Nude mouse system in the study of tumor biology, treatment strategies and progesterone receptor physiology in human endometrial carcinoma

A nude mouse system where the biological, morphological and biochemical characteristics of human endometrial carcinoma are maintained during serial transplantation has been previously described. Applications of this system to the study of (a) hormonal sensitivity (b) treatment strategies and (c) progesterone receptor physiology of human endometrial carcinomas are presented here.     

Role of fibroblast growth factor 8 in growth and progression of hormonal cancer

Hormonal cancers such as breast and prostate cancer arise from steroid hormone-regulated tissues. In addition to breast and prostate cancer hormonal regulation has also a role in endometrial, ovarian, testis and thyroid carcinomas. The effects of estrogens, androgens and progestagens on tumor growth are largely mediated by paracrine and autocrine target molecules which include growth factors and growth factor receptors. During cancer progression the hormonal growth regulation is often lost or overcome by an inappropriate activation of growth factor signaling cascades. One of the growth factors which have been associated with the regulation of growth and progression of hormonal cancer is fibroblast growth factor 8 (FGF8) which has also been recognized as an oncogene. FGF8 is widely expressed during embryonic development. It has been shown to mediate embryonic epithelial-mesenchymal transition and to have a crucial role in gastrulation and early organization and differentiation of midbrain/hindbrain, pharyngeal, cardiac, urogenital and limb structures. During adulthood FGF8 expression is much more restricted but in hormonal cancers it becomes frequently activated. High level of FGF8 expression in tumors is associated with a poor prognosis at least in prostate cancer. In experimental models FGF8 induces and facilitates prostate tumorigenesis and increases growth and angiogenesis of tumors. Several lines of evidence for autocrine and paracrine loops in the growth regulation of breast, prostate and ovarian cancer by FGF8 have been suggested.     

Application of functional genomics to primate endometrium: insights into biological processes

Endometrium is a dynamic tissue that responds on a cyclic basis to circulating levels of the ovarian-derived steroid hormones, estradiol and progesterone. Functional genomics has enabled a global approach to understanding gene regulation in whole endometrial tissue in the setting of a changing hormonal milieu. The proliferative phase of the cycle, under the influence of estradiol, has a preponderance of genes involved in DNA synthesis and cell cycle regulation. Interestingly, genes encoding ion channels and cell adhesion, as well as angiogenic factors, are also highly regulated in this phase of the cycle. After the LH surge, different gene expression profiles are uniquely observed in the early secretory, mid-secretory (window of implantation), and late secretory phases. The early secretory phase is notable for up-regulation of multiple genes and gene families involved in cellular metabolism, steroid hormone metabolism, as well as some secreted glycoproteins. The mid-secretory phase is characterized by multiple biological processes, including up-regulation of genes encoding secreted glycoproteins, immune response genes with a focus on innate immunity, and genes involved in detoxification mechanisms. In the late secretory phase, as the tissue prepares for desquamation, there is a marked up-regulation of an inflammatory response, along with matrix degrading enzymes, and genes involved in hemostasis, among others. This monograph reviews hormonal regulation of gene expression in this tissue and the molecular events occurring therein throughout the cycle derived from functional genomics analysis. It also highlights challenges encountered in using human endometrial tissue in translational research in this context.     

Effects of hormones on SBP mRNA levels in human cancer cells

The human plasma sex steroid binding protein (SBP) has been previously shown to be synthesized in liver cells. The hormonal regulation studies of hepatic SBP mRNA demonstrate that it is controlled by estradiol, antiestrogen tamoxifen, dihydrotestosterone, triiodothyronine and insulin in a similar way as secreted SBP. The metabolic inhibitor cycloheximide was unable to prevent the estrogen or thyroid hormone induced increase in SBP mRNA. The slight stimulation of SBP synthesis by estradiol suggests that non-steroidal factors may be involved in its regulation and that the estrogen regulatory mechanism could also be partly post-transcrptional. In endometrial (Ishikawa cells) and prostatic (LNCaP cells) carcinoma cells, SBP mRNA has been detected suggesting that SBP may play a role in the uptake and intracellular mechanism of action of sex steroid in target cells.     

Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism

Evidence for a role of estrogen metabolism in hormonal carcinogenesis was obtained with the Syrian hamster as an in vivo model system. Both natural and synthetic estrogens are capable of inducing a high incidence of renal carcinomas in this species. A high incidence of hepatocellular carcinomas can also be induced in the hamster with synthetic estrogens such as ethinyl estradiol or diethylstilbestrol, provided alpha-naphthoflavone (ANF) is present in the diet. Although steroid receptor-mediated hormonal events appear to be intimately involved in the process of in vivo cell transformation of both tissues, certain observations strongly suggest that nonhormonal events are also important. Despite their potent estrogenic activity at the doses used, ethinyl estradiol and alpha-zearalanol induce relatively low renal tumor incidences after 9.0 and 10.0 months of continuous treatment, respectively. A role for the metabolism of estrogens to reactive intermediates is also suggested by studies showing estrogen-induced renal tumorigenesis can be partially inhibited by concomitant administration of ANF or ascorbic acid. Consistent with this is the general correlation between the amount of catechol estrogen formed by a compound, as mediated by estrogen 2-/4-hydroxylase, and renal carcinogenicity data. Recently, additional supporting evidence has been obtained from studies involving the irreversible binding of reactive metabolites of steroidal or stilbene estrogens to hamster liver microsomal proteins.     

Acceleration of the Glycolytic Flux by Steroid Receptor Coactivator-2 Is Essential for Endometrial Decidualization

Early embryo miscarriage is linked to inadequate endometrial decidualization, a cellular transformation process that enables deep blastocyst invasion into the maternal compartment. Although much of the cellular events that underpin endometrial stromal cell (ESC) decidualization are well recognized, the individual gene(s) and molecular pathways that drive the initiation and progression of this process remain elusive. Using a genetic mouse model and a primary human ESC culture model, we demonstrate that steroid receptor coactivator-2 (SRC-2) is indispensable for rapid steroid hormone-dependent proliferation of ESCs, a critical cell-division step which precedes ESC terminal differentiation into decidual cells. We reveal that SRC-2 is required for increasing the glycolytic flux in human ESCs, which enables rapid proliferation to occur during the early stages of the decidualization program. Specifically, SRC-2 increases the glycolytic flux through induction of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3), a major rate-limiting glycolytic enzyme. Similarly, acute treatment of mice with a small molecule inhibitor of PFKFB3 significantly suppressed the ability of these animals to exhibit an endometrial decidual response. Together, these data strongly support a conserved mechanism of action by which SRC-2 accelerates the glycolytic flux through PFKFB3 induction to provide the necessary bioenergy and biomass to meet the demands of a high proliferation rate observed in ESCs prior to their differentiation into decidual cells. Because deregulation of endometrial SRC-2 expression has been associated with common gynecological disorders of reproductive-age women, this signaling pathway, involving SRC-2 and PFKFB3, promises to offer new clinical approaches in the diagnosis and/or treatment of a non-receptive uterus in patients presenting idiopathic infertility, recurrent early pregnancy loss, or increased time to pregnancy.     

Could DNA hydroxymethylation be crucial in influencing steroid hormone signaling in endometrial biology and endometriosis?

Endometriosis affects 10% of reproductive‐aged women. It is characterized by the growth of the endometrium, outside the uterus and is associated with infertility and chronic abdominal pain. Lack of noninvasive diagnostic tools and early screening tests results in delayed treatment and subsequently increased disease severity. Endometriosis is a disease associated with a deregulated hormonal response, therefore, understanding the molecular mechanisms that govern this hormonal interplay is of paramount importance. DNA methylation is an epigenetic mark that regulates gene expression and is often associated with genes that code for steroid receptors and enzymes associated with estrogen synthesis and metabolism in endometriosis. DNA hydroxymethylation, which is structurally similar to methylation but functionally different, is a biologically critical mechanism that is also known to regulate gene expression. Ten Eleven Translocation (TET) proteins mediate hydroxymethylation. However, the role of DNA hydroxymethylation or TETs in the endometrium remains relatively unexplored. Currently, the “gold standard” technique used to study methylation patterns is bisulfite genomic sequencing. This technique also detects hydroxymethylation but fails to distinguish between the two, thereby limiting our understanding of these two processes. The presence of TETs in the male and female reproductive tract and its contribution to endometrial cancer makes it an important factor to study in endometriosis. This review summarizes the role of DNA methylation in aberrant steroid hormone signaling and hypothesizes that hydroxymethylation could be a factor influencing hormonal instability seen in endometriosis.     

Human endometrial epithelial cell lines for studying steroid and cytokine actions

Summary Recent studies suggest that the proliferation and expression of HLA-DR molecules in endometrial epithelium may be regulated by systemic steroids and local cytokines. To test the interacting influences of cytokines and steroids on the expression of HLA-DR and proliferation of epithelial cells, an endometrial cell model is required that is sensitive to both signals. In this study, we characterize cells of carcinoma cell lines of endometrial lineage for their responsiveness to cytokines and steroids. Independently developed for its response to steroid hormones from a well-differentiated adenocarcinoma of human endometrium, EnCa101AE cell line is further cloned for the expression of progesterone receptor. Immunohistochemical localization using monoclonal antibodies demonstrates that both EnCa101AE cell line and cloned ECC1 cells are purely epithelial, as evidenced by the expression of cytokeratin and epithelial membrane antigen, express estrogen receptors, and concomitantly exhibit IFN-gamma receptor. Experiments using radioiodinated IL-1 reveal that these cell lines also possess high affinity receptors for IL-1. As indicated by the induction of HLA-DR molecules, and alterations in morphologic characteristics, these cell lines are sensitive to both IFN-gamma and IL-1 action. The class II molecules (HLA-DR, HLA-DP, and HLA-DQ) are differentially induced by IFN-gamma treatment in carcinoma cell lines, with HLA-DR being the prevailing induced molecule. IFN-gamma inhibits and estradiol-17β promotes growth of ECC1 cells in a dose-and time-dependent manner. These findings indicate that the interacting effect(s) of the cytokines and steroid hormones on endometrial epithelium may be studied in these unique steroid-and cytokine-sensitive epithelial cell lines.     

Morphology and DNA content of endometrial cancer nuclei under progestogen treatment.

Twelve endometrial cancers have been observed before and during progestogen treatment. Eight were well-differentiated and displayed an objective response. It was essentially characterized by a volumetric reduction of the nucleus and the disappearance of tetraploid nuclei. The DNA content remained diploid or near-diploid. Reduction of nuclear non DNA proteins under hormonal treatment is suggested.     

Variable responsiveness of hormone-inducible hybrid genes in different cell lines

Altered steroid responsiveness leads to various pathological conditions and is a particular problem for the treatment of cancers arising in steroid-sensitive cells. To develop cellular model systems for the analysis of the molecular mechanisms mediating altered steroid responses, we have analyzed the inducibility of a steroid-responsive promoter in different cell lines. In vitro constructs containing the mouse mammary tumor virus promoter fused to the herpes simplex virus thymidine kinase gene or the bacterial neo gene were transfected into four different cell lines [Rat-2, CHO chinese hamster ovary cells, F9, and T47D). Thymidine kinase+ clones and neo-resistant clones were selected in the presence of dexamethasone (dex) and/or other steroid hormones. We find that the mouse mammary tumor virus promoter activity is completely dependent on the presence of dex in Rat-2 cells but is constitutively active in CHO cells and is inactive in F9 teratocarcinoma cells in the presence and absence of dex. In the human breast cancer cell line T47D, we observe no response to dex but do observe an inducibility by progesterone. Examination of glucocorticoid receptors in these cell lines showed that Rat-2, CHO, and F9 cells contain sufficient receptors to allow a hormonal response, whereas in T47D cells several glucocorticoid binding activities appear to be present. Our results indicate that the presence of receptor in cells is not always sufficient to allow hormonal activation and that, in some cell lines, like CHO, other factors are present that can substitute for an activated steroid hormone receptor complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid hormones acutely regulate expression of a Nudix protein-encoding gene in the endometrial epithelium of sheep

Steroid hormones regulate endometrial gene expression to meet the needs of developing embryos. Our hypothesis is that steroid hormones transiently induce expression of genes in the endometrial epithelium to make the uterine environment different between the earliest days of pregnancy. We identified one such gene product using differential display-polymerase chain reactions. The gene product that was strongly induced in ewes between day 3 and 6 of the estrous cycle was cloned and sequenced to identify it as encoding a member of the Nudix family of hydrolase enzymes. Northern blot analyses indicated that NUDT16 mRNA concentrations were elevated 10-fold in the endometrium of sheep from day 5 to 9 of the estrous cycle and returned to basal levels by day 11. In assays of RNA samples from 15 different tissues from an adult ewe, the concentrations of NUDT16 mRNA were greatest in endometrium. In situ hybridization localized NUDT16 mRNA exclusively to the endometrial epithelial cells of the glands and uterine lumen. In ovariectomized ewes, NUDT16 mRNA was induced by a regimen of alternating estrogen and progesterone therapy designed to mimic the hormonal experiences of a ewe at day 6 of the estrous cycle. The final estrogen treatment in the regimen was critical to the expression of NUDT16 as well as progesterone receptor and estrogen receptor-beta genes. Characterization of the NUDT16 gene identified putative steroid hormone response elements, which can now be investigated to understand its unique pattern of regulation in the earliest days of pregnancy.     

Gonadal steroids regulate the expression of aggrecanases in human endometrial stromal cells in vitro

The human endometrium undergoes cyclic change during each menstrual cycle in response to gonadal steroids. Proteolysis of endometrial extracellular matrix (ECM) is necessary to prepare this dynamic tissue for pregnancy. Proteolytic enzymes such as matrix metalloproteinase (MMP) and closely related a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) have been assigned key roles in the highly regulated cyclic remodelling of the endometrial ECM. We have previously shown that ADAMTS‐1 undergoes spatiotemporal changes in human endometrial stromal cells under the regulation of gonadal steroids. This suggests that other ADAMTS subtypes, known as aggrecanases, may contribute to the ECM remodelling events that occur in female physiological cycles and in preparation for pregnancy. To determine whether progesterone (P4), 17β‐estradiol (E2), or dihydrotestosterone (DHT), alone or in combination, are capable of regulating ADAMTS‐4, ‐5, ‐8 or ‐9 expression in human endometrial stromal cells in vitro. Real‐time quantitative PCR and Western blot analysis were used to measure ADAMTSs mRNA and protein levels in primary cultures of human endometrial stromal cells (n = 12). P4, DHT but not E2 have regulatory effects on ADAMTS‐8, ‐9 and ‐5 expression. Combined treatment with gonadal steroids did not show any synergistic or antagonistic effects. However, the synthetic steroid antagonists RU486 and hydroxyflutamide specifically inhibited the P4‐ or DHT‐mediated regulatory effects on ADAMTS expression. These studies provide evidence that the regulation of aggrecanases by gonadal steroids in human endometrial stromal cells may play an important role during decidualization.     

Female sex steroid induced epithelial changes in the gallbladder of the ovariectomized Syrian hamster.

Ovariectomized Syrian hamsters treated by female sex steroids during a 1-month period show gallbladder surface epithelial changes in the fundic area consistent with apical bulging and decapitations of the epithelial cells. These events were detected in the infundibulum and the fundic or body regions of estrogen- and estrogen+progesterone-treated hamsters. In control hamsters, these events were restricted to the region in the vicinity of the bile duct. Following steroid treatment, intraluminal deposits detected resembled Ca-bilirubinate deposits described in previous studies while decapitations are similar to endometrial epithelium changes associated with hormonal physiological changes or treatments. Moreover some small electron-dense deposits are comparable to those found in human cholesterol gallstones. This report indicates that, besides an alteration in bile composition, cell fragments originating from the surface epithelium of the bile duct and/or of the gallbladder mucosal epithelium could participate in gallstone nucleation.     

In vitro release of PGF(2alpha) from the caprine uterus and the effect of ovarian steroids

The purpose of the study was to determine the in vitro release of prostaglandin F(2)alpha (PGF(2alpha)) from the uterine tissues of ovariectomized goats after steroid treatment. Strips of endometrial tissues (separated by trypsin treatment) and intact uterine tissues (with endometrium and myometrium) were maintained in organ culture and exposed to estradiol-17beta(E) at 50 ng/ml and to progesterone (P) at 250 pg/ml alone or in combination. The endometrial tissues, in general, continued to release PGF(2alpha) without steroids treatment throughout the 96-h incubation period; their output was consistently higher (based on ng/gm of wet tissue) than the corresponding intact uterine tissues. However, exposure to E reversed this trend and produced a three-fold increase from the intact uterine strips at 24 h. A combined E-plus-P treatment blocked the stimulatory response, whereas P treatment alone evoked a delayed stimulatory response at 48 h. Endometrial tissue reaction was similar to that of P treatment, but it exhibited a more moderated response to E, which could not be blocked by a combined E-plus-P treatment. The results suggest that both endometrial and myometrial tissues release PGF(2alpha) and that this release is regulated by ovarian steroid hormones.     

N-acetyllactosamine and sialosyl-N-acetyllactosamine in normal and malignant human endometrium

We have evaluated using immunohistochemistry the expression of the type 2 chain histo-blood group precursorsN-acetyllactosamine (Lac), sialosyl-Lac (S-Lac) and binary-sialosyl Lac (DS-Lac) in epithelial cells of normal non-secretory, gestational and malignant human endometrial tissues (n=120). Staining was assessed in relation to genetic (ABO, Lewis blood group and secretor status), morphologic and hormonal factors (serum levels of estrogens). The staining pattern for Lac, S-Lac and DS-Lac showed great variation and was not related to blood group or the secretor status. Staining for Lac showed a limited distribution in both normal and malignant endometrium and was most frequenly found in gestational and atrophic endometrium. S-Lac was strongly expressed, but only infrequently as DS-Lac structures in normal endometrium. Staining for both S-Lac and DS-Lac was most widespread in proliferating endometria. Endometrial carcinomas showed an increased staining for DS-Lac and a varied, and in most cases a reduced, staining for S-Lac, a pattern like that previously found in secretory endometrium. Staining scores for S-Lac showed a weak correlation with serum levels of free estradiol. Thus, the increased expression of DS-Lac in contrast to S-Lac structures in endometrial carcinomas is probably unrelated to both hormonal and genetic factors and may be considered a tumor-associated but not a tumor-specific change in endometrial cell glycosylation.     

Differential effects of progesterone treatment on the oxytocin-induced prostaglandin F2 alpha response and the levels of endometrial oxytocin receptors in ovariectomized ewes.

The oxytocin-induced uterine prostaglandin (PG) F2 alpha response and the levels of endometrial oxytocin receptors were measured in ovariectomized ewes after they had been given steroid pretreatment (SP) with progesterone and estrogen to induce estrus (day of expected estrus = Day 0) and had subsequently been treated with progesterone over Days 1-12 and/or PGF2 alpha over Days 10-12 postestrus. The uterine PGF2 alpha response was measured after an i.v. injection of 10 IU oxytocin on Days 13 and 14, using the PGF2 alpha metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), as an indicator for PGF2 alpha release. The levels of oxytocin receptors in the endometrium were measured on Day 14. During the treatment with progesterone, the peripheral progesterone concentrations were elevated and remained above 1.8 ng/ml until the morning of Day 14. The PGFM responses to oxytocin in untreated controls and SP controls were low on both Days 13 and 14 whereas the levels of endometrial oxytocin receptors in the same ewes were high. Treatment with progesterone either alone or in combination with PGF2 alpha significantly (p less than 0.04) increased the PGFM response on Day 14 and reduced the levels of endometrial oxytocin receptors; treatment with PGF2 alpha alone had no effect. It is concluded that progesterone promotes the PGFM response to oxytocin while simultaneously suppressing the levels of endometrial oxytocin receptors. PGF2 alpha treatment had no effect on either the uterine secretory response to oxytocin or the levels of oxytocin receptors in the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)