Unraveling the Molecular Landscape of Uterine Fibroids, Insights into HMGA2 and Stem Cell Involvement.

Uterine fibroids are prevalent benign tumors in women that exhibit considerable heterogeneity in clinical presentation and molecular characteristics, necessitating a deeper understanding of their etiology and pathogenesis. HMGA2 overexpression has been associated with fibroid development, yet its precise role remains elusive. Mutations in fibroids are mutually exclusive and largely clonal, suggesting that tumors originate from a single mutant cell. We explored a possible role for HMGA2 overexpression in differentiated myometrial cells, hypothesizing its potential to induce a stem cell-like or dedifferentiating phenotype and drive fibroid development. Myometrial cells were immortalized and transduced with an HMGA2 lentivirus to produce HMGA2hi cells. In vitro stem cell assays were conducted and RNA from HMGA2hi and control cells and fibroid-free myometrial and HMGA2 fibroid (HMGA2F) tissues were submitted for RNA-sequencing. HMGA2hi cells have enhanced self-renewal capacity, decreased proliferation, and have a greater ability to differentiate into other mesenchymal cell types. HMGA2hi cells exhibit a stem cell-like signature and share transcriptomic similarities with HMGA2F. Moreover, dysregulated extracellular matrix pathways are observed in both HMGA2hi cells and HMGA2F. Our findings suggest that HMGA2 overexpression drives myometrial cells to dedifferentiate into a more plastic phenotype and underscore a pivotal role for HMGA2 in fibroid pathogenesis.

Cysteine-rich intestinal protein 1 is a novel surface marker for human myometrial stem/progenitor cells.

Myometrial stem/progenitor cells (MyoSPCs) have been proposed as the cells of origin for uterine fibroids, but the identity of the MyoSPC has not been well established. We previously identified SUSD2 as a possible MyoSPC marker, but the relatively poor enrichment in stem cell characteristics of SUSD2+ over SUSD2- cells compelled us to find better markers. We combined bulk RNA-seq of SUSD2+/- cells with single cell RNA-seq to identify markers for MyoSPCs. We observed seven distinct cell clusters within the myometrium, with the vascular myocyte cluster most highly enriched for MyoSPC characteristics and markers. CRIP1 expression was found highly upregulated by both techniques and was used as a marker to sort CRIP1+/PECAM1- cells that were both enriched for colony forming potential and able to differentiate into mesenchymal lineages, suggesting that CRIP1+/PECAM1- cells could be used to better study the etiology of uterine fibroids.

PIK3CA mutation in endometriotic epithelial cells promotes viperin-dependent inflammatory response to insulin.

Endometrial epithelia are known to harbor cancer driver mutations in the absence of any pathologies, including mutations in PIK3CA. Insulin plays an important role in regulating uterine metabolism during pregnancy, and hyperinsulinemia is associated with conditions impacting fertility. Hyperinsulinemia also promotes cancer, but the direct action of insulin on mutated endometrial epithelial cells is unknown. Here, we treated 12Z endometriotic epithelial cells carrying the PIK3CAH1047R oncogene with insulin and examined transcriptomes by RNA-seq. While cells naively responded to insulin, the magnitude of differential gene expression (DGE) was nine times greater in PIK3CAH1047R cells, representing a synergistic effect between insulin signaling and PIK3CAH1047R expression. Interferon signaling and the unfolded protein response (UPR) were enriched pathways among affected genes. Insulin treatment in wild-type cells activated normal endoplasmic reticulum stress (ERS) response programs, while PIK3CAH1047R cells activated programs necessary to avoid ERS-induced apoptosis. PIK3CAH1047R expression alone resulted in overexpression (OE) of Viperin (RSAD2), which is involved in viral response and upregulated in the endometrium during early pregnancy. The transcriptional changes induced by insulin in PIK3CAH1047R cells were rescued by knockdown of Viperin, while Viperin OE alone was insufficient to induce a DGE response to insulin, suggesting that Viperin is necessary but not sufficient for the synergistic effect of PIK3CAH1047R and insulin treatment. We identified interferon signaling, viral response, and protein targeting pathways that are induced by insulin but dependent on Viperin in PIK3CAH1047R mutant cells. These results suggest that response to insulin signaling is altered in mutated endometriotic epithelial cells.

Cysteine-Rich Intestinal Protein 1 is a Novel Surface Marker for Myometrial Stem/Progenitor Cells.

Myometrial stem/progenitor cells (MyoSPCs) have been proposed as the cells of origin for uterine fibroids, which are benign tumors that develop in the myometrium of most reproductive age women, but the identity of the MyoSPC has not been well established. We previously identified SUSD2 as a possible MyoSPC marker, but the relatively poor enrichment in stem cell characteristics of SUSD2+ over SUSD2- cells compelled us to find better discerning markers for more rigorous downstream analyses. We combined bulk RNA-seq of SUSD2+/- cells with single cell RNA-seq to identify markers capable of further enriching for MyoSPCs. We observed seven distinct cell clusters within the myometrium, with the vascular myocyte cluster most highly enriched for MyoSPC characteristics and markers, including SUSD2. CRIP1 expression was found highly upregulated in both techniques and was used as a marker to sort CRIP1+/PECAM1- cells that were both enriched for colony forming potential and able to differentiate into mesenchymal lineages, suggesting that CRIP1+/PECAM1- cells could be used to better study the etiology of uterine fibroids.

Endometrial hyperplasia with loss of APC in a novel population of Lyz2-expressing mouse endometrial epithelial cells.

Loss of heterozygosity and promoter hypermethylation of APC is frequently observed in human endometrial cancer, which is the most common gynecological cancer in the USA, but its carcinogenic driver status in the endometrial epithelium has not been confirmed. We have identified a novel population of progenitor endometrial epithelial cells (EECs) in mice that express lysozyme M (LysM) and give rise to approximately 15% of all EECs in adult mice. LysM is a glycoside hydrolase that is encoded by Lyz2 and functions to protect cells from bacteria as part of the innate immune system. Its expression has been shown in a subset of hematopoietic stem cells and in specialized lung and small intestinal epithelial cells. Conditional deletion of Apc in LysM + EECs results in significantly more epithelial cells compared to wild-type mice. At 5 months of age, the ApccKO mice have enlarged uterine horns with pathology that is consistent with endometrial hyperplasia with cystic endometrial glands, non-villous luminal papillae and nuclear atypia. Nuclear accumulation of ß-catenin and ERα, both of which are known to induce endometrial hyperplasia, was observed in the EECs of the ApccKO mice. These results confirm that loss of APC in EECs can result in a phenotype similar to endometrial hyperplasia.

The Effects of Periostin Expression on Fibroid-Like Transition of Myometrial Cells.

Fibroids, benign tumors of the myometrium, are the most common tumors in women and are associated with spontaneous abortion, preterm birth, placenta abruption, and infertility, among others. The incidence of fibroids in reproductive aged women is 20-89%. Fibroids are characterized by high production of extracellular matrix (ECM), particularly collagens, which play a role in their growth. However, their pathogenesis is poorly understood. Recently, we and others have found periostin (POSTN), a regulatory ECM protein, to be overexpressed in the majority of fibroids analyzed. Periostin is an ECM protein that is a critical regulator and well-established biomarker for fibrosis in tissues such as the lung, skin, and kidney. Our hypothesis was that periostin plays a role in the fibrotic transition of myometrial cells to fibroid cells. To test this, we evaluated the effects of POSTN overexpression in myometrial cells. Telomerase-immortalized myometrial cells were transduced with control or POSTN-overexpression lentivirus particles, generating one control (dCas9-Mock) and two overexpression (dCas9-POSTN-01, dCas9-POSTN-02) cell lines. Overexpression of POSTN in immortalized myometrial cells resulted in a change in phenotype consistent with fibroid cells. They upregulated expression of key fibroid genes and had increased proliferation, adhesion, and migration in vitro. Here, we show a potential role for periostin in the transition of myometrial cells to fibroid cells, giving rationale for future investigation into the role of periostin in fibroid pathogenesis and its potential as a therapeutic target.

Children's outcomes in road traffic accidents: challenges for personal injury assessment.

Children represent a specific group of road traffic accident (RTA) victims. Performing a personal injury assessment (PIA) on a child presents a significant challenge, especially when assessing permanent disabilities and needs. However, medico-legal recommendations for PIA in such cases are lacking. The main objective of this study was to analyse the differences between children and a young- and middle-aged adult population of RTA victims to contribute to the development of relevant guidelines. Secondary objectives were to identify and characterize specifics of children's posttraumatic damages regarding: (i) temporary and permanent outcomes; and (ii) medico-legal damage parameters in the Portuguese context. We performed a retrospective study of RTA victims by comparing two groups (n = 114 each) matched for acute injury severity (SD = 0.01): G1 (children) and G2 (young- and middle-aged adults). Logistic regression was used to estimate the odds ratios. G1 presented a greater chance of evolving without or with less severe body, functional and situational outcomes (three-dimensional assessment methodology), and with lower permanent functional disability values than G2. Our findings suggest that childhood trauma generally has a better prognosis than trauma in young- and middle-aged adults. This study generated evidence on the subject and highlighted the most significant difficulties encountered by medico-legal experts when performing PIA in children. Key points: This retrospective study of PIA in child victims of RTA in Portugal considered outcomes in victims' real-life situations.Several significant differences between children and young- and middle-aged adults were observed.Children's cases presented better results in terms of the severity of body, functional and situational outcomes, and permanent damage parameters.The average time between the RTA and final PIA date and the consolidation time were longer for children because of the need to wait for the Children's next growth phase or final pubertal period (as applicable), which increased the time for PIA conclusion.There were several difficulties in the medical-legal evaluation of children's cases, which was a complex process because the trauma affected them in their growth phase.

Obesity alters the mouse endometrial transcriptome in a cell context-dependent manner.

Obesity impacts fertility and is positively correlated with endometrial hyperplasia and endometrial cancer occurrence. Endometrial epithelia often harbor disease driver-mutations, while endometrial stroma are highly regulative of neighboring epithelia. Here, we sought to determine distinct transcriptome changes occurring in individual cell types in the obese mouse uterus. Outbred CD-1 mice were fed high-fat or control diets for 18 weeks, estrous cycle staged, and endometrial epithelia, macrophages, and stroma isolated for transcriptomic analysis. High-fat diet mice displayed increased body mass and developed glucose intolerance, hyperinsulinemia, and fatty liver. Obese mouse epithelia displayed differential gene expression for genes related to innate immunity and leukocyte chemotaxis. The obese mouse stroma differentially expressed factors related to circadian rhythm, and expression of these genes correlated with glucose tolerance or body mass. We observed correlations between F4/80 + macrophage numbers, Cleaved Caspase 3 (CC3) apoptosis marker staining and glucose intolerance among obese mice, including a subgroup of obese mice with high CC3 + luminal epithelia. This subgroup displayed differential gene expression among all cell types, with pathways related to immune escape in epithelia and macrophages, while the stroma dysregulated pathways related to regulation of epithelia. These results suggest an important role for differential response of both the epithelia and stroma in their response to obesity, while macrophages are dysregulated in the context of apoptotic epithelia. The obesity-related gene expression programs in cells within the uterine microenvironment may influence the ability of the endometrium to function during pregnancy and influence disease pathogenesis.

Transcriptome and DNA methylome analyses reveal underlying mechanisms for the racial disparity in uterine fibroids.

Uterine fibroids (leiomyomas) affect Black women disproportionately compared with women of other races and ethnicities in terms of prevalence, incidence, and severity of symptoms. The causes of this racial disparity are essentially unknown. We hypothesized that myometria of Black women are more susceptible to developing fibroids, and we examined the transcriptomic and DNA methylation profiles of myometria and fibroids from Black and White women for comparison. Myometrial samples cluster by race in both their transcriptome and DNA methylation profiles, whereas fibroid samples only cluster by race in the latter. More differentially expressed genes (DEGs) were detected in the Black and White myometrial sample comparison than in the fibroid comparison. Leiomyoma gene set expression analysis identified 4 clusters of DEGs, including a cluster of 24 genes with higher expression in myometrial samples from Black women. One of the DEGs in this group, von Willibrands factor (VWF), was significantly hypomethylated in both myometrial samples from Black women and in all fibroids at 2 CpG probes that are near a putative enhancer site and that are correlated with VWF expression levels. These results suggest that the molecular basis for the disparity in fibroid disease between Black and White women could be found in the myometria before fibroid development and not in the fibroids themselves.

Obesity-induced follicular phase endometrial proteome dysregulation in a well-phenotyped population.

OBJECTIVE: Despite obesity's significant impact on reproduction, its influence on the physiology of the human endometrium is largely understudied. We hypothesized that endometrial proteomic differences exist between obese (OW; body mass index [BMI] ≥30 kg/m2) and normal-weight women (NWW; BMI, 18.5-24.9 kg/m2). DESIGN: Clinical cross-sectional study. SETTING: Academic Medical Center. PATIENT(S): Healthy, normally-cycling, 18 to 40-year-old women (n = 6 OW and n = 6 NWW). MAIN OUTCOME MEASURE(S): Participants underwent screening and midfollicular phase visits. Demographic and anthropometric characteristics, blood samples, ultrasounds, and follicular phase endometrial biopsies were collected. Proteomic analyses of endometrial samples (liquid chromatography-mass spectrometry) were performed. Proteins with ≥2-fold difference and a false discovery rate of <0.1 were considered statistically significant (Benjamini-Hochberg adjustment). RESULT(S): Reproductive hormone levels did not differ between the two groups. Mean BMI, serum leptin concentration, and bioelectrical impedance analysis indices of adiposity were higher in OW than in NWW. Histological examination of the endometrial samples confirmed normal-appearing endometrium in both OW and NWW. A total of 2,930 proteins were detected across all samples, with an average number of proteins per sample of 2,059 ± 482 in NWW and 2,437 ± 187 in OW. A total of 17 proteins were differentially expressed in OW vs. NWW; 2 were more abundant, whereas 15 were underexpressed in OW, including the progesterone receptor. CONCLUSION(S): In this well-phenotyped population of healthy women, obesity was associated with significant endometrial proliferative phase proteomic differences affecting the hormonal and immunologic pathways. These could contribute to an increased risk of menstrual bleeding abnormalities and create an altered environment for future luteinization.

Transcriptome Analyses of Myometrium from Fibroid Patients Reveals Phenotypic Differences Compared to Non-Diseased Myometrium.

Uterine fibroid tissues are often compared to their matched myometrium in an effort to understand their pathophysiology, but it is not clear whether the myometria of uterine fibroid patients represent truly non-disease control tissues. We analyzed the transcriptomes of myometrial samples from non-fibroid patients (M) and compared them with fibroid (F) and matched myometrial (MF) samples to determine whether there is a phenotypic difference between fibroid and non-fibroid myometria. Multidimensional scaling plots revealed that M samples clustered separately from both MF and F samples. A total of 1169 differentially expressed genes (DEGs) (false discovery rate < 0.05) were observed in the MF comparison with M. Overrepresented Gene Ontology terms showed a high concordance of upregulated gene sets in MF compared to M, particularly extracellular matrix and structure organization. Gene set enrichment analyses showed that the leading-edge genes from the TGFß signaling and inflammatory response gene sets were significantly enriched in MF. Overall comparison of the three tissues by three-dimensional principal component analyses showed that M, MF, and F samples clustered separately from each other and that a total of 732 DEGs from F vs. M were not found in the F vs. MF, which are likely understudied in the pathogenesis of uterine fibroids and could be key genes for future investigation. These results suggest that the transcriptome of fibroid-associated myometrium is different from that of non-diseased myometrium and that fibroid studies should consider using both matched myometrium and non-diseased myometrium as controls.

ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation.

Endometriosis affects 1 in 10 women and is characterized by the presence of abnormal endometrium at ectopic sites. ARID1A mutations are observed in deeply invasive forms of the disease, often correlating with malignancy. To identify epigenetic dependencies driving invasion, we use an unbiased approach to map chromatin state transitions accompanying ARID1A loss in the endometrium. We show that super-enhancers marked by high H3K27 acetylation are strongly associated with ARID1A binding. ARID1A loss leads to H3K27 hyperacetylation and increased chromatin accessibility and enhancer RNA transcription at super-enhancers, but not typical enhancers, indicating that ARID1A normally prevents super-enhancer hyperactivation. ARID1A co-localizes with P300 at super-enhancers, and genetic or pharmacological inhibition of P300 in ARID1A mutant endometrial epithelia suppresses invasion and induces anoikis through the rescue of super-enhancer hyperacetylation. Among hyperactivated super-enhancers, SERPINE1 (PAI-1) is identified as an essential target gene driving ARID1A mutant endometrial invasion. Broadly, our findings provide rationale for therapeutic strategies targeting super-enhancers in ARID1A mutant endometrium.

In Vivo Cell Fate Tracing Provides No Evidence for Mesenchymal to Epithelial Transition in Adult Fallopian Tube and Uterus.

The mesenchymal to epithelial transition (MET) is thought to be involved in the maintenance, repair, and carcinogenesis of the fallopian tube (oviduct) and uterine epithelium. However, conclusive evidence for the conversion of mesenchymal cells to epithelial cells in these organs is lacking. Using embryonal cell lineage tracing with reporters driven by mesenchymal cell marker genes of the female reproductive tract (AMHR2, CSPG4, and PDGFRß), we show that these reporters are also expressed by some oviductal and uterine epithelial cells at birth. These mesenchymal reporter-positive epithelial cells are maintained in adult mice across multiple pregnancies, respond to ovarian hormones, and form organoids. However, no labeled epithelial cells are present in any oviductal or uterine epithelia when mesenchymal cell labeling was induced in adult mice. Organoids developed from mice labeled in adulthood were also negative for mesenchymal reporters. Collectively, our work found no definitive evidence of MET in the adult fallopian tube and uterine epithelium.

Putative human myometrial and fibroid stem-like cells have mesenchymal stem cell and endometrial stromal cell properties.

STUDY QUESTION: Can endometrial stromal stem/progenitor cell markers, SUSD2 and CD146/CD140b, enrich for human myometrial and fibroid stem/progenitor cells? SUMMARY ANSWER: SUSD2 enriches for myometrial and fibroid cells that have mesenchymal stem cell (MSC) characteristics and can also be induced to decidualise. WHAT IS KNOWN ALREADY: Mesenchymal stem-like cells have been separately characterised in the endometrial stroma and myometrium and may contribute to diseases in their respective tissues. STUDY DESIGN, SIZE, DURATION: Normal myometrium, fibroids and endometrium were collected from hysterectomies with informed consent. Primary cells or tissues were used from at least three patient samples for each experiment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Flow cytometry, immunohistochemistry and immunofluorescence were used to characterise tissues. In vitro colony formation in normoxic and hypoxic conditions, MSC lineage differentiation (osteogenic and adipogenic) and decidualisation were used to assess stem cell activity. Xenotransplantation into immunocompromised mice was used to determine in vivo stem-like activity. Endpoint measures included quantitative PCR, colony formation, trichrome, Oil Red O and alkaline phosphatase activity staining. MAIN RESULTS AND THE ROLE OF CHANCE: CD146+CD140b+ and/or SUSD2+ myometrial and fibroid cells were located in the perivascular region and formed more colonies in vitro compared to control cells and differentiated down adipogenic and osteogenic mesenchymal lineages in vitro. SUSD2+ myometrial cells had greater in vitro decidualisation potential, and SUSD2+ fibroid cells formed larger tumours in vivo compared to control cells. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Markers used in this study enrich for cells with stem/progenitor cell activity; however, they do not distinguish stem from progenitor cells. SUSD2+ myometrial cells express markers of decidualisation when treated in vitro, but in vivo assays are needed to fully demonstration their ability to decidualise. WIDER IMPLICATIONS OF THE FINDINGS: These results suggest a possible common MSC for the endometrial stroma and myometrium, which could be the tumour-initiating cell for uterine fibroids. STUDY FUNDING/COMPETING INTEREST(S): These studies were supported by NIH grants to JMT (R01OD012206) and to ALP (F32HD081856). The authors certify that we have no conflicts of interest to disclose.

ARID1A and PI3-kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion.

ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. Here we show that monoallelic loss of ARID1A in the mouse endometrial epithelium is sufficient for vaginal bleeding when combined with PI3K activation. Sorted mutant epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT). We further show that ARID1A is bound to promoters with open chromatin, but ARID1A loss leads to increased promoter chromatin accessibility and the expression of EMT genes. PI3K activation partially rescues the mesenchymal phenotypes driven by ARID1A loss through antagonism of ARID1A target gene expression, resulting in partial EMT and invasion. We propose that ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates, and that coexistent ARID1A and PI3K mutations promote epithelial transdifferentiation and collective invasion. Broadly, our findings support a role for collective epithelial invasion in the spread of abnormal endometrial tissue.

Label-Retaining, Putative Mesenchymal Stem Cells Contribute to Murine Myometrial Repair During Uterine Involution.

Uterine remodeling during pregnancy is a fundamental, dynamic process required for successful propagation of eutherian species. The uterus can increase in size up to 40-fold during pregnancy, which is largely attributed to expansion of the myometrium by hyperplasia and hypertrophy. After pregnancy, the uterus repairs the remodeled or "damaged" tissue during uterine involution (INV). Little is known about this repair process, particularly the role of mesenchymal stem/progenitor cells. The objective of this study was to identify and characterize putative mesenchymal stem/progenitor cells in the murine myometrium using a combination of label retention and mesenchymal stem cell (MSC) marker expression and a pregnancy and uterine INV model. Tet-off transgenic mice with the Cre-lox system were used to specifically label mesenchymal cells (ie, myometrial and endometrial stromal cells) within the uterus while avoiding other cell types (eg, epithelial, immune, and endothelial cells) to identify slowly dividing cells and assess their stem cell qualities. We identified myometrial label-retaining cells (LRCs) that persisted for at least 3 months, expressed CD146 and CD140b (MSC markers), and proliferated at a higher rate during uterine INV compared with nonlabeled cells. The LRCs did not appear to express either estrogen receptor alpha or progesterone receptor, nor did the number of LRCs change at different estrous stages or in response to exogenous estradiol or progesterone administration, suggesting that LRCs were not involved in normal estrous cycling. The results from this study provide important insight into putative stem/progenitor cells in the myometrium and their possible role in uterine physiology.

Etripamil Nasal Spray for Rapid Conversion of Supraventricular Tachycardia to Sinus Rhythm.

BACKGROUND: There is no nonparenteral medication for the rapid termination of paroxysmal supraventricular tachycardia. OBJECTIVES: The purpose of this study was to assess the efficacy and safety of etripamil nasal spray, a short-acting calcium-channel blocker, for the rapid termination of paroxysmal supraventricular tachycardia (SVT). METHODS: This phase 2 study was performed during electrophysiological testing in patients with previously documented SVT who were induced into SVT prior to undergoing a catheter ablation. Patients in sustained SVT for 5 min received either placebo or 1 of 4 doses of active compound. The primary endpoint was the SVT conversion rate within 15 min of study drug administration. Secondary endpoints included time to conversion and adverse events. RESULTS: One hundred four patients were dosed. Conversion rates from SVT to sinus rhythm were between 65% and 95% in the etripamil nasal spray groups and 35% in the placebo group; the differences were statistically significant (Pearson chi-square test) in the 3 highest active compound dose groups versus placebo. In patients who converted, the median time to conversion with etripamil was <3 min. Adverse events were mostly related to the intranasal route of administration or local irritation. Reductions in blood pressure occurred predominantly in the highest etripamil dose. CONCLUSIONS: Etripamil nasal spray rapidly terminated induced SVT with a high conversion rate. The safety and efficacy results of this study provide guidance for etripamil dose selection for future studies involving self-administration of this new intranasal calcium-channel blocker in a real-world setting for the termination of SVT. (Efficacy and Safety of Intranasal MSP-2017 [Etripamil] for the Conversion of PSVT to Sinus Rhythm [NODE-1]; NCT02296190).

Nuclear PTEN Localization Contributes to DNA Damage Response in Endometrial Adenocarcinoma and Could Have a Diagnostic Benefit for Therapeutic Management of the Disease.

Endometrial adenocarcinoma (EndoCA) is the most common gynecologic cancer type in the United States, and its incidence is increasing. The majority of patients are disease-free after surgical resection of stage I tumors, which is often followed by radiotherapy, but most patients with advanced disease recur and have a poor prognosis, largely because the tumors become refractory to cytotoxic chemotherapies. PTEN, a commonly mutated tumor suppressor in EndoCAs, is well known for its ability to inhibit the AKT/mTOR signaling pathway. Nuclear functions for PTEN have been proposed as well, but whether those affect EndoCA development, progression, or outcomes is not well understood. Using immunohistochemistry, nuclear PTEN expression was observed in approximately half of EndoCA patient tumors, independent of grade and cytoplasmic PTEN expression. Higher levels of the DNA damage response (DDR) marker, γH2AX, were observed by immunohistochemistry and immunofluorescence in human EndoCA tumor sections that were PTEN-negative, in murine EndoCA tissues that were genetically modified to be PTEN-null, and in Ishikawa EndoCA cells, which do not express endogenous PTEN. Overexpression of exogenous PTEN-WT or PTEN-NLS, a modified PTEN with an added nuclear localization signal, significantly improved both DDR and G2-M transition in Ishikawa cells treated with a DNA-damaging agent. Whereas PARP inhibition with Olaparib was not as effective in Ishikawa cells expressing native or PTEN-NLS, inhibition with Talazoparib was not affected by PTEN overexpression. These results suggest that nuclear PTEN subcellular localization in human EndoCA could be diagnostic when considering DDR therapeutic intervention. Mol Cancer Ther; 17(9); 1995-2003. ©2018 AACR.

Pathogenic Anti-Müllerian Hormone Variants in Polycystic Ovary Syndrome.

Context: Polycystic ovary syndrome (PCOS), a common endocrine condition, is the leading cause of anovulatory infertility. Objective: Given that common disease-susceptibility variants account for only a small percentage of the estimated PCOS heritability, we tested the hypothesis that rare variants contribute to this deficit in heritability. Design, Setting, and Participants: Unbiased whole-genome sequencing (WGS) of 80 patients with PCOS and 24 reproductively normal control subjects identified potentially deleterious variants in AMH, the gene encoding anti-Müllerian hormone (AMH). Targeted sequencing of AMH of 643 patients with PCOS and 153 control patients was used to replicate WGS findings. Main Outcome Measures: Dual luciferase reporter assays measured the impact of the variants on downstream AMH signaling. Results: We found 24 rare (minor allele frequency < 0.01) AMH variants in patients with PCOS and control subjects; 18 variants were specific to women with PCOS. Seventeen of 18 (94%) PCOS-specific variants had significantly reduced AMH signaling, whereas none of 6 variants observed in control subjects showed significant defects in signaling. Thus, we identified rare AMH coding variants that reduced AMH-mediated signaling in a subset of patients with PCOS. Conclusion: To our knowledge, this study is the first to identify rare genetic variants associated with a common PCOS phenotype. Our findings suggest decreased AMH signaling as a mechanism for the pathogenesis of PCOS. AMH decreases androgen biosynthesis by inhibiting CYP17 activity; a potential mechanism of action for AMH variants in PCOS, therefore, is to increase androgen biosynthesis due to decreased AMH-mediated inhibition of CYP17 activity.