Decreased Progesterone Receptor Research Articles

EHD1 impaired decidualization of endometrial stromal cells in recurrent implantation failure: role of SENP1 in modulating progesterone receptor signalling†.

Recurrent implantation failure (RIF) patients exhibit poor endometrial receptivity and abnormal decidualization with reduced effectiveness and exposure to progesterone, which is an intractable clinical problem. However, the associated molecular mechanisms remain elusive. We found that EH domain containing 1 (EHD1) expression was abnormally elevated in RIF and linked to aberrant endometrial decidualization. Here we show that EHD1 overexpressed in human endometrial stromal cells significantly inhibited progesterone receptor (PGR) transcriptional activity and the responsiveness to progesterone. No significant changes were observed in PGR mRNA levels, while a significant decrease in progesterone receptor B (PRB) protein level. Indeed, EHD1 binds to the PRB protein, with the K388 site crucial for this interaction. Overexpression of EHD1 promotes the SUMOylation and ubiquitination of PRB, leading to the degradation of the PRB protein. Supplementation with the de-SUMOylated protease SENP1 ameliorated EHD1-repressed PRB transcriptional activity. To establish a functional link between EHD1 and the PGR signalling pathway, sg-EHD1 were utilized to suppress EHD1 expression in HESCs from RIF patients. A significant increase in the expression of prolactin and insulin-like growth factor-binding protein 1 was detected by interfering with the EHD1. In conclusion, we demonstrated that abnormally high expression of EHD1 in endometrial stromal cells attenuated the activity of PRB associated with progesterone resistance in a subset of women with RIF.

A Novel Surrogate Nomogram Capable of Predicting OncotypeDX Recurrence Score©.

Background: OncotypeDX Recurrence Score© (RS) is a commercially available 21-gene expression assay which estimates prognosis and guides chemoendocrine prescription in early-stage estrogen-receptor positive, human epidermal growth factor receptor-2-negative (ER+/HER2−) breast cancer. Limitations of RS testing include the cost and turnaround time of several weeks. Aim: Our aim is to develop a user-friendly surrogate nomogram capable of predicting RS. Methods: Multivariable linear regression analyses were performed to determine predictors of RS and RS > 25. Receiver operating characteristic analysis produced an area under the curve (AUC) for each model, with training and test sets were composed of 70.3% (n = 315) and 29.7% (n = 133). A dynamic, user-friendly nomogram was built to predict RS using R (version 4.0.3). Results: 448 consecutive patients who underwent RS testing were included (median age: 58 years). Using multivariable regression analyses, postmenopausal status (β-Coefficient: 0.25, 95% confidence intervals (CIs): 0.03–0.48, p = 0.028), grade 3 disease (β-Coefficient: 0.28, 95% CIs: 0.03–0.52, p = 0.026), and estrogen receptor (ER) score (β-Coefficient: −0.14, 95% CIs: −0.22–−0.06, p = 0.001) all independently predicted RS, with AUC of 0.719. Using multivariable regression analyses, grade 3 disease (odds ratio (OR): 5.67, 95% CIs: 1.32–40.00, p = 0.037), decreased ER score (OR: 1.33, 95% CIs: 1.02–1.66, p = 0.050) and decreased progesterone receptor score (OR: 1.16, 95% CIs: 1.06–1.25, p = 0.002) all independently predicted RS > 25, with AUC of 0.740 for the static and dynamic online nomogram model. Conclusions: This study designed and validated an online user-friendly nomogram from routinely available clinicopathological parameters capable of predicting outcomes of the 21-gene RS expression assay.

SCM-198 Prevents Endometriosis by Reversing Low Autophagy of Endometrial Stromal Cell via Balancing ERα and PR Signals.

BackgroundEndometriosis (EMS), an endocrine-related inflammatory disease, is characterized by estrogen and progesterone imbalance in ectopic lesions. However, its pathogenic mechanism has not been fully elucidated. While SCM-198 is the synthetic form of leonurine and has multiple pharmacological activities such as antioxidation and anti-inflammation, it remains unknown whether it could inhibit the progress of EMS by regulating estrogen signaling and inflammation.MethodsThe therapeutic effects of SCM-198 on EMS and its potential mechanism were analyzed by establishing EMS mouse models and performing an RNA sequencing (RNA-seq) assay. ELISA was performed to detect estrogen and tumor necrosis factor (TNF) -α concentrations in normal endometrial stromal cells (nESCs) and ectopic endometrial stromal cells (eESCs) with or without SCM-198 treatment. Western blotting, RNA silencing, and plasmid overexpression were used to analyze the relationship between inflammation, endocrine factors, and autophagy and the regulatory activity of SCM-198 on the inflammation-endocrine-autophagy axis.ResultsIncreased estrogen-estrogen receptor (ER) α signaling and decreased progesterone receptor isoform B (PRB) expression synergistically led to a hypo-autophagy state in eESCs, which further inhibited the apoptosis of eESCs. The high expression of TNF-α in eESCs enhanced the antiapoptotic effect mediated by low autophagy through the activation of the aromatase-estrogen-ERα signaling pathway. SCM-198 inhibited the growth of ectopic lesions in EMS mice and promoted the apoptosis of eESCs both in vivo and in vitro. The apoptotic effect of SCM-198 on eESCs was attained by upregulating the autophagy level via the inhibition of the TNF-α-activated aromatase-estrogen-ERα signal and the increase in PRB expression.ConclusionInflammation facilitated the progress of EMS by disrupting the estrogen regulatory axis. SCM-198 inhibited EMS progression by regulating the inflammation-endocrine-autophagy axis.

ARID1a INVOLVEMENT IN ENDOMETRIOSIS DISEASE PROGRESSION

Endometriosis is a chronic gynecologic disease affecting 10% of reproductive-aged women. While medical therapy and surgery are the cornerstones of treatment, each has associated failure rates. We have shown that non-responders (NRs) to progestins have decreased progesterone receptor (PR) expression; surprisingly estrogen receptors (ERs) alpha and beta were often decreased as well. The decreases in ERs and PR levels in endometriosis may be analogous to receptor negative breast cancer, suggesting other non-hormonal mechanisms are involved in disease progression.

Adenomyosis pathogenesis: insights from next-generation sequencing.

BACKGROUNDAdenomyosis, characterized by the presence of islands of endometrial tissue surrounded by hypertrophic smooth muscle cells within the myometrium, is one of the most challenging uterine disorders in terms of diagnosis and management. Adenomyosis presents with pelvic pain, excessive uterine bleeding, anemia and infertility. The relative contributions of abnormal endometrial tissue and myometrial smooth muscle cells to the development and growth of adenomyosis are not well understood. Moreover, there is continuing debate on the origins of adenomyosis; two competing theories describe the invagination of basal endometrium into the myometrium or the metaplastic differentiation of remnant endometrial stem/progenitor cells within the myometrium.OBJECTIVE AND RATIONALEA recent series of next-generation sequencing (NGS) studies have provided the best scientific evidence thus far regarding the cellular origins of adenomyosis and the contributions of new signaling pathways to its pathogenesis, survival, and growth. These seminal studies on endometrium, adenomyosis and endometriosis demonstrate or support the following key points. (i) Mutations of KRAS map to both intracavitary endometrial tissue and proximally located adenomyotic samples, supporting the invagination theory of pathogenesis. Driver mutations found in smooth muscle cells of uterine fibroids are absent in adenomyosis. (ii) KRAS and other less frequent mutations are limited to endometrial-type epithelial cells. They are also observed in endometriosis, indicating that the disease process in adenomyosis is similar to that in endometriosis and distinct from that of uterine fibroids. (iii) Activating mutations of KRAS stimulate specific pathways to increase cell survival and proliferation and are associated with progesterone resistance in adenomyosis. Together, these findings suggest that distinct cell populations in eutopic endometrial tissue play key roles in the etiology of adenomyosis. Dependence on ovarian steroids and ovulatory cycles for disease severity is a unique feature of adenomyosis. In this context, common patterns of aberrant gene expression have been reported both in adenomyosis and endometriosis. These include pathways that favor increased estrogen biosynthesis, decreased estradiol metabolism, a unique estrogen receptor beta (ESR2)-driven inflammatory process, and progesterone resistance due to decreased progesterone receptor expression. Since adenomyosis exhibits a uniquely estrogen-driven inflammatory process and progesterone resistance, we discuss the interactions between these molecular characteristics and signaling pathways induced by the newly discovered KRAS mutations.SEARCH METHODSWe conducted a comprehensive search using PubMed for human and animal studies published until 2020 in the following areas: adenomyosis, endometriosis, endometrium, NGS, whole-exome sequencing, whole-genome sequencing, RNA sequencing, targeted deep sequencing, epigenetics, driver mutation, KRAS, progesterone resistance, estrogen action and steroid production.OUTCOMESTargeted deep sequencing analyses of epithelial cells in adenomyosis and adjacent basalis endometrial glands demonstrated recurring KRAS mutations in both cell types. This finding suggests that adenomyosis originates from basalis endometrium. Epithelial cells of the endometrium, adjacent adenomyosis and co-occurring endometriosis also share identical KRAS mutations. These findings suggest both adenomyosis and endometriosis are oligoclonal tissues that arise from endometrial cell populations carrying a specific driver mutation that most commonly affects the KRAS gene.WIDER IMPLICATIONSAdenomyosis usually follows an event such as pregnancy that has disrupted the integrity of the endometrial–myometrial junction followed by repetitious menstrual episodes that increase the likelihood of the entrapment of the basalis endometrium within the myometrium. Glandular epithelial cells carrying KRAS mutations and located within the deep crypts of basalis endometrium may become entrapped and invade myometrial tissue to give rise to adenomyosis. Evidence suggests that KRAS mutations may be responsible, in part, for previously observed phenomena such as prolonged cell survival and progesterone resistance in adenomyosis.

The Notch Family Transcription Factor, RBPJκ, Modulates Glucose Transporter and Ovarian Steroid Hormone Receptor Expression During Decidualization.

During decidualization, endometrial stromal cells differentiate into a secretory phenotype to modulate the uterine microenvironment and promote embryo implantation. This highly metabolic process relies on ovarian steroid receptors and glucose transporters. Canonical Notch signaling is mediated by the transcription factor Recombination Signal Binding Protein for Immunoglobulin Kappa J Region (RBPJ). Loss of RBPJ in the mouse uterus (Pgrcre/+Rbpjflox/flox; Rbpj c-KO) results in subfertility in part due to an abnormal uterine-embryonic axis during implantation and, as described herein, decidualization failure. Induced in vivo decidualization in Rbpj c-KO mice was impaired with the downregulation of decidual markers and decreased progesterone receptor (Pgr) signaling. Consistent with in vivo mouse data, RBPJ knockdown during in vitro Human uterine fibroblast (HuF) cell decidualization results in the reduced expression of decidual marker genes along with PGR. Expression of the glucose transporter, SLC2A1, was decreased in the RBPJ-silenced HuF cells, which corresponded to decreased Slc2a1 in the secondary decidual zone of Rbpj c-KO mouse uteri. Exogenous administration of pyruvate, which bypasses the need for glucose, rescues PRL expression in RBPJ-deficient HuF cells. In summary, Notch signaling through RBPJ controls both ovarian steroid receptor PGR and glucose transporter SLC2A1 expression during decidualization, and this dysregulation likely contributes to embryo implantation failure.

Bisphenol A impairs decidualization of human uterine stromal fibroblasts

This study examined the effect of bisphenol A (BPA) exposure on human uterine stromal fibroblast cells (HuF) undergoing decidualization. HuF cells were isolated and cultured for eight days in the presence of a decidualization-inducing cocktail, while concurrently exposed to physiological and supra-physiologic doses of BPA (1ng/mL, 10ng/mL, 0.5μg/mL, 10μg/mL and 20μg/mL). Decidualization markers, steroid hormone receptors and cell cycle gene expression were detected by qRT-PCR and cellular proliferation was assessed by KI-67 immunofluorescent staining and MTS assay. BPA impaired decidualization at 10μg/mL and 20μg/mL, but not at lower doses. Additionally, BPA at 20μg/mL decreased progesterone receptor and estrogen receptor-alpha compared to controls. The highest dose of BPA also reduced cellular proliferation and cyclin D2 expression compared to controls. These findings demonstrate that BPA disrupts in vitro decidualization of uterine stromal fibroblasts by altering steroid hormone receptor expression at higher concentrations but not at lower physiological doses.

Age-associated gene expression changes in the arcuate nucleus of male rhesus macaques.

The hypothalamic arcuate nucleus (ARC) represents a major component of the neuroendocrine reproductive axis and plays an important role in controlling the onset of puberty as well as age-associated reproductive senescence. Although significant gene expression changes have been observed in the ARC during sexual maturation, it is unclear what changes occur during aging, especially in males. Therefore, in the present study, we profiled the expression of reproduction-related genes in the ARC of young and old male rhesus macaques, as well as old males that had received 6 months of hormone supplementation (HS) in the form of daily testosterone and dehydroepiandrosterone; we also compared morning vs night ARC gene expression in the old males. Using Affymetrix gene microarrays, we found little evidence for age-associated expression changes for genes associated with the neuroendocrine reproductive axis, whereas using qRT-PCR, we detected a similar age-associated decrease in PGR (progesterone receptor) that we previously observed in postmenopausal females. We also detected a sex-steroid-dependent and age-associated decrease in androgen receptor (AR) expression, with highest AR levels being expressed at night (i.e., coinciding with the natural peak in daily testosterone secretion). Finally, unlike previous observations made in females, we did not find a significant age-associated increase in KISS1 (Kisspeptin) or TAC3 (Neurokinin B) expression in the ARC of males, most likely because the attenuation of circulating sex-steroid levels in the males was much less than that in postmenopausal females. Taken together, the data highlight some similarities and differences in ARC gene expression between aged male and female nonhuman primates.

Endometrial Cancers With Activating KRas Mutations Have Activated Estrogen Signaling and Paradoxical Response to MEK Inhibition.

The aims of this study were to determine if activating KRas mutation alters estrogen signaling in endometrial cancer (EC) and to explore the potential therapeutic impact of these alterations. The Cancer Genome Atlas was queried for changes in estrogen-regulated genes in EC based on KRas mutation status. In vitro studies were conducted to evaluate estrogen receptor α (ERα) phosphorylation changes and related kinase changes in KRas mutant EC cells. The resulting effect on response to MEK inhibition, using trametinib, was evaluated. Immunohistochemistry was performed on KRas mutant and wild-type EC tumors to test estrogen signaling differences. KRas mutant tumors in The Cancer Genome Atlas showed decreased progesterone receptor expression (P = 0.047). Protein analysis in KRas mutant EC cells also showed decreased expression of ERα (P < 0.001) and progesterone receptor (P = 0.001). Although total ERα is decreased in KRas mutant cells, phospho-ERα S118 was increased compared with wild type. Treatment with trametinib in KRas mutant cells increased phospho-ERα S167 and increased expression of estrogen-regulated genes. While MEK inhibition blocked estradiol-stimulated phosphorylation of ERK1/2 and p90RSK in wild-type cells, phospho-ERK1/2 and phospho-p90RSK were substantially increased in KRas mutants. KRas mutant EC tumor specimens showed similar changes, with increased phospho-ERα S118 and phospho-ERα S167 compared with wild-type EC tumors. MEK inhibition in KRas mutant cells results in activation of ER signaling and prevents the abrogation of signaling through ERK1/2 and p90RSK that is achieved in KRas wild-type EC cells. Combination therapy with MEK inhibition plus antiestrogen therapy may be necessary to improve response rates in patients with KRas mutant EC.

Immunohistochemical Phenotype of Breast Cancer during 25-Year Follow-up of the Royal Marsden Tamoxifen Prevention Trial.

The randomized, double-blinded Royal Marsden Tamoxifen Breast Cancer Prevention Trial in healthy high-risk women started in 1986 and is still blinded. Eligible participants (n = 2,471) were randomly assigned to tamoxifen (20 mg/d) or placebo for 8 years. Analysis in 2006 showed a 30% risk reduction of estrogen receptor (ER)-positive invasive breast cancer mostly in the posttreatment period. Biomarker analysis in this population may identify any subgroup-specific preventive effects tamoxifen. After a median follow-up of 18.4 years, 242 patients had developed invasive cancer, 134 on placebo and 108 on tamoxifen. From these, 180 tissue blocks were available and ER, progesterone receptor (PgR), Ki67, HER2, and EGFR were immunohistochemically analyzed. A 32% reduction in ER+ and PgR+ invasive cancers resulted after 8 years of treatment. Quantitative levels of ER and PgR were lower in the tamoxifen-treated group, significantly so for ER (P = 0.001). These lower ER levels were restricted to the posttreatment period (P = 0.018). Among the ER+ group, there was a similar proportional decrease in PgR+ and PgR- tumors by tamoxifen. The median levels of Ki67 were similar in both arms. The numbers of HER2-positive and EGFR-positive cancers were higher in the tamoxifen arm but not significantly so. In conclusion, the preventive effects of tamoxifen result in reduced ER-positive but not ER-negative tumors and reduced ER expression in the ER-positive cases largely confined to the posttreatment period. Overall reductions in PgR expression are explained by lower frequency of ER-positive cases. Impact on Ki67, HER2, and EGFR was modest. Cancer Prev Res; 10(3); 171-6. ©2017 AACR.

Decreased epithelial progesterone receptor A at the window of receptivity is required for preparation of the endometrium for embryo attachment.

The precise timing of progesterone signaling through its cognate receptor, the progesterone receptor (PGR), is critical for the establishment and maintenance of pregnancy. Loss of PGR expression in the murine uterine epithelium during the preimplantation period is a marker for uterine receptivity and embryo attachment. We hypothesized that the decrease in progesterone receptor A (PGRA) expression is necessary for successful embryo implantation. To test this hypothesis, a mouse model constitutively expressing PGRA (mPgrALsL/+) was generated. Expression of PGRA in all uterine compartments (Pgrcre) or uterine epithelium (Wnt7acre) resulted in infertility with defects in embryo attachment and stromal decidualization. Expression of critical PGRA target genes, indian hedgehog, and amphiregulin (Areg), was maintained through the window of receptivity while the estrogen receptor target gene, the leukemia inhibitory factor (Lif), a key regulator of embryo receptivity, was decreased. Transcriptomic and cistromic analyses of the mouse uterus at day 4.5 of pregnancy identified an altered group of genes regulating molecular transport in the control of fluid and ion levels within the uterine interstitial space. Additionally, LIF and its cognate receptor, the leukemia inhibitory factor receptor (LIFR), exhibited PGR-binding events in regions upstream of the transcriptional start sites, suggesting PGRA is inhibiting transcription at these loci. Therefore, downregulation of the PGRA isoform at the window of receptivity is necessary for the attenuation of hedgehog signaling, transcriptional activation of LIF signaling, and modulation of solutes and fluid, producing a receptive environment for the attaching embryo.

Decreased Progesterone Receptor B/A Ratio in Endometrial Cells by Tumor Necrosis Factor-Alpha and Peritoneal Fluid from Patients with Endometriosis.

PurposeProgesterone resistance is thought to be a major factor that contributes to progression of endometriosis. However, it is not clear what causes progesterone resistance in endometriosis. This study aimed to assess whether cytokines or peritoneal fluid can affect progesterone receptor (PR) expression in endometrial cells and to verify whether PR expression is reduced in endometriosis.Materials and MethodsThe PR-B/A ratio was measured via real-time polymerase chain reaction after in vitro culture, in which endometrial cells were treated with either tumor necrosis factor-alpha (TNF-α), interleukin-1 beta, or peritoneal fluid obtained from women with advanced-stage endometriosis. Immunohistochemistry was performed to compare PR-B expression between eutopic and ectopic endometrial tissues from women with and without advanced-stage endometriosis.ResultsThe PR-B/A ratio was significantly decreased by treatment with either TNF-α (p=0.011) or peritoneal fluid from women with advanced-stage endometriosis (p=0.027). Immunoreactivity of PR-B expression was significantly lower during the secretory phase than during the proliferative phase in endometrial tissues from control subjects (p<0.001). PR-B expression was significantly reduced in the eutopic endometrium (p=0.031) and ovarian endometrioma (p=0.036) from women with advanced-stage endometriosis compared with eutopic endometrium tissues from control subjects.ConclusionProgesterone resistance in endometriosis may be caused by proinflammatory conditions in the pelvic peritoneal microenvironment.

Transforming growth factor β1 (TGFβ1) and progesterone regulate matrix metalloproteinases (MMP) in human endometrial stromal cells.

Menstruation is preceded by progesterone withdrawal and endometrial matrix remodeling predominantly through induction of matrix metalloproteinases (MMP) and recruitment of invading neutrophils. Using endometrial tissues from women during various phases of the menstrual cycle, we found that MMP2, MMP9, and MMP11 were up-regulated in the late secretory phase/premenstrual phase. Because TGFβ-responsive genes were also up-regulated in endometrium during this time, we tested the hypothesis that TGFβ1 and progesterone regulate expression of MMP in human endometrial stromal cells (HESC). Treatment of HESC with TGFβ1 resulted in marked increases in MMP2 and MMP11 mRNA and pro- and active MMP2 activity. Progesterone inhibited TGFβ1-induced stimulation of MMP2 and MMP11 through its nuclear hormone receptors. Interestingly, TGFβ1 also decreased progesterone receptor (PR)-A and PR-B in HESC with a more pronounced effect on PR-A. These data support the hypothesis that TGFβ1 has endogenous anti-progestational effects in HESC and that the opposing effects of progesterone and TGFβ1 are important in regulation of matrix integrity in human endometrium.

A polymorphism in a let-7 microRNA binding site of KRAS in women with endometriosis.

Endometriosis is found in 5–15% of women of reproductive age and is more frequent in relatives of women with the disease. Activation of KRAS results in de novo endometriosis in mice, however, activating KRAS mutations have not been identified in women. We screened 150 women with endometriosis for a polymorphism in a let-7 microRNA (miRNA) binding site in the 3'-UTR of KRAS and detected a KRAS variant allele in 31% of women with endometriosis as opposed to 5% of a large diverse control population. KRAS mRNA and protein expression were increased in cultured endometrial stromal cells of women with the KRAS variant. Increased KRAS protein was due to altered miRNA binding as demonstrated in reporter assays. Endometrial stromal cells from women with the KRAS variant showed increased proliferation and invasion. In a murine model, endometrial xenografts containing the KRAS variant demonstrated increased proliferation and decreased progesterone receptor levels. These findings suggest that an inherited polymorphism of a let-7 miRNA binding site in KRAS leads to abnormal endometrial growth and endometriosis. The LCS6 polymorphism is the first described genetic marker of endometriosis risk.

Decreased progesterone receptor isoform expression in luteal phase fallopian tube epithelium and high-grade serous carcinoma

We previously reported that BRCA1/2-mutated fallopian tube epithelium (FTE) collected during the luteal phase exhibits gene expression profiles more closely resembling that of high-grade serous carcinoma (HGSC) specimens than FTE collected during the follicular phase or from control patients. Since the luteal phase is characterised by high levels of progesterone, we determined whether the expression of progesterone receptor (PR) and PR-responsive genes was altered in FTE obtained from BRCA mutation carriers during the luteal phase of the menstrual cycle. RT-qPCR confirmed a decreased expression of PR mRNA in FTE during the luteal phase relative to follicular phase, in both BRCA1/2 mutation carriers and control patients. Immunohistochemistry using isoform-specific antibodies confirmed a low level of both PR-A and PR-B in HGSC and a lower level of staining in FTE samples obtained during the luteal phase compared with the follicular phase. No significant difference in PR-A or PR-B staining was found based on patient BRCA mutation status. Analysis of our previously reported gene expression profiles based upon known PR-A- and PR-B-specific target genes did not partition samples by BRCA mutation status, indicating that overall FTE PR response is not altered in BRCA mutation carriers. HGSC samples grouped separately from other samples, consistent with the observed loss of PR expression. These findings indicate no overall difference in PR signalling in FTE as a function of BRCA mutation status. Thus, the molecular similarity of BRCA1/2-mutated luteal phase FTE and HGSC likely results from an altered response to luteal phase factors other than progesterone.

Progesterone receptor antagonist CDB-4124 increases depression-like behavior in mice without affecting locomotor ability

Progesterone withdrawal has been proposed as an underlying factor in premenstrual syndrome and postpartum depression. Progesterone withdrawal induces forced swim test (FST) immobility in mice, a depression-like behavior, but the contribution of specific receptors to this effect is unclear. The role of progesterone's GABA(A) receptor-modulating metabolite allopregnanolone in depression- and anxiety-related behaviors has been extensively documented, but little attention has been paid to the role of progesterone receptors. We administered the classic progesterone receptor antagonist mifepristone (RU-38486) and the specific progesterone receptor antagonist CDB-4124 to mice that had been primed with progesterone for five days, and found that both compounds induced FST immobility reliably, robustly, and in a dose-dependent fashion. Although CDB-4124 increased FST immobility, it did not suppress initial activity in a locomotor test. These findings suggest that decreased progesterone receptor activity contributes to depression-like behavior in mice, consistent with the hypothesis that progesterone withdrawal may contribute to the symptoms of premenstrual syndrome or postpartum depression.

Biological Evidence for Dual Antiangiogenic-Antiaromatase Activity of the VEGFR Inhibitor PTK787/ZK222584In vivo

Targeting vascular endothelial growth factor (VEGF) and estrogen receptor signaling pathways concomitantly may enhance benefit in estrogen receptor-positive breast cancer. We had shown previously that the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) is a competitive aromatase inhibitor in vitro. Here we investigated (a) whether PTK/ZK shows both antiangiogenic and antiaromatase inhibitory properties in vivo, and (b) whether the combination of PTK/ZK and letrozole is superior to letrozole alone. Estrogen-dependent human breast cancer cells engineered to express aromatase (MCF7 AROM 1 and BT474 AROM) were used. Mice were treated with vehicle, PTK/ZK (25, 50, or 100 mg/kg), letrozole, or PTK/ZK in combination with letrozole. In MCF7 AROM 1 tumors, all treatments induced growth suppression and were associated with a reduction in cell turnover index, a composite measurement of both proliferation and apoptosis. PTK/ZK significantly reduced vessel density. Whereas letrozole caused tumor regression, PTK/ZK stabilized tumor volumes. The growth suppressive and antiangiogenic effects of PTK/ZK were confirmed in BT474 AROM xenografts. The addition of PTK/ZK did not enhance the growth-suppressive effects of letrozole. However, PTK/ZK decreased progesterone receptor (PgR) and TFF1 expression and uterine weight, indicating that PTK/ZK decreases 17beta-estradiol (E2) signaling in vivo. The VEGF receptor inhibitor PTK/ZK showed effects on E2-dependent gene expression consistent with aromatase inhibition as well as antiangiogenesis in xenograft models of breast cancer. The combination with letrozole was not superior to letrozole alone. Overall, these results provide further support for a potential therapeutic approach of dual inhibition of VEGF and E2 signaling using a single agent.

Cyclin D1 Enhances the Response to Estrogen and Progesterone by Regulating Progesterone Receptor Expression

Estrogen and progesterone are the defining hormones of normal female development, and both play critical roles in breast carcinogenesis. Cyclin D1 is a breast cancer oncogene whose amplification is linked to poor prognosis in estrogen and progesterone receptor-positive breast cancers. Here we report that cyclin D1 regulates progesterone receptor expression, consequently enhancing responses to estrogen and progesterone. Estrogen treatment of cyclin D1 transgenic mice increased progesterone receptor expression and induced mammary hyperplasias that were stimulated by progesterone and blocked by a progesterone antagonist. Progesterone receptor levels decreased in cyclin D1 knockout mice. Cyclin D1 regulated progesterone receptor expression through a novel estrogen- and cyclin D1-responsive enhancer in DNA encoding part of the 3' untranslated region of the progesterone receptor gene. Small inhibitory RNAs for cyclin D1 decreased progesterone receptor expression and estrogen receptor binding to the 3' enhancer region in human breast cancer cells. Since estrogen and progesterone regulate cyclin D1, our results suggest that cyclin D1's participation in a feed-forward loop could contribute to increased breast cancer risks associated with estrogen and progesterone combinations. Additionally, its regulation of the progesterone receptor identifies a novel role for cyclin D1 in ovarian hormone control of breast development and breast carcinogenesis.

Abstract 1469: Cyclin D1 plays a critical role in the response of mammary gland to estrogen and progesterone by regulating progesterone receptor expression

Abstract Estrogen and progesterone are important hormones in normal female development, yet both have critical roles in breast carcinogenesis. Cyclin D1 is a breast cancer oncogene whose amplification is linked to poor prognosis in estrogen and progesterone receptor positive breast cancers. Here we show that cyclin D1 regulates progesterone receptor expression, consequently enhancing responses to estrogen and progesterone in breast development and carcinogenesis. Estrogen treatment of cyclin D1 transgenic mice caused mammary hyperplasias expressing increased progesterone receptors, which were stimulated by progesterone and blocked by a progesterone antagonist. Cyclin D1 regulated progesterone receptor expresion through a novel estrogen- and cyclin D1-responsive enhancer in DNA encoding part of the 3’ untranslated region of the progesterone receptor gene. Small inhibitory RNAs for cyclin D1 decreased progesterone receptor expression in human breast cancer cells, which sensitized them to progesterone-antagonists. Since estrogen and progesterone regulate cyclin D1, our results suggest that cyclin D1's participation in a feed-forward loop could contribute to increased breast cancer risks associated with estrogen and progesterone combinations. Additionally, its regulation of the progesterone receptor identifies a novel role for cyclin D1 in ovarian hormone control of breast development and breast carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1469.

The Synergistic Effect of ConditionalPtenLoss and OncogenicK-rasMutation on Endometrial Cancer Development Occurs via Decreased Progesterone Receptor Action

Endometrial cancer is the most common gynecological cancer. Estrogen-dependent endometrioid carcinoma is the most common type of endometrial cancer, and alterations in the expression of PTEN and K-ras have been associated with this disease. To study the roles of Pten and K-ras in endometrial cancer, we generated Pten ablation and oncogenic K-ras mutation in progesterone receptor positive cells (PR cre/+ Pten f/f K-ras G12D). Double mutant mice dramatically accelerated the development of endometrial cancer compared to a single mutation of either gene. Histological analysis showed that all of the 1-month old double mutant female mice developed endometrial cancer with myometrial invasion. The expression of PR was downregulated in double mutant mice compared to a single mutation of either gene which resulted in decreased suppression of estrogen signaling. Therefore, these results suggest a synergistic effect of dysregulation of the Pten and K-ras signaling pathways during endometrial tumorigenesis.