Global expression analysis of endometrial cancer cells in response to progesterone identifies new therapeutic targets.

Progesterone prevents development of endometrial cancers through its receptor (PR) although the molecular mechanisms have yet to be fully characterized. In this study, we performed a global analysis of gene regulation by progesterone using human endometrial cancer cells that expressed PR endogenously or exogenously. We found progesterone strongly inhibits multiple components of the platelet derived growth factor receptor (PDGFR), Janus kinase (JAK), signal transducer and activator of transcription (STAT) pathway through PR. The PDGFR/JAK/STAT pathway signals to control numerous downstream targets including AP-1 transcription factors Fos and Jun. Treatment with inhibitors of the PDGFR/JAK/STAT pathway significantly blocked proliferation in multiple novel patient-derived organoid models of endometrial cancer, and activation of this pathway was found to be a poor prognostic signal for the survival of patients with endometrial cancer from The Cancer Genome Atlas. Our study identifies this pathway as central to the growth-limiting effects of progesterone in endometrial cancer and suggests that inhibitors of PDGFR/JAK/STAT should be considered for future therapeutic interventions.

Microbial Communities in Gynecological Cancers and Their Association with Tumor Somatic Variation.

There are strong correlations between the microbiome and human disease, including cancer. However, very little is known about potential mechanisms associated with malignant transformation in microbiome-associated gynecological cancer, except for HPV-induced cervical cancer. Our hypothesis is that differences in bacterial communities in upper genital tract epithelium may lead to selection of specific genomic variation at the cellular level of these tissues that may predispose to their malignant transformation. We first assessed differences in the taxonomic composition of microbial communities and genomic variation between gynecologic cancers and normal samples. Then, we performed a correlation analysis to assess whether differences in microbial communities selected for specific single nucleotide variation (SNV) between normal and gynecological cancers. We validated these results in independent datasets. This is a retrospective nested case-control study that used clinical and genomic information to perform all analyses. Our present study confirms a changing landscape in microbial communities as we progress into the upper genital tract, with more diversity in lower levels of the tract. Some of the different genomic variations between cancer and controls strongly correlated with the changing microbial communities. Pathway analyses including these correlated genes may help understand the basis for how changing bacterial landscapes may lead to these cancers. However, one of the most important implications of our findings is the possibility of cancer prevention in women at risk by detecting altered bacterial communities in the upper genital tract epithelium.

Particles and Prejudice: Nanomedicine Approaches to Reducing Health Disparities in Endometrial Cancer.

Endometrial cancer is the most common gynecological malignancy worldwide and unfortunately has a much higher mortality rate in Black women compared with White women. Many potential factors contribute to these mortality rates, including the underlying effects of systemic and interpersonal racism. Furthermore, other trends in medicine have potential links to these rates including participation in clinical trials, hormone therapy, and pre-existing health conditions. Addressing the high incidence and disparate mortality rates in endometrial cancer requires novel methods, such as nanoparticle-based therapeutics. These therapeutics have been growing in increasing prevalence in pre-clinical development and have far-reaching implications in cancer therapy. The rigor of pre-clinical studies is enhanced by the likeness of the model to the human body. In systems for 3D cell culture, for example, the extracellular matrix mimics the tumor more closely. The increasing emphasis on precision medicine can be applied to cancer using nanoparticle-based methods and applied to pre-clinical models by using patient-derived model data. This review highlights the intersections of nanomedicine, precision medicine, and racial disparities within endometrial cancer and provides insights into reducing health disparities using recent scientific advances on the nanoscale.

FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways.

BACKGROUND: Mutations in the receptor tyrosine kinase gene fibroblast growth factor receptor 2 (FGFR2) occur at a high frequency in endometrial cancer (EC) and have been linked to advanced and recurrent disease. However, little is known about how these mutations drive carcinogenesis. METHODS: Differential transcriptomic analysis and two-step quantitative real-time PCR (qRT-PCR) assays were applied to identify genes differentially expressed in two cohorts of EC patients carrying mutations in the FGFR2 gene as well as in EC cells harbouring mutations in the FGFR2. Candidate genes and target signalling pathways were investigated by qRT-PCR assays, immunohistochemistry and bioinformatics analysis. The functional roles of differently regulated genes were analysed using in vitro and in vivo experiments, including 3D-orthotypic co-culture systems, cell proliferation and migration protocols, as well as colony and focus formation assays together with murine xenograft tumour models. The molecular mechanisms were examined using CRISPR/Cas9-based loss-of-function and pharmacological approaches as well as luciferase reporter techniques, cell-based ectodomain shedding assays and bioinformatics analysis. RESULTS: We show that common FGFR2 mutations significantly enhance the sensitivity to FGF7-mediated activation of a disintegrin and metalloprotease (ADAM)17 and subsequent transactivation of the epidermal growth factor receptor (EGFR). We further show that FGFR2 mutants trigger the activation of ADAM10-mediated Notch signalling in an ADAM17-dependent manner, highlighting for the first time an intimate cooperation between EGFR and Notch pathways in EC. Differential transcriptomic analysis in EC cells in a cohort of patients carrying mutations in the FGFR2 gene identified a strong association between FGFR2 mutations and increased expression of members of the Notch pathway and ErbB receptor family. Notably, FGFR2 mutants are not constitutively active but require FGF7 stimulation to reprogram Notch and EGFR pathway components, resulting in ADAM17-dependent oncogenic growth. CONCLUSIONS: These findings highlight a pivotal role of ADAM17 in the pathogenesis of EC and provide a compelling rationale for targeting ADAM17 protease activity in FGFR2-driven cancers.

Using Genomic Variation to Distinguish Ovarian High-Grade Serous Carcinoma from Benign Fallopian Tubes.

The preoperative diagnosis of pelvic masses has been elusive to date. Methods for characterization such as CA-125 have had limited specificity. We hypothesize that genomic variation can be used to create prediction models which accurately distinguish high grade serous ovarian cancer (HGSC) from benign tissue. METHODS: In this retrospective, pilot study, we extracted DNA and RNA from HGSC specimens and from benign fallopian tubes. Then, we performed whole exome sequencing and RNA sequencing, and identified single nucleotide variants (SNV), copy number variants (CNV) and structural variants (SV). We used these variants to create prediction models to distinguish cancer from benign tissue. The models were then validated in independent datasets and with a machine learning platform. RESULTS: The prediction model with SNV had an AUC of 1.00 (95% CI 1.00-1.00). The models with CNV and SV had AUC of 0.87 and 0.73, respectively. Validated models also had excellent performances. CONCLUSIONS: Genomic variation of HGSC can be used to create prediction models which accurately discriminate cancer from benign tissue. Further refining of these models (early-stage samples, other tumor types) has the potential to lead to detection of ovarian cancer in blood with cell free DNA, even in early stage.

Integrated Clinical and Genomic Models to Predict Optimal Cytoreduction in High-Grade Serous Ovarian Cancer.

Advanced high-grade serous (HGSC) ovarian cancer is treated with either primary surgery followed by chemotherapy or neoadjuvant chemotherapy followed by interval surgery. The decision to proceed with surgery primarily or after chemotherapy is based on a surgeon's clinical assessment and prediction of an optimal outcome. Optimal and complete cytoreductive surgery are correlated with improved overall survival. This clinical assessment results in an optimal surgery approximately 70% of the time. We hypothesize that this prediction can be improved by using biological tumor data to predict optimal cytoreduction. With access to a large biobank of ovarian cancer tumors, we obtained genomic data on 83 patients encompassing gene expression, exon expression, long non-coding RNA, micro RNA, single nucleotide variants, copy number variation, DNA methylation, and fusion transcripts. We then used statistical learning methods (lasso regression) to integrate these data with pre-operative clinical information to create predictive models to discriminate which patient would have an optimal or complete cytoreductive outcome. These models were then validated within The Cancer Genome Atlas (TCGA) HGSC database and using machine learning methods (TensorFlow). Of the 124 models created and validated for optimal cytoreduction, 21 performed at least equal to, if not better than, our historical clinical rate of optimal debulking in advanced-stage HGSC as a control. Of the 89 models created to predict complete cytoreduction, 37 have the potential to outperform clinical decision-making. Prospective validation of these models could result in improving our ability to objectively predict which patients will undergo optimal cytoreduction and, therefore, improve our ovarian cancer outcomes.

TP53 Sequencing and p53 Immunohistochemistry Predict Outcomes When Bevacizumab Is Added to Frontline Chemotherapy in Endometrial Cancer: An NRG Oncology/Gynecologic Oncology Group Study.

PURPOSE: The status of p53 in a tumor can be inferred by next-generation sequencing (NGS) or by immunohistochemistry (IHC). We examined the association between p53 IHC and sequence and whether p53 IHC alone, or integrated with TP53 NGS, predicts the outcome. METHODS: From GOG-86P, a randomized phase II study of chemotherapy combined with either bevacizumab or temsirolimus in advanced endometrial cancer, 213 cases had p53 protein expression data measured by IHC and TP53 NGS data. An analysis was designed to integrate p53 expression by IHC with the presence or absence of a TP53 mutation. These variables were further correlated with progression-free survival (PFS) and overall survival (OS) in the chemotherapy plus bevacizumab arms versus the chemotherapy plus temsirolimus arm. RESULTS: In the analysis of p53 IHC, the most striking treatment effect favoring bevacizumab was in cases where p53 was overexpressed (PFS hazard ratio [HR]: 0.46, 95% CI, 0.26 to 0.88; OS HR: 0.31, 95% CI, 0.16 to 0.62). On integrated analysis, patients with TP53 missense mutations and p53 protein overexpression had a similar treatment effect on PFS (HR: 0.41, 95% CI, 0.22 to 0.83) and OS (HR: 0.28, 95% CI, 0.14 to 0.59) favoring bevacizumab plus chemotherapy relative to temsirolimus plus chemotherapy. Concordance between TP53 NGS and p53 IHC was 88%. Concordance was 92% when cases with TP53 mutations and POLE mutations or mismatch repair deficiency were removed. CONCLUSION: IHC for p53 alone or when integrated with sequencing for TP53 identifies a specific, high-risk tumor genotype/phenotype for which bevacizumab is particularly beneficial in improving outcomes when combined with chemotherapy.

Blocking autophagy overcomes resistance to dual histone deacetylase and proteasome inhibition in gynecologic cancer.

Histone deacetylase (HDAC) inhibitors and proteasome inhibitors have been approved by the FDA for the treatment of multiple myeloma and lymphoma, respectively, but have not achieved similar activity as single agents in solid tumors. Preclinical studies have demonstrated the activity of the combination of an HDAC inhibitor and a proteasome inhibitor in a variety of tumor models. However, the mechanisms underlying sensitivity and resistance to this combination are not well-understood. This study explores the role of autophagy in adaptive resistance to dual HDAC and proteasome inhibition. Studies focus on ovarian and endometrial gynecologic cancers, two diseases with high mortality and a need for novel treatment approaches. We found that nanomolar concentrations of the proteasome inhibitor ixazomib and HDAC inhibitor romidepsin synergistically induce cell death in the majority of gynecologic cancer cells and patient-derived organoid (PDO) models created using endometrial and ovarian patient tumor tissue. However, some models were not sensitive to this combination, and mechanistic studies implicated autophagy as the main mediator of cell survival in the context of dual HDAC and proteasome inhibition. Whereas the combination of ixazomib and romidepsin reduces autophagy in sensitive gynecologic cancer models, autophagy is induced following drug treatment of resistant cells. Pharmacologic or genetic inhibition of autophagy in resistant cells reverses drug resistance as evidenced by an enhanced anti-tumor response both in vitro and in vivo. Taken together, our findings demonstrate a role for autophagic-mediated cell survival in proteasome inhibitor and HDAC inhibitor-resistant gynecologic cancer cells. These data reveal a new approach to overcome drug resistance by inhibiting the autophagy pathway.

The Synthetic Curcumin Analog HO-3867 Rescues Suppression of PLAC1 Expression in Ovarian Cancer Cells.

Elevated expression of placenta-specific protein 1 (PLAC1) is associated with the increased proliferation and invasiveness of a variety of human cancers, including ovarian cancer. Recent studies have shown that the tumor suppressor p53 directly suppresses PLAC1 transcription. However, mutations in p53 lead to the loss of PLAC1 transcriptional suppression. Small molecules that structurally convert mutant p53 proteins to wild-type conformations are emerging. Our objective was to determine whether the restoration of the wild-type function of mutated p53 could rescue PLAC1 transcriptional suppression in tumors harboring certain TP53 mutations. Ovarian cancer cells OVCAR3 and ES-2, both harboring TP53 missense mutations, were treated with the p53 reactivator HO-3867. Treatment with HO-3867 successfully rescued PLAC1 transcriptional suppression. In addition, cell proliferation was inhibited and cell death through apoptosis was increased in both cell lines. We conclude that the use of HO-3867 as an adjuvant to conventional therapeutics in ovarian cancers harboring TP53 missense mutations could improve patient outcomes. Validation of this conclusion must, however, come from an appropriately designed clinical trial.

Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy.

Angiogenesis plays a crucial role in tumor development and metastasis. Both bevacizumab and cediranib have demonstrated activity as single anti-angiogenic agents in endometrial cancer, though subsequent studies of bevacizumab combined with chemotherapy failed to improve outcomes compared to chemotherapy alone. Our objective was to compare the efficacy of cediranib and bevacizumab in endometrial cancer models. The cellular effects of bevacizumab and cediranib were examined in endometrial cancer cell lines using extracellular signal-related kinase (ERK) phosphorylation, ligand shedding, cell viability, and cell cycle progression as readouts. Cellular viability was also tested in eight patient-derived organoid models of endometrial cancer. Finally, we performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab versus cediranib. Cediranib but not bevacizumab blocked ligand-mediated ERK activation in endometrial cancer cells. In both cell lines and patient-derived organoids, neither bevacizumab nor cediranib alone had a notable effect on cell viability. Cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib + chemotherapy, consistent with the abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Phosphoproteomic analysis revealed that bevacizumab and cediranib had both similar and unique effects on cell signaling that underlie their shared versus individual actions as anti-angiogenic agents. An anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy.

Successful Patient-Derived Organoid Culture of Gynecologic Cancers for Disease Modeling and Drug Sensitivity Testing.

Developing reliable experimental models that can predict clinical response before treating the patient is a high priority in gynecologic cancer research, especially in advanced or recurrent endometrial and ovarian cancers. Patient-derived organoids (PDOs) represent such an opportunity. Herein, we describe our successful creation of 43 tumor organoid cultures and nine adjacent normal tissue organoid cultures derived from patients with endometrial or ovarian cancer. From an initial set of 45 tumor tissues and seven ascites fluid samples harvested at surgery, 83% grew as organoids. Drug sensitivity testing and organoid cell viability assays were performed in 19 PDOs, a process that was accomplished within seven days of obtaining the initial surgical tumor sample. Sufficient numbers of cells were obtained to facilitate testing of the most commonly used agents for ovarian and endometrial cancer. The models reflected a range of sensitivity to platinum-containing chemotherapy as well as other relevant agents. One PDO from a patient treated prior to surgery with neoadjuvant trastuzumab successfully predicted the patient's postoperative chemotherapy and trastuzumab resistance. In addition, the PDO drug sensitivity assay identified alternative treatment options that are currently used in the second-line setting. Our findings suggest that PDOs could be used as a preclinical platform for personalized cancer therapy for gynecologic cancer patients.

Identification of Novel Fusion Transcripts in High Grade Serous Ovarian Cancer.

Fusion genes are structural chromosomal rearrangements resulting in the exchange of DNA sequences between genes. This results in the formation of a new combined gene. They have been implicated in carcinogenesis in a number of different cancers, though they have been understudied in high grade serous ovarian cancer. This study used high throughput tools to compare the transcriptome of high grade serous ovarian cancer and normal fallopian tubes in the interest of identifying unique fusion transcripts within each group. Indeed, we found that there were significantly more fusion transcripts in the cancer samples relative to the normal fallopian tubes. Following this, the role of fusion transcripts in chemo-response and overall survival was investigated. This led to the identification of fusion transcripts significantly associated with overall survival. Validation was performed with different analytical platforms and different algorithms to find fusion transcripts.

Bacterial, Archaea, and Viral Transcripts (BAVT) Expression in Gynecological Cancers and Correlation with Regulatory Regions of the Genome.

Bacteria, archaea, and viruses are associated with numerous human cancers. To date, microbiome variations in transcription have not been evaluated relative to upper female genital tract cancer risk. Our aim was to assess differences in bacterial, archaea, and viral transcript (BAVT) expression between different gynecological cancers and normal fallopian tubes. In this case-control study we performed RNA sequencing on 12 normal tubes, 112 serous ovarian cancers (HGSC) and 62 endometrioid endometrial cancers (EEC). We used the centrifuge algorithm to classify resultant transcripts into four indexes: bacterial, archaea, viral, and human genomes. We then compared BAVT expression from normal samples, HGSC and EEC. T-test was used for univariate comparisons (correcting for multiple comparison) and lasso for multivariate modelling. For validation we performed DNA sequencing of normal tubes in comparison to HGSC and EEC BAVTs in the TCGA database. Pathway analyses were carried out to evaluate the function of significant BAVTs. Our results show that BAVT expression levels vary between different gynecological cancers. Finally, we mapped some of these BAVTs to the human genome. Numerous map locations were close to regulatory genes and long non-coding RNAs based on the pathway enrichment analysis. BAVTs may affect gynecological cancer risk and may be part of potential targets for cancer therapy.

Creation and validation of models to predict response to primary treatment in serous ovarian cancer.

Nearly a third of patients with high-grade serous ovarian cancer (HGSC) do not respond to initial therapy and have an overall poor prognosis. However, there are no validated tools that accurately predict which patients will not respond. Our objective is to create and validate accurate models of prediction for treatment response in HGSC. This is a retrospective case-control study that integrates comprehensive clinical and genomic data from 88 patients with HGSC from a single institution. Responders were those patients with a progression-free survival of at least 6 months after treatment. Only patients with complete clinical information and frozen specimen at surgery were included. Gene, miRNA, exon, and long non-coding RNA (lncRNA) expression, gene copy number, genomic variation, and fusion-gene determination were extracted from RNA-sequencing data. DNA methylation analysis was performed. Initial selection of informative variables was performed with univariate ANOVA with cross-validation. Significant variables (p < 0.05) were included in multivariate lasso regression prediction models. Initial models included only one variable. Variables were then combined to create complex models. Model performance was measured with area under the curve (AUC). Validation of all models was performed using TCGA HGSC database. By integrating clinical and genomic variables, we achieved prediction performances of over 95% in AUC. Most performances in the validation set did not differ from the training set. Models with DNA methylation or lncRNA underperformed in the validation set. Integrating comprehensive clinical and genomic data from patients with HGSC results in accurate and robust prediction models of treatment response.

Mutated p53 portends improvement in outcomes when bevacizumab is combined with chemotherapy in advanced/recurrent endometrial cancer: An NRG Oncology study.

BACKGROUND: Successfully combining targeted agents with chemotherapy is an important future goal for cancer therapy. However, an improvement in patient outcomes requires an enhanced understanding of the tumor biomarkers that predict for drug sensitivity. NRG Oncology/Gynecologic Oncology Group (GOG) Study GOG-86P was one of the first attempts to combine targeted agents (bevacizumab or temsirolimus) with chemotherapy in patients with advanced endometrial cancer. Herein we performed exploratory analyses to examine the relationship between mutations in TP53, the most commonly mutated gene in cancer, with outcomes on GOG-86P. METHODS: TP53 mutational status was determined and correlated with progression-free survival (PFS) and overall survival (OS) on GOG-86P. RESULTS: Mutations in TP53 were associated with improved PFS and OS for patients that received bevacizumab as compared to temsirolimus (PFS: HR 0.48, 95% CI 0.31, 0.75; OS: HR: 0.61, 95% CI 0.38, 0.98). By contrast, there was no statistically significant difference in PFS or OS between arms for cases with WT TP53. CONCLUSIONS: This exploratory study suggests that combining chemotherapy with bevacizumab, but not temsirolimus, may enhance PFS and OS for patients whose tumors harbor mutant p53. These data set the stage for larger clinical studies evaluating the potential of TP53 mutational status as a biomarker to guide choice of treatment for endometrial cancer patients. Clintrials.gov: NCT00977574.

Genomic characterization of five commonly used endometrial cancer cell lines.

Recently, the compilation of massive amounts of genetic and genomic information on a wide variety of human cancer types, collectively known as The Cancer Genome Atlas (TCGA), has revealed a wealth of descriptive classification schemes both within and between different types and sources of cancer. In endometrial cancer, TCGA analyses have produced a post hoc scheme composed of four clusters: DNA polymerase ε catalytic subunit A (POLE) ultra­mutated (cluster 1), microsatellite instability (MSI) hypermutated (cluster 2), copy­number low (endometrioid, cluster 3) and copy­number high (serous­like, cluster 4). Given that cultured cells are the pre­clinical platform of cancer research, it was questioned how representative endometrial cancer cultured cell lines are in the context of TCGA­driven classification scheme. To address this issue in endometrial cancer cell lines, the present study investigated five commonly used cell lines: Ishikawa, ECC­1, Hec50co, KLE And RL95­2. The histology, mutation profile, MutL homolog 1 promoter methylation, copy­number variation, homologous recombination repair and microsatellite instability in each of these cell lines was assessed. The result of this characterization was that none of the cell lines fits neatly into any one of TCGA classes but are still useful models for groups of endometrial tumors. Furthermore, the contention that the ECC­1 cell line is actually Ishikawa was addressed using additional data. It was confirmed that ECC­1 cells likely no longer exist as ECC­1 but that they are not exactly Ishikawa either. For this reason, ECC­1 cells are suggested to be used in vitro but with this caveat in mind. Finally, we compiled a database of 127 endometrial cancer cell lines, including the five reported on here. The wide range of variation found in these cell lines highlights the need to further characterize these cells to select models that are more representative of the various histological and genomic aspects of endometrial cancer.

Detargeting Lentiviral-Mediated CFTR Expression in Airway Basal Cells Using miR-106b.

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.

Loss of progesterone receptor through epigenetic regulation is associated with poor prognosis in solid tumors.

BACKGROUND: Hormonal therapy using progestins, acting through the progesterone receptor (PR), is a well-established method to treat uterine endometrial hyperplasia and carcinoma. Recent population studies indicate that progestin exposure significantly reduces the incidence of ovarian, pancreatic and lung cancers in addition to endometrial cancer in women. This unexpected differentiating function of progestin in organs outside of the reproductive system led us to hypothesize that progestins/PR are protective against cancer development and progression in many tumor types. METHODS: The Cancer Genome Atlas, Oncomine and Prognostic Databases were searched to determine the relative expression of PR in tumors from multiple sites, and clinical outcomes linked to PR expression were determined. In addition, mRNA and protein expression were evaluated using real-time PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was performed to understand the PR downregulation mechanisms in tumor cells and patient samples. Methylation-specific PCR was conducted to survey the PR methylation status. The Student's t-test were performed to determine significance. Flow cytometry was used to quantify apoptotic cells. RESULTS: Low PR expression levels were consistently linked to less favorable clinical outcomes in endometrial, pancreatic, ovarian and non-small cell lung cancers. Clinical specimens and cell lines from these cancers demonstrate low levels of PR, and we now report that the mechanism for loss of PR is mediated through epigenetic repression. However, PR silencing can be overcome with epigenetic modulators. Histone deacetylase inhibitor (LBH589) and hypomethylating agent (5-aza-decitabine) restored functional PR expression at both the mRNA and protein levels and promoted marked cell death through induction of apoptosis in the presence of progesterone. CONCLUSIONS: Our studies support the possibility that progestin therapy in combination with epigenetic modulators, a concept we term "molecularly enhanced progestin therapy", is an approach worthy of study for malignancies originating from tissues outside of the reproductive tract.

Integration of Clinical and Molecular Features into Prediction Models for Outcomes in Endometrial Cancer.

Endometrial cancer recurrence carries a poor prognosis. The rising incidence of endometrial cancer calls for improvements in treatment of advanced and recurrent diseases. Efforts have been made to molecularly characterize endometrial cancer with the goal of improving therapies. The study presented here describes the utilization of molecular features of endometrial cancer tumors that are likely to recur, along with clinical characteristics utilized together to predict recurrence. This work further studies recurrent endometrial cancers to group them into "clusters" based on the tumor's molecular makeups with the ultimate aim to focus therapy on the molecular pathways potentially leading to recurrence.

Characterization of a TP53 Somatic Variant of Unknown Function From an Ovarian Cancer Patient Using Organoid Culture and Computational Modeling.

In our proof-of-concept study of 1 patient with stage IIIC carcinosarcoma of the ovary, we discovered a rare mutation in the tumor suppressor, TP53, that results in the deletion of N131. Immunofluorescence imaging of the organoid culture revealed hyperstaining of p53 protein. Computational modeling suggests this residue is important for maintaining protein conformation. Drug screening identified the combination of a proteasome inhibitor with a histone deacetylase inhibitor as the most effective treatment. These data provide evidence for the successful culture of a patient tumor and analysis of drug response ex vivo.