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

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.

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

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

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.

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.

Uterine leiomyomas (UL), benign tumors of the myometrium, are the number one indication for hysterectomies in the U.S. due to a lack of effective therapy. ULs show increased phosphorylation of the pro-survival protein AKT compared to normal myometrium indicating that AKT signaling is increased in UL. However, substantial data directly linking AKT to UL cell survival is lacking. We hypothesized that AKT promotes UL cell survival and that it is a viable drug target for inhibiting UL growth. We utilized the AKT inhibitor MK-2206 (MK), currently in phase II trials, on cultured primary human UL cells. MK-2206 effectively reduced phosphorylation of AKT and PRAS40, an AKT substrate. MK-2206 did not affect cell proliferation but caused cell death. We observed a lack of apoptosis in dead UL cells by Annexin V staining. Additionally, MK-2206 was able to induce UL cell death in the presence of pan-caspase inhibitor, Z-VAD-FMK, indicating that MK-2206 mediated cell death is caspase independent and non-apoptotic. Given that caspases did not modulate cell death, we explored necrosis. MK-2206 induced HMGB1 cytoplasmic localization and mitochondrial disruption indicating possible leiomyoma necrosis. We next explored programmed necrosis, a form of non-apoptotic cell death mediated by RIP kinases (RIPK). However, MK-2206 was able to induce cell death in the presence of RIPK inhibitors, necrostatin-1 and necrosulfonamide, indicating that programmed necrosis is not the mechanism of MK-2206 mediated UL cell death. We are actively exploring alternate necrotic cell death pathways as the mechanisms of MK-2206 induced UL cell death. Lastly, in-vivo MK-2206 treatments reduced tumor volume of human UL cells implanted under the kidney capsule of NSG mice. Xenograft sections from mice treated with MK-2206-2206 contained dying cells in the absence of cleaved caspase 3 supporting the in-vitro results. This is the first report of non-apoptotic cell death in UL and the first time that AKT has been targeted in-vivo for UL therapy. Our data provides molecular evidence for the involvement of AKT in UL cell survival and suggest that AKT inhibition by MK-2206 may be a viable option for the future treatment of UL. This work was funded by NIH P01HD057877. Citation Format: Elizabeth Sefton, Wenan Qiang, Vanida Serna, Takeshi Kurita, Julie Kim. AKT inhibitor MK-2206 induces necrotic cell death of uterine leiomyoma cells in vitro and reduces tumor volume in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1047. doi:10.1158/1538-7445.AM2013-1047

Mutations in the PTEN gene are the most common genetic defects in endometrioid endometrial carcinoma and seen in more than 50% of tumors. PTEN, a tumor suppressor gene, negatively regulates PI3K/Akt-driven cell growth and survival. Previously, we have shown that levels of the forkhead protein, FOX01, a direct target of AKT, were significantly lower in endometrial carcinoma and overexpression of this gene in endometrial cancer cell lines resulted in decreased cell proliferation and increased apoptosis. Interestingly, the function of FOX01 in this context was determined by the progesterone receptor isoform that was expressed, strongly implicating the regulatory role of the progesterone receptor predictably at the transcription level. In this study, we aimed to look at the effects of an AKT inhibitor (APi59) on endometrial cancer cells and the influence of PRA or PRB on its efficacy. The cell viability assay revealed a decrease in the number of viable PRA and PRB cells treated with APi59 for 48 hours, while the progestin, R5020, alone did not affect cell viability. In addition, more cell death was observed in the PRA cells compared to the PRB cells. APi59 treatment promoted apoptosis, as measured by cleaved PARP levels, in both PRA and PRB cells, with increased apoptosis in PRA cells compared to the PRB cells. R5020 treatment alone did not cause apoptosis as compared to control. In order to further investigate the increased levels of apoptosis in the PRA cells, a real time PCR array focused on genes associated with apoptosis was used to identify potential genes involved in this differential response. The most highly upregulated gene specifically expressed in PRB cells in response to R5020 was BIRC3 (ciap2). BIRC3 was upregulated 13-fold with R5020 treatment in PRB cells where its expression in PRA cells was unaffected. Given that BIRC3 is a member of the inhibitor of apoptosis genes (IAP), we hypothesized that the increased expression of BIRC3 in PRB cells attenuated APi59-mediated apoptosis. Thus, PR was silenced using transient transfection of siRNA specific to PR and cells were treated with APi59 and R5020. Upon PR silencing, there was an increase in apoptosis as measured by cleaved PARP in APi59 treated cells along with a concomitant decrease in BIRC3 protein. To determine if the attenuation in apoptosis through BIRC3 occurred with other cytotoxic agents, PRB cells were treated with 10nm paclitaxel and R5020. Again, apoptosis in response to paclitaxel was attenuated, as shown by lower cleaved PARP levels when R5020 was added. In conclusion, not only can the AKT inhibitor, APi59, promote apoptosis in endometrial cancer cells, but liganded PRB can blunt the apoptotic response through increased expression of BIRC3, an inhibitor of apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1196.

Objectives: SNCG is a novel prognostic biomarker in UPSC. We have shown that its overexpression correlates with aggressive cellular properties and resistance to paclitaxel in a UPSC cell line (SPEC2) as well as poor prognosis in a pilot set of UPSC patients. The goal of this study was to determine the mechanism of SNCG-associated taxane resistance by evaluating the effect of SNCG on the mitotic checkpoint and its propensity to modulate extracellular signal-regulated protein kinase 1/2 (ERK 1/2) signaling pathways. Methods: A stably transfected SPEC2 cell line was created using shSNCG versus shControl oligonucleotides. Similarly, Ishikawa cells were transiently transfected with an SNCG expression vector versus empty vector. Differences in cell proliferation were measured using a cell viability assay and BrdU incorporation assay after treatment with paclitaxel. Cell cycle analysis was used to study the effect of SNCG on the mitotic checkpoint. Differences in expression of the checkpoint kinase BubR1 due to SNCG were studied using real time PCR, Western blot, 26S ubiquitin-proteasome inhibition, and siRNA to BubR1. ERK and phospho-ERK levels were measured in SPEC2 cells with and without SNCG expression by Western blot. SPEC2 cells were treated with paclitaxel with and without the MEK inhibitor UO126 and apoptosis was measured using cleaved PARP analysis. Results: SNCG overexpression lead to acquired, reversible paclitaxel resistance in endometrial cancer cell lines. SNCG was noted to interfere with mitotic checkpoint function by causing degradation of the critical checkpoint protein BubR1 thereby allowing cells to escape paclitaxel-induced mitotic arrest. SPEC2 cells exhibited sustained activation of ERK which was attenuated by silencing SNCG. When SPEC2 cells were exposed to paclitaxel combined with the MEK inhibitor UO126, decreased cell viability and increased apoptosis was noted compared to either agent alone. SNCG knockdown further enhanced paclitaxel and UO126 sensitivity. Cell cycle analysis revealed that silencing SNCG in SPEC2 cells and adding UO126 acted synergistically in overcoming paclitaxel resistance. Conclusions: SNCG overexpression in UPSC leads to taxane resistance by interacting with the mitotic checkpoint kinase BubR1 causing its degradation. SNCG is also associated with sustained ERK activation. Targeting SNCG in conjunction with MEK inhibition may represent a novel therapeutic strategy to overcome taxane resistance in these cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3548.