Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth.

Estrogen receptor-α (ERα) positive breast cancer frequently responds to inhibitors of ERα activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ERα in ERα-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ERα over-expression in ERα-positive breast cancer cells, we over-expressed ERα in the MCF-7 breast cancer cell line using adenovirus gene transduction. ERα over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ERα transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ERα-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ERα remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ERα could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.

Mechanisms of endometrial progesterone resistance.

Throughout the reproductive years, the rise and fall in ovarian hormones elicit in the endometrium waves of cell proliferation, differentiation, recruitment of inflammatory cells, apoptosis, tissue breakdown and regeneration. The activated progesterone receptor, a member of the superfamily of ligand-dependent transcription factors, is the master regulator of this intense tissue remodelling process in the uterus. Its activity is tightly regulated by interaction with cell-specific transcription factors and coregulators as well as by specific posttranslational modifications that respond dynamically to a variety of environmental and inflammatory signals. Endometriosis, a chronic inflammatory disorder, disrupts coordinated progesterone responses throughout the reproductive tract, including in the endometrium. This phenomenon is increasingly referred to as 'progesterone resistance'. Emerging evidence suggests that progesterone resistance in endometriosis is not just a consequence of perturbed progesterone signal transduction caused by chronic inflammation but associated with epigenetic chromatin changes that determine the intrinsic responsiveness of endometrial cells to differentiation cues.

Risks of adverse pregnancy outcome in endometriosis.

Bleeding from endometriotic implants is now an established cause of acute hemoperitoneum in pregnancy. However, the adverse impact of pelvic endometriosis on uterine function before conception may also interfere with subsequent deep placentation, accounting for the increased risk of obstetrical complications, including preterm birth and antepartum hemorrhage.

NADPH oxidase-derived reactive oxygen species mediate decidualization of human endometrial stromal cells in response to cyclic AMP signaling.

Differentiation of human endometrial stromal cells into specialized decidual cells is critical for embryo implantation and survival of the conceptus. Initiation of this differentiation process is strictly dependent on elevated cAMP levels, but the signal intermediates that control the expression of decidual marker genes, such as prolactin (PRL) and IGFBP1, remain poorly characterized. Here we show that cAMP-dependent decidualization can be attenuated or enhanced upon treatment of primary cultures with a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenylen iodonium) or activator (apocynin), respectively. Time-course analysis demonstrated that cAMP enhances endogenous reactive oxygen species production, apparent after 12 h of stimulation, which coincides with a dramatic increase in decidual PRL and IGFBP1 expression. Knockdown of the Rho GTPase RAC1, which disables activation of the NADPH oxidase homologs NADPH oxidase (NOX)-1, NOX-2, and NOX-3, had no effect on PRL or IGFBP1 expression. In contrast, silencing of NOX-4, or its cofactor p22(PHOX), inhibited the expression of both decidual markers. Finally, we show that the NOX-4/p22(PHOX) complex regulates the DNA-binding activity of CCAAT/enhancer binding protein-ß, a key regulator of human endometrial stromal cell differentiation. Thus, NOX-4 activation and reactive oxygen species signaling play an integral role in initiating the endometrial decidual response in preparation of pregnancy.

Deregulation of the serum- and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure.

Infertility and recurrent pregnancy loss (RPL) are prevalent but distinct causes of reproductive failure that often remain unexplained despite extensive investigations. Analysis of midsecretory endometrial samples revealed that SGK1, a kinase involved in epithelial ion transport and cell survival, is upregulated in unexplained infertility, most prominently in the luminal epithelium, but downregulated in the endometrium of women suffering from RPL. To determine the functional importance of these observations, we first expressed a constitutively active SGK1 mutant in the luminal epithelium of the mouse uterus. This prevented expression of certain endometrial receptivity genes, perturbed uterine fluid handling and abolished embryo implantation. By contrast, implantation was unhindered in Sgk1-/- mice, but pregnancy was often complicated by bleeding at the decidual-placental interface and fetal growth retardation and subsequent demise. Compared to wild-type mice, Sgk1-/- mice had gross impairment of pregnancy-dependent induction of genes involved in oxidative stress defenses. Relative SGK1 deficiency was also a hallmark of decidualizing stromal cells from human subjects with RPL and sensitized these cells to oxidative cell death. Thus, depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility, or predisposes to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage.