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Background: Progesterone (P) and progestin are instrumental in the breast cancer risk associated with reproductive hormones. Progesterone receptor (PR) signaling has been shown to be inhibited by the wild-type BRCA1 protein and it is hypothesized that if this inhibition is lost, due to gene mutation, potentially tumorigenic consequences may result. Our efforts are focused on understanding the role PR and P play in BRCA1 mutation carriers. The purpose of this study was to determine if the presence of a BRCA1 mutation effects alternative splicing (AS) events. Methods: Mammary organoids from 12 patients, six with a BRCA1 mutation and six without (WT, control), were cultured in vitro. The organoids from each patient were divided into two groups and treated over the course of 28 days to mimic a normal menstrual cycle. One group was treated was estradiol and progesterone (EP) while the other was treated with EP and the PR antagonist telapristone acetate (TPA) to identify PR-mediated effects. After treatment, RNA was extracted from both groups and RNA-sequencing performed. A statistical model, “replicate Multivariate Analysis of Transcript Splicing” (rMATS) was employed to identify AS events. Output from rMATS was then entered into “RNA Map Analysis And Plotting Server” (rMAPS) to identify RNA-binding proteins (RBP) motifs in the region of the splices. Droplet digital PCR (ddPCR) from Bio-Rad was employed, with the use of Evagreen intercalating dye, to validate a subset of the AS events identified by the RNA-Seq data. RNA from eight of the EP treated samples was reverse transcribed and ddPCR performed. The data was analyzed using QuantaSoft Analysis Pro (Bio-Rad). The identified AS events were validated additionally using data generated from solid tissue normals (STN) from premenopausal, BRCA1mut and BRCA1WT patients, who developed breast cancer, which was available for download from The Cancer Genome Atlas (TCGA). Results: Genes involved in the epithelial-mesenchymal-transition (EMT), progesterone metabolism, and cellular proliferation are significantly differentially spliced in the EP treated BRCA1mut tissue (p-value < 0.01 and FDR < 5%) and absent in those with TPA treatment, suggesting the AS is a PR-mediated effect. CD44, associated with cellular proliferation and migration, NCOR2 associated with tamoxifen resistance, and AKR1C2 associated with progesterone metabolism all showed significant skipped exon (SE) events. Exon 11 in CD44 and exon 45 in NCOR2 were skipped in both the BRCA1 organoids as well as in BRCA1 STN from TCGA. Skipping of Exon 4 in AKR1C2 was validated using the ddPCR: the BRCA1mut organoids displayed two isoforms, indicative of a skipped exon. Conclusions: PR-mediated alternative splicing events differ in mammary organoids of BRCA1 carriers compared to non-carriers. We hypothesize that the skipping of Exon 4 of AKR1C2 in BRCA1mut results in a structural alteration that decreases protein activity, leading to increased concentrations of P4 and 5α-pregnane-3,20-dione. This may explain the previously reported high median luteal phase serum P levels (p=0.00034) in BRCA1mut/BRCA2mut (PMID: 24140203). Citation Format: Gannon Cottone, Batzaya Davaadelger, Shivangi Yadav, J Julie Kim, Seema A. Khan, Susan Clare. Alternative splicing events from progesterone exposure differ based on BRCA1 mutation status [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-09.

Endometrial cancer is the most commonly diagnosed type of gynecological cancer, and obesity is a major risk factor for this disease. Despite rising rates of obesity and endometrial cancer, it is still unclear how obesity alters the benign endometrium and the endometrial hormonal response to promote carcinogenesis. We used an endometrial organoid model to investigate how adiposity predisposes women to develop endometrial cancer. We hypothesized that increased adiposity would directly alter endometrial gene expression and diminish the protective progesterone response of the benign endometrium. Scaffold-free, three-dimensional endometrial organoids consisting of both epithelial and stromal cells from benign endometrial tissue were generated. Organoids were cocultured with different numbers of adipocyte spheroids to represent different levels of adiposity and were treated with a menstrual cycle level of hormones (E2 + P4 +T) over 14 days. Endometrial organoid gene expression was investigated using RNA-seq. Coculturing endometrial organoids with 30 adipocyte spheroids in the presence of E2, P4, and T caused significant differential expression of pathways involved in metabolism, angiogenesis, chromatin modification, and metal ion response, compared to endometrial organoids cultured without adipocyte spheroids. We investigated the metal ion response pathways that were altered and found that many of the most significantly differentially regulated genes in the metal ion response pathways belonged to the metallothionein (MT) family of genes. The expression of MT1A, MT1M, MT1X, and MT2A decreased approximately 50% in endometrial organoids cocultured with 30 adipocyte spheroids. Furthermore, although the results were not statistically significant, we observed a downregulation of the progesterone-responsive genes HSD17B2, IGFBP1, PAEP, PRL, FOXO1, and SPP1 in the endometrial organoids cultured with 30 adipocyte spheroids. Our results show that culturing endometrial organoids in conditions of increased adiposity alters endometrial gene expression and may change the way the endometrium responds to progesterone. Metallothioneins, which are downregulated in the presence of adipocyte spheroids, are important modulators of zinc and copper ions and reactive oxygen species in the cell. Progesterone plays an important protective role against endometrial cancer. In summary, our data show that adipocytes may influence the progesterone response of the benign endometrium. Further investigation into the role of progesterone-driven metallothioneins in the endometrium is currently underway. Citation Format: Alina R. Murphy, J. Julie Kim. Understanding the influence of adiposity on the endometrium using human primary endometrial organoids [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO030.

Background Patient derived xenografts (PDX) are generated by transplanting the original patient’s tumor tissue into immune-deficient mice. Unlike xenograft models derived from cell lines, PDX models can better preserve the histopathology from the original patient and molecular pathways. High-grade serous carcinoma (HGSC) is a deadly form of ovarian/fallopian tube cancer whose response to current chemotherapies varies widely due to patient variability. Therefore, a PDX model can provide a valuable tool to study and test treatment options for each individual patient. Methods In this study, over 200 PDX tumors from nine HGSC were analyzed to investigate the nature and behavior of PDX tumors originating from HGSC. PDX tumors were serially passaged (from P0 to P4) and tumors were grafted orthotopically under the ovarian bursa or subcutaneously. Results Comparative analysis of the histology and molecular markers of tumors from over 200 PDX tumor-bearing mice, revealed that the tumors maintained similar histologies, stem cell populations, and expression for the majority of the tested oncogenic markers, compared to the primary tumors. However, a significant loss of steroid hormone receptors and altered expression of immunoresponsive genes in PDX tumors were also noted. Conclusion Our findings provide substantial new information about PDX tumor characteristics from HGSC which will be valuable towards the development of personalized therapy and new drug development for HGSC. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0318-6) contains supplementary material, which is available to authorized users.

Fenretinide is a synthetic retinoid analogue that promotes apoptosis but has decreased toxicity when compared to other retinoids. We have previously shown that retinoic acid (RA) production in endometriotic tissue is decreased, resulting in reduced estrogen metabolism and apoptotic resistance. We hypothesize fenretinide may induce apoptosis in endometriotic cells and tissues, thereby reducing disease burden. Primary endometriotic stromal cells were collected, isolated, cultured, and treated with fenretinide in doses from 0 to 20 p,mol/L. Cell count, viability, and immunoblots were per-formed to examine apoptosis. Quantitative reverse transcription-polymerase chain reaction from endometriotic cells treated with fenretinide was used to examine expression of genes involved in RA signaling including stimulated by RA 6 (STRA6), cellular RA binding protein 2 (CRABP2), and fatty acid binding protein 5 (FABP5). Endometriotic tissue was xenografted subcutaneously into the flanks of mice which were treated with fenretinide for 2 weeks, after which the mice were killed and lesion volumes calculated. Statistical analysis was performed using ttestand analysis ofvariance. Treatment with fenretinide significantly decreased total cell count (doses 5-20 p,L) and viability (doses 10-20 p,mol/L). Fenretinide increased protein levels of the apoptotic marker poly (ADP ribose) polymerase (starting at 10 p,mol/L) and decreased proliferation marker proliferating cell nuclear antigen (10 μmol/L, starting at 8-day treatment). Examination of genes involved in retinoid uptake and action showed that treatment induced STRA6 expression while expression of CRABP2 and FABP5 remained unchanged. Fenretinide also significantly decreased the endometriotic lesion xenograft volume. Fenretinide increases STRA6 expression thereby potentially reversing the pathological loss of retinoid availability. Treatment with this Compound induces apoptosis. In vivo treatments decrease lesion volume. Targeting the RA signaling pathway may be a promising novel treatment for women with endometriosis.

Study question Do interactions between human fallopian tube epithelium and murine follicles occur during an artificial reproductive cycle in a co-culture system in vitro? Summary answer In a co-culture system, human fallopian tissues responded to the menstrual cycle mimetic by changes in morphology and levels of secreted factors, and increasing murine corpus luteum progesterone secretion. What is known already The entire fallopian tube epithelium, including ciliated and secretory cells, can be regulated in the reproductive cycle. Currently, there are no in vitro culture models that can monitor fallopian tissues in real time in response to factors produced by the ovary. In addition, there are no reports on the impact of fallopian tissue on ovarian function during the menstrual cycle. Study design, samples/materials, methods Human fallopian tissue (n = 24) was obtained by routine hysterectomies from women (aged 26-50 years, mean age = 43.6) who had not undergone exogenous hormonal treatment for at least 3 months prior to surgery. CD1 female mice were used for ovarian follicle isolation. The human fallopian epithelium layers were either co-cultured with five murine multilayer secondary follicles (150-180 μm follicles, encapsulated in one alginate gel bead) for 15 days or received stepwise steroid hormone additions for 13 days. The fallopian tissue morphology and cilia beating rate, as measured by an Andor Spinning Disk Confocal, were investigated. Oviduct-specific glycoprotein 1 (OVGP1), human insulin-like growth factor 1 (hIGF1), vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL8) as biological functional markers were measured either by ELISA or western blot to indicate dynamic changes in the fallopian epithelium during the reproductive cycle generated by mouse follicles or by stepwise steroid hormone induction. Three or four patients in each experiment were recruited for replicates. Data were presented as mean ± SD and further analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. Main results and the role of chance The cultured fallopian tube epithelium responded to exogenous steroid hormone stimulation, as demonstrated by enhanced cilia beating rate (~25% increase, P = 0.04) and an increase in OVGP1 secretion (P = 0.02) in response to 1 nM estradiol (E2) treatment when compared with 0.1 nM E2. Conversely, 10 nM progesterone plus 1 nM E2 suppressed cilia beating rate by ~30% (P = 0.008), while OVGP1 secretion was suppressed by 0.1 nM E2 plus 50 nM progesterone (P = 0.002 versus 1 nM E2 alone). Human fallopian tube epithelium was co-cultured with murine secondary follicles to mimic the human menstrual cycle. OVGP1 and VEGF-A secretion from fallopian tissue was similar with stepwise hormone treatment and when cultured with murine follicles. However, the secretion patterns of hIGF1 and IL8 differed in the luteal phase when comparing steroid treatment with follicle co-culture. In co-culture, hIGF1 secretion was suppressed in the luteal versus follicular phase (P = 0.005) but stepwise hormone treatment had no effect on hIGF1. In co-culture, IL8 secretion was also suppressed on luteal phase day 15 (P = 0.013) versus follicular phase day 7, but IL8 secretion increased continuously under high E2/progesterone treatment (P = 0.003 for D13 versus D3). In the co-culture system, the corpus luteum continuously produced progesterone in the presence of fallopian tube tissue until Day 18 while, without fallopian tissue, progesterone started to drop from Day 13. Limitations, reasons for caution One limitation of this study is that murine follicles were used to mimic the human menstrual cycle. However, although secretion patterns of peptide hormones such as inhibins and activins differ in mice and humans, the co-culture system used here did reveal interactions between the tissues that govern reproductive function. Wider implications of the findings In vitro co-culture models of fallopian reproductive tissues with ovarian follicles can provide an important tool for understanding fertility and for uncovering the mechanisms responsible for reduced fertility. In addition, the role of oviductal secretions and how they influence ovarian function, such as the production of progesterone during the menstrual cycle, can be uncovered using this model. Large-scale data None. Study funding and competing interests This work was funded by grants from the NIH (UH3TR001207), the American Cancer Society (RSG-12-230-01-TBG) and NIH (R01EB014806). The authors declare no competing financial interest.

Objective To determine the presence, differential expression, and regulation of epidermal growth factor–containing fibulin-like extracellular matrix protein 1 (EFEMP1) in uterine leiomyomas. Design Laboratory in vivo and in vitro study with the use of human leiomyoma and myometrial tissue and primary cells. Setting Academic medical center. Sample(s) Leiomyoma and myometrial tissue samples and cultured cells. Intervention(s) 5-Aza-2′-deoxycytidine (5-aza-dC) treatment. Main Outcome Measure(s) Fold-change difference between EFEMP1 and fibulin-3 expression in leiomyoma tissue and cells compared with matched myometrial samples, and fold-change difference in EFEMP1 expression with 5-Aza-dC treatment. Result(s) In vivo, EFEMP1 expression was 3.19-fold higher in myometrial tissue than in leiomyoma tissue. EFEMP1 expression in vitro was 5.03-fold higher in myometrial cells than in leiomyoma cells. Western blot and immunohistochemistry staining of tissue and cells confirmed similar findings in protein expression. Treatment of leiomyoma cells with 5-Aza-dC resulted in increased expression of EFEMP1 in vitro. Conclusion(s) The EFEMP1 gene and its protein product, fibulin-3, are both significantly down-regulated in leiomyoma compared with myometrium when studied both in vivo and in vitro. The increase in EFEMP1 expression in leiomyoma cells with 5-Aza-dC treatment suggest that differential methylation is responsible, in part, for the differences seen in gene expression.

Progestins have long been used clinically for the treatment of endometrial cancers, however, the response rates to progestin therapy vary and the molecular mechanisms behind progestin insensitivity are poorly understood. We hypothesized that in PTEN mutated endometrial cancers, hyperactive Akt signaling downregulates Progesterone Receptor B (PRB) transcriptional activity, leading to overall impaired progestin responses. We report that inhibition of Akt with the Akt inhibitor, MK-2206 (MK), in conjunction with progestin (R5020) treatment, is sufficient to upregulate a subset of PRB target genes in Ishikawa cells stably expressing PRB (PRB-Ishikawa). Through gene ontology analysis of Akt-regulated PRB target genes, angiogenesis was found to be the principle process regulated by Akt-PRB. To further interrogate the mechanism by which Akt modulates PRB transcriptional activity, ChIP-Mass Spectrometry was performed to identify potential cofactors that differentially interact with PRB in the presence of the R5020 and MK+R5020. 14-3-3σ was identified as a protein enriched in the MK+R5020 dataset, and it was demonstrated that 14-3-3σ is required for the upregulation in PRB target gene expression following inhibition of Akt. In order to determine the ramifications of MK+R5020 treatment on angiogenesis, in vitro assays were performed and combinatorial MK+R5020 treatment significantly decreased endothelial cell invasion and tube formation more than MK or R5020 treatment alone. Furthermore, we found that combinatorial MK-2206+Progesterone treatments decreased angiogenesis and proliferation in the Ptend/d conditional mouse model of endometrial cancer. Taken together, these findings suggest that a combinatorial therapeutic approach utilizing Akt inhibitors with progestins may improve the efficacy of progestin therapy for the treatment of endometrial cancer.

Introduction Racial disparities in breast cancer (BrCa) mortality are well documented. We previously reported that African American (AA) women with estrogen receptor-positive (ER+) BrCa have a 4-fold higher rate of death compared to non-Hispanic whites (white) after controlling for stage at diagnosis and treatment initiation. This suggests that racial differences in tumor biology may contribute to the survival disparity. In preliminary work we found that AA BrCa cases have higher serum levels of C-reactive protein (CRP) compared to whites, and that increased CRP was associated with worse survival, suggesting that increased systemic inflammation in AA women drives aggressive tumor biology and contributes to the racial disparity in survival. The goal of this study is to identify specific inflammatory pathways that may be involved. Experimental Procedures Pre-treatment blood samples from women with stage I-III ER+ BrCa (AA, n=40; white, n=55) and from healthy controls (AA, n=51; white, n=38) were analyzed with the Olink® targeted proteomic assay, which measures 92 inflammatory proteins. Normalized data was log2 transformed, and generalized linear models compared the normalized intensities with covariates of interest. The fit of the model was tested with empirical Bayes methods. All p-values are corrected for multiple testing with the false discovery rate (FDR) method of Benjamini and Hochberg. Findings After adjustment for site of enrollment, plasma levels of 13 inflammatory cytokines showed a log2 fold-change (log2FC) ≥ 1.0 for AA compared to white women, i.e. at least a two-fold increase in the mean value on a linear scale (Table). The racial difference was evident among both cases and controls, and remained after adjusting for body mass index (BMI). There were no differences between AA cases and controls, indicating that cytokine elevation in AA BrCa patients is not an effect of the tumor. Several of the cytokines are known to promote tumor progression and aggressive biological features in breast tumors. ProteinLog2FCProteinLog2FCCXCL52.42**CCL81.13***CXCL12.06***EIF4EBP11.12**STAMBP1.91*CCL201.10**CXCL111.89**TNF141.06*CCL31.45**TGF beta1.04**CXCL61.40**CCL71.03***CCL131.16**FDR corrected p-values: * < 0.05, **< 0.01, ***< 0.001 Conclusion Systemic elevation of biologically relevant inflammatory cytokines in AA women with ER+ BrCa does not appear to be an effect of the tumor. This inflammatory phenotype may activate inflammatory programs in ER+ breast tumors that develop in AA women, leading to disproportionately aggressive tumor biology and worse outcomes. Supported by grants U54CA203000; U54CA2022995; and U54CA2022997 from the National Institutes of Health. Citation Format: Oana C. Danciu, George Chlipala, Zeynep Madak-Erdogan, Hariyali Patel, Landan Banks, Ayesha Zaidi, Ashlie Santaliz-Casiano, Lauren Schulte, Lauren Weller, Deanna Taiym, Natalie Pulliam, Elona Liko Hazizi, Elonia Martin, Anita Fareeduddin, Archana Bargaje, Carlos Garcia, Scott Hegerty, Sarah Friedewald, Seema Khan, J Julie Kim, William Gradishar, Garth Rauscher, Jonna Frasor, Kent F. Hoskins. Racial differences in circulating inflammatory cytokines and breast cancer disparities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1176.

Background Ovarian cancer remains a major health problem for women as it is often diagnosed at a late stage with metastatic disease. There are limited therapeutic agents and survival rates remain poor. The perinucleolar compartment (PNC) has been shown to be associated with malignancy and is considered a surrogate phenotypic marker for metastatic cancer cells. A small molecule, ML246, was derived from a screen against PNCs. In this study, the effect of ML246 on ovarian cancer growth and spread was investigated. Methods SKOV3 or OVCAR3 cells were treated with ML246 in vitro and PNC was visualized with immunofluorescent staining. Cell invasion was assessed using Matrigel-coated transwell systems. SKOV3 cells were xenografted orthotopically under the ovarian bursa of immunocompromised mice. Additionally, a patient derived ovarian cancer cell line was grafted subcutaneously. Mice were treated with ML246 and tumor growth and spread was assessed. Results PNCs were prevalent in the ovarian cancer cell lines OVCAR3 and SKOV3 with higher prevalence in OVCAR3 cells. Treatment with ML246 significantly reduced PNC prevalence in OVCAR3 and SKOV3 cells. Moreover, the invasive activity of both cell lines was significantly inhibited in vitro. Orthotopic implantation of SKOV3 cells resulted in growth of the tumor on the ovary as well as spread of tumor tissues outside of the primary site on organs into the abdominal cavity. Treatment with ML246 decreased the incidence of tumors outside of the ovary. In addition, a patient-derived xenograft (PDX) line was grafted subcutaneously to monitor tumor growth. ML246 significantly attenuated growth of tumors over a 5-week treatment period. Conclusions PNC’s are present in ovarian cancer cells and treatment with ML246 decreases invasion in vitro and tumor growth and spread in vivo. Additional studies are warranted to determine the efficacy of ML246 as an inhibitor of metastatic disease in ovarian cancer and to determine its precise mechanism of action.

Uterine leiomyomas (ULM) are histologically and molecularly heterogeneous and clinically they grow at vastly different rates. Several driver gene mutations have been identified in ULM, including MED12 mutations, HMGA2 overexpression, and biallelic FH inactivation. ULM with different driver mutant genes may use different molecular pathways, but currently no clear correlation between gene mutations and growth related pathways has been established. To better define this relationship, we collected ULM with MED12 (n = 25), HMGA2 (n = 15), and FH (n = 27) mutations and examined the sex steroid hormone, cell cycle, and AKT pathway genes by immunohistochemistry. While ER and PR were highly expressed in all types of ULM, FH ULM showed lower ER expression and higher PR expression. HMGA2 tumors had significantly higher levels of AKT signaling and mitogenic activity than other ULM types. HMGA2 activated AKT signaling through upregulation of IGF2BP2. Silencing HMGA2 in ULM cells resulted in downregulation of AKT and upregulation of p16 and p21, which eventually led to cell senescence. HMGA2 overexpression in ULM is not only related to tumor development but also plays a role in controlling cellular proliferation through the AKT pathway.

Uterine leiomyomas (ULM) grow under high oxidative stress due to a hypoxic microenvironment and defects in redox metabolism. AKT is one major pathway activated by reactive oxygen species (ROS) that maintains ULM growth and survival. We previously reported that AKT inactivated by AKT inhibitors can significantly induce cellular senescence in ULM cells. Since some miRNAs are induced by AKT inhibitors in an ROS-dependent manner, we proposed that these miRNAs may modulate AKT function and cellular senescence in ULM. We therefore established ex vivo models of a three-dimensional ULM spheroid culture system to study the role of miRNAs in cellular senescence. Four miRNAs, miR-29b, miR-181a, miR-182, and miR-200c, were found to induce cellular senescence in primary ULM and myometrium spheroid cultures when stably overexpressed. miR-181a and miR-182 were found to repress AKT3 and CCND2, respectively. Correspondingly, RNAi of AKT3 or CCND2 also induced cellular senescence and G0/G1 arrest. Thus, miR-181a and miR-182 may drive cellular senescence in ULM by repressing AKT3 and CCND2 activity, respectively. We further demonstrated that senescent ULM cells can be effectively removed by BH3 mimetic ABT263, which provides a new therapeutic venue for the treatment of ULM. Our findings suggest that miRNAs are potent modulators in regulating the ROS-AKT-cell cycle axis in uterine leiomyoma.A subset of oxidative stress-induced miRNAs is involved in AKT signaling in uterine leiomyoma. Overexpression of miR-181a and miR-182 resulted in cellular senescence in leiomyoma through repression of AKT3 and CCND2, respectively. Silencing of AKT3 and CCND2 drives leiomyoma cell into senescence and cycle arrest. Application of our newly developed 3D leiomyoma spheroids can provide a quick and reliable ex vivo model for cytopathologic and functional analysis. BH3 mimetics can effectively reduce the viability of miRNA-mediated senescent cells in leiomyoma.

Cancer-associated fibroblasts have been shown to inhibit or stimulate tumor growth depending on stage, grade, and tumor type. It remains unclear, however, the effect of endometrial-cancer-associated fibroblasts on hormone-driven responses in endometrial cancer. In this study, we investigated the effect of normal and cancer-associated stromal cells from patients with and without endometrial cancer on endometrial tumor growth in response to estradiol (E2) and progesterone (P4). Compared to benign endometrial stromal cells, the low-grade and high-grade cancer-associated stromal cells exhibited a blunted hormone response for proliferation as well as IGFBP1 secretion. Additional analysis of the influence of stromal cells on hormone-driven tumor growth was done by mixing stromal cells from benign, low-grade, or high-grade tumors, with Ishikawa cells for subcutaneous tumor formation. The presence of both benign and high-grade cancer-associated stromal cells increased estradiol-driven xenografted tumor growth compared to Ishikawa cells alone. Low-grade cancer-associated stromal cells did not significantly influence hormone-regulated tumor growth. Addition of P4 attenuated tumor growth in Ishikawa + benign or high-grade stromal cells, but not in Ishikawa cells alone or with low-grade stromal cells. Using an angiogenesis focused real-time array TGFA, TGFB2 and TGFBR1 and VEGFC were identified as potential candidates for hormone-influenced growth regulation of tumors in the presence of benign and high-grade stromal cells. In summary, endometrial-cancer-associated cells responded differently to in vitro hormone treatment compared to benign endometrial stromal cells. Additionally, presence of stromal cells differentially influenced hormone-driven xenograft growth in vivo depending on the disease status of the stromal cells.

Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo.The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells.Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells.We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability.CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.

The endocervix has both anatomical and biological functions that participate in the delicate balance between tolerance necessary for conception and protection from pathogens. Our goal was to develop a robust 3-dimensional (3D) endocervix model that was a reliable representation of the in vivo tissues and to identify the physiological responses to changing levels of steroid hormones during a 28-day time period. Human endocervical cells were grown on polystyrene scaffolds, and the morphologic and hormonal responses of cultured cells were assessed in response to fluctuating levels of estradiol (E2) or progesterone (P4). Morphologically, the 3D cultures were composed of a mixed population of cells, including epithelial and stromal cells. Treatment with E2 and P4 (d 28) increased cell growth and proliferation as compared with no treatment control. Cells expressed estrogen receptor and P4 receptor and produced both neutral and acidic mucins, including Mucin 16. In addition, a 45-plex Luminex assay identified numerous factors secreted and regulated by hormones. Specifically, IL-1β and leukemia inhibitory factor significantly decreased in the presence of E2 and P4 as compared with the no hormone control at day 26. Cotreatment with RU486 (mifepristone) attenuated the inhibition of IL-1β and leukemia inhibitory factor secretion. In summary, a robust, novel 3D endocervical culture was developed, and physiologic responses to the menstrual cycle mimic of E2 and P4 levels for a period of 28 days were identified.

Objective Ovarian cancer is the most lethal gynecological malignancy that affects women. Recent data suggests that the disease may originate in the fallopian fimbriae; however, the anatomical origin of ovarian carcinogenesis remains unclear. This is largely driven by our lack of knowledge regarding the structure and function of normal fimbriae and the relative paucity of models that accurately recapitulate the in vivo fallopian tube. Therefore, a human three-dimensional (3D) culture system was developed to examine the role of the fallopian fimbriae in serous tumorigenesis. Methods Alginate matrix was utilized to support human fallopian fimbriae ex vivo . Fimbriae were cultured with factors hypothesized to contribute to carcinogenesis, namely; H 2 O 2 (1mM) a mimetic of oxidative stress, insulin (5μg/ml) to stimulate glycolysis, and estradiol (E 2 , 10nM) which peaks before ovulation. Cultures were evaluated for changes in proliferation and p53 expression, criteria utilized to identify potential precursor lesions. Further, secretory factors were assessed after treatment with E 2 to identify if steroid signaling induces a pro-tumorigenic microenvironment. Results 3D fimbriae cultures maintained normal tissue architecture up to 7days, retaining both epithelial subtypes. Treatment of cultures with H 2 O 2 or insulin significantly induced proliferation. However, p53 stabilization was unaffected by any particular treatment, although it was induced by ex vivo culturing. Moreover, E 2 -alone treatment significantly induced its canonical target PR and expression of IL8, a factor linked to poor outcome. Conclusions 3D alginate cultures of human fallopian fimbriae provide an important microphysiological model, which can be further utilized to investigate serous tumorigenesis originating from the fallopian tube.

A deeper understanding of the pathways that drive uterine leiomyoma (ULM) growth and survival requires model systems that more closely mimic the in vivo tumors. This would provide new insights into developing effective therapeutic strategies for these common benign tumors of childbearing-aged women. In this study, we examined the role of BCL-2 in mediating ULM survival in the context of increased protein kinase B (AKT) and oxidative stress using a three-dimensional (3D), spheroid-based model that more closely resembles the native ULM tumor microenvironment. Human primary cells from matched myometrium (MM) and ULM tissues were used to establish spheroid cultures in vitro. Histological and immunohistochemical methods were used to assess the spheroid architecture and characteristics. Viability assays for 3D cultures were used to evaluate their response to BH3 mimetics and the superoxide inducer, paraquat (PQ). Primary MM and ULM cells formed spheroids in culture. Notably, ULM spheroids exhibited low proliferation, increased oxidative stress, and secretion of interstitial collagen. Knockdown studies revealed that AKT sustained BCL-2 expression in ULM. The targeting of BCL-2 with BH3 mimetics effectively reduced viability and induced apoptosis in a subset of ULM spheroids. ULM spheroids that did not respond to BH3 mimetics alone responded to combination treatment with PQ. In conclusion, BCL-2 mediates AKT survival of ULM, providing compelling evidence for further evaluation of BH3 mimetics for ULM treatment. ULM spheroids recapitulated intrinsic features of the native ULM tumor microenvironment and can be used as a model for preclinical testing of potential therapeutic options for ULM.

Microphysiologic systems (MPS), including new organ-on-a-chip technologies, recapitulate tissue microenvironments by employing specially designed tissue or cell culturing techniques and microfluidic flow. Such systems are designed to incorporate physiologic factors that conventional 2D or even 3D systems cannot, such as the multicellular dynamics of a tissue–tissue interface or physical forces like fluid sheer stress. The female reproductive system is a series of interconnected organs that are necessary to produce eggs, support embryo development and female health, and impact the functioning of non-reproductive tissues throughout the body. Despite its importance, the human reproductive tract has received less attention than other organ systems, such as the liver and kidney, in terms of modeling with MPS. In this review, we discuss current gaps in the field and areas for technological advancement through the application of MPS. We explore current MPS research in female reproductive biology, including fertilization, pregnancy, and female reproductive tract diseases, with a focus on their clinical applications. Impact statement This review discusses existing microphysiologic systems technology that may be applied to study of the female reproductive tract, and those currently in development to specifically investigate gametes, fertilization, embryo development, pregnancy, and diseases of the female reproductive tract. We focus on the clinical applicability of these new technologies in fields such as assisted reproductive technologies, drug testing, disease diagnostics, and personalized medicine.

The endometrium maintains complex controls on proliferation and apoptosis as part of repetitive menstrual cycles that prepare the endometrium for the window of implantation and pregnancy. The reliance on inflammatory mechanisms for both implantation and menstruation, creates the opportunity in the setting of endometriosis for the establishment of chronic inflammation that is disruptive to endometrial receptivity, causing both infertility and abnormal bleeding. Clinically, there can be little doubt that the endometrium of women with endometriosis is less receptive to embryo implantation, and strong evidence exists to suggests that endometrial changes are associated with decreased cycle fecundity as a result of this disease. Here we provide unifying concepts regarding those changes and how they are coordinated to promote progesterone resistance and estrogen dominance through aberrant cell signaling pathways and reduced expression of key homeostatic proteins in eutopic endometrium of women with endometriosis.

Three-dimensional (3D) in vitro models have been established to study the physiology and pathophysiology of the endometrium. With emerging evidence that the native extracellular matrix (ECM) provides appropriate cues and growth factors essential for tissue homeostasis, we describe, a novel 3D endometrium in vitro model developed from decellularized human endometrial tissue repopulated with primary endometrial cells. Analysis of the decellularized endometrium using mass spectrometry revealed an enrichment of cell adhesion molecules, cytoskeletal proteins, and ECM proteins such as collagen IV and laminin. Primary endometrial cells within the recellularized scaffolds proliferated and remained viable for an extended period of time in vitro. In order to evaluate the hormonal response of cells within the scaffolds, the recellularized scaffolds were treated with a modified 28-day hormone regimen to mimic the human menstrual cycle. At the end of 28 days, the cells within the endometrial scaffold expressed both estrogen and progesterone receptors. In addition, decidualization markers, IGFBP-1 and prolactin, were secreted upon addition of dibutyryl cyclic AMP indicative of a decidualization response. This 3D model of the endometrium provides a new experimental tool to study endometrial biology and drug testing.

The purpose of this study was to test the effect of MK2206, an allosteric inhibitor of AKT, on the growth and invasion of patient-derived xenografts (PDX) of endometrial cancer. Three PDX lines, USC1 (uterine serous), EEC2 (endometrioid grade 2) and EEC4 (endometrioid grade 3) of endometrial cancer were grafted under the renal capsule of NSG mice. After 2 weeks of tumor growth the mice were treated with vehicle or 120mg/kg MK2206 twice a week for 3 weeks. Growth of all 3 PDX lines of different type and grade was significantly inhibited in response to MK2206 compared with vehicle control. Histological analysis revealed invasion and spread of EEC2 and EEC4 tumors were significantly decreased with MK2206 treatment. Immunohistochemical analysis showed a decrease in Ki67 in EEC2 upon MK2206 treatment, while USC1 and EEC4 tumors did not show differences in Ki67 levels. PR levels were evident in EEC2 which dramatically increased upon MK2206 treatment. In vitro analysis of EEC4 and AN3CA cells showed a dose-dependent decrease in p(Ser473)-AKT and p(Thr308)-AKT with MK2206. Invasion of EEC4 and AN3CA cells also significantly decreased after 36h and 72h of MK2206 treatment. PDX tumors provide an appropriate model for the testing of compounds that incorporates the heterogeneous nature of endometrial cancer. Further studies to determine efficacy of MK2206 alone or in combination with other compounds can also identify predictors of response to these pathway inhibitors.

BACKGROUND: Uterine smooth muscle tumors (USMTs) constitute a group of histologic, genetic, and clinical heterogeneous tumors that include at least 6 major histologically defined tumor types: leiomyoma (ULM), mitotically active leiomyoma (MALM), cellular leiomyoma (CLM), atypical leiomyoma (ALM), uncertain malignant potential (STUMP), and leiomyosarcoma (LMS). Apart from ULM and LMS, the nature of these variants is not well defined. METHODS: A total of 167 cases of different USMT variants were collected, reviewed, and diagnostically confirmed based on the World Health Organization and Stanford schemes. These included 38 cases of LMS, 18 cases of STUMP, 42 cases of ALM, 22 cases of CLM, 7 cases of MALM, and 40 cases of ULM. Molecular analysis included selected microRNAs (miRNAs), oncogenes, and tumor suppressors that are highly relevant to USMT. RESULTS: Overall, 49% (17/35) of LMS cases and 7% (1/14) of STUMP cases died due to their USMT, but no deaths were attributed to ALM. miRNA profiling revealed that ALM and LMS shared similar miRNA signatures. P53 mutations and PTEN deletions were significantly higher in LMS, ALM, and STUMP compared with other USMT variants (P 74%) but were significantly less common (

The pathogenesis of endometriosis remains unclear, and relatively little is known about the mechanisms that promote establishment and survival of the disease. Previously, we demonstrated that v-akt murine thymoma viral oncogene homolog (AKT) activity was increased in endometriosis tissues and cells from ovarian endometriomas and that this increase promoted cell survival as well as decreased levels of progesterone receptor. The objective of this study was to demonstrate a role for AKT in the establishment of ectopic lesions. First, a dose-dependent inhibition of AKT in stromal cells from human ovarian endometriomas (OSIS) as well as endometrial stromal cells from disease-free patients (ESC) with the allosteric AKT inhibitor MK-2206 was demonstrated by decreased levels of phosphorylated (p)(Ser473)-AKT. Levels of the AKT target protein, p(Ser256)-forkhead box O1 were increased in OSIS cells, which decreased with MK-2206 treatment, whereas levels of p(Ser9)-glycogen synthase kinase 3β did not change in response to MK-2206. Although MK-2206 decreased viability of both OSIS and ESC in a dose-dependent manner, proliferation of OSIS cells was differentially decreased significantly compared with ESC. Next, the role of hyperactive AKT in the establishment of ectopic lesions was studied using the bigenic, PRcre/+Ptenf/+ heterozygous mouse. Autologous implantation of uterine tissues was performed in these mice. After 4 weeks, an average of 4 ± 0.33 lesions per Ptenf/+ mouse and 7.5 ± 0.43 lesions in the PRcre/+Ptenf/+ mouse were found. Histological examination of the lesions showed endometrial tissue-like morphology, which was similar in both the Ptenf/+ and PRcre/+Ptenf/+ mice. Treatment of mice with MK-2206 resulted in a significantly decreased number of lesions established. Immunohistochemical staining of ectopic lesions revealed decreased p(Ser473)-AKT and the proliferation marker Ki67 from MK-2206–treated mice compared with vehicle-treated mice. Furthermore, levels of FOXO1 and progesterone receptor increased in lesions of mice receiving MK-2206. These results demonstrate that heightened AKT activity plays an active role in the establishment of ectopic endometrial tissues.

Endometriosis is characterized by progesterone resistance and hyperactivity of the AKT and MAPK pathways. Kinases can cause posttranslational modifications of the progesterone receptor (PR) to influence cellular localization and protein stability.The objective of this study was to determine whether the increased AKT or MAPK kinase-1/2 (MEK1/2) activity observed in endometriotic stromal cells (OSIS) from ovarian endometriomas influences levels of PR protein. In turn, the effects of inhibiting AKT or MEK1/2 in the presence of the progestin R5020 on cell viability were investigated.Inhibiting AKT with MK-2206 or MEK1/2 with U0126 for 24 hours in the absence of R5020 increased total and nuclear PRA and PRB protein levels in OSIS but not in eutopic endometrial stromal cells from disease-free patients from disease-free patients. MK-2206 and R5020 decreased OSIS viability and increased apoptosis. Trends toward decreased volumes of sc grafted endometriosis tissues were demonstrated with MK-2206 and progesterone.Inhibition of AKT or MEK1/2 increased total and nuclear PR protein in OSIS. MK-2206 and R5020 decreased OSIS viability and increased apoptosis. The AKT and MAPK pathways may be potential molecular targets for the treatment of endometriosis.

Acetylation-mediated inactivation of MnSOD triggers activation of prosurvival AKT signaling in uterine smooth muscle tumors., AKT signaling promotes cell growth and survival and is often dysregulated via multiple mechanisms in different types of cancer, including uterine leiomyomas (ULMs). ULMs are highly prevalent fibrotic tumors that arise from the smooth muscular layer of the uterus, the myometrium (MM). ULMs pose a major public health issue because they can cause severe morbidity and poor pregnancy outcomes. ‬We investigate the mechanisms driving ULM growth and survival via aberrant activation of AKT. We demonstrate that an acetylation-mediated impairment of the manganese superoxide dismutase (MnSOD) activity is prevalent in ULM cells compared to the normal-matched MM from the same patients. This impairment increases the levels of superoxide and oxidative stress, which activate AKT via oxidative inactivation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Redox activation of AKT promotes ULM cell survival under conditions of moderate but persistent oxidative stress that are compatible with ULM’s prooxidative microenvironment. Moreover, because of impaired MnSOD activity, ULM cells are sensitive to high levels of reactive oxygen species (ROS) and superoxide-generating compounds, resulting in decreased ULM cell viability. On the contrary, MM cells with functional MnSOD are more resistant to high levels of oxidants. This study demonstrates a causative role of acetylation-mediated MnSOD dysfunction in activating prosurvival AKT signaling in ULMs. The specific AKT and redox states of ULM cells provide a potential novel therapeutic rationale to selectively target ULM cells because of their defective ROS-scavenging system.‬‬‬‬‬‬‬‬

Background Synuclein gamma (SNCG) expression is associated with advanced disease and chemoresistance in multiple solid tumors. Our goal was to determine if SNCG protein expression in ovarian cancer was correlated with clinicopathologic variables and patient outcomes. Methods Tissue microarrays from primary tumors of 357 ovarian, fallopian tube, and primary peritoneal cancer patients, who underwent primary surgery at Roswell Park Cancer Institute between 1995 and 2007, were immunohistochemically stained for SNCG. A pathologist blinded to patient data scored tumors as positive if ≥10 % of the sample stained for SNCG. Medical records were reviewed for clinicopathologic and demographic variables. Between the positive and negative groups, Wilcoxon rank-sum test was used to compare the median ages and Fisher’s exact test was used to compare groups in categorical variables. Cox proportional hazard models examined associations between SNCG and overall and progression-free survival. Results The median follow-up was 36 months, median overall survival was 39 months, and median progression-free survival was 18 months. SNCG presence was associated with clinical variables of serous histology, grade 3 disease, suboptimal debulking, ascites at surgery, FIGO stage III-IV cancer, or initial CA-125 level >485. There was no significant difference in overall survival (HR 1.06 95 % CI 0.81–1.39 P 0.69) or progression-free survival (HR 1.16 95 % CI 0.89–1.50 P 0.28) for patients with or without SNCG expression. Conclusions SNCG expression in ovarian cancer is frequent in patients with high-risk features, but it does not correlate with chemotherapy response, overall survival, or progression-free survival. Electronic supplementary material The online version of this article (doi:10.1186/s13048-016-0281-4) contains supplementary material, which is available to authorized users.

The endocrine system dynamically controls tissue differentiation and homeostasis, but has not been studied using dynamic tissue culture paradigms. Here we show that a microfluidic system supports murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single, dual and multiple unit microfluidic platforms (Solo-MFP, Duet-MFP and Quintet-MPF, respectively). These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver, with a sustained circulating flow between all tissues. The reproductive tract tissues and peripheral organs integrated into a microfluidic platform, termed EVATAR, represents a powerful new in vitro tool that allows organ–organ integration of hormonal signalling as a phenocopy of menstrual cycle and pregnancy-like endocrine loops and has great potential to be used in drug discovery and toxicology studies.

The endocervix plays an important role in providing appropriate protective mechanisms of the upper female reproductive tract (FRT) while at the same time providing the appropriate milieu for sperm transport. Hormone fluctuations throughout the menstrual cycle contribute to changes in the mucosal environment that render the FRT vulnerable to infectious diseases. The objective of this study was to identify genes in human endocervix tissues that were differentially expressed in the follicular versus the luteal phases of the menstrual cycle using gene expression profiling. A microarray using the IIlumina platform was performed with eight endocervix tissues from follicular and four tissues from luteal phases of the menstrual cycle. Data analysis revealed significant differential expression of 110 genes between the two phases, with a P value

Background The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from these agents will be a critical factor for their clinical success. Methods We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines. Results TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites. Conclusions By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2355-5) contains supplementary material, which is available to authorized users.

Uterine leiomyomas are benign tumors that originate from the myometrium. Evidence points to ovarian steroid hormones, in particular, progesterone as major promoters of leiomyoma development and growth. While progesterone action in leiomyomas involves the classical nuclear receptor effects on gene regulation, there is growing evidence that signaling pathways are directly activated by the progesterone receptor (PR) and that PR can interact with growth factor signaling systems to promote proliferation and survival of leiomyomas. Studies investigating the genomic and non-genomic actions of PR and its role in leiomyoma growth are summarized here. Studies testing various selective progesterone receptor modulators for the treatment of leiomyomas are also highlighted. An increased understanding of the mechanisms associated with progesterone-driven growth of leiomyomas is critical in order to develop more efficient and targeted therapies for this prevalent disease.

Progesterone is a growth inhibitory hormone in the endometrium. While progestins can be used for the treatment of well-differentiated endometrial cancers, resistance to progestin therapy occurs for reasons that remain unclear. We have previously demonstrated that progesterone receptors (PR) A and B differentially regulate apoptosis in response to overexpression of the forkhead transcription factor, FOXO1. In this study, we further examined the PR-isoform-dependent cellular response to the AKT pathway. Treatment of PRA and PRB-expressing Ishikawa cells (PRA14, PRB23), with an AKT inhibitor API-59CJ-OMe (API-59) promoted apoptosis in the presence and absence of the ligand, R5020 preferentially in PRA14 cells. Upon PR knockdown using small interfering RNA, an increase in apoptosis was observed in PRB23 cells treated with API-59 with or without R5020 while there was no influence in PRA14 cells. Using an apoptosis-focused real-time PCR array, genes regulated by API-59 and R5020 were identified both common and unique to PRA14 and PRB23 cells. BIRC3 was identified as the only gene regulated by R5020 which occurred only in PRB cells. Knockdown of BIRC3 in PRB23 cells promoted a decrease in cell viability in response to API-59 + R5020. Furthermore, the important role of inhibitors of apoptosis (IAPs) in the PRB23 cells to promote cell survival was demonstrated using an antagonist to IAPs, a second mitochondria-derived activator of caspase (Smac also known as DIABLO) mimetic. Treatment of PRB23 cells with Smac mimetic increased apoptosis in response to API-59 + R5020. In summary, our findings indicate a mechanism by which PRB can promote cell survival in the setting of high AKT activity in endometrial cancer cells.

Progesterone is a steroid hormone that plays an important role in the breast. Progesterone exerts its action through binding to Progesterone Receptor (PR), a transcription factor that belongs to the nuclear receptor family. Deregulation of the progesterone signaling pathway is implicated in the formation, development and progression of breast cancer. Next generation selective progesterone receptor modulators (SPRMs) have potent anti-progestin activity and are selective for PR, reducing the off-target effects on other nuclear receptors. To date, there is limited information on how the newer generation of SPRM's such as Telapristone acetate (TPA), affect PR function at the molecular level. In this study we investigated the molecular mechanisms by which TPA antagonizes PR action in T47D breast cancer cells. First, we performed ChiP sequencing to obtain a global analysis of the effect of TPA on the PR cistrome. We observed that TPA decreases PR genome wide recruitment to the chromatin. Approximately 19,865 and 19,892 PR binding peaks were identified in the progestin, R5020 or R5020 + TPA data sets, respectively, with 83% (18,113) of overlapping PR binding regions. HOMER motif analysis indicated similar enriched motifs between R5020 and R5020+TPA groups, identifying progesterone response elements as the most significant motif. We validated our ChiP-seq data with PR ChIP by examining the promoter regions of known PR target genes. PR occupancy was decreased at these sites with the addition of TPA. In addition, TPA decreased R5020- driven expression of these genes. To further investigate the mechanism by which TPA inhibits PR transcriptional activity we performed Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). TPA significantly influenced co-regulator recruitment to PR. After applying a stringent cut-off to our data set, 3 unique proteins were identified in the R5020+TPA group: TRPS1, LASP1 and AP1G1. TRPS1 has been shown to be a transcriptional corepressor and is overexpressed in majority of human breast cancer. Follow-up studies revealed an increase in the levels of PR-TRPS1 complex with TPA treatment. In addition, silencing TRPS1 with siRNA increased PR occupancy to the known PR gene promoters, suggesting that TRPS1 inhibited PR binding to these regions upon TPA treatment. In addition, knockdown of TRPS1 attenuated the inhibition of ACSL1, NOTCH2, PACSIN1 and GREB1 gene expression after TPA treatment. Taken together our results show that TPA decreases recruitment of PR to chromatin, recruits co-repressors such as TRPS1 to the PR complex thereby regulates PR target gene expression. This is the first study showing TRPS1 complexes with PR and regulates its activity. Ongoing studies are focused on the potential role of TRPS1 as a predictive biomarker of SPRM response in breast cancer. Citation Format: Batzaya Davaadelger, Seema A. Khan, J Julie Kim. Mechanism of telapristone acetate (CDB4124) on progesterone receptor action in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1812.

Uterine leiomyoma is the most common tumor of the female genital tract and the leading cause of hysterectomy. Although progesterone stimulates the proliferation of uterine leiomyoma cells, the mechanism of progesterone action is not well understood. We used chromatin immunoprecipitation (ChIP)–cloning approach to identify progesterone receptor (PR) target genes in primary uterine leiomyoma smooth muscle cells. We identified 18 novel PR-binding sites, one of which was located 20.5 kb upstream of the transcriptional start site of the Krüppel-like transcription factor 11 (KLF11) gene. KLF11 mRNA levels were minimally downregulated by progesterone but robustly upregulated by the progesterone antagonist RU486. Luciferase reporter assays showed significant baseline and RU486-inducible promoter activity in the KLF11 basal promoter or distal PR-binding region, both of which contained multiple Sp1-binding sequences but lacked classic progesterone response elements. RU486 stimulated recruitment of Sp1, RNA polymerase II, PR, and the coactivators SRC-1 and SRC-2 to the distal region and basal promoter. siRNA knockdown of PR increased KLF11 expression, whereas knockdown of KLF11 increased leiomyoma cell proliferation and abolished the antiproliferative effect of RU486. In vivo, KLF11 expression was significantly lower in leiomyoma tissues compared with adjacent myometrial tissues. Taken together, using a ChIP-cloning approach, we uncovered KLF11 as an integrator of PR signaling and proliferation in uterine leiomyoma cells. Cancer Res; 70(4); 1722–30

Breast cancer research has historically relied on two model systems: two-dimensional cell culture systems and mouse models. These models, although very convenient, lack the functional phenotypic architecture of human tissue. Extrapolation of hormonal responses from models to human relevance also requires further investigation due to the anatomical, physiological, and biological differences. Three-dimensional spheroids provide alternative models as they better represent the architecture and maintain the histomorphological, functional, and micro-environmental features of the in vivo human tumor tissue. We established and characterized tumor spheroids in vitro from the breast cancer cell lines T47D and MCF7. The cells were seeded at densities of 5000 cells per well in low adhesive 96-well plates using specific growth media that sustains spheroid growth. After 10 days of culture, the spheroids were stained to verify spheroid viability using Calcein AM [green] and Ethidium homodimer-1 [red] (top row). Spheroids were stained with E-cadherin [yellow] and DAPI [blue] (bottom row) to verify the 3D architecture, and then imaged using a Leica spinning-disk confocal microscope. We noticed that MCF7 cells (left images) tended to form multiple spheroids in a single well, whereas the T47D cels (right images) formed single spheroids. Both spheroids expressed E-cadherin, demonstrating cell-to-cell adhesion and remained viable after 10 days of culture. More importantly the spheroids formed every time they were cultured in low-adhesive plates with appropriate growth media, demonstrating the effectiveness, reproducibility, and robustness of this in vitro model system. This article is protected by copyright. All rights reserved.

WOS: 000273921100006, PubMed ID: 20104427, Loss of progesterone signaling in the endometrium may be a causal factor in the development of endometriosis, and progesterone resistance is commonly observed in women with this disease. In endometriotic stromal cells, the levels of progesterone receptor (PR), particularly the PR-B isoform, are significantly decreased, leading to a loss of paracrine signaling. PR deficiency likely underlies the development of progesterone resistance in women with endometriosis who no longer respond to progestin therapy. Here we review the complex epigenetic and transcriptional mechanisms leading to PR deficiency. The initial event may involve deficient methylation of the estrogen receptor (ER)beta promoter resulting in pathologic overexpression of ER beta in endometriotic stromal cells. We speculate that alterations in the relative levels of ER beta and ER alpha in endometrial tissue dictate E2-regulated PR expression, such that a decreased ER alpha-ER beta ratio may result in suppression of PR. In this review, we propose a molecular model that may be responsible for changes in ER beta and ER alpha leading to PR loss and progesterone resistance in endometriosis., NICHD [HD040093]; Friends of Prentice, This article was supported by grants from the NICHD (HD040093) and Friends of Prentice ( both to S. E. B.).

Much effort has been made to increase the survival rate of cancer patients through the development of early disease detection and the improvement of prognoses to guide appropriate therapeutic interventions. Although these strategies have improved survival rates significantly in cancer patients, the current prognostic and predictive markers used to determine treatment strategy remain insufficiently accurate in forecasting the outcome of individual patients. Because cancers are highly heterogeneous diseases, there often are exceptions to the current prognostic criteria, leading to the under treatment or over treatment of patients.1,2 Oncogenesis and cancer progression are complex processes involving a combination of many genetic and epigenetic insults. Cells undergo extensive biochemical and structural alterations during the process of malignant transformation because of these insults. Recent advances in cellular and molecular techniques have fostered a better understanding of the specific genetic and epigenetic changes that take place during cancer development and progression. From these changes, a large number of molecular tumor markers have been identified; however, only a few them have been shown to be reproducible and clinically relevant.3,4 In addition, most molecular markers are developed for specific types of tumors, thus allowing only a small portion of cancer patients to benefit from each type of marker. Phenotypic markers, such as histologic grading and staging, have been very useful tools for indicating the progression of cancer across a broad range of tumor types; however, these markers have limited prognostic capabilities, because there are often exceptions to the generally accepted trends of disease progression and inconsistent scoring of samples.5,6 Therefore, an easily detectable phenotypic marker that can be scored accurately and precisely in a broad range of tumor types would be ideal to increase the accuracy of prognosis for many cancer patients. In the current study, we describe the potential for developing the perinucleolar compartment (PNC) into such a prognostic marker. The PNC is a nuclear substructure that is associated with, but structurally distinct from, the nucleolus (Fig. 1A).7 The PNC is a generally heritable trait, and the structure is stable through interphase, disassembles during mitosis, and reassembles in early G1 phase.7 The PNC is concentrated with newly synthesized RNA polymerase III transcripts and RNA binding proteins,8–14 implicating the involvement of PNCs in RNA metabolism.15 However, the complete molecular composition and function of the PNC remain to be elucidated. Initial examination of the PNC in 13 human cell lines demonstrated that the PNC prevalence (the percentage of nonapoptotic and nonmitotic cells with 1 or more PNC) was invariably low in normal cell lines but was higher, yet hetrogenous, among cancerous and transformed cell lines,7 suggesting that the PNC forms preferentially in cancer cells. Investigations using human breast tissue samples demonstrated that PNC prevalence is 0% in normal breast epithelium, increases in parallel with disease progression (as determined by staging), and reaches nearly 100% in distant metastases, demonstrating that the PNC prevalence is associated with the malignancy of breast cancer. Multivariate analysis also demonstrated that PNC prevalence contains additional prognostic information over grading and tumor size for patients with stage I disease.16 FIGURE 1 Survey of perinucleolar compartment (PNC) prevalence (the percentage of cells with 1 or more PNC) in primary cells and cell lines. (A) Immunofluorescent staining of HeLa cells with a polypyrimide tract binding protein (PTB) antibody was used to mark the ... Although PNC prevalence has potential to be developed clinically as a prognostic marker for breast cancer,16 it is not clear whether the association of PNC prevalence with malignancy can be extended to tumors other than those originating in the breast. An association with malignancy in a broad range of tissue types would indicate that PNC prevalence has the potential to be a pancancer biomarker. To explore this possibility and to determine the specificity of the association between PNC and malignant behavior, we examined a large number of cells lines, primary cells, and human tumor samples derived from a broad spectrum of tissues for PNC prevalence and evaluated the selective association of PNC prevalence with metastatic capacity under various experimental conditions.

Homeobox (HOX) A10 is essential for fertility as demonstrated in transgenic mice, specifically affecting implantation and decidualization. Its role in human decidualization, however, remains unknown. In this study, we used gene silencing followed by microarray analysis to decipher the role of HOXA10 during decidualization of human endometrial stromal cells (HESCs). HOXA10 was knocked down using siRNA oligonucleotide transfection and cells were treated with estradiol, medroxyprogesterone acetate and dibutyryl cAMP (H + cAMP) to induce decidualization. Genes significantly regulated were identified using the Affymetrix microarray chip. With this method, 2361 transcripts were significantly altered by 1.5-fold or higher (P < 0.05) with H + cAMP treatment only. Of these genes, 258 were significantly up-regulated by HOXA10 knockdown and 236 transcripts were significantly down-regulated by more than 1.5-fold, totaling 494 genes that were regulated by HOXA10 during decidualization. Data analysis using the Ingenuity System revealed that many of the genes regulated by HOXA10 knockdown during H + cAMP treatment were associated with cell cycle. Real-time PCR was used to confirm that HOXA10 knockdown decreased expression of the cell cycle genes CDC2 and CCNB2. In addition, a higher percentage of cells were arrested in the G2/M phase. Next, we observed that cell proliferation as measured by BrdU incorporation was decreased upon HOXA10 knockdown and H + cAMP treatment. Apoptosis, on the other hand, as measured by Annexin V staining was not influenced by siHOXA10 in decidualizing cells. Together, these data demonstrate that during decidualization of HESC, HOXA10 is actively involved in promoting cell proliferation through the regulation of hundreds of genes.

Local estrogen biosynthesis is a major factor in the pathogenesis of endometriosis. Aberrant expression of steroidogenic acute regulatory protein (StAR) and aromatase in endometriotic tissue leads to an up-regulation of estrogen production. The transcription factor steroidogenic factor-1 (SF-1) activates the promoters of both StAR and aromatase in endometriotic tissue. We investigated differences in SF-1 expression in endometriotic tissue and normally located endometrium to elucidate the mechanism underlying increased StAR and aromatase activities in endometriosis. Serial deletion and site-directed mutants of the SF-1 promoter showed that an E-box sequence was critical for its activity in endometriotic stromal cells. EMSAs showed that the upstream stimulatory factor (USF) 1 and 2 in nuclear extracts from endometrial and endometriotic stromal cells bound to the E-box. Chromatin-immunoprecipitation-PCR assay, however, demonstrated in intact cells that binding activity of USF2 to the SF-1 promoter was strikingly higher than that of USF1 in endometriotic stromal cells and that USF1 or USF2 binding activity was hardly detectable in endometrial stromal cells. Moreover, knockdown of USF2 but not USF1 resulted in robust and consistent down-regulation of SF-1 and its target genes StAR and aromatase in endometriotic stromal cells. USF2 but not USF1 mRNA and protein levels were significantly higher in endometriotic vs. endometrial stromal cells. In vivo, USF2 mRNA and immunoreactive USF2 levels in endometriotic tissues were strikingly higher than those in endometrium. Taken together, the elevated levels of USF2 in endometriosis account for, in part, the aberrant expression of SF-1 and its target gene StAR and aromatase.

In many type I endometrial cancers, the PTEN gene is inactivated, which ultimately leads to constitutively active Akt and the inhibition of Forkhead box O1 (FOXO1), a member of the FOXO subfamily of Forkhead/winged helix family of transcription factors. The expression, regulation, and function of FOXO1 in endometrial cancer were investigated in this study. Immunohistochemical analysis of 49 endometrial tumor tissues revealed a decrease of FOXO1 expression in 95.9% of the cases compared with the expression in normal endometrium. In four different endometrial cancer cell lines (ECC1, Hec1B, Ishikawa, and RL95), FOXO1 mRNA was expressed at similar levels; however, protein levels were low or undetectable in Ecc1, Ishikawa, and RL95 cells. Using small interfering RNA technology, we demonstrated that the low levels of FOXO1 protein were due to the involvement of Skp2, an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex, given that silencing Skp2 increased FOXO1 protein expression in Ishikawa cells. Inhibition of Akt in Ishikawa cells also increased nuclear FOXO1 protein levels. Additionally, progestins increased FOXO1 protein levels, specifically through progesterone receptor B (PRB) as determined by using stably transfected PRA-specific and PRB-specific Ishikawa cell lines. Finally, overexpression of triple mutant (Tm) FOXO1 in the PR-specific Ishikawa cell lines caused cell cycle arrest and significantly decreased proliferation in the presence and absence of the progestin, R5020. Furthermore, TmFOXO1 overexpression induced apoptosis in PRB-specific cells in the presence and absence of ligand. Taken together, these data provide insight into the phosphoinositide-3-kinase/Akt/FOXO pathway for the determination of progestin responsiveness and the development of alternate therapies for endometrial cancer.

Uterine leiomyomas (fibroids) represent the most common class of benign tumors in women. Multiple leiomyomas usually arise from the uterus of a symptomatic woman. These tumors cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are responsible for more than 200,000 hysterectomies in the United States annually. Each leiomyoma seems to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a mutation. Tumor expansion is sustained by cell proliferation together with the production of large amounts of extracellular matrix. Estrogen and progesterone stimulate the growth of leiomyomas. Estrogen, together with its receptor ERα, enables progesterone action via induction of progesterone receptor (PR) expression. Progesterone induces the growth of leiomyoma by regulation of a set of key genes that control proliferation and apoptosis. A distinct cell population with stem-progenitor properties is indispensable for progesterone-dependent growth of leiomyomas. This stem-progenitor cell population is deficient in ERα and PR and dependent on the much higher levels of these steroid receptors in surrounding mature leiomyoma or myometrial cells. Progesterone sends paracrine signals from these mature cells to stem cells. The WNT/β-catenin pathway comprises a key component of this paracrine signaling system. The majority of medical treatments currently available for leiomyoma works by inhibiting estrogen or progesterone production or action, but tumors tend to regrow once treatment is stopped. Targeting stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma may lead to the development of more effective therapeutics.

The forkhead box O1A (FOXO1A) has been identified as one gene that is up-regulated early in the decidualization process. To further investigate the role of FOXO1A during this process, six genes, IGFBP1, PRL, TIMP3, LAMB1, CNR1, and DCN, shown to be up-regulated during decidualization, were chosen as potential targets of FOXO1A action. Treatment of human endometrial stromal cells with hormones (estradiol and medroxyprogesterone acetate) plus dibutyryl cAMP (H+dbcAMP) for 48 h increased expression of IGFBP1, PRL, TIMP3, CNR1, and DCN but not LAMB1, as measured by real-time PCR. Silencing of FOXO1A using small interfering RNA oligonucleotides decreased IGFBP1 and DCN levels and increased CNR1, TIMP3, and PRL levels. LAMB1 was not affected. When FOXO1A was overexpressed in human endometrial stromal cells, expression of IGFBP1, DCN, and PRL increased, whereas levels of TIMP3 and CNR1 decreased. Addition of H+dbcAMP caused an increased expression of IGFBP1, PRL, and DCN beyond that of FOXO1A alone. TIMP3 and CNR1 levels decreased even further in response to H+dbcAMP compared with FOXO1A alone. LAMB1, which was unresponsive to FOXO1A, decreased when H+dbcAMP was added. Overexpressing FOXO1A also caused a change in cell shape, in that the stromal fibroblasts acquired a rounded, epithelioid appearance. Finally, reporter studies showed that cotransfection of FOXO1A significantly increased PRL promoter activity but not TIMP3 promoter activity. Addition of H+dbcAMP resulted in a significant increase in PRL promoter activity and a significant decrease in TIMP3 promoter activity. In summary, this study demonstrates the versatile nature of FOXO1A in the regulation of a number of decidualization-specific genes.

Endometriosis is the most common cause of pelvic pain and affects an estimated 5 million women in the US. The biologically active estrogen estradiol (E2) is the best-defined mitogen for the growth and inflammation processes in the ectopic endometriotic tissue that commonly resides on the pelvic organs. Progesterone and progestins may relieve pain by limiting growth and inflammation in endometriosis but a portion of patients with endometriosis and pelvic pain do not respond to treatment with progestins. Moreover, progesterone-induced molecular changes in the eutopic (intrauterine) endometrial tissue of women with endometriosis are either blunted or undetectable. These in vivo observations are indicative of resistance to progesterone action in endometriosis. The molecular basis of progesterone resistance in endometriosis may be related to an overall reduction in the levels of progesterone receptors (PRs) and the lack of the PR isoform named progesterone receptor B (PR-B). In normal endometrium, progesterone acts on stromal cells to induce secretion of paracrine factor(s). These unknown factor(s) act on neighboring epithelial cells to induce the expression of the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD-2), which metabolizes the biologically active estrogen E2 to estrone (E1). In endometriotic tissue, progesterone does not induce epithelial 17beta-HSD-2 expression due to a defect in stromal cells. The inability of endometriotic stromal cells to produce progesterone-induced paracrine factors that stimulate 17beta-HSD-2 may be due to the lack of PR-B and very low levels of progesterone receptor A (PR-A) observed in vivo in endometriotic tissue. The end result is deficient metabolism of E2 in endometriosis giving rise to high local concentrations of this local mitogen. The cellular and molecular mechanisms underlying progesterone resistance and failure to metabolize E2 in endometriosis are reviewed.

Insulin-like growth factor-binding protein-1 (IGFBP1) is a major secretory product of the decidualized endometrium. In the present study, we investigated the role of two transcription factors, progesterone receptor (PGR) and a member of the forkhead box class O family of transcription factors (FOXO1A), in the regulation of the IGFBP1 gene in endometrial cells. Human endometrial fibroblasts (HuF) expressed FOXO1A, progesterone receptor A (PGRA), and progesterone receptor B (PGRB) proteins, whereas the endometrial adenocarcinoma cell line, HEC1B cells, expressed only FOXO1A and no detectable PGR proteins. When FOXO1A expression was silenced using small interference RNA, IGFBP1 expression decreased in both HuF and HEC-1B cells. Using the chromatin immunoprecipitation technique, we demonstrated that liganded PGR was recruited to the IGFBP1 promoter region (2358 to 249). In addition, immunoprecipitation of HuF nuclear proteins with a PGR antibody followed by immunoblotting with anti-FOXO1A revealed that these two proteins interact in these cells. Reporter studies demonstrated that whereas liganded PGRA or PGRB increased a progesterone response element-linked reporter construct, pPRE/ GRE.E1b.Luc, coexpression of FOXO1A inhibited the PGRB response in HuF and synergistically increased PGRA and PGRB response in HEC-1B cells. Furthermore, in HEC-1B cells, FOXO1A increased IGFBP1 promoter activity, and coexpression of PGRA or PGRB further increased the promoter activity in a cooperative manner. In HuF, the response to FOXO1A and PGR was not additive; in fact, it was lower than the sum of the individual responses. Thus, FOXO1A and PGR associate with one another, and each influences the transactivating potential of the other. The cell type-dependent responses strongly implicate the involvement of other cofactors. decidua, female reproductive tract, implantation, progesterone receptor, uterus

Uterine smooth muscle tumors (USMTs) constitute a group of histologic, genetic, and clinical heterogeneous tumors that include at least 6 major histologically defined tumor types: leiomyoma (ULM), mitotically active leiomyoma (MALM), cellular leiomyoma (CLM), atypical leiomyoma (ALM), uncertain malignant potential (STUMP), and leiomyosarcoma (LMS). Apart from ULM and LMS, the nature of these variants is not well defined.A total of 167 cases of different USMT variants were collected, reviewed, and diagnostically confirmed based on the World Health Organization and Stanford schemes. These included 38 cases of LMS, 18 cases of STUMP, 42 cases of ALM, 22 cases of CLM, 7 cases of MALM, and 40 cases of ULM. Molecular analysis included selected microRNAs (miRNAs), oncogenes, and tumor suppressors that are highly relevant to USMT.Overall, 49% (17/35) of LMS cases and 7% (1/14) of STUMP cases died due to their USMT, but no deaths were attributed to ALM. miRNA profiling revealed that ALM and LMS shared similar miRNA signatures. P53 mutations and PTEN deletions were significantly higher in LMS, ALM, and STUMP compared with other USMT variants (P .01). In contrast, MED12 mutations were extremely common in ULM and MALM (74%) but were significantly less common (15%) in CLM, ALM, STUMP, and LMS (P .01).Six types of USMT have different gene mutation fingerprints. ALM shares many molecular alterations with LMS. Our findings suggest that ALM may be a precursor lesion of LMS or have similar genetic changes during its early stage.

Uterine leiomyomas (fibroids) are a major public health problem. Current medical treatments with GnRH analogs do not provide long-term benefit. Thus, permanent shrinkage or inhibition of fibroid growth via medical means remains a challenge. The AKT pathway is a major growth and survival pathway for fibroids. We propose that AKT inhibition results in a transient regulation of specific mechanisms that ultimately drive cells into cellular senescence or cell death. In this study, we investigated specific mechanisms of AKT inhibition that resulted in senescence. We observed that administration of MK-2206, an allosteric AKT inhibitor, increased levels of reactive oxygen species, up-regulated the microRNA miR-182 and several senescence-associated genes (including p16, p53, p21, and β-galactosidase), and drove leiomyoma cells into stress-induced premature senescence (SIPS). Moreover, induction of SIPS was mediated by HMGA2, which colocalized to senescence-associated heterochromatin foci. This study provides a conceivable molecular mechanism of SIPS by AKT inhibition in fibroids.

ObjectiveMost endometrial cancers are detected early and have a good prognosis, while some endometrial cancers are highly invasive, metastasize early, and respond suboptimally to therapy. Currently, appropriate model systems to study the aggressive nature of these tumors are lacking. The objective of this study was to establish a mouse xenograft model of endometrial tumors derived from patients in order to study the biological aggressive characteristics that underlie invasion and metastasis.MethodsEndometrial tumor tissue fragments (1.5 mm × 1.5 mm) from patients undergoing surgery, were transplanted under the renal capsule of NOD scid gamma mice. After 6-8 weeks, tumors were excised and serially transplanted into additional mice for propagation. Immunohistochemical analysis of the tumors was done for various tumor markers.ResultsFour cases of different subtypes of endometrial cancer were grown and propagated in mice. Three of the four tumor cases invaded into the kidneys and to adjacent organs. While all tumors exhibited minimal to no staining for estrogen receptor α, progesterone receptor staining was observed for tumor grafts. In addition, levels and localization of E-cadherin, cytokeratin and vimentin varied depending on subtype. Finally, all tumor xenografts stained positively for urokinase plasminogen activator while 3 tumor xenografts, which showed invasive characteristics, stained positively for urokinase plasminogen activator receptor.ConclusionEndometrial tumors transplanted under the renal capsule exhibit growth, invasion and local spread. These tumors can be propagated and used to study aggressive endometrial cancer.

Objective To establish a novel xenograft model using a severely immunocompromised host that is more convenient for uterine leiomyoma research compared with a pre-existing model using nonobese diabetic/severe combined immunodeficient (NOD/SCID) IL-2Rγ-null mice. Design Experimental study. Setting University and an attached animal facility. Animal(s) NOD/SCID, SCID, BALB/c nude, and NOD/SCID IL-2Rγ-null mice. Intervention(s) Xenografts consisting of primary cultured leiomyoma and myometrial cells in the subrenal and subcutaneous (SC) spaces in ovariectomized mice, followed by sex steroids (estrogen and P) administration. Main Outcome Measure(s) Viability, volume, histology, and sex steroid receptor expression of xenografts in response to sex steroid administration, to evaluate feasibility of the model; and messenger RNA expression levels of 12 genes representative of leiomyoma in the xenografts, to characterize the model. Result(s) Leiomyoma xenografts increased in volume at the highest frequency (55.1%) in response to sex steroids in NOD/SCID mice. Xenografts reproduced the histology and maintained expression of sex steroids receptors and representative genes of the original tissues. Subrenal xenografts were significantly larger than the SC xenografts, whereas those consisting of myometrial cells never increased. Conclusion(s) The modified NOD/SCID murine subrenal leiomyoma xenograft model reproduced most characteristics of the original leiomyoma tissue. Our model provides a more convenient research tool to investigate the pathogenesis of uterine leiomyoma.

The female reproductive tract produces hormones for reproductive function and cardiovascular, bone and sexual health; the tract supplies a finite number of gametes, and it supports fetal development. Diseases that affect each of the female reproductive tract organs, along with treatments that have direct, deleterious effects on the reproductive tract (for example, chemotherapeutics), are understudied due to the lack of model systems that phenocopy in vivo function. This review describes a path toward developing female reproductive tract mimics. The models use isolated primary support cells cultured onto a biological scaffold and within a microfluidic system to create a niche and support the desired differentiation of epithelia, germ and somatic cells from patient-derived induced pluripotent stem cells. Improving our fund of knowledge about reproductive tract biology and creating reproductive organs for patients who have lost gonadal, uterine or vaginal/ cervical function is a major step forward in women's health and an important advancement in personalized medicine.

Blastocyst implantation in the baboon usually occurs between 8 and 10 days post ovulation. Changes that occur within this window of receptivity and immediately following implantation can be divided into three distinct phases. The first phase, regulated by estrogen and progesterone, is characterized primarily by changes in both the luminal and glandular epithelial cells in preparation for blastocyst apposition and attachment. The second phase is the further modulation of these steroid induced changes in both epithelial and stromal cells by embryonic signals. The final phase is associated with trophoblast invasion and the remodeling of the endometrial stromal compartment. During the initial phase, the actions of estrogen and progesterone are dependent on the presence of specific receptors. Estrogen up-regulates both its own receptor (ER) and the progesterone receptor (PR), while progesterone down-regulates this expression pattern. However, the pattern of progesterone-induced down-regulation of ER and PR is confined to the epithelial cells and demonstrates a gradient effect from the functionalis to the basalis. What is most intriguing is that the loss of epithelial PR is closely correlated with the establishment of uterine receptivity. Coincident with the changes in ER and PR expression, epithelial cells undergo alterations in their cytoskeletal architecture and secretory profile. These changes can be counteracted by PR antagonist treatment during the luteal phase. Although estrogen and progesterone play a critical role in establishing the initial phase of uterine receptivity, it is becoming increasingly evident that the embryo induces functional receptivity in ruminants and rodents. In our studies in the primate, we demonstrate that chorionic gonadotrophin when infused in a manner that mimics blastocyst transit, has physiological effects on the three major cell types in the uterine endometrium. The luminal epithelium undergoes endoreplication and distinct epithelial plaques are evident. The glandular epithelium responds by inducing transcriptional and post-translational modifications in the major secretory product, glycodelin. The stromal fibroblasts initiate their differentiation process into a decidual phenotype and are characterized by the expression of actin filaments. In phase three, blastocyst attachment to the surface epithelium and subsequent implantation is associated with local remodeling of the maternal stroma, smooth muscle, and endothelium of the blood vessels by the trophoblast. In addition, there is a gradual diminution of the epithelial plaques on the luminal surface although the glandular epithelium remains highly secretory. The most dramatic effect is on the stromal fibroblasts, which in response to embryonic stimuli, differentiate into decidual cells, the major cell type of the gestational endometrium. This differentiation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1) in these cells. The cytokine IL-1 beta is one possible embryonic signal. COX-2 is the rate-limiting enzyme for prostaglandin biosynthesis and transcription of this enzyme in response to the embryonic stimulus (IL-1 beta) results in an increase in prostaglandin biosynthesis in stromal fibroblasts at the site of implantation. Prostaglandins and PGE2 in particular, binds to its specific receptor (EP2 or EP4) and activates adenyl cyclase. The resulting increase in intracellular levels of cAMP can now activate IGFBP-1 gene transcription at the site of implantation. In summary, our studies have demonstrated that chorionic gonadotrophin, when infused into non-pregnant baboons during the window of uterine receptivity can induce epithelial responses that are similar to those observed in a fertile cycle. Stromal differentiation is initiated; however, decidualization requires a signal from the conceptus.