Consistent and effective method to define the mouse estrous cycle stage by a deep learning-based model.

The mouse estrous cycle is divided into four stages: proestrus (P), estrus (E), metestrus (M), and diestrus (D). The estrous cycle affects reproductive hormone levels in a wide variety of tissues. Therefore, to obtain reliable results from female mice, it is important to know the estrous cycle stage during sampling. The stage can be analyzed from a vaginal smear under a microscope. However, it is time-consuming, and the results vary between evaluators. Here, we present an accurate and reproducible method for staging the mouse estrous cycle in digital whole-slide images (WSIs) of vaginal smears. We developed a model using a deep convolutional neural network (CNN) in a cloud-based platform, Aiforia Create. The CNN was trained by supervised pixel-level multiclass semantic segmentation of image features from 171 hematoxylin-stained samples. The model was validated by comparing the results obtained by CNN with those of four independent researchers. The validation data included three separate studies comprising altogether 148 slides. The total agreement attested by the Fleiss kappa value between the validators and the CNN was excellent (0.75), and when D, E, and P were analyzed separately, the kappa values were 0.89, 0.79, and 0.74, respectively. The M stage is short and not well defined by the researchers. Thus, identification of the M stage by the CNN was challenging due to the lack of proper ground truth, and the kappa value was 0.26. We conclude that our model is reliable and effective for classifying the estrous cycle stages in female mice.

Reversal of increased mammary tumorigenesis by valproic acid and hydralazine in offspring of dams fed high fat diet during pregnancy.

Maternal or paternal high fat (HF) diet can modify the epigenome in germ cells and fetal somatic cells leading to an increased susceptibility among female offspring of multiple generations to develop breast cancer. We determined if combined treatment with broad spectrum DNA methyltransferase (DNMT) inhibitor hydralazine and histone deacetylase (HDAC) inhibitor valproic acid (VPA) will reverse this increased risk. C57BL/6 mouse dams were fed either a corn oil-based HF or control diet during pregnancy. Starting at age 7 weeks, female offspring were administered 3 doses of 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary cancer. After last dose, offspring started receiving VPA/hydralazine administered via drinking water: no adverse health effects were detected. VPA/hydralazine reduced mammary tumor multiplicity and lengthened tumor latency in HF offspring when compared with non-treated HF offspring. The drug combination inhibited DNMT3a protein levels and increased expression of the tumor suppressor gene Cdkn2a/p16 in mammary tumors of HF offspring. In control mice not exposed to HF diet in utero, VPA/hydralazine increased mammary tumor incidence and burden, and elevated expression of the unfolded protein response and autophagy genes, including HIF-1α, NFkB, PERK, and SQSTM1/p62. Expression of these genes was already upregulated in HF offspring prior to VPA/hydralazine treatment. These findings suggest that breast cancer prevention strategies with HDAC/DNMT inhibitors need to be individually tailored.

The role of early life genistein exposures in modifying breast cancer risk.

Review of the existing literature suggests that consumption of soy foods or an exposure to a soy isoflavone genistein during childhood and adolescence in women, and before puberty onset in animals, reduces later mammary cancer risk. In animal studies, an exposure that is limited to the fetal period or adult life does not appear to have the same protective effect. A meta-analysis of human studies indicates a modest reduction in pre- and postmenopausal risk when dietary intakes are assessed during adult life. These findings concur with emerging evidence indicating that timing may be vitally important in determining the effects of various dietary exposures on the susceptibility to develop breast cancer. In this review, we address the mechanisms that might mediate the effects of an early life exposure to genistein on the mammary gland. The focus is on changes in gene expression, such as those involving BRCA1 and PTEN. It will be debated whether mammary stem cells are the targets of genistein-induced alterations and also whether the alterations are epigenetic. We propose that the effects on mammary gland morphology and signalling pathways induced by pubertal exposure to genistein mimic those induced by the oestrogenic environment of early first pregnancy.

Progesterone enhances branching morphogenesis in the mouse mammary gland by increased expression of Msx2.

Branching morphogenesis within the peripubertal mouse mammary gland is directed by progesterone (P). A role for the homeobox-containing transcription factor, Msx2, during branching morphogenesis is suggested from its ontogenic expression profile and hormonal regulation. Herein, we define the spatio-temporal control of Msx2 expression, the regulation of its expression by P and its direct role in ductal branching morphogenesis. P induces Msx2 in the presence of estrogen (E) both in vitro and in vivo while absence of the P receptor (PR) decreased Msx2 expression. Stable transfection of PR into mouse mammary epithelial cells increased the endogenous expression of Msx2 and their ability to undergo branching morphogenesis in vitro. Furthermore, normal mammary cells stably-transfected with Msx2 demonstrated increased branching morphogenesis in vitro while transgenic mice expressing Msx2 in their mammary glands demonstrated enhanced lateral branching during early development. The action of P on branching morphogenesis appears to involve Bmp2/4. Together, these data demonstrate that P, acting through PR-A and the Bmp2/4 pathway, induces Msx2 to enhance ductal branching in the mammary glands.

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network "Phytohealth" (QLKI-2002-2453).

Phytoestrogens are naturally occurring plantderived polyphenols with estrogenic potency. They are ubiquitous in diet and therefore, generally consumed. Among Europeans, the diet is rich in multiple putative phytoestrogens including flavonoids, tannins, stilbenoids, and lignans. These compounds have been suggested to provide beneficial effects on multiple menopause-related conditions as well as on development of hormone-dependent cancers, which has increased the interest in products and foods with high phytoestrogen content. However, phytoestrogens may as well have adverse estrogenicity related effects similar to any estrogen. Therefore, the assessment of estrogenic potency of dietary compounds is of critical importance. Due to the complex nature of estrogenicity, no single comprehensive test approach is available. Instead, several in vitro and in vivo assays are applied to evaluate estrogenic potency. In vitro estrogen receptor (ER) binding assays provide information on the ability of the compound to I) interact with ERs, II) bind to estrogen responsive element on promoter of the target gene as ligand-ER complex, and III) interact between the co-activator and ERs in ligand-dependent manner. In addition, transactivation assays in cells screen for ligand-induced ERmediated gene activation. Biochemical in vitro analysis can be used to test for possible effects on protein activities and E-screen assays to measure (anti)proliferative response in estrogen responsive cells. However, for assessment of estrogenicity in organs and tissues, in vivo approaches are essential. In females, the uterotrophic assay is applicable for testing ERa agonistic and antagonistic dietary compounds in immature or adult ovariectomized animals. In addition, mammary gland targeted estrogenicity can be detected as stimulated ductal elongation and altered formation of terminal end buds in immature or peripubertal animals. In males, Hershberger assay in peri-pubertal castrated rats can be used to detect (anti)androgenic/ (anti)estrogenic responses in accessory sex glands and other hormone regulated tissues. In addition to these short-term assays, sub-acute and chronic reproductive toxicity assays as well as two-generation studies can be applied for phytoestrogens to confirm their safety in long-term use. For reliable assessment of estrogenicity of dietary phytoestrogens in vivo, special emphasis should be focused on selection of the basal diet, route and doses of administration, and possible metabolic differences between the species used and humans. In conclusion, further development and standardization of the estrogenicity test methods are needed for better interpretation of both the potential benefits and risks of increasing consumption of phytoestrogens from diets and supplements.

Estrogen receptor beta in breast cancer.

Estrogen is essential for normal growth and differentiation in the mammary gland. It also supports growth of approximately 50% of primary breast cancers. For this reason, removal of estrogen or blocking of its action with the anti-estrogen, tamoxifen, is the main treatment for estrogen receptor alpha (ERalpha)-positive tumors. In 1996, when oncologists became aware of a second ER, ERbeta, there was some doubt as to whether this receptor would be of importance in breast cancer because the clinical consensus was that responsiveness to tamoxifen is related to the presence of ERalpha in breast cancer. Today we know that ERalpha and ERbeta have distinct cellular distributions, regulate separate sets of genes and can oppose each other's actions on some genes. We also know that ERbeta is widely expressed in both the normal and malignant breast and that there are proliferating cells in the breast which express ERbeta. In this review we summarize what is known about ERbeta in breast cancer and examine the possibility that ERbeta-selective ligands may well represent a useful class of pharmacological tools with a novel target, namely proliferating cells expressing ERbeta.

Altered structure and function of reproductive organs in transgenic male mice overexpressing human aromatase.

Aromatization of androgens is a key step in estrogen production, and it regulates the delicate balance between estrogens and androgens in the gonads and sex steroid target tissues. In the present study, we generated transgenic mice (AROM(+)) bearing the human ubiquitin C promoter/human P450 aromatase fusion gene. AROM(+) male mice are characterized by an imbalance in sex hormone metabolism, resulting in elevated serum E(2) concentrations, combined with significantly reduced testosterone and FSH levels, and elevated levels of PRL and corticosterone. AROM(+) males present a multitude of severe structural and functional alterations in the reproductive organs, such as cryptorchidism associated with Leydig cell hyperplasia, dysmorphic seminiferous tubules, and disrupted spermatogenesis. The males also have small or rudimentary accessory sex glands with abnormal morphology; a prominent prostatic utricle with squamous epithelial metaplasia, and edema in the ejaculatory ducts and vas deferens. In addition, the abdominal muscle wall is thin, and the adrenal glands are enlarged, with cortical hyperplasia. Some of the abnormalities, such as undescended testes and undeveloped prostate, resemble those observed in animals exposed perinatally to high levels of exogenous estrogen, indicating that the elevated aromatase activity results in excessive estrogen exposure during early phases of development. Some of the disorders in the reproductive organs, furthermore, can be explained by the fact that AROM(+) males are hypoandrogenic, and have elevated levels of serum PRL and corticosterone. Thus, the AROM(+) mouse model provides a novel tool to investigate the consequences of a prolonged increase in conversion of androgens to estrogens which results in complex hormonal disturbances altering the structure and function of various male reproductive organs.

Uptake and metabolism of hydroxymatairesinol in relation to its anticarcinogenicity in DMBA-induced rat mammary carcinoma model.

The chemopreventive effects of hydroxymatairesinol (HMR), a lignan extracted from Norway spruce (Picea abies), on the development of mammary carcinoma induced by 7,12-dimethylbenz[a]anthracene (DMBA) was studied in rats. HMR administered via diet in an average daily dose of 4.7 mg/kg body wt starting before DMBA induction reduced tumor volume and tumor growth, but no significant reduction in tumor multiplicity (number of tumors/rat) was observed. The predominant histological type in the control group was type B (well-differentiated adenocarcinoma, 78%). The proportion of type B tumors decreased to 35% in the HMR group, while the type A (poorly differentiated) and type C (atrophic) tumor proportions increased. Anticarcinogenic effects of dietary HMR (4.7 mg/kg) were also evident when the administration started after DMBA induction and was seen as growth inhibition of established tumors. Dietary HMR supplementation significantly increased serum and urinary enterolactone and HMR concentrations but had no significant effect on the uterine weight, suggesting that HMR or its major metabolite enterolactone did not have an antiestrogenic effect. Further studies are warranted to further clarify and verify HMR action and the associated mechanisms in mammary tumorigenesis.

Hydroxymatairesinol, a novel enterolactone precursor with antitumor properties from coniferous tree (Picea abies).

The potential for the extraction of the plant lignan hydroxymatairesinol (HMR) in large scale from Norway spruce (Picea abies) has given us the opportunity to study the metabolism and biological actions of HMR in animals. HMR, the most abundant single component of spruce lignans, was metabolized to enterolactone (ENL) as the major metabolite in rats after oral administration. The amounts of urinary ENL increased with the dose of HMR (from 3 to 50 mg/kg), and only minor amounts of unmetabolized HMR isomers and other lignans were found in urine. HMR (15 mg/kg body wt po) given for 51 days decreased the number of growing tumors and increased the proportion of regressing and stabilized tumors in the rat dimethylbenz[a]anthracene-induced mammary tumor model. HMR (50 mg/kg body wt) did not exert estrogenic or antiestrogenic activity in the uterine growth test in immature rats. HMR also showed no antiandrogenic responses in the growth of accessory sex glands in adult male rats. Neither ENL nor enterodiol showed estrogenic or antiestrogenic activity via a classical alpha- or beta-type estrogen receptor-mediated pathway in vitro at < 1.0 microM. HMR was an effective antioxidant in vitro.

Anti-oestrogen stimulation of ERBB2 ectodomain shedding from BT-474 human breast cancer cells with ERBB2 gene amplification.

Oestrogen has previously been shown to downregulate the expression of ERBB2 oncogene in human breast cancer cells, which contain a normal non-amplified ERBB2 gene. However, amplified ERBB2 seems to escape from hormonal regulation. We studied shedding of the extracellular domain (ectodomain, ECD) of the ERBB2 encoded protein in BT-474 human breast cancer cells treated with oestrogen or anti-oestrogen. Oestrogen-responsiveness of these cells has been previously demonstrated by stimulation of cell growth and expression of pS2, a marker gene known to be regulated by oestrogen receptor at transcriptional level. The concentration of the soluble ECD in the culture medium was increased by the anti-oestrogen toremifene as a function of time. In contrast, the level of ERBB2 mRNA and protein in cell lysates was not stimulated, but was transiently suppressed by toremifene. In the presence of oestrogen, the level of ECD remained low. The increased shedding of ECD in the presence of toremifene, without parallel change in ERBB2 transcripts (4.8 and 2.3 kb) and in cellular ERBB2 protein level, suggests that toremifene specifically contributes to the shedding of the ERBB2 ectodomain. These results show that shedding of ECD is an additional level of regulation of ERBB2 by the anti-oestrogen toremifene. This may contribute to resistance to growth inhibition by anti-oestrogens of breast cancers which overexpress ERBB2.

Mitotic activity, apoptosis and TRPM-2 mRNA expression in DMBA-induced rat mammary carcinoma treated with anti-estrogen toremifene.

Anti-estrogen toremifene inhibits growth of the 7,12-dimethylbenz(a)anthracene(DMBA)-induced rat mammary carcinoma. The changes in proliferation and cell death were studied in detail. Proliferation was measured by counting mitotic figures in histologic sections, expressed by volume-corrected mitotic index (M/V INDEX). Respective volume corrected apoptotic index (A/V INDEX) was measured by counting apoptotic nuclei in the same sections. The presence of apoptotic cells was confirmed by transmission electron microscopy. In the untreated tumors, both mitosis and apoptosis were frequent. In the toremifene-treated tumors, the mean M/V INDEX was about half of the mean M/V INDEX in the untreated control tumors. The mean A/V INDEX in the toremifene-treated tumors was about 3/4 of the mean A/V INDEX in the controls. In toremifene-treated tumors, A/V INDEX was strongly correlated with TRPM-2-gene expression, which was also enhanced when compared with the controls. TRPM-2 gene has been associated with programmed cell death induced by hormonal ablation in prostate- and breast-cancer cells. No such correlation was seen in control tumors. These findings suggest that, in the DMBA-tumor model, toremifene affects the cell turnover by inhibiting mitotic activity and modifying abundant spontaneous apoptosis. In this model, the inhibition of tumor growth results mostly from reduction of mitotic activity.

Apoptosis in toremifene-induced growth inhibition of human breast cancer cells in vivo and in vitro.

BACKGROUND: Antiestrogens inhibit the stimulative effects of estrogens on breast cancer growth, but the mechanism(s) by which they trigger tumor regression are not completely understood. Growth retardation and tumor regression can be achieved by enhanced cell death and/or arrested cell proliferation. PURPOSE: Our aim was to investigate the effect of a new antiestrogen, toremifene, on human breast cancer cells grown either in culture or as tumors in nude mice. METHODS: The growth and morphology of in vitro cultured cells of the human breast cancer cell line MCF-7 were monitored by time-lapse video. MCF-7 cells and ZR-75-1 human breast cancer cells were grown as tumors in nude mice and subsequently examined by electron microscopy. The integrity of DNA isolated from these cells was determined by standard gel electrophoretic techniques. Northern blot hybridization analysis was used to determine the steady-state levels of the mRNAs for testosterone-repressed prostatic message-2 (TRPM-2), tumor growth factor beta-1 (TGF beta 1), and pS2 (a small, cysteine-rich protein of unknown function). RESULTS: Time-lapse video microscopy of the cell cultures indicated that treatment with 7.5 microM toremifene for 3 days caused approximately 60% of the cells to exhibit morphologic characteristics typical of cells undergoing programmed death, or apoptosis. The number of mitoses gradually decreased to zero over a 3- to 4-day period. Estrogen withdrawal for the same length of time resulted in an approximately equal number of apoptoses and mitoses. These changes were not associated with the pattern of DNA fragmentation, detectable as ladders in agarose gels, that is characteristic of the DNA of cells undergoing apoptosis. Elevated levels of TRPM-2 and TGF beta 1 mRNAs were observed in in vitro or in vivo grown tumor cells treated with 5-10 microM toremifene. Elevated levels of TRPM-2, but not TGF beta 1, mRNA were observed in the tumor cells after estrogen withdrawal. The steady-state level of pS2 mRNA in the tumor cells dropped in response to either toremifene treatment or estrogen withdrawal. CONCLUSION: Toremifene causes growth inhibition of estrogen-sensitive breast cancer cells by inducing some cells to undergo apoptosis and by inhibiting other cells from entering mitosis. The higher than normal amounts of TRPM-2 and TGF beta 1 protein that would likely result from the elevated levels of TRPM-2 and TGF beta 1 mRNAs measured in these cells after toremifene treatment may have an important role in the growth inhibition process. IMPLICATION: Apoptosis as an active, targeted process provides a potential new therapeutic approach for treating breast cancer.

Analysis of tyrosine kinase mRNAs including four FGF receptor mRNAs expressed in MCF-7 breast-cancer cells.

The MCF-7 cell line is a hormone-responsive human breast-cancer cell line, which has been extensively used in studies of estrogen regulation of cell growth. These studies have indicated that the growth stimulation of the MCF-7 cells by estrogens may be effected by an autocrine mechanism involving several growth factors, such as EGF, TGF alpha and IGF-I and their receptors. We have amplified and cloned tyrosine-kinase-related sequences from the MCF-7 cell mRNA using the polymerase chain reaction and characterized the partial cDNAs obtained by nucleic acid sequencing. Nine tyrosine kinase cDNAs and one serine/threonine kinase cDNA were identified among the amplified sequences. Four different tyrosine kinase genes encoding receptors for fibroblast growth factors (FGFs) were found to be expressed by the MCF-7 cells. In addition, differences were observed in the expression of these members of FGF receptor family in different breast-cancer cells. A putative tyrosine-kinase receptor and a novel serine/threonine kinase were preferentially expressed in estrogen-responsive tumor cell lines. However, no estrogen-dependent regulation of any of the novel tyrosine-kinase receptor mRNAs was found in any of the cell lines including the MCF-7 or ZR-75-I cells, where the expression of the neu proto-oncogene mRNA was decreased during estrogen treatment. The expression of several FGF receptors by breast-cancer cells suggests that FGFs may be involved in their growth regulation and tumorigenesis.

Estrogen suppression of erbB2 expression is associated with increased growth rate of ZR-75-1 human breast cancer cells in vitro and in nude mice.

Amplification and enhanced expression of the erbB2/HER-2/neu gene has been associated with an increased growth rate and poor prognosis of human breast cancer. We have studied the relationship between erbB2 expression and the regulation of cell growth by estrogen and anti-estrogens in the human breast cancer cell line ZR-75-1 in vitro and in athymic nude mice, pS2 being used as a marker gene for estrogen-stimulated gene expression. Only low amounts of erbB2 mRNA were seen in the cells grown in vitro in the presence of estrogen which stimulated the cells to proliferate rapidly and induced the expression of pS2 mRNA. Upon hormone withdrawal, erbB2 mRNA and protein increased, while pS2 mRNA declined to an undetectable level and cell proliferation slowed down. Opposite but more rapid changes were observed upon estrogen addition. The anti-estrogens toremifene and tamoxifen inhibited estrogen induction of pS2 expression, down-regulation of erbB2 expression and proliferation of the ZR-75-I cells in a concentration-dependent manner. Similar results were obtained in nude mice. ZR-75-I cells formed tumors only in mice carrying estrogen pellets. In these tumors little erbB2 mRNA was seen. Concomitant administration of toremifene or tamoxifen increased erbB2 mRNA and abolished pS2 mRNA. Our results show that enhanced expression of erbB2 is associated with hormone deprivation and growth arrest of the estrogen-dependent breast cancer cell line ZR-75-I. Thus, in mammary epithelial cells, erbB2 may have important estrogen-regulated functions which are not related to cell proliferation.

Effect of toremifene on the activity of NK-cells in NZB/NZW mice.

The effect of toremifene on NK-cells isolated from the spleen of NZB/NZW mice was studied in comparison to tamoxifen and estradiol. Unlike estradiol but like tamoxifen, toremifene did not influence the activity of NK-cells. Low doses (0.1 and 10.0 mg/kg) of toremifene did not suppress, but a high dose of toremifene and tamoxifen (50 mg/kg for 6 weeks) suppressed the stimulating effect of human interferon alpha on the cells.

Expression of the c-Ha-ras and neu oncogenes in DMBA-induced, anti-estrogen-treated rat mammary tumors.

Dimethylbenzanthracene (DMBA)-induced rat mammary tumors were analyzed for the structure and expression of the oncogenes c-Ha-ras and neu and the effects of anti-estrogen treatment. Tumor samples were divided into 3 groups, the first consisting of untreated tumors, the second of anti-estrogen (toremifene)-treated unresponsive (growing) tumors, and the third of toremifene-treated responsive (regressing) tumors. DNA and RNA derived from normal tissues of the same experimental animals were also analyzed. In Southern blot analysis of genomic DNAs, 2 tumors out of 23 contained a new Xbal site in the Ha-ras gene, indicating a point mutation in the second nucleotide of codon 61. Both of these tumors belonged to the group that had not received toremifene. No amplifications of the Ha-ras or the neu genes were observed. Although greatly variable, the levels of Ha-ras mRNA were highest in untreated tumors, lower in toremifene-treated, unresponsive tumors and even lower in toremifene-treated, regressing tumors, corresponding approximately to the levels detected in normal liver and uterus of untreated animals. Expression of the neu mRNA was variable and considerably lower than that of Ha-ras mRNA. It was similar in all 3 groups and somewhat elevated than in several non-malignant control tissues. Localization of c-Ha-ras expression by in situ hybridization revealed a relatively even distribution of the mRNA throughout the mammary tissue. The results suggest that mechanisms other than activation of the c-Ha-ras or neu genes are important for progression and regression of DMBA-induced rat mammary carcinomas.