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Racial disparities exist in cancer patients both in incidence and death rates. In endometrial cancer, Black patients are reported to have higher incidence of aggressive endometrial cancer subtypes and higher death rates than White women. To date, diagnostic and prognostic biomarkers associated with race-specific methylation driven genes have yet to be identified. The objective of this study was to explore DNA methylation patterns in endometrial tumor samples from White and Black women.Differentially methylated CpGs (DMCs) and differentially methylated regions (DMRs) were identified in White tumor samples compared to Black tumor samples using Endometrial Carcinoma (EC) methylation and clinical data from The Cancer Genome Atlas (TCGA). Survival analysis was performed using survival R package and results were visualized using Kaplan-Meier plots. To access the correlation between changes in methylation and gene expression, we downloaded raw RNA-sequencing by Expectation-Maximization (RSEM) counts data from The Cancer Genome Atlas (TCGA) using TCGABiolinks package (v2.18.0).Our analysis revealed 704 differentially methylated CpGs in tumors from Black and White women. These differentially methylated genes showed strong negative correlation with gene expression and statistically significant enrichment in regulatory regions such as DNase I hypersensitivity sites (DHSs) and transcription factor binding sites (TFBSs). Increased variability in methylation occurred in genes such as the insulin signaling pathway in Black tumor samples.By using two independent statistical method based on means (DMR, DMCs) and variances (DVCs) on the endometrial carcinoma TCGA data, we showed that endometrial tumors from Black women are hypomethylated and more hypervariable than tumors from White women. In-depth investigation of these methylation driven markers can aid in successful management of endometrial cancer disparities and improved overall survival in Black and White populations.

STUDY QUESTION Are there differences in Mediator Complex Subunit 12 mutations (MED12) mutation, transcriptomics, and protein expression in uterine myometrium and leiomyomas of Black and White women? SUMMARY ANSWER RNA sequencing, tissue microarray, and immunohistochemistry data revealed that Black and White women have significant differences in their myometrium and leiomyoma profiles. WHAT IS KNOWN ALREADY Black women develop uterine leiomyoma earlier than White women, and are more likely to be anemic, have multiple tumors, undergo hysterectomy at an earlier age, have a higher uterine weight, and report very severe pelvic pain. STUDY DESIGN, SIZE, DURATION Uterine tissues were collected from premenopausal women undergoing hysterectomy or myomectomy at Northwestern University Prentice Women’s Hospital (Chicago, IL) from 2010 to 2021. Tissues were collected from a total of 309 women, including from 136 Black women, 135 White women, and 38 women from other racial groups. A total of 529 uterine leiomyomas (290 from Black women, 184 from White women, and 55 from women of other racial groups) were subjected to molecular analysis. Leiomyoma and matched myometrium from a total of 118 cases including 60 Black women and 58 White women, were used for tissue microarrays, along with 34 samples of myometrium without leiomyoma from White women. PARTICIPANTS/MATERIALS, SETTING, METHODS Tissues from the above patient cohorts were analyzed by tissue microarray, immunohistochemistry, RNA sequencing, and mutation analysis. MAIN RESULTS AND THE ROLE OF CHANCE The results indicated that leiomyoma from Black women have a higher rate of MED12 mutations (79.0%) than those from White women (68.5%) (*P ≤ 0.05). RNA-sequencing analysis in myometrium revealed differentially expressed genes (270 upregulated, 374 downregulated) dependent on race, wherein reactive oxygen species, hypoxia, and oxidative phosphorylation pathways were positively correlated with samples derived from Black patients. The levels of proteins associated with oxidative DNA damage and repair, 8-hydroxyguanosine (8-OHdG), 8-oxoguanine glycosylase (OGG1), heme oxygenase-1 (HO-1), and kelch-like ECH-associated protein 1 (KEAP1), were higher in leiomyoma and matched myometrium, particularly those from Black patients, compared to the control myometrium (with leiomyoma) (***P ≤ 0.001). LARGE SCALE DATA The datasets are available in the NCBI (The BioProject number: PRJNA859428). LIMITATIONS, REASONS FOR CAUTION Myometrium without leiomyoma derived from White patients was used as a control in the tissue microarray analysis, as myometrium without leiomyoma from Black patients was not accessible in large numbers. The RNA sequencing was performed on myometrium tissue with leiomyoma present from 10 White and 10 Black women. However, one sample from a Black woman yielded low-quality RNA-sequencing data and was excluded from further analysis. WIDER IMPLICATIONS OF THE FINDINGS Women with symptomatic leiomyomas have a considerable loss in their quality of life. This study provides information on underlying genetic and molecular defects that may be necessary for future therapeutics targeted at leiomyomas. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from NCI (R01CA254367) and NICHD (P01HD057877). The authors declare no conflict of interest.

Supplemental Figure 5. Ki67 Change was not associated with drug concentration in Tumor samples. Tumor samples were available for quantitation from 22 women.

Supplemental Figure 2. Ki67 Change relative to treatment duration. There was no significant correlation between change in Ki67 and treatment duration, in either TPA or placebo arms.

Supplemental Figure 4. Plots showing changes in serum estradiol (left panel) and serum progesterone (right panel) in three subgroups: placebo, TPA-treated Ki67 responders and TPA-treated Ki67-nonresponders.

Supplemental Figure 3. Validation of RNA seq results of responders by Nanostring nCounter gene expression assay

Purpose. Wnt signaling regulates the fine balance between stemness and differentiation. Here, the role of Wnt signaling to maintain the balance between estrogen-induced proliferation and progesterone-induced differentiation during the menstrual cycle, as well as during the induction of hyperplasia and carcinogenesis of the endometrium, was investigated.Experimental Design: Endometrial gene expression profiles from estradiol (E2) and E2 + medroxyprogesterone acetate–treated postmenopausal patients were combined with profiles obtained during the menstrual cycle (PubMed; GEO DataSets). Ishikawa cells were transfected with progesterone receptors and Wnt inhibitors dickkopf homologue 1 (DKK1) and forkhead box O1 (FOXO1), measuring Wnt activation. Expression of DKK1 and FOXO1 was inhibited by use of sequence-specific short hairpins. Furthermore, patient samples (hormone-treated endometria, hyperplasia, and endometrial cancer) were stained for Wnt activation using nuclear β-catenin and CD44.Results: In vivo, targets and components of the Wnt signaling pathway (among them DKK1 and FOXO1) are regulated by E2 and progesterone. In Wnt-activated Ishikawa cells, progesterone inhibits Wnt signaling by induction of DKK1 and FOXO1. Furthermore, using siRNA-mediated knockdown of both DKK1 and FOXO1, progesterone inhibition of Wnt signaling was partly circumvented. Subsequently, immunohistochemical analysis of the Wnt target gene CD44 showed that progesterone acted as an inhibitor of Wnt signaling in hyperplasia and in well-differentiated endometrial cancer.Conclusion: Progesterone induction of DKK1 and FOXO1 results in inhibition of Wnt signaling in the human endometrium. This Wnt inhibitory effect of progesterone is likely to play a rate-limiting role in the maintenance of endometrial homeostasis and, on its loss, in tumor onset and progression toward malignancy. (Clin Cancer Res 2009;15(18):5784–93)

Supplemental table 1. Differential gene expression and pathway enrichment analysis in TPA responders (excel file) -uploading separately.

Supplemental table 3. Nanostring nCounter Differential gene expression (excel file) -uploading separately.

Purpose:Selective progesterone receptor modulators (SPRMs) show preclinical activity against hormone-sensitive breast cancer, but have not been tested in patients with early, treatment-naïve tumors.Patients and Methods:In a double-blind presurgical window trial of oral telapristone acetate (TPA) 12 mg daily versus placebo, 70 patients with early-stage breast cancer were randomized 1:1 (stratified by menopause) and treated for 2 to 10 weeks. The primary endpoint was change in Ki67 between diagnostic biopsy and surgical specimens. Gene expression pre- and posttherapy was assessed using RNA-sequencing and gene set enrichment analysis was performed to determine pathways enriched in response to TPA and placebo treatments.Results:Among 61 evaluable women (29 placebo and 32 telapristone acetate), 91% of tumors were ER/PR positive. The mean Ki67 declined by 5.5% in all women treated with telapristone acetate (P = 0.003), and by 4.2% in all women treated with placebo (P = 0.04). After menopausal stratification, the Ki67 decline remained significant in 22 telapristone acetate–treated premenopausal women (P = 0.03). Differential gene expression analysis showed no significant modulation overall. However, in a subset of tumors that demonstrated ≥30% relative reduction in Ki67 in the telapristone acetate group, genes related to cell-cycle progression, and those in the HER2 amplicon were significantly downregulated. In contrast, no significantly enriched pathways were identified in the placebo group.Conclusions:Patients treated with telapristone acetate whose Ki67 decreased by ≥30% demonstrated a selective antiproliferative signal, with a potentially important effect on HER2 amplicon genes. Evaluation of SPRMs in a neoadjuvant trial is merited, with attention to predictors of response to SPRM therapy, and inclusion of pre- and postmenopausal women.

Supplementary Data from Progesterone Inhibition of Wnt/β-Catenin Signaling in Normal Endometrium and Endometrial Cancer

Supplemental Materials Supplemental Table 4. Changes in serum sex hormone concentrations according to the treatments. Supplemental Table 5. Summary of BESS Quality of Life Assessment by symptom clusters according to the treatments Supplemental Table 6. Summary of Adverse Effects.

Supplemental Figure 1. Validation of RNA seq results of responders by Nanostring nCounter gene expression assay.

Supplementary Figure Legend from Transcription Factor KLF11 Integrates Progesterone Receptor Signaling and Proliferation in Uterine Leiomyoma Cells

Supplementary Table 1 from Transcription Factor KLF11 Integrates Progesterone Receptor Signaling and Proliferation in Uterine Leiomyoma Cells

Supplementary Figure 1 from Transcription Factor KLF11 Integrates Progesterone Receptor Signaling and Proliferation in Uterine Leiomyoma Cells

Supplementary Table 2 from Transcription Factor KLF11 Integrates Progesterone Receptor Signaling and Proliferation in Uterine Leiomyoma Cells

Pharmacological approaches to breast cancer risk-reduction for BRCA1 mutation carriers would provide an alternative to mastectomy. BRCA1-deficiency dysregulates progesterone signaling, promoting tumorigenesis. Selective progesterone receptor (PR) modulators (SPRMs) are therefore candidate prevention agents. However, their efficacy varies in different BRCA1-deficient mouse models. We examined chemopreventive efficacy of telapristone acetate (TPA), ulipristal acetate (UPA) and mifepristone (MFP) in mice with a conditional knockout of the Brca1 C-terminal domain. The SPRMs displayed a spectrum of efficacy: UPA was most effective, TPA less, and MFP ineffective. Compared to no-treatment controls, UPA reduced tumorigenesis (p = 0.04), and increased tumor latency (p = 0.03). In benign mammary glands, UPA decreased Ki67 (p 0.001) and increased PR expression (p 0.0001). RNA sequencing analysis revealed distinct gene expression in response to UPA and MFP. UPA downregulated glycolysis and extracellular matrix-inflammation genes (Fn1, Ptgs2, Tgfb2, Tgfb3) whereas MFP downregulated claudin genes and upregulated amino acid metabolism and inflammation genes. The anti-glucocorticoid effects of MFP appeared not to be tumor-protective, while altering estrogen receptor signaling and NF-kB activation. Our study points to an important role of epithelial PR and its paracrine action on the microenvironment in BRCA1-deficient mammary tumorigenesis, and prevention.

Each month during a woman's reproductive years, the endometrium undergoes vast changes to prepare for a potential pregnancy. Diseases of the endometrium arise for numerous reasons, many of which remain unknown. These endometrial diseases, including endometriosis, adenomyosis, endometrial cancer and Asherman syndrome, affect many women, with an overall lack of efficient or permanent treatment solutions. The challenge lies in understanding the complexity of the endometrium and the extensive changes, orchestrated by ovarian hormones, that occur in multiple cell types over the period of the menstrual cycle. Appropriate model systems that closely mimic the architecture and function of the endometrium and its diseases are needed. The emergence of organoid technology using human cells is enabling a revolution in modelling the endometrium in vitro. The goal of this Review is to provide a focused reference for new models to study the diseases of the endometrium. We provide perspectives on the power of new and emerging models, from organoids to microfluidics, which have opened up a new frontier for studying endometrial diseases.

For patients diagnosed with ovarian, uterine, or cervical cancer, race impacts expected outcome, with black women suffering worse survival than white women for all three malignancies. Moreover, outcomes for black women have largely worsened since the 1970s. In this narrative review, we first provide an updated summary of the incidence and survival of ovarian, uterine, and cervical cancer, with attention paid to differences between white and black patients. We then offer a theoretical framework detailing how racial disparities in outcomes for each of the gynecologic malignancies can be explained as the sum result of smaller white–black differences in experience of preventive strategies, implementation of screening efforts, early detection of symptomatic disease, and appropriate treatment. Much research has been published regarding racial disparities in each of these domains, and with this review, we seek to curate the relevant literature and present an updated understanding of disparities between black and white women with gynecologic malignancies.

Background More than 70% of leiomyomas (LM) harbor MED12 mutations, primarily in exon 2 at c.130–131(GG). The cause of MED12 mutations in myometrial cells remains largely unknown. We hypothesized that increased ROS promotes MED12 mutations in myometrial cells through the oxidation of guanine nucleotides followed by misrepair. Methods Genomic oxidative burden (8-OHdG) was evaluated in vitro and in vivo by immunohistochemistry. MED12 mutations were examined by Sanger sequencing and deep sequencing. Transcriptome examined by RNA-seq was performed in myometrium with and without LM, in primary myometrial cells treated with ROS. 8-OHdG mediated misrepair was analyzed by CRISPR/Cas9. Results Uteri with high LM burden had a significantly higher rate of MED12 mutations than uteri with low LM burden. Compelling data suggest that the uterus normally produces reactive oxidative species (ROS) in response to stress, and ROS levels in LM are elevated due to metabolic defects. We demonstrated that genomic oxidized guanine (8-OHdG) was found at a significantly higher level in the myometrium of uteri that had multiple LM compared to myometrium without LM. Transcriptome and pathway analyses detected ROS stress in myometrium with LM. Targeted replacement of guanine with 8-OHdG at MED12 c.130 by CRISPR/Cas9 significantly increased the misrepair of G > T. Exposure of primary myometrial cells to oxidative stress in vitro increased misrepair/mutations as detected by duplex sequencing. Conclusions Together, our data identified a clear connection between increased myometrial oxidative stress and a high rate of MED12 mutations that may underlie the risk of LM development and severity in women of reproductive age.

Objective To study the role of HMGA2 in promoting angiogenesis in uterine leiomyoma (LM). Design This study involved evaluation of vessel density and angiogenic factors in leiomyomas with HMGA2 overexpression; examining angiogenic factor expression and AKT signaling in myometrial (MM) and leiomyoma cells by introducing HMGA2 overexpression in vitro; and exploring vessel formation induced by HMGA2 overexpression both in vitro and in vivo. Setting University research laboratory. Patients None. Interventions None. Main Outcome Measures The main outcome measures include vessel density in leiomyomas with HMGA2 (HMGA2-LM) or MED12 (MED12-LM) alteration; angiogenic factor expression in primary leiomyoma and in vitro cell line model; and vessel formation in leiomyoma cells with HMGA2 overexpression in vitro and in vivo. Results Angiogenic factors and receptors were significantly upregulated at mRNA and protein levels in HMGA2-LM. Specifically, HMGA2-LM exhibited increased expression of VEGFA, EGF, bFGF, TGFα, VEGFR1, and VEGFR2 compared to MED12-LM and myometrium. Overexpression of HMGA2 in MM and LM cell lines resulted in increased secretion of angiogenesis-associated factors. Secreted factors promoted human umbilical vein endothelial cell (HUVEC) migration, tube formation, and wound healing. HMGA2 overexpression upregulated IGF2BP2 and pAKT, and silencing the IGF2BP2 gene reduced pAKT levels and reduced HUVEC migration. Myometrial cells with stable HMGA2 overexpression exhibited increased colony formation and cell growth in vitro and formed xenografts with increased blood vessels. Conclusions HMGA2-LM have a high vasculature density, which likely contributes to tumor growth and disease burden of this leiomyoma subtype. HMGA2 plays an important role in angiogenesis and the involvement of IGF2BP2-mediated pAKT activity in angiogenesis, which provides a potential novel target for therapy for this subtype of LM.

STUDY QUESTION How does exposure to a testosterone rich environment affect the function and gene expression of human fallopian tube epithelium (hFTE)? SUMMARY ANSWER Elevated testosterone level alters several gene transcripts that regulate cilia expression and negatively impacts the rate of cilia beating. WHAT IS KNOWN ALREADY The presence of estrogen in the follicular phase of the menstrual cycle increases the human fallopian tube ciliary beating frequency. The luteal phase, triggered by ovulation and increasing progesterone, is marked by a decrease in ciliary beating. Women with polycystic ovarian syndrome (PCOS) may have twice the serum level of testosterone than ovulatory women. To date, the effect of elevated androgens on the function of the human fallopian tube is not well-understood. We chose to examine the impact of elevated testosterone on hFTE. STUDY DESIGN, SIZE, DURATION A prospective basic science study of human fallopian tube specimens from reproductive-aged women undergoing benign gynecologic surgery was performed. Fallopian tube removal at a large US academic center was collected and provided to us to continue with epithelium isolation and culturing. A total of 12 patients were analyzed in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS Fallopian tube epithelium was isolated and exposed to two different conditions: normal with low testosterone concentration of 0.8 nM and PCOS-like, with high testosterone concentration of 2 nM. The study was conducted in both static and dynamic conditions in microfluidic devices for a total of 14 days, after which the tissue was collected for processing including RNA extraction, quantitative PCR and immunohistochemistry. After the first 7 days of each experiment, a sample of tissue from each condition was imaged to quantify cilia beating frequency. MAIN RESULTS AND THE ROLE OF CHANCE hFTE exposed to the 2 nM testosterone displayed slower cilia beating, inhibited estrogen signaling and decreased expression of the ciliary marker FOXJ1 when compared to stimulation with 0.8 nM testosterone. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION The in vivo response to elevated testosterone may differ from in vitro studies. RNA amount was limited from tissue cultured in the microfluidic devices as compared to static culture. WIDER IMPLICATIONS OF THE FINDINGS Understanding elevated testosterone in tubal function may explain an additional contribution to subfertility in women with PCOS and other hyper-androgen disorders, aside from oligo-ovulation. Furthermore, this adds to the body of literature of fallopian tube function using a microfluidic device. STUDY FUNDING/COMPETING INTEREST(S) NIH grants: UH3 ES029073 and R01 CA240301. There are no competing interests.

Since the beginning of clinical medicine, the human uterus has held the fascination of clinicians and researchers, given its critical role in the reproduction of our species. The endometrial lining provides residence for the embryo; however, this symbiotic interaction can be disrupted if the timing is not correct and the endometrium is not receptive. Diseases associated with the endometrium interfere with the reproductive process and cause a life-altering burden of pain and even death. With the advancement of technologies and new insights into the biology of the endometrium, much has been uncovered about the dynamic and essential changes that need to occur for normal endometrial function, as well as aberrations that lead to endometrial diseases. As expected, the more that is uncovered, the more the complexity of the endometrium is made evident. In this study, we bring together three areas of scientific advancement that remain in their infancy, but which together have the potential to mirror this complexity and enable understanding. Studies on induced pluripotent stem cells, three-dimensional tissue mimics, and microfluidic culture platforms will be reviewed with a focus on the endometrium. These unconventional approaches will provide new perspectives and appreciation for the elegance and complexity of the endometrium. IMPACT STATEMENT: The ability of the human endometrium to regenerate on a monthly basis for ∼4 decades of reproductive years exemplifies its complexity as well as its susceptibility to disease. Restrictions on the types of research that can be done in the human endometrium motivate the development of new technologies and model systems. The three areas of technological advancement reviewed here—induced pluripotent stem cells, three-dimensional model systems, and microfluidic culture systems—will highlight some of the tools that can be applied to studying the human endometrium in ways that have not been done before.

Purpose: Selective progesterone receptor modulators (SPRMs) show preclinical activity against hormone-sensitive breast cancer, but have not been tested in patients with early, treatment-naïve tumors. Patients and Methods: In a double-blind presurgical window trial of oral telapristone acetate (TPA) 12 mg daily versus placebo, 70 patients with early-stage breast cancer were randomized 1:1 (stratified by menopause) and treated for 2 to 10 weeks. The primary endpoint was change in Ki67 between diagnostic biopsy and surgical specimens. Gene expression pre- and posttherapy was assessed using RNA-sequencing and gene set enrichment analysis was performed to determine pathways enriched in response to TPA and placebo treatments. Results: Among 61 evaluable women (29 placebo and 32 telapristone acetate), 91% of tumors were ER/PR positive. The mean Ki67 declined by 5.5% in all women treated with telapristone acetate (P = 0.003), and by 4.2% in all women treated with placebo (P = 0.04). After menopausal stratification, the Ki67 decline remained significant in 22 telapristone acetate–treated premenopausal women (P = 0.03). Differential gene expression analysis showed no significant modulation overall. However, in a subset of tumors that demonstrated ≥30% relative reduction in Ki67 in the telapristone acetate group, genes related to cell-cycle progression, and those in the HER2 amplicon were significantly downregulated. In contrast, no significantly enriched pathways were identified in the placebo group. Conclusions: Patients treated with telapristone acetate whose Ki67 decreased by ≥30% demonstrated a selective antiproliferative signal, with a potentially important effect on HER2 amplicon genes. Evaluation of SPRMs in a neoadjuvant trial is merited, with attention to predictors of response to SPRM therapy, and inclusion of pre- and postmenopausal women.

The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. A lack of suitable 3D in vitro models permitting the study of interactions between cell types and the microenvironment is a contributing factor. To address this limitation, we developed endometriotic organoids (EO) to explore the role of epithelial-stromal interactions and model peritoneal cell invasion associated with lesion development. Using a non-adherent microwell culture system, spherical organoids were generated with endometriotic epithelial cells (12Z) combined with immortalized endometriotic stromal cells (iEc-ESC) or immortalized uterine stromal cells (iHUF). Organoids self-organized with stromal cells occupying the center and epithelial cells on the periphery of the organoid. Endometriotic organoids (EO), containing iEc-ESC, resulted in the development of stratified 12Z epithelial cells compared to those with iHUF where the 12Z cells developed as a single layered epithelium. Transcriptomic analysis found 4,522 differentially expressed genes (DEG) between EO and 12Z/iHUF organoids, and the top DEG included increased expression of interleukins and prostaglandin synthase enzymes. An overlap of the EO DEG with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed using EO and extracellular matrix containing human peritoneal mesothelial cells (LP9). Invasion of EO into the extracellular matrix-LP9 layer was increased in presence of estrogen or THP1-derived proinflammatory macrophages. Taken together, our results strongly support the concept that EO are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.One Sentence SummaryEndometriotic organoids are an appropriate model to study epithelial-stromal interactions and model cell invasion associated with lesion development.

// Joseph F. Peevey 1 , Brandon-Luke L. Seagle 2 , Kruti P. Maniar 1 and J. Julie Kim 3 1 Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 2 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 3 Division of Reproductive Science in Medicine, Department of Obstetrics and Gynecology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Correspondence to: J. Julie Kim, email: j-kim4@northwestern.edu Keywords: endometrial cancer, PR, ER, 14-3-3, obesity Received: January 26, 2017 Accepted: April 06, 2017 Published: April 18, 2017 ABSTRACT Obesity is a prominent risk factor for endometrial cancer (EC) and can impede on surgical and hormonal treatments. Markers of EC, estrogen receptor (ER), progesterone receptor (PR), phospho(Ser473)-AKT (pAKT) and 14-3-3 sigma (14-3-3σ) were measured in EC tissues in both the tumor and stroma and grouped by body mass index (BMI). Immunohistochemical scoring of 82 cases of Type 1 and Type II EC tissues revealed a significantly increased tumor expression of ER, PR and 14-3-3σ in women with Type I (BMI 40 compared to BMI < 40. Type II EC showed increased expression of ER in the stroma only between high and low BMI. Analysis of the TCGA RNA-Seq mRNA expression of ER, PR, PIK3CA, PTEN and SFN (gene for 14-3-3σ) confirmed increased PR expression in EC of obese women. In conclusion, ER, PR and 14-3-3σ are differentially regulated in Type I compared to Type II EC while PR is dysregulated in obese women with Type I EC. These findings have potential implications for efficacy of progestin treatment in obese women.

ContextPolycystic ovary syndrome (PCOS) is a prevalent disorder in reproductive aged women associated with a number of endocrine and metabolic complications, including increased risk of endometrial cancer.ObjectiveTo study the effect of the characteristic increased androgen levels in PCOS on the endometrium, a novel scaffold-free multicellular endometrial organoid was established.DesignHuman endometrial organoids were constructed using primary endometrial epithelial and stromal cells from endometrial tissues. Organoids were treated for 14 days with physiologic levels of estradiol and testosterone to mimic a normal follicular phase or PCOS hormone profiles. Organoids were harvested for immunostaining and ribonucleic acid sequencing.SettingAcademic institution.PatientsEndometrial tissues from 10 premenopausal women undergoing hysterectomy for benign pathologies were obtained following written consent.Main Outcome MeasuresOrganoid architecture, cell specific markers, functional markers, proliferation, and gene expression were measured.ResultsA method to generate scaffold-free endometrial organoids containing epithelial and stromal cells was established. These organoids exhibited distinct organization with epithelial cells lining the outer surface and stromal cells in the center of the organoids. Epithelial cells were polarized, organoids expressed cell type specific and functional markers, as well as androgen, estrogen, and progesterone receptors. Treatment with PCOS hormones increased cell proliferation and dysregulated genes in endometrial organoids.ConclusionsA new multicellular, scaffold-free endometrial organoid system was established that resembled physiology of the native endometrium. Excess androgens in PCOS promoted cell proliferation in endometrial organoids, revealing new mechanisms of PCOS-associated with risk of endometrial neoplasia.

Cellular senecence is an important biologic endpoint. Naturally occuring (aging) senescence is common in uterine leiomyoma (ULM). AKT is one of major pathways in promoting ULM growth and survial. Inactivation of AKT by MK2206 in ULM resulted in stress-induced senescence in vitro. Study of the senescent phenotypes and molecular changes in ULM may greatly facilitate the understanding of the tumor biology and potential clinical therapy for this common disease associated with high morbidity. To study senescence in a model system that closely resembles primary ULM in vivo, we applied an ex vivo model of three dimensional (3D) spheroid culture system which maintained the molecular and cellular characteristics of primary ULM and matched myometrium as seen in vivo. Gene expression profiling done on ULM induced to undergo replication (passaging) or stress-induced (MK2206) senescence revealed that ROS and hypoxic related genes were upregulated in the two types of senescences. Overexpression of two selected genes, WIPI1 and SLITKR4, induced cellular senescence in in ULM spheroids. Additionally, administration of ABT263 (a BH3 mimetic) effectively reduced the senescent cells induced in ULM spheroids. This study identified novel genes associated with senescence in ULM and demonstrated a BH3 mimetic to act as a senolytic to remove senscent cells.

© 2020 The Authors Women's health concerns are generally underrepresented in basic and translational research, but reproductive health in particular has been hampered by a lack of understanding of basic uterine and menstrual physiology. Menstrual health is an integral part of overall health because between menarche and menopause, most women menstruate. Yet for tens of millions of women around the world, menstruation regularly and often catastrophically disrupts their physical, mental, and social well-being. Enhancing our understanding of the underlying phenomena involved in menstruation, abnormal uterine bleeding, and other menstruation-related disorders will move us closer to the goal of personalized care. Furthermore, a deeper mechanistic understanding of menstruation—a fast, scarless healing process in healthy individuals—will likely yield insights into a myriad of other diseases involving regulation of vascular function locally and systemically. We also recognize that many women now delay pregnancy and that there is an increasing desire for fertility and uterine preservation. In September 2018, the Gynecologic Health and Disease Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a 2-day meeting, “Menstruation: Science and Society” with an aim to “identify gaps and opportunities in menstruation science and to raise awareness of the need for more research in this field.” Experts in fields ranging from the evolutionary role of menstruation to basic endometrial biology (including omic analysis of the endometrium, stem cells and tissue engineering of the endometrium, endometrial microbiome, and abnormal uterine bleeding and fibroids) and translational medicine (imaging and sampling modalities, patient-focused analysis of menstrual disorders including abnormal uterine bleeding, smart technologies or applications and mobile health platforms) to societal challenges in health literacy and dissemination frameworks across different economic and cultural landscapes shared current state-of-the-art and future vision, incorporating the patient voice at the launch of the meeting. Here, we provide an enhanced meeting report with extensive up-to-date (as of submission) context, capturing the spectrum from how the basic processes of menstruation commence in response to progesterone withdrawal, through the role of tissue-resident and circulating stem and progenitor cells in monthly regeneration—and current gaps in knowledge on how dysregulation leads to abnormal uterine bleeding and other menstruation-related disorders such as adenomyosis, endometriosis, and fibroids—to the clinical challenges in diagnostics, treatment, and patient and societal education. We conclude with an overview of how the global agenda concerning menstruation, and specifically menstrual health and hygiene, are gaining momentum, ranging from increasing investment in addressing menstruation-related barriers facing girls in schools in low- to middle-income countries to the more recent “menstrual equity” and “period poverty” movements spreading across high-income countries.

It is unclear how endometrial cancer risk factors such as obesity, high serum testosterone, and high serum levels of the endocrine-disrupting compound bisphenol-A (BPA) influence hormone action to promote carcinogenesis. We hypothesized that obesity, high testosterone, and BPA exposure alters the protective progesterone response in the benign endometrium. Primary human benign endometrial organoids, consisting of both epithelial and stromal cells, were exposed to each of these risk factors in vitro in the presence of cyclic levels of estradiol, progesterone, and testosterone for 14 days. Progesterone response genes HSD17B2, IGFBP1, PAEP, and PRL were measured by real-time qPCR and IHC. First, to simulate obesity, endometrial organoids were cocultured with increasing numbers of human adipocyte spheroids during the hormone treatment. Real-time qPCR analysis revealed dysregulation of expression of HSD17B2 and IGFBP1 by approximately 20% when cocultured with 30 adipocyte spheroids. In addition, PRL protein levels were significantly lower in the stroma of the endometrial organoids. Second, increasing concentrations of BPA and 3nM testosterone individually or in combination were added to endometrial organoids together with the 14-day menstrual cycle hormones. Treatment with 0.6 ng/mL of BPA decreased expression of HSD17B2, IGFBP1, and PAEP by 50% to 80%. However, this effect was not seen in the context of high testosterone, indicating that there may be crosstalk between these two risk factors. In summary, this study demonstrated that adipocytes, BPA exposure, and high testosterone directly alter progesterone action in benign endometrial organoids, suggesting a diminution of the protective effects of progesterone and an increased risk of endometrial cancer.

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. 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 antiprogestin 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 SPRMs, specifically telapristone acetate (TPA), affect PR function at the molecular level. In this study, T47D breast cancer cells were used to investigate the molecular mechanism by which TPA antagonizes PR action. Global profiling of the PR cistrome and interactome was done with chromatin immunoprecipitation sequencing (ChIP-seq) and rapid immunoprecipitation mass spectrometry. Validation studies were done on key genes and interactions. Our results demonstrate that treatment with the progestin (R5020) alone resulted in robust PR recruitment to the chromatin, and addition of TPA reduced PR recruitment globally. TPA significantly changed coregulator recruitment to PR compared with R5020. Upon conservative analysis, three proteins (TRPS1, LASP1, and AP1G1) were identified in the R5020+TPA-treated group. Silencing TRPS1 with small interfering RNA increased PR occupancy to the known PR regulatory regions and attenuated the inhibition of gene expression after TPA treatment. TRPS1 silencing alleviated the inhibition of proliferation by TPA. In conclusion, TPA decreases PR occupancy on chromatin and recruits coregulators such as TRPS1 to the PR complex, thereby regulating PR target gene expression and associated cellular responses.

Leiomyoma with bizarre nuclei (LM-BN), is a variant of uterine smooth muscle tumor with atypical histological features. Although some LM-BN share several significant genetic alterations with leiomyosarcoma (LMS), including p16 and p53, the underlying tumorigenesis of LM-BN remains largely unknown. As we previous reported, LM-BN can be divided into two subtypes, type I and type II, based on different nuclear features. Type I LM-BN have similar histologic features as uterine smooth muscle tumors with fumarate hydratase (FH) alterations. In this study, we examined FH expression and FH mutations in 77 LM-BN (40 type I cases and 37 type II cases). FH expression was examined by immunohistochemistry (IHC) using S-(2-succino)-cysteine antibodies (2SC, a protein modification associated with FH inactivation and subsequent fumarate accumulation) and FH antibodies (fumarate hydratase gene products). Seventy-two LM-BN tumors underwent Sanger sequencing to detect FH mutations. We found that 51% (39/77) of LM-BN showed FH alterations detected by immunohistochemistry with both 2SC and FH. Mutational analysis showed that 21% (15/72) of LM-BN harbored FH gene mutations. Further analysis revealed that 85% (34/40) of those with FH alterations were type I LM-BN while 19% (7/37) were type II LM-BN. Our findings suggest that over half of histologically-diagnosed LM-BN may be related to FH alterations or FH mutations and the majority of these have the characteristic histologic features of type I LM-BN.

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.

Background: Over 30% of US adults are obese (body mass index (BMI) of ≥ 30 kg/m2). Obesity is considered a significant and modifiable risk factor for ER+ postmenopausal breast cancer (BC). However, the effect on pre-menopausal breast cancer risk is less clear. Bariatric surgery is performed on obese patients who have a BMI of >35. It has been associated with reduced risk of BC, but the mechanisms behind this are unknown. Studies in obese patients have revealed multiple changes in the physiology of the microenvironment that contribute to BC development. The molecular changes in the stroma and epithelium from metabolic syndrome, excess inflammation, and ectopic hormone synthesis likely play key role in obesity related BC. Therefore, understanding how we can alter the risk posed will have an impact on decreasing the incidence of BC. We hypothesize that bariatric surgery will reduce the incidence of postmenopausal ER+ BC by reducing estradiol availability and ER activity. Methods: We recruited 30 women undergoing bariatric surgery, who underwent core needle biopsy at the time of surgery and after they had lost 25% of their excess body weight. Changes in hormone concentration by LCMS/MS in serum and breast tissue, protein expression in breast tissue by IHC and the altered gene expression in breast epithelium and stroma by RNA seq were measured. Results: When segregated by menopausal status, Androstenedione (p=0.02), Testosterone (p < 0.0001) and 27-hydroxycholesterol (p=0.017) showed significant reduction in the plasma post-surgery in premenopausal women. There were no significant differences in the hormone levels in the breast tissue and in postmenopausal women post-surgery. We observed that African Americans (AA) were half as likely to show a reduction in tissue concentration compared to Caucasian women. IHC was performed to examine protein expression of Ki67, leptin R, aromatase, CYP27A1, Stat3, adiponectin, CYP27B1 and ER in the breast core biopsy. Aromatase expression (p < 0.0001) was significantly reduced in both the epithelial and stromal compartments. In addition, Stat3 levels (p=0.044) were significantly reduced in the stroma. RNA -seq analysis revealed distinct differentially expressed genes pre and post-surgery. Specifically, genes associated with metabolic process, epithelial development, extracellular space, immune and inflammatory response pathways. Furthermore, known ER target genes TFF1, AGR2 expression were significantly decreased in Caucasian women, but not in AA women, post- surgery. Conclusions: Taken together, these data show that bariatric surgery decreased aromatase expression, some hormones in pre-menopausal women and reduced estrogen availability and ER activity. Further analysis of differentially regulated genes due to bariatric surgery can allow us to explore previously unknown pathways related to breast cancer risk reduction.

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.

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.