Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 383 C. ER� and oncogene-induced tumorigenesis: Wnt-1/�ERKO mice Several lines of evidence indicate that breast cancer in humans is strongly correlated with the extent of lifetime exposure to estrogen. Most notably, breast cancer almost exclusively occurs in females and is never seen before puberty but rather only after several years into the reproductive life span (283, 284). Furthermore, an increased length of a woman’s reproductive years, i.e., early menarche and late menopause, has been associated with an elevated risk of breast cancer (284), whereas women who have experienced premature menopause due to natural causes or castration appear to be at a much lower risk (283). Furthermore, studies have indicated that women who circulate higher levels of active estrogens may also be at greater risk of developing breast cancer (283). In apparent contrast, early pregnancy tends to provide a protective effect, although it is obviously associated with an increased level of steroid hormone exposure. In addition, several years of research have indicated little positive correlation with the risk of breast cancer and prolonged use of contraceptive pills composed of synthetic estrogens and progestins, although this issue remains controversial (reviewed in Refs. 285 and 286). Still, a large portion of chemotherapeutics for breast cancer are aimed at either blocking estrogen action or reducing estrogen levels (283). Therefore, although an association between estrogen action and breast cancer is apparent, it involves less understood yet critical mechanisms, including the periodicity and cyclicity of hormone exposure as well as the sensitivity of the end organ (283). This is further complicated by the influence that environmental exposures, geography, diet, body weight, and genetics also play in individual risk of developing breast cancer (283). Numerous studies have been carried out concerning the levels of ER and PR in neoplastic breast tissue and the prognostic value that these parameters may provide (reviewed in Refs. 287–291). Reports indicate that more than 70% of primary breast tumors are ER�-positive and exhibit estrogendependent growth (291). However, the most malignant mammary tumors are often ER�-negative and exhibit estrogen-independent aggressive growth, but are thought to progress from a once ER�-positive cell population (287). In addition, the role of local aromatase activity and estrogen production in breast cancer is receiving increased attention (reviewed in Ref. 292). Added complexity is introduced by the detection and description of numerous variants of the ER� transcript in breast cancer tissues, although their possible role in the etiology of the disease remains speculative (reviewed in Refs. 28 and 293). Recent reports also described the detection of ER� transcripts in multiple immortalized human breast cancer cell lines, normal human breast tissue, and human breast tumors (64, 73, 101, 102, 294). Vladusic et al. (73) also characterized an ER� mRNA variant detected in normal as well as malignant human breast tissue (73). It is clear from the severely underdeveloped mammary gland of the �ERKO that estradiol acting via the ER� is a potent mitogen in the breast. To gain insight into the potential role of ER� in the induction and promotion of mammary gland carcinogenesis, we crossed the �ERKO mice with the MMTV-Wnt-1 mice, a transgenic line that is highly susceptible to mammary adenocarcinoma. The family of Wnt genes encode a series of secretory glycoproteins that act in autoand paracrine pathways to stimulate cell proliferation and differentiation (reviewed in Ref. 295). The MMTV-Wnt-1 mice possess a transgene designed for targeted overexpression of the Wnt-1 protooncogene in the mammary gland and exhibit a nearly 100% and 15% incidence of mammary hyperplasia and lobuloalveolar adenocarcinoma by 1 yr of age in females and males, respectively (295, 296). Therefore, breeding of the two transgenic lines allowed for the generation of animals that possessed the Wnt-1 transgene on either a wild-type or �ERKO background and thereby allowed for the assessment of the role of ER� in the initiation and promotion of protooncogene-induced mammary tumors (297). At 6 months of age, virgin wild-type Wnt-1 females exhibit extensive hyperplasia of the ductal epithelium and aberrant lobuloalveolar development that occupies the entire fat pad. This was the expected phenotype based on that described previously in the original line of Wnt-1 females derived from a different mouse strain (296). Interestingly, a similar phenotype of lobuloalveolar hyperplasia was observed in the rudimentary duct of the �ERKO-Wnt-1 females, although the extent of ductal growth was much reduced compared with that seen in the wild-type (269). Nonetheless, the rudimentary ductal structure previously described in the �ERKO female was obviously induced to proliferate by the presence of ectopic Wnt-1 expression. However, comparison of mammary glands from a series of age-matched animals indicated that the ductal growth observed in the mammary gland of a 6-month-old �ERKO-Wnt-1 female remained confined to the nipple region and approximated that seen in a 2.5-month-old wild-type-Wnt-1 female, illustrating a significant delay in the proliferation of the �ERKO epithelium (269). Interestingly, the mammary hyperplasia in the �ERKO-Wnt-1 females did not markedly progress into the inguinal fat pad, but rather remained confined to the area of the nipple. Therefore, although the lobuloalveolar phenotype characteristic of Wnt-1 overexpression was evident, ductal elongation did not occur in the �ERKO-Wnt-1 female, indicating that the hyperplastic action of ectopic Wnt-1 expression cannot substitute for ER�mediated terminal end bud formation and ductal morphogenesis (297). In the males, Wnt-1-induced epithelial hyperplasia was obvious in both the wild-type and �ERKO animals as well, but no distinct difference in growth rates between the two genotypes was evident (269). The incidence of lobuloalveolar carcinoma in the Wnt-1 mice mirrored the observations described above for the epithelial hyperplasia. Wild-type-Wnt-1 and heterozygous- ER�/Wnt-1 females developed mammary tumors at a rapid rate, reaching an incidence of 50% by 6 months of age (297). Ectopic expression of the Wnt-1 gene was also able to induce tumorigenesis in the �ERKO females and therefore did not require the presence of functional ER� (297). However, a 50% incidence in tumors in the �ERKO-Wnt-1 females was not observed until 12 months of age, twice the time required for the wild-type-Wnt-1 colony (269). Ribonuclease protection assays indicated that the level of Wnt-1 transgene expression was relatively equal in the two ER� genotypes. Prepubertal ovariectomy had no effect on the overall incidence of tumors
384 COUSE AND KORACH Vol. 20, No. 3 in either genotype, although it resulted in a delayed incidence of palpable tumors in both, i.e., wild-type-Wnt-1 at 50% at 10.5 momths and a further delay in the �ERKO-Wnt-1 at 50% at 15 months (269). Therefore, ovarian factors clearly accelerated the growth rate of the Wnt-1-induced adenocarcinoma in both the wild-type and �ERKO females. However, multiple pregnancies had no significant effect on the time of tumor onset in the wild-type-Wnt-1 females (269). These studies indicate that ER� signaling is not involved in the induction of hyperplasia and tumorigenesis in the mammary gland that results from overexpression of the Wnt-1 protooncogene, but indeed plays a promotional role in these phenotypes. Similar findings of a promotional role for the ER� in uterine carcinogenesis were reported in transgenic mice that overexpressed an ER� gene (298). A possible explanation for the observed tumor latency may be drawn from an inherent difference between the wild-type and �ERKO mammary glands, i.e., because the �ERKO-Wnt-1 glands possess a reduced amount of ductal morphogenesis and proliferating epithelia due to the lack of ER�, the number of cells most susceptible to neoplastic transformation may be decreased (297). Interestingly, the growth rates of the ductal hyperplasia and ultimate adenocarcinomas were also significantly reduced when the animals were ovariectomized, even in those lacking functional ER�. A possible explanation for this finding may be that ovarian estradiol is acting via non- ER�-mediated pathways, suggesting a possible role for ER�. However, in contrast to reports in other species, ER� mRNA remains difficult to detect by standard assays in the mouse mammary gland, including the glands of the �ERKO (93) and Wnt-1 females (297). Furthermore, ovariectomy also results in the loss of other gonadal hormones in addition to estrogen. Remarkably, overexpression of the Wnt-1 gene in the �ERKO mammary gland resulted in significant increases in PR mRNA levels compared with the low basal levels detected in control �ERKO glands, when assayed by ribonuclease protection assay (297). Recently, Shyamala et al. (299) showed that misregulation of the PR gene resulting in increased levels of PR A through transgenic methodologies may be tumorigenic in the mammary gland. Therefore, the ability of Wnt-1 to compensate for the reduced PR levels generated by the loss of ER� action may provide a pathway by which the role of ER� in tumor induction and promotion may be overridden. In addition, if the PR pathway was involved in the promotion of the Wnt-1 lobuloalveolar carcinomas, ovariectomy would be expected to result in a delay in their growth. In summary, the �ERKO/Wnt-1 mouse model has demonstrated that artificially induced hyperplasia and tumorigenesis of the mammary gland can take place in the absence of ER� signaling. These findings have potential importance in understanding the etiology of human breast cancer by illustrating that oncogenic induction of mammary neoplasia can occur in an ER�-negative tissue. Extending these observations further may indicate that hormone-independent breast tumors can originate from a cell population lacking ER� as well as those that are ER� positive. The characterization of mice produced from crosses of other overexpressing transgenic models, e.g., neu, with the �ERKO are cur- rently being carried out in a fashion similar to those studies described above. V. Reproductive Tract Phenotypes of the Male More than 40 yr ago, Jost employed a series of classical organ ablation studies to demonstrate that development of the male reproductive system was dependent on the secretion of testosterone and the peptide, anti-Müllerian hormone, by the fetal testes (107). We now know that development of the undifferentiated fetal gonad to a testis is determined by the presence of testis-determining factors or genes localized to the Y chromosome, e.g., the SRY gene (300). Testosterone produced from the Leydig cells of the fetal testes is crucial to the survival of the Wolffian duct, the primordial structure of the male reproductive tract, and its differentiation into the male reproductive structures (143, 192). In turn, secretion of anti-Müllerian hormone from the Sertoli cells of the fetal testis induces regression of the primordial female reproductive structures in the developing fetus (143, 192). Differentiation of the epididymis, ductus deferens, and seminal vesicle from the Wolffian duct is stimulated by testosterone, whereas development of the lower structures, the prostate, bulbourethral glands, urethra, scrotum, and penis, is primarily influenced by the more potent testosterone metabolite, 5�-dihydrotestosterone (DHT) (143). The importance of androgen action in the development of the male reproductive tract is evident by the feminized phenotype that results in human males with complete androgen insensitivity syndrome (cAIS), caused by a naturally occurring mutation that results in the lack of functional AR. The phenotypes of the cAIS human male and the analogous testicular feminized male (Tfm) rodent are characterized by the complete absence of either male or female internal reproductive structures except for the presence of inguinal testes (37, 301). External genitalia in the cAIS human and Tfm mouse are indistinguishable from the normal wild-type female, but a short and often blunt-ended vagina is present (37, 301). During puberty and the later stages of sexual maturation in the male, virulization of the external genitalia is dependent on the actions of DHT (143, 302). This is illustrated in human males lacking sufficient 5�-reductase activity and DHT synthesis, who exhibit normal internal reproductive structures and testosterone levels but severely undervirulized external structures (reviewed in Ref. 37). Once again, the critical role of androgens in the development of the male reproductive tract was reiterated by the complete lack of gonadal development in mice homozygous for a disruption of the gene encoding SF-1 (137, 138). Therefore, androgen action is critical to the development and function of the tissues of the male reproductive tract from fetal stages through adulthood, whereas a defined role for estrogen remains unclear. Although there is no apparent role for estrogen action in the development of the male reproductive tract, studies have reported significant effects of early exposure to estrogen agonists and/or antagonists on the adult male reproductive system. However, it was generally concluded that such effects were caused by estrogen actions at the hypothalamicpituitary level that ultimately resulted in decreased stimu-
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Estrogen Receptor Null Mice - Endocrine Reviews

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