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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.

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.

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.

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.

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.

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.

CD55 is a membrane complement regulatory protein that attenuates complement-mediated cytotoxicity. Saygin et al. elucidate a new role for CD55 as a signaling hub for cancer stem cell self-renewal and cisplatin resistance pathways in endometrioid tumors and open a new line of research into chemotherapeutic-refractory cancers., Effective targeting of cancer stem cells (CSCs) requires neutralization of self-renewal and chemoresistance, but these phenotypes are often regulated by distinct molecular mechanisms. Here we report the ability to target both of these phenotypes via CD55, an intrinsic cell surface complement inhibitor, which was identified in a comparative analysis between CSCs and non-CSCs in endometrioid cancer models. In this context, CD55 functions in a complement-independent manner and required lipid raft localization for CSC maintenance and cisplatin resistance. CD55 regulated self-renewal and core pluripotency genes via ROR2/JNK signaling and in parallel cisplatin resistance via lymphocyte-specific protein tyrosine kinase (LCK) signaling, which induced DNA repair genes. Targeting LCK signaling via saracatinib, an inhibitor currently undergoing clinical evaluation, sensitized chemoresistant cells to cisplatin. Collectively, our findings identify CD55 as a unique signaling node that drives self-renewal and therapeutic resistance through a bifurcating signaling axis and provides an opportunity to target both signaling pathways in endometrioid tumors.

The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL-induced activation of the transcription factor STAT5 results in the up-regulation of numerous genes implicated in breast cancer pathogenesis. However, the molecular mechanisms that enable STAT5 to access the promoters of these genes are not well understood. Here, we show that PRL signaling induces chromatin decompaction at promoter DNA, corresponding with STAT5 binding. The chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) specifically promotes STAT5 accessibility at promoter DNA by facilitating the dissociation of the linker histone H1 in response to PRL. Knockdown of H1 rescues the decrease in PRL-induced transcription following HMGN2 knockdown, and it does so by allowing increased STAT5 recruitment. Moreover, H1 and STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling.

BackgroundWomen, who carry a germline BRCA1 gene mutation, have a markedly increased risk of developing breast cancer during their lifetime. While BRCA1 carriers frequently develop triple-negative, basal-like, aggressive breast tumors, hormone signaling is important in the genesis of BRCA1 mutant breast cancers. We investigated the hormone response in BRCA1-mutated benign breast tissue using an in vitro organoid system.MethodsScaffold-free, multicellular human breast organoids generated from benign breast tissues from non-carrier or BRCA1 mutation carriers were treated in vitro with a stepwise menstrual cycle hormone regimen of estradiol (E2) and progesterone (P4) over the course of 28 days.ResultsBreast organoids exhibited characteristics of the native breast tissue, including expression of hormone receptors, collagen production, and markers of luminal and basal epithelium, and stromal fibroblasts. RNA sequencing analysis revealed distinct gene expression in response to hormone treatment in the non-carrier and BRCA1-mutated organoids. The selective progesterone receptor modulator, telapristone acetate (TPA), was used to identify specifically PR regulated genes. Specifically, extracellular matrix organization genes were regulated by E2+P4+TPA in the BRCA1-mutated organoids but not in the non-carrier organoids. In contrast, in the non-carrier organoids, known PR target genes such as the cell cycle genes were inhibited by TPA.ConclusionsThese data show that BRCA1 mutation influences hormone response and in particular PR activity which differs from that of non-carrier organoids. Our organoid model system revealed important insights into the role of PR in BRCA1-mutated benign breast cells and the critical paracrine actions that modify hormone receptor (HR)-negative cells. Further analysis of the molecular mechanism of BRCA1 and PR crosstalk is warranted using this model system.

Women, who carry a germline mutation in the breast cancer-associated gene 1 (BRCA1) have an increased risk (60-85%) of developing breast cancer during their lifetime. While BRCA1 mutated patients usually develop triple-negative, basal like, aggressive breast tumors, hormone signaling is important in the genesis of BRCA1 mutant breast cancers. Estrogen and progesterone promote carcinogenesis in the breast. Progesterone promotes proliferation of both benign epithelial cells and cancer cells and can expand the mammary stem cell pool. It is unclear how PR functions in the background of BRCA1 mutation in normal non-cancerous mammary epithelial cells before breast cancer develops. In order to determine whether BRCA1 mutation affects hormone response in benign mammary cells, human mammospheres from patients with wild type (WT; n=6) or BRCA1 mutation (n=6) were treated in vitro with a stepwise menstrual cycle hormone regimen of estrogen (E2) and progesterone (P4) over the course of 28 days. During the last 14 days of treatment, the SPRM, telapristal acetate (TPA) was added to the mammospheres. The mammospheres grown from benign breast tissues retained the expression of hormone receptors (ER and PR) even after the 28 day hormone treatment in vitro. Human mammary epithelial cells expressed aldehyde dehydrogenese-1 ALDH-1 with increasing levels observed in the BRCA1 mutated mammospheres compared to the WT mammospheres. RNA sequencing (RNA-seq) analysis revealed distinct differentially expressed genes between the WT and BRCA1 groups. Specifically, genes associated with extracellular matrix (ECM) organization were differentially regulated by TPA in the BRCA1 mutation group but not in the WT group. In fact, these ECM genes were differentially expressed in response to E2+P4 in WT versus BRCA1 mammospheres. In addition, cell cycle genes were responsive to TPA but in the WT group only. Taken together, these data show that BRCA1 mutation influences the hormone response and in particular to TPA compared to WT cells. Our physiologically relevant model systems have provided important insights into the role of progesterone in BRCA1 mutated breast cells. Further analysis of the molecular mechanism of BRCA1 and PR crosstalk is warranted using this model system.

BACKGROUND Synuclein-γ (SNCG) is highly expressed in advanced solid tumors, including uterine serous carcinoma (USC). The objective of the current study was to determine whether SNCG protein was associated with survival and clinical covariates using the largest existing collection of USCs from the Gynecologic Oncology Group (GOG-8023). METHODS High-density tissue microarrays (TMAs) of tumor tissues from 313 patients with USC were stained by immunohistochemistry for SNCG, p53, p16, FOLR1, pERK, pAKT, ER, PR, and HER2/neu. Associations of SNCG and other tumor markers with overall and progression-free survival were assessed using log-rank tests and Cox proportional-hazards models, which also were adjusted for age, race, and stage. RESULTS The overall survival at 5 years was 46% for women with high SNCG expression and 62% for those with low SNCG expression (log-rank P = .021; hazard ratio [HR], 1.31; 95% confidence interval [CI], 0.91-1.9 in adjusted Cox model). The progression-free survival rate at 5 years was worse for women who had high SNCG expression, at 40%, compared with 56% for those who had low SNCG expression (log-rank P = .0081; HR, 1.36; 95% CI, 0.96-1.92 in adjusted Cox model). High levels of both p53 and p16 were significantly associated with worse overall survival (p53: HR, 4.20 [95% CI, 1.54-11.45]; p16: HR, 1.95 [95% CI, 1.01-3.75]) and progression-free survival (p53: HR, 2.16 [95% CI, 1.09-4.27]; p16: HR, 1.53 [95% CI, 0.87-2.69]) compared with low levels. CONCLUSIONS This largest collection of USCs to date demonstrates that SNCG was associated with poor survival in univariate analyses. SNCG does not predict survival outcome independent of p53 and p16 in models that jointly consider multiple markers. Cancer 2017;123:1144-1155. © 2016 American Cancer Society.

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.

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.

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.

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.

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.

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.

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.

Context Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. Objective To define differential gene expression and signaling pathways in leiomyoma cell populations. Design Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Setting Research laboratory. Patients Eight African American women. Interventions None. Main Outcomes Measures Gene expression patterns, cell proliferation, and differentiation. Results A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. Conclusions IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas.

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.

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 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.

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

Progesterone is a key hormone in the endometrium that opposes estrogen-driven growth. Insufficient progesterone will result in unopposed estrogen action that could lead to the development of endometrial hyperplasia and adenocarcinoma. Although these endometrial neoplasias can regress in response to progestin treatment, this does not occur in all instances. To understand this resistance to progesterone and to improve on existing hormonal therapies, it is imperative that the molecular mechanisms of progesterone action through its receptor be deciphered in endometrial cancer. This review highlights what is known thus far regarding the efficacy of progestin therapy in the clinic and the role of progesterone in endometrial cancer cell behavior and gene regulation.

Context Progesterone has been associated with promoting growth of uterine leiomyomas. The mechanisms involved remain unclear. Objective In this study we investigated the activation of the AKT pathway and its downstream effectors, GSK3b and FOXO1 by progesterone as a mechanism of proliferation and survival of leiomyoma cells. Inhibitors of the AKT pathway were used to demonstrate the role of PI3K, AKT and FOXO1 in contributing to cell proliferation and apoptosis. Results Treatment of leiomyoma cells with R5020 over a period of 72h resulted in higher cell numbers compared to untreated cells. When cells were treated with 100nM R5020 for 1h and 24h, the levels of phospho(Ser 473)-AKT increased. This increase was inhibited when cells were co-treated with RU486. Treatment of leiomyoma cells with a PI3K inhibitor, LY294 dramatically decreased levels of phospho(Ser 473)-AKT, despite R5020 treatment. In addition to increased phospho(Ser 473)-AKT levels, R5020 treatment resulted in an increase in phospho(Ser 256)-FOXO1 and phospho-GSK3b. Inhibition of AKT using API-59 decreased proliferation and cell viability even in the presence of R5020. Higher concentrations of API-59 induced apoptosis of leiomyoma cells even in the presence of R5020. Psammaplysene A increased nuclear FOXO1 levels and did not affect cell proliferation but induced apoptosis of leiomyoma cells. Conclusions The progestin, R5020, can rapidly activate the AKT pathway. Inhibition of the AKT pathway inhibits cell proliferation and promotes apoptosis of leiomyoma cells.

Background: Several lines of evidence suggest that progesterone signaling is important in the breast cancer development, particularly in young women. Therefore, we sought to establish the effects of the antiprogestin CDB4124 (telapristone), and to identify PR related signature genes in hormone receptor positive (ER+ PR+) breast cancer cells. Methods: The PR expressing breast cancer cell line T47D was used to evaluate responses to PR ligands (P4, MPA and R5020, 10nM) alone or in combination with estradiol (E2, 1nM). The effects of the antiprogestin CDB4124 (0.1 or 1μM) were tested using varying hormonal conditions. The effect on (a) PRE promoter activity by dual luciferase assay; (b) cell proliferation using the MTT assay; (c) cell cycle by flow cytometry; (d) determination of gene expression signatures related to active PR responses. For the gene array experiment, cells were treated with either vehicle or R5020 (10nM) or R5020 (10nM) plus CDB4124 (1μM) in triplicate for 24hr. Total RNA was isolated and converted to cDNA and human Illumina chip microarray was performed. Data obtained from the microarray was further analyzed by Metacore Gene GO and Ingenuity Pathway Analysis. Real time PCR was performed in triplicate to confirm the expression of those genes related to the cell cycle and proliferation. ANOVA analysis and post-hoc Sidak test were used to determined the statistical significance of the data. Results: The PRE reporter activity resulting from P4, MPA and R5020 stimulation was inhibited by 80-90% in the presence of CDB4124 at 10 to 1000nM (p< 0.001). Cell proliferation was increased by PR ligands (P4, MPA and R5020) in the presence of E2; the addition of CDB4124 caused 50% inhibition of proliferation (p< 0.01) at 72 hours. Cell cycle analysis of T47D cells treated with P4, MPA and R5020 alone or in combination with E2 showed significant increases in S and G2/M phase and decreases in G0/G1. These were blocked by CDB4124 at 0.1 or 1.0μM (p1.5 fold upregulation (p Conclusion: These data demonstrate that PR mediated cell proliferation occurs upon treatment with three different ligands of PR (P4, MPA and R5020); that PR actively engages key genes involved in the G2/M phase of the cell cycle to drive proliferation of ER+ and PR+ cells; and that the antiprogestin, CBD4124 is a potent transcriptional inhibitor for blockade of PR mediated cell proliferation in hormone receptor positive breast cancer cells. Citation Format: Akash Gupa, Mi Ran Choi, Susan E Clare, Jun Wang, Oukseub Lee, David Z Ivancic, J Julie Kim, Seema A Khan. Progesterone receptor (PR) blockade by antiprogestin, CDB4124 in hormone receptor positive breast cancer cells leads to significant inhibition of G2/M cell cycle genes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-10.

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.

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) 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.

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.

Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/β-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of β-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of β-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/β-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.

Uterine leiomyomas (ULs), benign tumors of the myometrium, are the number one indication for hysterectomies in the United States due to a lack of an effective alternative therapy. ULs show activation of the pro-survival AKT pathway compared with normal myometrium; however, substantial data directly linking AKT to UL cell survival are lacking. We hypothesized that AKT promotes UL cell survival and that it is a viable target for inhibiting UL growth. We used the investigational AKT inhibitor MK-2206, currently in phase II trials, on cultured primary human UL and myometrial cells, immortalized leiomyoma cells, and in leiomyoma grafts grown under the kidney capsule in mice. MK-2206 inhibited AKT and PRAS40 phosphorylation but did not regulate serum- and glucocorticoid-induced kinase and ERK1/2, demonstrating its specificity for AKT. MK-2206 reduced UL cell viability and decreased UL tumor volumes. UL cells exhibited disruption of mitochondrial structures and underwent cell death that was independent of caspases. Additionally, mammalian target of rapamycin and p70S6K phosphorylation were reduced, indicating that mammalian target of rapamycin complex 1 signaling was compromised by AKT inhibition in UL cells. MK-2206 also induced autophagy in UL cells. Pretreatment of primary UL cells with 3-methyladenine enhanced MK-2206-mediated UL cell death, whereas knockdown of ATG5 and/or ATG7 did not significantly influence UL cell viability in the presence of MK-2206. Our data provide molecular evidence for the involvement of AKT in UL cell survival and suggest that AKT inhibition by MK-2206 may be a viable option to consider for the treatment of ULs.

Endometrial cancer is the fourth most common malignancy among women and is a major cause of morbidity contributing to approximately 8,200 annual deaths in the USA. Despite advances to the understanding of endometrial cancer, novel interventions for the disease are necessary given that many tumors become refractory to therapy. As a strategy to identify novel therapies for endometrial carcinoma, in this study, we examined the contribution of the peroxisome proliferator-activated receptor β/δ (PPARβ/δ) to endometrial cancer cell proliferation and apoptosis. We found that when activated with the highly selective PPARβ/δ agonists, GW0742 and GW501516, PPARβ/δ inhibited the proliferation and markedly induced the apoptosis of three endometrial cancer cell lines. The specificity of the PPARβ/δ-induced effects on cell proliferation and apoptosis was demonstrated using PPARβ/δ-selective antagonists and PPARβ/δ small interfering RNA in combination with PPARβ/δ-selective agonists. Furthermore, we showed that PPARβ/δ activation increased phosphatase and tensin homolog expression, which led to protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) dephosphorylation, and increased β-catenin phosphorylation associated with its degradation. Overall, our data suggest that the antitumorigenic effect of PPARβ/δ activation in endometrial cancer is mediated through the negative regulation of the AKT/GSK3β/β-catenin pathway. These findings warrant further investigation of PPARβ/δ as a therapeutic target in endometrial cancer.

Objective: Epithelioid trophoblastic tumor (ETT) is a rare variant of gestational trophoblastic neoplasia (GTN) that develops from chorionic-type intermediate trophoblasts, simulates carcinoma, often presents many years following a pregnancy event, is associated with low or normal human chorionic gonadotropin (hCG) levels, and is relatively resistant to chemotherapy. Our aim was to identify the role of surgery in the management of both localized and metastatic ETT. Methods: A retrospective review of women with ETT treated at a gestational trophoblastic disease center from 2010-2015 identified five women. Medical records were abstracted for demographic data, clinical presentation, hCG levels, prior pregnancy information, disease sites, and treatment with chemotherapy and surgery. Results: The five women with ETT ranged in age from 28 to 48 years. Clinical presentation was abnormal uterine bleeding prompting dilation and curettage, which established the diagnosis of ETT in three women. One woman presented with repeated spontaneous pneumothorax requiring thoracoscopic resection, which identified a 6 cm ETT in the lung. Another woman was initially treated with methotrexate for a presumed ectopic pregnancy without a response. Two women had no identifiable antecedent pregnancy, two women had spontaneous abortions 19 and 72 months prior to the diagnosis of ETT, and one woman had a normal term delivery 48 months earlier. Four of five women had metastatic disease to the lungs. Three of these women underwent pulmonary wedge resection as well as hysterectomy followed by paclitaxel-cisplatin/paclitaxel-etoposide (TP/TE) chemotherapy, while the other woman was treated with etoposide, methotrexate, actinomycin D/etoposide, cisplatin (EMA/EP) chemotherapy and hysterectomy. The one woman with nonmetastatic disease was treated with hysterectomy only. All five women are currently without evidence of disease. Conclusions: Surgery, including hysterectomy and resection of metastatic disease, is an important component in the treatment of women with ETT. Adjuvant chemotherapy with a platinum-containing regimen, such as TP/TE or EMA/EP, should be used in women with metastatic disease. All five women with ETT in this series were cured using this approach, including the four with metastatic disease. Citation Format: Abigail Winder, Janelle Sobecki-Rausch, Kruti Maniar, Karen Novak, Julie Kim, John Lurain. The role of surgery in the treatment of epithelioid trophoblastic tumor: A single institution case series. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2592.

Background Progesterone, via its nuclear receptor (PR), exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma) and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. Principal Findings ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs), whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2), a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. Conclusions Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in uterine fibroids and breast cancer.

Context: Endometriosis affects approximately 10% of women in the United States and causes pain and infertility. Decidualization of endometrial stromal cells from women with endometriosis is aberrant. Objective: The objective of this study was to investigate a potential mechanism for the inadequate decidual response in stromal cells from ovarian endometriomas. Design: Stromal cells of the endometrium from women without endometriosis (HSC) or from ovarian endometriomas (OsisSC) were grown in culture and treated with 10 μm LY294002 or 250 nm MK2206, 100 nm medroxyprogesterone acetate (M), and 0.5 mm dibutyryl cAMP (A) or infection with 100 multiplicity of infection adenoviral constructs containing wild-type Forkhead box O1 or triple-mutant FOXO1. Real-time PCR was used to measure the expression of FOXO1, IGF binding protein-1 (IGFBP1), and prolactin (PRL) mRNA, and Western blot and immunohistochemical staining were used to detect the levels of progesterone receptor (PR), FOXO1, AKT, and p(Ser473)-AKT protein in vitro or in vivo. Results: Expression of the decidua-specific genes, IGFBP1 and PRL, were significantly lower in OsisSC compared with normal HSC in response to M+A treatment. Basal expression levels of PRA, PRB, and FOXO1 proteins were dramatically lower in OsisSC. Overexpression of triple-mutant FOXO1 increased mRNA levels of IGFBP1 and PRL in OsisSC in the presence of M+A, whereas the overexpression of wild-type FOXO1 had no effect. AKT was highly phosphorylated in OsisSC compared with HSC and inhibition of phosphatidylinositol 3-kinase, with LY294002, increased levels of FOXO1 protein as well as IGFBP1 mRNA in the presence of M+A. Moreover, inhibition of AKT with MK2206, an allosteric AKT inhibitor, dramatically increased the accumulation of nuclear FOXO1 as well as expression of IGFBP1. Finally, immunohistochemical staining demonstrated higher p(Ser473)-AKT and lower FOXO1 levels in endometriosis tissues, compared with normal endometrial tissues. Conclusions: In endometriotic stromal cells, overactivation of the phosphatidylinositol 3-kinase/AKT signaling pathway contributes to the reduced expression of the decidua-specific gene, IGFBP1, potentially through reduced levels of nuclear FOXO1.

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)

Endometrial cancer is the most common type of gynecologic cancer in the United States. In this study, we propose that a marine sponge compound, psammaplysene A (PsA) induces apoptosis in endometrial cancer cells through forced nuclear expression of FOXO1.Ishikawa and ECC1 cells were treated with varying doses of PsA. FOXO1 protein localization was observed using immunofluorescent staining of cells. The effects of PsA on cell viability and proliferation were assessed using a cell viability assay and a BrdU incorporation assay respectively. Cell cycle analysis was performed using flow cytometry. To assess the role of FOXO1 in PsA-induced apoptosis, FOXO1 was silenced in ECC1 cells using siRNA technique, and overexpressed in Ishikawa cells using an adenovirus containing FOXO1 cDNAs. Western blots were used to measure levels of FOXO1 and cleaved PARP proteins.Treatment of both ECC1 and Ishikawa cells with PsA caused an increase in nuclear FOXO1 protein, a dramatic decrease in cell viability of approximately 5-fold (p0.05) and minimal effect on proliferation. Furthermore, treatment of cells with PsA doubled the percentage of cells in the G2/M phase (p0.05). PsA induced apoptosis in endometrial cancer cells. When FOXO1 was silenced in ECC1 cells and treated with PsA, the incidence of apoptosis decreased. In addition, overexpression of FOXO1 with PsA treatment increased apoptosis.Increasing nuclear FOXO1 function is important for the induction of apoptosis of endometrial cancer cells by PsA.

Progesterone is indispensable for differentiation of human endometrial stromal cells (HESCs) into decidual cells, a process that critically controls embryo implantation. We now show an important role for androgen receptor (AR) signaling in this differentiation process. Decreased posttranslational modification of the AR by small ubiquitin-like modifier (SUMO)-1 in decidualizing cells accounted for increased responsiveness to androgen. By combining small interfering RNA technology with genome-wide expression profiling, we found that AR and progesterone receptor (PR) regulate the expression of distinct decidual gene networks. Ingenuity pathway analysis implicated a preponderance of AR-induced genes in cytoskeletal organization and cell motility, whereas analysis of AR-repressed genes suggested involvement in cell cycle regulation. Functionally, AR depletion prevented differentiation-dependent stress fiber formation and promoted motility and proliferation of decidualizing cells. In comparison, PR depletion perturbed the expression of many more genes, underscoring the importance of this nuclear receptor in diverse cellular functions. However, several PR-dependent genes encode for signaling intermediates, and knockdown of PR, but not AR, compromised activation of WNT/β-catenin, TGFβ/SMAD, and signal transducer and activator of transcription (STAT) pathways in decidualizing cells. Thus, the nonredundant function of the AR in decidualizing HESCs, centered on cytoskeletal organization and cell cycle regulation, implies an important role for androgens in modulating fetal-maternal interactions. Moreover, we show that PR regulates HESC differentiation, at least in part, by reprogramming growth factor and cytokine signal transduction.