNA) targeting p53, expression of both p53 and ER was significantly reduced (>60%) by 24 h. Induction of ER by DNA-damaging agents was p53 dependent as either ionizing radiation or doxorubicin failed to up-regulate ER after treatment with p53-targeting siRNA. To further investigate whether p53 directly regulates transcription of the ER gene promoter, MCF-7 cells were transiently transfected with a wild-type (WT) p53 expression vector along with a luciferase reporter containing the proximal promoter of ER. In cells transfected with WT p53, transcription from the ER promoter was increased 8-fold. Chromatin immunoprecipitation assays showed that p53 was recruited to the ER promoter along with CARM1, CBP, c-Jun, and Sp1 and that this multifactor complex was formed in a p53-dependent manner. These data show that p53 regulates ER expression through transcriptional control of the ER promoter, accounting for their concordant expression in human breast cancer.

Preclinical Evidence for Therapeutic Efficacy of Selective Estrogen receptor Modulators for Uterine Leiomyoma

Objective: Uterine leiomyoma are the most common gynecologic neoplasm and a primary cause of hysterectomy in premenopausal women. Preclinical studies were conducted in the Eker rat model to investigate the potential efficacy of selective estrogen receptor modulators (SERMs) as therapeutic agents for this tumor. Methods: Twelve-month-old Eker rats were randomized into five treatment arms including tamoxifen, placebo, LY 326315, vehicle, and no treatment. Additional animals received ovariectomy or sham surgery at 4 months of age to determine the effect of ovarian ablation on tumor development. The study was terminated after 2 to 4 months of treatment, and tumor incidence, size, proliferative and apoptotic indices were determined. Size and incidence data were subjected to chi-square analysis. One-way analysis of variance and Fishers least significant difference tests were used to compare proliferative and apoptotic indices. Results: Ovariectomy virtually ablated leiomyoma development, indicating that these tumors were dependent on ovarian hormones for growth and development. Treatment with tamoxifen or raloxifene analog LY 326315 reduced leiomyoma incidence by 40-60% and reduced the size of remaining tumors. The effect of SERMs on leiomyomas was mediated by a decrease in cell proliferation without a decrease in apoptotic index. Conclusion: SERMs have been shown to be therapeutically efficacious against breast cancer and to reduce tumor incidence in women at increased risk for this disease. The present data indicate that therapeutic efficacy may also be extended to uterine leiomyoma and demonstrate the utility of this animal model for preclinical studies to identify new therapeutic modalities.

Localization of immunoreactive epidermal growth factor receptor in neonatal and adult rat hippocampus.

The regional and developmental expression of epidermal growth factor (EGF) receptor in rat hippocampus was investigated utilizing immunocytochemical techniques at the light and electron microscopic levels. EGF receptor immunoreactivity in adult hippocampus was compared to that found at postnatal day 7 (P7). While the receptor was observed in P7 hippocampus, immunostaining was more prominent in the adult hippocampus, especially in the pyramidal CA2 field. Ultrastructural analysis of this region revealed that the receptor was localized to the cell bodies of both P7 and adult neurons rather than the axons or dendrites. The expression of EGF receptor in selected regions of the adult brain was verified by Western blotting. These results demonstrate the presence of EGF receptor in rat hippocampus as early as P7, localize the receptor to the pyramidal cell body, and establish the hippocampal formation, particularly CA2, as a major site of EGF receptor expression in rat brain.

Paracrine Overexpression of Insulin-Like Growth Factor-1 Enhances Mammary Tumorigenesis in Vivo

Insulin-like growth factor-1 (IGF-1) stimulates proliferation, regulates tissue development, protects against apoptosis, and promotes the malignant phenotype in the breast and other organs. Some epidemiological studies have linked high circulating levels of IGF-1 with an increased risk of breast cancer. To study the role of IGF-1 in mammary tumorigenesis in vivo, we used transgenic mice in which overexpression of IGF-1 is under the control of the bovine keratin 5 (BK5) promoter and is directed to either the myoepithelial or basal cells in a variety of organs, including the mammary gland. This model closely recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 seen in women. Histologically, mammary glands from transgenic mice were hyperplastic and highly vascularized. Mammary glands from prepubertal transgenic mice had significantly increased ductal proliferation compared with wild-type tissues, although this difference was not maintained after puberty. Transgenic mice also had increased susceptibility to mammary carcinogenesis, and 74% of the BK5.IGF-1 mice treated with 7,12-dimethylbenz[a]anthracene (20 microg/day) developed mammary tumors compared with 29% of the wild-type mice. Interestingly, 31% of the vehicle-treated BK5.IGF-1 animals, but none of the wild-type animals, spontaneously developed mammary cancer. The mammary tumors were moderately differentiated adenocarcinomas that expressed functional, nuclear estrogen receptor at both the protein and mRNA levels. These data support the hypothesis that tissue overexpression of IGF-1 stimulates mammary tumorigenesis.

Effects of raloxifene in a guinea pig model for leiomyomas

OBJECTIVE Chronic exposure of oophorectomized guinea pigs to 17beta-estradiol causes leiomyoma formation. Our aims were to determine whether these leiomyomas can become estradiol independent after exposure to estradiol and if raloxifene inhibits leiomyoma growth when given concomitantly with estradiol. STUDY DESIGN To induce leiomyoma development, 6 oophorectomized animals received two estradiol implants for 140 days. Next, the estradiol implants were replaced with empty implants in 3 animals, whereas the other 3 received 2 new estradiol implants and raloxifene given per os 10 mg/kg per day for 60 days. Tumor size was monitored biweekly by ultrasonography. RESULTS On estradiol removal, abdominal wall leiomyomas regressed within 15 to 30 days; when estradiol implants were reintroduced, leiomyomas redeveloped. Within 30 days on raloxifene, all abdominal leiomyomas (n = 9) regressed as determined by ultrasonography and verified at laparotomy. Serum raloxifene and estradiol levels were 432 +/- 46 pg/mL and 78 +/- 13 pg/mL (mean +/- SEM, n = 3), respectively, after 60 days of treatment. CONCLUSIONS Leiomyomas did not become estradiol independent, even after long exposure to estradiol; ultrasonography allowed frequent, noninvasive assessment of leiomyoma size, and raloxifene rapidly regressed leiomyomas in this animal model.

Exercise Effects on Tumorigenesis in a p53-deficient Mouse Model of Breast Cancer

PURPOSE Physically active women have a reduced risk of breast cancer, but the dose of activity necessary and the role of energy balance and other potential mechanisms have not been fully explored in animal models. We examined treadmill and wheel running effects on mammary tumorigenesis and biomarkers in p53-deficient (p53(+/-)):MMTV-Wnt-1 transgenic mice. METHODS Female mice (9 wk old) were randomly assigned to the following groups in experiment 1: treadmill exercise 5 d x wk(-1), 45 min x d(-1), 5% grade at 20 m x min(-1), approximately 0.90 km x d(-1) (TREX1, n = 20) or at 24 m x min(-1), approximately 1.08 km x d(-1) (TREX2, n = 21); or a nonexercise control (CON-TREX, n = 22). In experiment 2, mice were randomly assigned to voluntary wheel running (WHL, n = 21, 2.46 +/- 1.11 km x d(-1) (mean +/- SD)) or to a nonexercise control (CON-WHL, n = 22). Body composition was measured at approximately 9 wk and serum insulin-like growth factor 1 (IGF-1) at two to three monthly time points beginning at approximately 9 wk on study. Mice were sacrificed when tumors reached 1.5 cm, mice became moribund, or there was only one mouse per treatment group remaining. RESULTS TREX1 (24 wk) and TREX2 (21 wk) had shorter median survival times than CON-TREX (34 wk; P < 0.01), whereas those of WHL and CON-WHL were similar (23 vs 24 wk; P = 0.32). TREX2 had increased multiplicity of mammary gland carcinomas compared with CON-TREX; WHL had a higher tumor incidence than CON-WHL. All exercising animals were lighter than their respective controls, and WHL had lower body fat than CON-WHL (P < 0.01). There was no difference in IGF-1 between groups (P > 0.05). CONCLUSIONS Despite beneficial or no effects on body weight, body fat, or IGF-1, exercise had detrimental effects on tumorigenesis in this p53-deficient mouse model of spontaneous mammary cancer.

An in vivo/in vitro model to assess endocrine disrupting activity of xenoestrogens in uterine leiomyoma.

Abstract: Xenoestrogens with endocrine disrupting activity have been associated with the dysregulation of reproductive function and promotion of malignancies in experimental animals and human populations. The high incidence of uterine leiomyomas, a benign estrogen‐responsive neoplasm of the uterine myometrium, calls into question the potential influence of xenoestrogens in the pathogenesis of these tumors. An in vivo/in vitro animal model, the Eker rat, that can be used to assess the estrogen‐like agonist activity of potential endocrine disruptors in the uterine myometrium is discussed. Using this model, several in vitro assays are developed that demonstrate that compounds from three major classes of xenoestrogens can mimic the effect of estrogen on leiomyoma cells and act as estrogen receptor (ER) agonists: phytoestrogens, organochlorine pesticides and pharmacologic agents. These compounds can stimulate transcription via the ER and upregulate the expression of an estrogen‐responsive gene in uterine leiomyoma cells. The use of these in vitro assays has also advanced our ability to predict the agonist activity of potential therapeutic agents in the uterine myometrium. Selective estrogen receptor modulators (SERMs), while able to act as agonists in some tissues such as the bone and uterine endometrium, act as antagonists in vivo in the uterine myometrium and in our in vitro assays. This antagonist activity in the uterine myometrium suggests that SERMs may be useful in the treatment of uterine leiomyoma.