Sara Antonia Li

Estrogen mediates Aurora-A overexpression, centrosome amplification, chromosomal instability, and breast cancer in female ACI rats

Estrogens play a crucial role in the causation and development of sporadic human breast cancer (BC). Chromosomal instability (CIN) is a defining trait of early human ductal carcinoma in situ (DCIS) and is believed to precipitate breast oncogenesis. We reported earlier that 100% of female ACI (August/Copenhagen/Irish) rats treated with essentially physiological serum levels of 17β-estradiol lead to mammary gland tumors with histopathologic, cellular, molecular, and ploidy changes remarkably similar to those seen in human DCIS and invasive sporadic ductal BC. Aurora-A (Aur-A), a centrosome kinase, and centrosome amplification have been implicated in the origin of aneuploidy via CIN. After 4 mo of estradiol treatment, levels of Aur-A and centrosomal proteins, γ-tubulin and centrin, rose significantly in female ACI rat mammary glands and remained elevated in mammary tumors at 5–6 mo of estrogen treatment. Centrosome amplification was initially detected at 3 mo of treatment in focal dysplasias, before DCIS. At 5–6 mo, 90% of the mammary tumor centrosomes were amplified. Comparative genomic hybridization revealed nonrandom amplified chromosome regions in seven chromosomes with a frequency of 55–82% in 11 primary tumors each from individual rats. Thus, we report that estrogen is causally linked via estrogen receptor α to Aur-A overexpression, centrosome amplification, CIN, and aneuploidy leading to BC in susceptible mammary gland cells.

Induction of chromosone aberrations in Syrian hamster renal cortical cells by various estrogens

Estrogens, both natural and synthetic, have been implicated in carcinogenesis at different organ sites in a variety of animals, including man, for more than six decades. However, the molecular mechanism(s) involved in the carcinogenic action of estrogens still remains both controversial and elusive. Cytogenetic damage in the hamster kidney has been studied after in vivo treatment with either potent or weak estrogens for varying periods. Compared to age-matched untreated control, diethylstilbestrol (DES) treatment resulted in significant increases in the number of chromatid gaps and breaks, chromosome breaks, and endoreduplicated cells in hamster renal cortical cells. These chromosomal aberrations (CA) were cumulative with continued hormone exposure from 1.0 to 5.0 months. However, chromosome exchanges as a result of the breaks were not elevated. After 5.0 months of hormone treatment, potent estrogens such as 17 beta-estradiol and Moxestrol exhibited similar frequencies of CA in the hamster kidney to that found for DES, whereas weak estrogens such as 17 alpha-estradiol and beta-dienestrol exhibited CA frequencies that were not significantly different from untreated levels. Ethinylestradiol treatment for a similar period resulted in significant increases in chromatid gaps, although these did not evolve into increases in either chromatid or chromosome breaks, and in a rise in endoreduplicated cells. These results raise the possibility that the CA generated after estrogen treatment may be involved in renal tumorigenic processes.

Ploidy differences between hormone- and chemical carcinogen–induced rat mammary neoplasms: Comparison to invasive human ductal breast cancer*

To ascertain differences between solely hormone– and chemical carcinogen–induced murine mammary gland tumors (MGTs), a direct comparison of their ploidy status was assessed. Nuclear image cytometry (NIC) was used to evaluate ploidy in ductal carcinoma in situ (DCIS) and MGTs induced solely by 17β‐estradiol (E2) in female A‐strain Copenhagen Irish hooded gene rats (ACI) and E2 plus testosterone propionate in male Noble rats. These results were compared to ploidy data from primary MGTs induced by two synthetic carcinogens, 7,12‐dimethylbenz[a]antracene and nitrosomethylurea in female Brown Lewis Norway rats and an environmental carcinogen, 6‐nitrochrysene, in female Sprague‐Dawley rats. Both DCIS and primary MGTs induced solely by hormones were highly aneuploid (> 84%), whereas MGTs induced by either synthetic or environmental carcinogens were primarily diploid (> 85%). Examination of 76 metaphase plates obtained from eight individual E2‐induced ACI female rat MGTs revealed the following consistent chromosome alterations: gains in chromosomes 7, 11, 12, 13, 19, and 20 and loss of chromosome 12. On Southern blot analysis, six of nine ACI female rat primary E2‐induced MGTs (66%) exhibited amplified copy numbers (range: 3.4–6.9 copies) of the c‐myc gene. Fluorescence in situ hybridization (FISH) analysis of these MGTs revealed specific fluorescent hybridization signals for c‐myc (7q33) on all three homologs of a trisomy in chromosome 7. NIC analysis of 140 successive nonfamilial sporadic invasive human ductal breast cancers (BCs) showed an aneuploid frequency of 61%, while 31 DCISs revealed a 71% aneuploid frequency. These results clearly demonstrate that the female ACI rat E2‐induced MGTs more closely resemble invasive human DCIS and ductal BC in two pertinent aspects: they are highly aneuploid compared with chemical carcinogen–induced MGTs and exhibit a high frequency of c‐myc amplification.

Aurora A and B overexpression and centrosome amplification in early estrogen-induced tumor foci in the Syrian hamster kidney: Implications for chromosomal instability, aneuploidy, and neoplasia

Estrogen-induced Syrian hamster tumors in the kidney represent a useful model to gain insight into the role of estrogens in oncogenic processes. We provided evidence that early tumor foci in the kidney arise from interstitial ectopic uterine-like germinal stem cells, and that early tumor foci and well-established tumors are highly aneuploid (92-94%). The molecular mechanisms whereby estrogens mediate this process are unclear. Here, we report that estrogen treatment induced significant increases in Aurora A protein expression (8.7-fold), activity (2.6-fold), mRNA (6.0-fold), and Aurora B protein expression (4.6-fold) in tumors, compared with age-matched cholesterol-treated kidneys. Immunohistochemistry revealed that this increase in Aurora A and B protein expression was essentially confined to cells within early and large tumor foci at 3.5 and 6 months of estrogen treatment, respectively. Upon estrogen withdrawal or coadministration of tamoxifen for 10 days, a 78% to 79% and 81% to 64% reduction in Aurora A and B expression, respectively, were observed in primary tumors compared with tumors continuously exposed to estrogens. These data indicate that overexpressed Aurora A and B in these tumors are under estrogen control via estrogen receptor alpha. Aurora A coenriched with the centrosome fraction isolated from tumors in the kidney. Centrosome amplification (number and area/cell) was detected in early tumor foci and large tumors but not in adjacent uninvolved or age-matched control kidneys. Taken together, these data indicate that persistent overexpression of Aurora A and B is under estrogen control, and is coincident with centrosome amplification, chromosomal instability, and aneuploidy, and represent an important mechanism driving tumorigenesis.

Serum and tissue levels of estradiol during estrogen-induced renal tumorigenesis in the Syrian hamster

The estrogen-induced renal tumor in the hamster has emerged as a major animal model in hormonal carcinogenesis. However, a fundamental aspect of this experimental model has as yet not been investigated. In the present study, comparisons between the serum and tissue 17 beta-estradiol (E2) levels in cyclic female hamsters and corresponding hormone levels in E2-treated castrated male hamsters have been made. Data is provided concerning the concentration of estrogenic hormones in the serum and target tissue typically required to elicit renal tumorigenesis in this species. Serum E2 levels in the cyclic female hamster average 79 pg/ml on days 1-2 and 311 pg/ml on days 3-4, attaining a maximum of 358 pg/ml on day 4 of the cycle. Elevation in uterine, renal and hepatic E2 tissue levels during days 3-4 of the cycle reflect increases in serum E2 levels which were 3.0-, 2.0-, and 2.6-fold higher when compared to day 1 of the cycle in these tissues. As expected, serum E2 levels of untreated castrated male hamsters did not appreciably vary over a 6 month period of aging and averaged about 32 pg/ml. Under conditions which produced essentially 100% renal tumor incidence, a rapid rise in serum E2 levels, averaging 71.0-fold higher than untreated castrated levels, was seen. A steady state serum E2 level of 2400 to 2700 pg/ml was maintained from 45-180 days of continuous estrogen treatment. Compared to kidneys of untreated hamsters, renal E2 levels in E2-treated hamsters rose only on average 5.4-fold between 15-180 days of hormone exposure. Serum levels of E2-treated hamsters were 5.7- to 8.0-fold higher than those observed in cyclic female hamsters on days 3 and 4. However, at these higher E2-treated serum levels there was no apparent effect either on weight loss or mortality of the animals.

Comparative genomic hybridization of estrogen-induced ectopic uterine-like stem cell neoplasms in the hamster kidney: nonrandom chromosomal alterations.

Karyotype and comparative genomic hybridization (CGH) analyses were performed to identify nonrandom/consistent chromosomal alterations in solely estrogen (E)‐induced primary ectopic uterine‐like stem cell tumors in the kidney (EULTK) of the Syrian hamster, using a criterion of ≥20% frequency for nonrandom occurrence. Employing this criterion, EULTK karyotype analysis showed consistent gains in chromosomes 3, 6, 11, 14, 16, 20, and 21. Consistent trisomies were seen in all of these nonrandomly gained chromosomes. Only chromosomes 3 and 6 exhibited appreciable tetrasomies. Chromosome losses were observed consistently in chromosomes 7, 12, 17, and 19. Employing the same criterion, CGH analysis of primary EULTKs showed nonrandom amplified sequences at 1pa1–a4, 2cen–pter, 3pa1–a4, 6qb2–b4, 20qa1–a4, 21qa1–a2, Xqa3–qter and regional consistent losses at 1qc1‐qter, 2qb1–c1, 3qa2–a7, 11qb5‐qter, 15qa2–a5, 18qa2–a4, and 21pa. Moreover, 88% of the EULTKs examined exhibited amplification of the 6qb2–b4 region, where c‐myc resides. The data presented lend credence to the supposition that chromosomal instability (CIN) is elicited by the upstream overexpression and subsequent amplification of c‐myc by Es in multipotential interstitial uterine stem cells present in the kidney, thus leading to neoplastic development.

Synthetic Estrogens and Liver Cancer: Risk Analysis of Animal and Human Data

In 1971, Baum et al. (1) reported a causal relationship between estrogen exposure and hepatic tumors in women. At that time, there was little or no evidence available indicating that liver tumors could be induced following prolonged estrogen treatment in experimental animals, even at high doses (2–5). Nevertheless, over the years, epidemiologic evidence continued to accumulate that supported this initial causal association in humans following the therapeutic use of estrogens in the liver (6–13). The present report summarizes the human data, largely in women, regarding the association of liver tumor incidences and estrogen intake. In addition, we present heretofore unpublished observations concerning liver tumor incidences in male and female hamsters exposed to various natural and synthetic estrogens in the absence of any other intervening agent.

Cytogenetic Changes in Renal Neoplasms and During Estrogen-Induced Renal Tumorigenesis in Hamsters

Cytogenetic changes have been implicated in tumor development. Such changes were studied in male Syrian hamster kidneys after chronic exposure to either diethylstilbestrol(DES) or 17s-estradiol(E2). The frequency of aneuploidy and micronuclei was elevated in estrogen-induced renal tumors. The frequency of aneuploidy in DES- and E2-induced tumors was 8.5-fold and 8.0-fold higher than in untreated kidneys, respectively. The number of micronuclei also increased 3.8-to 4.3-fold in these estrogen-induced tumors. Nonrandom numerical chromosomal abnormalities were +1, +2, +3, +11, +13, +16, +20, +21, and −8, −19, −20 in the DES- and 1, +2, +3, +6, +11, +16, +20, +21, +X and −14,−20 in the E2-induced tumors. Structural changes were observed only in E2-induced tumors. Endomitosis, telomeric associations, and breaks were also seen in these kidney tumors. The frequency of chromosomal abnormalities was higher in 3.5 months estrogen-treated hamster proximal tubules after culture. The findings suggest that chronic estrogen exposure induces genetic instability in the hamster kidney and it may be associated with renal tumorigenesis. (NCI, NIH. CA22008)

Estrogen-induced Breast Cancer in Female ACI Rats

It has become increasingly evident that neither physical (i.e. ionizing radiation) nor environmental agents (carcinogens, xeno-estrogens) can appreciably account for the gradual and persistent rise in breast cancer (BC) incidence since 1940. In the USA, it is estimated that 1:8 women will develop BC over their lifetime (1–3). It is now realized that estrogens (Es) and progesterone (P), to some extent, are critically involved in the etiology, promotion, and progression of human BC. Most, if not all, of the major risk factors associated with invasive sporadic BC, which comprises 90 to 95% of BCs, are associated with sex hormones, particularly Es. Well-established BC risk factors include: Early age at menarche, late age at menopause, nulliparity, late age at first pregnancy, obesity, and hormone replacement therapy (4–7). Even weaker indirect risk factors for BC, such as dietary fat and alcohol intake, appear to be associated with increase E levels (8, 9).

Metabolism of Moxestrol in the Hamster Kidney: Significance for Estrogen Carcinogenesis

Moxestrol [11β-methoxy-17-ethinyl-1, 3, 5 (10)-estratriene-3,17β-diol or R 2858] is a potent estrogen in both animals and humans (1,2) and is used in Europe as a postmenopausal agent. Its estrogenic potency, depending on the biologic or biochemical parameters used, is approximately 10 to 100 times higher than 17β-estradiol (E2) (1) and about 5 times more potent than ethynylestradiol (EE2) (2). Its hormonal effectiveness has been attributed to the stability of the complex it forms with the estrogen receptor (3), its lower affinity for serum plasma proteins (1), and also the degree to which it is metabolized (4). In the hamster estrogeninduced-renal adenocarcinoma model, Moxestrol and EE2 exhibited similar estrogenic activities. Moxestrol, however, displayed potent tumorigenic activity at this organ site, eliciting 100% tumor incidence (5,6). In contrast, its parent compound EE2 exhibits only modest 10% renal tumor incidence when similarly administered. Since metabolism has been postulated by us (7) and others (8–10) to play a significant if not crucial role in neoplastic transformation of the hamster kidney, it becomes evidently pertinent to investigate the metabolism of Moxestrol in the kidney and liver of this species.