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Background This study assesses the feasibility of minimally invasive surgery (MIS) for well-selected epithelial ovarian cancer (EOC) patients. Methods We performed a review of data prospectively collected from a single center from 2017 to 2022. Only patients with histologically confirmed EOC, with a tumor diameter of less than 10 cm, were eligible. We also performed a meta-analysis of similar studies comparing the outcomes of laparoscopy and laparotomy. We used MINORS (Methodological Index for Non-Randomized Studies) to assess the risk of bias and calculated the odds ratio or mean difference. Results Eighteen patients were included; 13 in re-staging group, four in PDS group, and one in IDS group. All achieved complete cytoreduction. One case was converted to laparotomy. The median number of removed pelvic lymph nodes was 25 (range 16–34), and 32 (range 19–44) for para-aortic nodes. There were two (15.4%) intraoperative urinary tract injuries. The median follow-up was 35 months (range 1–53). Recurrence was observed in one case (7.7%). Thirteen articles for early-stage ovarian cancer were included in our meta-analysis. Analysis of the pooled results found that MIS had a higher frequency of spillage (OR, 2.15; 95% CI 1.27–3.64). No differences were observed in recurrence, complications, or up-staging. Conclusions Our experience supports the possibility of conducting MIS for EOC in well-selected patients. Except for spillage, our meta-analysis findings are consistent with previous reports, the majority of which were also retrospective. Ultimately, randomized clinical trials will be needed to authenticate the safety.

Dose finding in vivo study of IMD-0354.

Epithelial ovarian cancer (EOC) cells metastasize within the peritoneal cavity and directly encounter human peritoneal mesothelial cells (HPMC) as the initial step of metastasis. The contact between ovarian cancer cells and the single layer of mesothelial cells involves direct communications that modulate cancer progression but the mechanisms are unclear. One candidate mediating cell–cell communications is exosomes, 30–100 nm membrane vesicles of endocytic origin, through the cell–cell transfer of proteins, mRNAs, or microRNAs. Therefore, the goal was to mechanistically characterize how EOC-derived exosomes modulate metastasis. Exosomes from ovarian cancer cells were fluorescently labeled and cocultured with HPMCs which internalized the exosomes. Upon exosome uptake, HPMCs underwent a change in cellular morphology to a mesenchymal, spindle phenotype. CD44, a cell surface glycoprotein, was found to be enriched in the cancer cell–derived exosomes, transferred, and internalized to HPMCs, leading to high levels of CD44 in HPMCs. This increased CD44 expression in HPMCs promoted cancer invasion by inducing the HPMCs to secrete MMP9 and by cleaning the mesothelial barrier for improved cancer cell invasion. When CD44 expression was knocked down in cancer cells, exosomes had fewer effects on HPMCs. The inhibition of exosome release from cancer cells blocked CD44 internalization in HPMCs and suppressed ovarian cancer invasion. In ovarian cancer omental metastasis, positive CD44 expression was observed in those mesothelial cells that directly interacted with cancer cells, whereas CD44 expression was negative in the mesothelial cells remote from the invading edge. This study indicates that ovarian cancer–derived exosomes transfer CD44 to HPMCs, facilitating cancer invasion.Implications: Mechanistic insight from the current study suggests that therapeutic targeting of exosomes may be beneficial in treating ovarian cancer. Mol Cancer Res; 15(1); 78–92. ©2016 AACR.

Supplemental Figure Legend.

The prolongation of progression-free survival (PFS) in patients with advanced ovarian cancer by antiangiogenic therapy has been shown in several clinical trials. However, although an anti-VEGF antibody (bevacizumab) is the only option currently available, its efficacy is limited and it is not cost effective for use in all patients. Therefore, the development of a novel antiangiogenic drug, especially composed of small-molecule compounds, could be a powerful armament for ovarian cancer treatment. As NF-κB signaling has the potential to regulate VEGF expression, we determined to identify whether VEGF expression is associated with NF-κB activation and to investigate the possibility of a novel IKKβ inhibitor, IMD-0354 (IMMD Inc.), as an antiangiogenic drug. Tissue microarrays from 94 ovarian cancer tissues were constructed and immunohistochemical analyses performed. We revealed that IKK phosphorylation is an independent prognostic factor (PFS: 26.1 vs. 49.8 months, P = 0.011), and is positively correlated with high VEGF expression. In in vitro analyses, IMD-0354 robustly inhibited adhesive and invasive activities of ovarian cancer cells without impairing cell viabilities. IMD-0354 significantly suppressed VEGF production from cancer cells, which led to the inhibition of angiogenesis. In a xenograft model, the treatment of IMD-0354 significantly inhibited peritoneal dissemination with a marked reduction of intratumoral blood vessel formation followed by the inhibition of VEGF expression from cancer cells. IMD-0354 is a stable small-molecule drug and has already been administered safely to humans in other trials. Antiangiogenic therapy targeting IKKβ is a potential future option to treat ovarian cancer. Mol Cancer Ther; 14(4); 909–19. ©2015 AACR.

Characteristics of 94 ovarian cancer patients.

Multivariate analysis for progression free survival of patients.

Supplementary Figure 1. MMP-9 activity is involved in EOC invasion through HPMC. Supplementary Figure 2. Inhibition of exosome secretion attenuates CD44 uptake by HPMCs and suppresses TYK-nu cell invasion. Supplementary Figure 3. Inhibition of exosome uptake attenuates CD44 expression in HPMCs. Supplementary Figure 4. Immunofluorescent analyses. Supplementary Figure 5. Not only CD44 but MMP-9 are strongly expressed in peritoneal mesothelial cells interacting with ovarian cancer cells. Supplementary Figure 6. Acquisition of CD44 of mouse omental tissues from ovarian cancer cells is dose-dependent. Supplementary Table 1. Clinical characteristics of ovarian cancer patients with stage III-IV consecutively treated at Osaka University Hospital between 2011 and 2015.

Supplementary Data from Exosomal CD47 Plays an Essential Role in Immune Evasion in Ovarian Cancer

Ovarian cancer is largely diagnosed at advanced stages upon detection of multiple peritoneal dissemination, resulting in poor outcomes. CD47 is overexpressed in tumors, facilitates tumor immune evasion, and is located on exosomes. We aimed to investigate the role of exosomal CD47 in ovarian cancer progression. Prognostic significance of CD47 expression in ovarian cancer was examined using a public database including 1,435 patients and validated with 26 patients at our institution. CD47 expression was associated with poor progression-free survival and inversely correlated with macrophage infiltration in ovarian cancer tissues. Exosomes were collected from ovarian cancer cell lines, and CD47 expression on exosomes was confirmed via flow cytometry. Inhibition of exosome secretion with GW4869 and exosome uptake with 5-(N-ethyl-N-isopropyl)-amiloride inhibited the surface CD47 expression on ovarian cancer cells and promoted phagocytosis by macrophages. RAB27A (a key regulator of exosome release) knockdown inhibited exosome secretion and led to CD47 downregulation in ovarian cancer cells. In a xenograft mouse model, suppression of the release of tumor-derived exosomes by GW4869 or RAB27A knockdown suppressed tumor progression and enhanced M1 macrophage phagocytosis in cancer tissues. Collectively, CD47 expression was correlated with poor prognoses in patients with ovarian cancer, suggesting the importance of immune evasion. CD47 was expressed on exosomes and the inhibition of exosome secretion and/or uptake enhanced cancer cell phagocytosis by macrophages, and thus, suppressed peritoneal dissemination. This suggests the potential of a novel immune checkpoint therapeutic agent that focuses on exosomes.Implications:Mechanistic insight from the current study suggests that exosomal CD47 may be an advantageous therapeutic target in ovarian cancer.

We aimed to evaluate the response to definitive radiotherapy (RT) for cervical cancer based on histological subtypes and investigate prognostic factors in adenocarcinoma (AC). Of the 396 patients treated with definitive RT between January, 2010 and July, 2020, 327 patients met the inclusion criteria, including 275 with squamous cell carcinoma (SCC) and 52 with AC restaged based on the 2018 International Federation of Gynecology and Obstetrics staging system. Patient characteristics, response to RT, and prognoses of SCC and AC were evaluated. The complete response (CR) rates were 92.4% and 53.8% for SCC and AC, respectively (p < 0.05). Three-year overall survival and progression-free survival (PFS) rates of SCC were significantly higher than those of AC (88.6% vs. 74.1%, p < 0.05 and 76.3% vs. 59.3%, p < 0.05, respectively). Among the AC population, univariate and multivariate analyses were performed to examine prognostic factors associated with non-complete response (CR). In the multivariate analysis, gastric-type adenocarcinoma (GAS) was associated with non-CR in AC (adjusted odds ratio, 12.2; 95% confidence interval 1.0–145.6; p < 0.05). The 3-year PFS rate in patients with GAS was significantly lower than that in patients with other histological types of AC (44.4% vs. 66.7%, p < 0.05). Definitive RT for cervical cancer was significantly less effective for AC than for SCC. GAS was the only independent prognostic factor associated with non-CR in AC.

The pathogenesis of endometriosis remains unclear. Endometrial cells in retrograde menstruation are considered the source of endometriosis; therefore, we hypothesized that the eutopic endometrium may provide clues regarding the pathogenesis. We aimed to clarify the role of eutopic endometrial cells in endometriosis development.Eutopic endometrial tissues were obtained from patients with or without endometriosis, and expression of cell surface molecules in eutopic endometrial stromal cells (ESCs) was evaluated via iTRAQ-based proteomic analysis. Based on the results, we focused on galectin-3. Galectin-3 expression in clinical samples was confirmed by immunohistochemistry and Western blot analysis. The concentration of secreted galectin-3 was measured using enzyme-linked immunosorbent assays. Adhesion and migration of ESCs were evaluated by in vitro adhesion and wound healing assays. The cytotoxicity of natural killer cells was measured via calcein release assays. Cell proliferation was measured using the CyQUANT Cell Proliferation Assay Kit.iTRAQ analysis revealed that galectin-3 expression was specifically elevated in the ESCs from endometriosis patients. Immunohistochemistry confirmed galectin-3 overexpression in the eutopic endometrium of endometriosis, irrespective of the menstrual phase. Galectin-3 was overexpressed and secreted by the eutopic ESCs from patients with endometriosis compared to that from patients without endometriosis. Galectin-3 expression in ESCs increased adhesion and migration, whereas galectin-3 inhibitors impaired these processes. Galectin-3 reduced the cytotoxicity of natural killer cells toward ESCs, while not affecting cell proliferation.Galectin-3 promotes peritoneal engraftment of ESCs due to impaired immune surveillance in the peritoneal cavity and increases ESCs adhesion and migration to the peritoneum.

Background: Preoperative anemia affects perioperative outcomes and often causes fatigue and psychological disorders. Therefore, anemia should be treated before a patient undergoes surgery. Ninjin’yoeito (NYT), a Japanese Kampo medicine composed of ginseng and Japanese angelica root with the other 10 herbs, is administered for anemia, fatigue and anxiety; however, there are a few reports that have prospectively examined the effects of NYT before surgery for gynecological diseases. Hence, we tended to investigate its efficacy and safety.Methods: In this open-label randomized trial, women with gynecological diseases accompanied by preoperative anemia (defined as Results: 40 patients were enrolled of whom 30 patients were finally analyzed after allocating 15 to each group. There was no difference in the characteristics between both groups. Hb significantly increased in both groups (iron supplementation group, 9.9 ± 0.8 g/dL vs. 11.9 ± 1.6 g/dL; NYT group, 9.8 ± 1.0 g/dL vs. 12.0 ± 1.0 g/dL); the difference in the elevations in Hb between both groups was statistically insignificant (P = 0.72). Contrarily, CFS (17.9 ± 10.2 vs. 8.1 ± 5.2) and VAS-A (56 mm (50–70) vs. 23 mm (6–48)) scores were significantly decreased only in the NYT group and these changes were greater in the NYT group (∆CFS, P = 0.015; ∆VAS-A, P = 0.014). Liver dysfunction occurred in one patient of the NYT group.Conclusions: For treating preoperative anemia in women with gynecological conditions, NYT administration along with iron supplementation safely and efficiently improved the preoperative anemia, fatigue and anxiety.Trial registration: jRCT1051190012 (28/April/2019, retrospectively registered).

This study aimed to elucidate the effects of neuraxial analgesia on labor in women classified based on the Robson classification system.We retrospectively reviewed the clinical data of singleton cephalic nulliparous deliveries in labor at term between January 2018 and December 2021 and compared obstetrical outcomes between deliveries with and without neuraxial analgesia in women of Robson group 1 (spontaneous labor) and group 2a (induced labor). Statistical analyses were performed using the Wilcoxon ranked-sum test, Fisher's exact test, and logistic regression model. Statistical significance was set at p 0.05.We identified 2726 deliveries during the period, of which 387 deliveries (215 with analgesia and 172 without analgesia) were in Robson group 1 and 502 deliveries (258 with analgesia and 244 without analgesia) in Robson 2a. In Robson group 1 pregnancies, the cesarean section (CS) rate was higher in those who received analgesia (15%) than in those who did not (3%) (p = 0.0001). Multivariate analysis revealed that labor with analgesia was a significant risk factor for CS (p 0.0001). Similarly, in Robson group 2a pregnancies, we observed a higher CS rate in those with analgesia than in those without analgesia (18 vs. 11%, p = 0.042).A higher CS rate was observed in deliveries with analgesia than in those without analgesia both in the Robson group 1 and group 2a pregnancies.

Paclitaxel is a first-line drug for treating epithelial ovarian cancer (EOC). However, prognosis for patients with advanced stage cancer remains poor due to primary or acquired drug resistance. Therefore, overcoming chemoresistance is one of the greatest challenges in treating EOC. In this study, we identified microRNAs (miRNA) that regulate paclitaxel resistance and tested their potential utility as therapeutic targets. Paclitaxel-resistant cell lines were established using two EOC cell lines: SKVO3ip1 and HeyA8. miRNA PCR arrays showed that miR-194-5p was downregulated in paclitaxel-resistant cells. Forced expression of miR-194-5p resensitized resistant cells to paclitaxel. Conversely, miR-194-5p inhibition induced paclitaxel resistance in parental cells. In silico analysis and luciferase reporter assay revealed that MDM2 is a direct target of miR-194-5p. MDM2 was upregulated in paclitaxel resistant cells compared with parental cells. MDM2 inhibition also resensitized resistant cells to paclitaxel and forced MDM2 induced paclitaxel resistance in parental cells. miR-194-5p induced p21 upregulation and G1 phase arrest in resistant cells by downregulating MDM2. Furthermore, a public database showed that high MDM2 expression was associated with a shorter progression-free survival in EOC patients treated with paclitaxel. Collectively, our results show that restoring miR-194-5p expression resensitizes EOCs to paclitaxel, and this may be exploited as a therapeutic option.

Ovarian cancer is largely diagnosed at advanced stages upon detection of multiple peritoneal dissemination, resulting in poor outcomes. CD47 is overexpressed in tumors, facilitates tumor immune evasion, and is located on exosomes. We aimed to investigate the role of exosomal CD47 in ovarian cancer progression. Prognostic significance of CD47 expression in ovarian cancer was examined using a public database including 1,435 patients and validated with 26 patients at our institution. CD47 expression was associated with poor progression-free survival and inversely correlated with macrophage infiltration in ovarian cancer tissues. Exosomes were collected from ovarian cancer cell lines, and CD47 expression on exosomes was confirmed via flow cytometry. Inhibition of exosome secretion with GW4869 and exosome uptake with 5-(N-ethyl-N-isopropyl)-amiloride inhibited the surface CD47 expression on ovarian cancer cells and promoted phagocytosis by macrophages. RAB27A (a key regulator of exosome release) knockdown inhibited exosome secretion and led to CD47 downregulation in ovarian cancer cells. In a xenograft mouse model, suppression of the release of tumor-derived exosomes by GW4869 or RAB27A knockdown suppressed tumor progression and enhanced M1 macrophage phagocytosis in cancer tissues. Collectively, CD47 expression was correlated with poor prognoses in patients with ovarian cancer, suggesting the importance of immune evasion. CD47 was expressed on exosomes and the inhibition of exosome secretion and/or uptake enhanced cancer cell phagocytosis by macrophages, and thus, suppressed peritoneal dissemination. This suggests the potential of a novel immune checkpoint therapeutic agent that focuses on exosomes. Implications: Mechanistic insight from the current study suggests that exosomal CD47 may be an advantageous therapeutic target in ovarian cancer.

The risk of uterine rupture in subsequent pregnancy is 1%‐12% in patients with prior classical uterine incision. Management of mild/moderate abdominal pain without an obvious abnormal finding before 36 weeks is challenging owing to fetal immaturity.

MicroRNA (miRNA) plays a pivotal role in cancer biology. Therefore, tumor suppressor (TS) miRNAs are an attractive target for cancer therapy. However, clinical trials have failed due to the difficulties in miRNA delivery, warranting the development of a novel drug delivery system (DDS). Exosomes are stable in circulation and selectively picked up by cancer cells, indicating that they can serve as a miRNA carrier. The aim of this study was to explore the possibility of exosomes as a carrier for miRNA replacement therapy for ovarian cancer (OC). First, exosomes were purified from primary-cultured omental fibroblasts of OC patients. miR-199a-3p was selected as a TS miRNA, and the synthesized miR-199a-3p was loaded into exosomes by electroporation. Treatment with miR199a-3p-loaded-exosomes (miR-199a-3p-Exo) drastically increased miR-199a-3p expression level in OC cell lines (CaOV3; 8592-, SKOV3; 67188-, and OVCAR3; 2280-fold). miR-199a-3p-Exo suppressed c-Met expression, a direct target of miR-199a-3p, and thereby inhibited cell proliferation and invasion. In a xenograft study, miR-199a-3p-Exo also drastically inhibited peritoneal dissemination in OC mice model, and diminished c-Met expression, ERK phosphorylation, and MMP2 expression in tumors. These results suggest that miRNA replacement therapy using exosomes shows promise for treatment of OC. Given that omental fibroblasts can be obtained from most OC patients, patient-derived exosomes can be utilized as a DDS for future molecular-targeted therapies.

SUMMARY To improve our understanding of ovarian cancer, we performed genome-wide analyses of 45 ovarian cancer cell lines. Given the challenges of genomic analyses of tumors without matched normal samples, we developed approaches for detection of somatic sequence and structural changes and integrated these with epigenetic and expression alterations. Alterations not previously implicated in ovarian cancer included amplification or overexpression of ASXL1 and H3F3B, deletion or underexpression of CDC73 and TGF-beta receptor pathway members, and rearrangements of YAP1-MAML2 and IKZF2-ERBB4. Dose-response analyses to targeted therapies revealed unique molecular dependencies, including increased sensitivity of tumors with PIK3CA and PPP2R1A alterations to PI3K inhibitor GNE-493, MYC amplifications to PARP inhibitor BMN673, and SMAD3/4 alterations to MEK inhibitor MEK162. Genome-wide rearrangements provided an improved measure of sensitivity to PARP inhibition. This study provides a comprehensive and broadly accessible resource of molecular information for the development of therapeutic avenues in ovarian cancer., In Brief The overall survival of patients with late-stage ovarian cancer is dismal. To identify therapeutic opportunities, Papp et al. integrate genomic, epigenomic, and expression analyses to provide a resource of molecular abnormalities in ovarian cancer cell lines and use these to identify tumors sensitive to PARP, MEK, and PI3K inhibitors., Graphical Abstract

Background microRNAs (miRNAs) stably exist in circulating blood and are encapsulated in extracellular vesicles such as exosomes. The aims of this study were to identify which exosomal miRNAs are highly produced from epithelial ovarian cancer (EOC) cells, to analyze whether serum miRNA can be used to discriminate patients with EOC from healthy volunteers, and to investigate the functional role of exosomal miRNAs in ovarian cancer progression. Methods Exosomes were collected from the culture media of serous ovarian cancer cell lines, namely TYK-nu and HeyA8 cells. An exosomal miRNA microarray revealed that several miRNAs including miR-99a-5p were specifically elevated in EOC-derived exosomes. Expression levels of serum miR-99a-5p in 62 patients with EOC, 26 patients with benign ovarian tumors, and 20 healthy volunteers were determined by miRNA quantitative reverse transcription-polymerase chain reaction. To investigate the role of exosomal miR-99a-5p in peritoneal dissemination, neighboring human peritoneal mesothelial cells (HPMCs) were treated with EOC-derived exosomes and then expression levels of miR-99a-5p were examined. Furthermore, mimics of miR-99a-5p were transfected into HPMCs and the effect of miR-99a-5p on cancer invasion was analyzed using a 3D culture model. Proteomic analysis with the tandem mass tag method was performed on HPMCs transfected with miR-99a-5p and then potential target genes of miR-99a-5p were examined. Results The serum miR-99a-5p levels were significantly increased in patients with EOC, compared with those in benign tumor patients and healthy volunteers (1.7-fold and 2.8-fold, respectively). A receiver operating characteristic curve analysis showed with a cut-off of 1.41 showed sensitivity and specificity of 0.85 and 0.75, respectively, for detecting EOC (area under the curve = 0.88). Serum miR-99a-5p expression levels were significantly decreased after EOC surgeries (1.8 to 1.3, p = 0.002), indicating that miR-99a-5p reflects tumor burden. Treatment with EOC-derived exosomes significantly increased miR-99a-5p expression in HPMCs. HPMCs transfected with miR-99a-5p promoted ovarian cancer invasion and exhibited increased expression levels of fibronectin and vitronectin. Conclusions Serum miR-99a-5p is significantly elevated in ovarian cancer patients. Exosomal miR-99a-5p from EOC cells promotes cell invasion by affecting HPMCs through fibronectin and vitronectin upregulation and may serve as a target for inhibiting ovarian cancer progression. Electronic supplementary material The online version of this article (10.1186/s12885-018-4974-5) contains supplementary material, which is available to authorized users.

Summary: CCNE1-amplified ovarian cancers (OVCAs) and endometrial cancers (EMCAs) are associated with platinum resistance and poor survival, representing a clinically unmet need. We hypothesized that dysregulated cell-cycle progression promoted by CCNE1 overexpression would lead to increased sensitivity to low-dose WEE1 inhibition and ataxia telangiectasia and Rad3-related (ATR) inhibition (WEE1i-ATRi), thereby optimizing efficacy and tolerability. The addition of ATRi to WEE1i is required to block feedback activation of ATR signaling mediated by WEE1i. Low-dose WEE1i-ATRi synergistically decreases viability and colony formation and increases replication fork collapse and double-strand breaks (DSBs) in a CCNE1 copy number (CN)-dependent manner. Only upon CCNE1 induction does WEE1i perturb DNA synthesis at S-phase entry, and addition of ATRi increases DSBs during DNA synthesis. Inherent resistance to WEE1i is overcome with WEE1i-ATRi, with notable durable tumor regressions and improved survival in patient-derived xenograft (PDX) models in a CCNE1-level-dependent manner. These studies demonstrate that CCNE1 CN is a clinically tractable biomarker predicting responsiveness to low-dose WEE1i-ATRi for aggressive subsets of OVCAs/EMCAs.

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer.

Epithelial ovarian cancer (EOC) cells metastasize within the peritoneal cavity and directly encounter human peritoneal mesothelial cells (HPMC) as the initial step of metastasis. The contact between ovarian cancer cells and the single layer of mesothelial cells involves direct communications that modulate cancer progression but the mechanisms are unclear. One candidate mediating cell–cell communications is exosomes, 30–100 nm membrane vesicles of endocytic origin, through the cell–cell transfer of proteins, mRNAs, or microRNAs. Therefore, the goal was to mechanistically characterize how EOC-derived exosomes modulate metastasis. Exosomes from ovarian cancer cells were fluorescently labeled and cocultured with HPMCs which internalized the exosomes. Upon exosome uptake, HPMCs underwent a change in cellular morphology to a mesenchymal, spindle phenotype. CD44, a cell surface glycoprotein, was found to be enriched in the cancer cell–derived exosomes, transferred, and internalized to HPMCs, leading to high levels of CD44 in HPMCs. This increased CD44 expression in HPMCs promoted cancer invasion by inducing the HPMCs to secrete MMP9 and by cleaning the mesothelial barrier for improved cancer cell invasion. When CD44 expression was knocked down in cancer cells, exosomes had fewer effects on HPMCs. The inhibition of exosome release from cancer cells blocked CD44 internalization in HPMCs and suppressed ovarian cancer invasion. In ovarian cancer omental metastasis, positive CD44 expression was observed in those mesothelial cells that directly interacted with cancer cells, whereas CD44 expression was negative in the mesothelial cells remote from the invading edge. This study indicates that ovarian cancer–derived exosomes transfer CD44 to HPMCs, facilitating cancer invasion. Implications: Mechanistic insight from the current study suggests that therapeutic targeting of exosomes may be beneficial in treating ovarian cancer. Mol Cancer Res; 15(1); 78–92. ©2016 AACR.

Objective The aim of this study was to "humanize" ovarian cancer patient-derived xenograft (PDX) models by autologous transfer of patient-matched tumor infiltrating lymphocytes (TILs) to evaluate immunotherapies. Methods Orthotopic high-grade serous ovarian cancer (HGSOC) PDX models were established from three patient donors. Models were molecularly and histologically validated by immunohistochemistry. TILs were expanded from donor tumors using a rapid expansion protocol. Ex vivo TIL and tumor co-cultures were performed to validate TIL reactivity against patient-matched autologous tumor cells. Expression of TIL activation markers and cytokine secretion was quantitated by flow cytometry and ELISA. As proof of concept, the efficacy of anti-PD-1 monotherapy was tested in autologous TIL/tumor HGSOC PDX models. Results Evaluation of T-cell activation in autologous TIL/tumor co-cultures resulted in an increase in HLA-dependent IFNγ production and T-cell activation. In response to increased IFNγ production, tumor cell expression of PD-L1 was increased. Addition of anti-PD-1 antibody to TIL/tumor co-cultures increased autologous tumor lysis in a CCNE1 amplified model. Orthotopic HGSOC PDX models from parallel patient-matched tumors maintained their original morphology and molecular marker profile. Autologous tumor-reactive TIL administration in patient-matched PDX models resulted in reduced tumor burden and increased survival, in groups that also received anti-PD-1 therapy. Conclusions This study validates a novel, clinically relevant model system for in vivo testing of immunomodulating therapeutic strategies for ovarian cancer, and provides a unique platform for assessing patient-specific T-cell response to immunotherapy.

ObjectiveAberrant activation of nuclear factor-kappa β (NF-κB) signaling has been correlated with poor outcome among patients with ovarian cancer. Although the therapeutic potential of NF-κB pathway disruption in cancers has been extensively studied, most classical NF-κB inhibitors are poorly selective, exhibit off-target effects, and have failed to be applied in clinical use. IMD-0560,N-[2,5-bis (trifluoromethyl) phenyl]-5-bromo-2-hydroxybenzamide, is a novel low-molecular-weight compound that selectively inhibits the IκB kinase complex and works as an inhibitor of NF-κB signaling. The aim of this study was to assess the therapeutic potential of IMD-0560 against ovarian cancer in vitro and in vivo.MethodsNF-κB activity (phosphorylation) was determined in 9 ovarian cancer cell lines and the inhibitory effect of IMD-0560 on NF-κB activation was analyzed by Western blotting. Cell viability, cell cycle, vascular endothelial growth factor (VEGF) expression, and angiogenesis were assessed in vitro to evaluate the effect of IMD-0560 on ovarian cancer cells. In vivo efficacy of IMD-0560 was also investigated using an ovarian cancer xenograft mouse model.ResultsThe NF-κB signaling pathway was constitutively activated in 8 of 9 ovarian cancer cell lines. IMD-0560 inhibited NF-κB activation and suppressed ovarian cancer cell proliferation by inducing G1 phase arrest. IMD-0560 decreased VEGF secretion from cancer cells and inhibited the tube formation of human umbilical vein endothelial cells. IMD-0560 significantly inhibited peritoneal metastasis and prolonged the survival in an ovarian cancer xenograft mice model. Immunohistochemical staining of excised tumors revealed that IMD-0560 suppressed VEGF expression, tumor angiogenesis, and cancer cell proliferation.ConclusionsIMD-0560 showed promising therapeutic efficacy against ovarian cancer xenograft mice by inducing cell cycle arrest and suppressing VEGF production from cancer cells. IMD-0560 may be a potential future option in regimens for the treatment of ovarian cancer.

Purpose: Clear-cell ovarian cancer (CCOC) is often resistant to standard chemotherapy and has limited treatment options in the advanced or recurrent setting. ARID1A is the most prevalent mutation, with approximately 50% of all CCOC harboring this mutation. ARID1A, a member of the SWI/SNF family, regulates transcription and has a major role in the repair of DNA lesions, directly facilitating DNA accessibility on the chromatin or indirectly by facilitating the functions of DNA repair proteins. We propose that CCOCs unique genomic alterations (e.g., ARID1A frameshift or nonsense mutations) will increase dependency on chromatin remodeling and DNA repair (e.g., ATR/CHK1/WEE1) pathways for survival. We hypothesize that combination of low-dose small-molecule inhibitors of the BET family (BRD4i) and the DNA damage repair pathway (ATR/CHK1/WEE1), will especially target ARID1A mutant cancer cells promoting mitotic catastrophe, apoptosis, and tumor regression, sparing normal cells. Method: We compared the sensitivity of BRD4i + ATRi/WEE1i combinations in ARID1A knockdown and knockout cells compared with isogenic parental lines. The effects on transcription, kinase signaling, cell cycle, apoptosis, and DNA damage were evaluated. RNAseq/ATACseq and Reverse Phase Protein Array (RPPA) were also performed. A CCOC preclinical drug development platform was established and BRD4i combinations were evaluated in ARID1A mutant and ARID1A wild-type (ARID1AMUT and ARID1AWT) PDX models. Results: Low-dose BRD4i in combination with DNA damage repair inhibitors, ATRi/WEE1i (BDR4i-ATRi or BRD4i-WEE1i), were synergistic in decreasing survival and colony formation with an increase in apoptosis in ARID1AMUT cells compared to ARID1AWT. BRD4i combinations caused a robust G1 arrest with a decrease pCDC6 levels along with an increase in DNA double-strand breaks in the ARID1AMUT or knockout cells. Combination BRD4i-ATRi showed significant tumor regression and increased overall survival compared to standard chemotherapy or monotherapy in several ARID1AMUT PDX models but not in an ARID1AWT PDX. Among BRD4i-DDRi combinations, BRD4i-ATRi is superior to BRD4i-WEE1i in terms of antitumor effect and drug tolerability. Conclusion: Our studies identify a novel drug combination targeting a genetic alteration (e.g., ARID1A) common in CCOC that is highly effective and tolerable. BRD4i in combination with ATRi or WEE1i was synergistic, decreasing survival, colony formation with an increase in DNA damage and apoptosis in ARID1AMUT models. Using our novel CCOC drug development PDX platform, we demonstrated that BRD4i-ATRi combination therapy is more effective than standard chemotherapy or monotherapy alone with acceptable toxicity in ARID1AMUTPDXs. This abstract is also being presented as Poster B24. Citation Format: Yasuto Kinose, Hyoung Kim, Haineng Xu, Sergey Medvedev, Erin George, Sarah Gitto, Margaret Whicker, Dorothy Hallberg, Lauren Schwartz, Gordon Mills, Victor E. Velculescu, Tian-Li Wang, Ronny Drapkin, Fiona Simpkins. Dual blockade of BRD4 and the ATR/WEE1 pathway exploits ARID1A loss in clear-cell ovarian cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR11.

Figure S1. ROC analysis discriminating specific EOC histologic types from other subtypes. (PPTX 143 kb)

During the dissemination of ovarian cancer cells, the cells float in the peritoneal cavity without access to a vascular supply and so are exposed to hypoxic conditions, which may cause the ovarian cancer cells to acquire a more aggressive and malignant phenotype. In this study, we screened microRNAs (miRNAs) to identify those that displayed altered expression patterns under hypoxic conditions and then analyzed their functional roles in ovarian cancer progression. miRNA PCR arrays performed on cells from 2 ovarian cancer cell lines (CaOV3 and RMUG-S) revealed miR-199a-3p as one of the miRNAs that are downregulated under hypoxia. In silico analyses indicated that MET is one of the target genes for miR-199a-3p; subsequently, miR-199a-3p expression was found to be inversely correlated with c-Met expression in ovarian cancer. Transfection of precursor miR-199a-3p into ovarian cancer cells reduced c-Met expression and inhibited the phosphorylation of c-Met, extracellular signal-regulated kinase, and AKT; in addition, proliferation, adhesion, and invasiveness were inhibited. Moreover, overexpression of miR-199a-3p in cancer cells significantly suppressed peritoneal dissemination in a xenograft model. In summary, the hypoxia-related microRNA miR-199a-3p drastically inhibits ovarian cancer progression through the downregulation of c-Met expression. Therefore, miR-199a-3p is a potential target for treating ovarian cancer dissemination.

Ovarian cancer is the most lethal of malignant gynecological tumors. Its lethality may be due to difficulties in detecting it at an early stage and lack of effective treatments for patients with an advanced or recurrent status. Therefore, there is a strong need for prognostic and predictive markers to diagnose it early and to help optimize and personalize treatment. MicroRNAs are noncoding RNAs that regulate target genes posttranscriptionally. They are involved in carcinogenesis, cell cycle, apoptosis, proliferation, invasion, metastasis, and chemoresistance. The dysregulation of microRNAs is involved in the initiation and progression of human cancers including ovarian cancer, and strong evidence that microRNAs can act as oncogenes or tumor suppressor genes has emerged. Several microRNA signatures that are unique to ovarian cancer have been proposed, and serum-circulating microRNAs have the potential to be useful diagnostic and prognostic biomarkers. Various microRNAs such as those in the miR-200 family, the miR-199/214 cluster, or the let-7 paralogs have potential as therapeutic targets for disseminated or chemoresistant ovarian tumors. Although many obstacles need to be overcome, microRNA therapy could be a powerful tool for ovarian cancer prevention and treatment. In this review, we discuss the emerging roles of microRNAs in various aspects of ovarian cancer.

Ovarian cancer lacks therapies that yield complete tumor regression resulting in low cure rates. Cyclin E (CCNE1) is an oncogenic driver that is amplified in 25% of high grade serous ovarian cancers (HGSOC). Cyclin E overexpression (CCNE1HIGH) is associated with poor survival and platinum resistance in these cancers. Effective therapies for CCNE1HIGH ovarian cancers yielding complete and durable responses are lacking. Aberrant expression of Cyclin E leads to unscheduled entry into S phase, premature origin firing, nucleotide depletion, and replication fork stress. The imbalances created by this abnormal progression leads to an increased reliance on cell cycle checkpoint regulators, such as WEE1 and ATR, which counter some of the untoward effects of Cyclin E overexpression. WEE1 is a dual specificity kinase that regulates cell cycle progression by inhibiting both CDK2 and CDK1, thereby inhibiting progression from G1 to S and G2 to M phases, respectively. ATR kinase protects the replication fork from collapse thereby inhibiting G2/M progression so DNA can repair. Additionally, mutations in another G1-S phase checkpoint regulator, TP53, may accentuate dependence on WEE1 and ATR by eliminating an alternative checkpoint pathway that limits CCNE1-CDK2 activity. TP53 mutations are also ubiquitous in HGSOC, further implying the promise of WEE1i and ATRi in selective killing CCNE1HIGH tumors as a rational therapeutic strategy. We tested drug effects on survival, colony formation, cell cycle and apoptosis in vitro and in patient-derived xenograft (PDX) models. Induction of Cyclin E expression in immortalized human fallopian tube secretory epithelial cells (FTSEC) upregulates pChk1, a downstream protein of ATR. Combination of WEE1i with ATRi (WEE1i-ATRi) synergistically decreases cell viability and colony formation in CCNE1HIGH HGSOC cells. Selective inhibition of ATR and WEE1 with siRNA was synergistic supporting that ATR and WEE1 are critical to survival. WEE1i-ATRi dramatically increased cell apoptosis, decreased S phase cells and arrested cells at G2/M phase. Combination inhibition of WEE1 and ATR induced gH2AX and phosphorylation of RPA32, indicating increased double strand DNA break and replication stress. Also, combination inhibition of WEE1 and ATR promoted mitotic catastrophe, as indicated by increased pHH3. Finally, combination WEE1-ATRi is tolerable and results in a 4-fold increase in survival compared to standard chemotherapy or monotherapy in a CCNE1 amplified HGSOC PDX model. In addition, sequential treatment is as effective and less toxic as concomitant WEE1i with ATRi warranting further study. Our studies developed a novel combination treatment approach for targeting Cyclin E over-expressing ovarian cancers. Citation Format: Haineng Xu, Yasuto Kinose, Hyoung Kim, Sergey Medvedev, Erin George, Ronny Drapkin, Eric Brown, Gordon Mills, Fiona Simpkins. A NOVEL TREATMENT APPROACH FOR TARGETING CYCLIN E OVER-EXPRESSING OVARIAN CANCERS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-060.

PURPOSE: Ovarian cancer is a heterogeneous disease with multiple histological subtypes and a wide range of genetic aberrations. While high-grade serous carcinoma (HGSC) is the most common type, clear cell carcinoma (CCC) of the ovary is notoriously the most challenging to treat and exhibits very low response rates to standard therapies. However, efforts to understand CCC and develop new therapeutic approaches to this subtype have been limited because it represents a minority of ovarian cancer cases in the United States and Europe. In contrast, CCC accounts for more than 30% of all ovarian cancer in Japan. To improve the survival of patients with ovarian CCC, a deeper understanding of the molecular features of available model systems is needed. Our goal is to characterize a panel of CCC cell lines genomically and functionally and identify those that can serve as tractable model systems for future in vivo drug discovery studies. METHOD: First, we characterize 9 CCC cell lines (ES-2, TOV21G, OVTOKO, OVMANA, OCI-C5x, JHOC-5, JHOC-7, JHOC-9, and OVISE) with whole exome sequence and proteomics approaches (reverse phase protein array; RPPA). Secondly, we performed in vitro assays, including soft-agar colony formation assays and MTT assays using standard chemotherapies. Thirdly, to test in vivo tumorigenic potential, by injecting 5 million cells of luciferized CCC lines in NSG female mice using both the subcutaneous route and the intraperitoneal route. Imaging was performed weekly using the In Vivo Imaging System. RESULTS: Among the 9 CCC lines, seven (TOV21G, OVTOKO, OVMANA, OCI-C5x, JHOC-5, JHOC-9, and OVISE) harbor ARID1A mutation, which is the most prevalent mutation in CCC (50%). The second common mutation in CCC (40%), PIK3CA mutation, and was detected in 4 cells (TOV21G, OVMANA, OCI-C5x, and OVISE). The ES-2 line has TP53 and BRAF mutation and its genomic profile is inconsistent with CCC. A BRCA2 mutation was found in OVTOKO and JHOC-5, finding not previously reported. Principal component analysis of RPPA data showed distinct clusters between the 9 CCC lines and the 6 HGSC lines. Interestingly, we also observed two distinct clusters within the CCC lines. Consistent with our genomic analysis, ES-2 correlated with HGSC lines based on RPPA data. In our in vitro drug studies, OVTOKO, OCI-C5x, and OVISE exhibited a Carboplatin/Paclitaxel resistance phenotype. In xenograft study, 4 cell lines (ES-2, TOV21G, OVTOKO, and OCI-C5x) formed tumor within a month, suggesting they are useful tools for in vivo studies. In contrast, OVMANA, JHOC-7, JHOC-9, and OVISE take more than 100 days to form the tumors. Soft-agar colony formation results correlated with how long it took to develop xenograft tumor in each line. CONCLUSION: Our genomic studies identified aberrations in CCC lines not previously described. The proteomics data revealed two clusters within the CCC lines that might represent functionally distinct groups. Finally, we identified four cell lines (ES-2, TOV21G, OVTOKO, and OCI-C5x) that readily form tumors in mice and could be used for future in vivo drug studies. However, ES-2 appears to cluster more closely with HGSC and may not represent the CCC histotype. Citation Format: Yasuto Kinose, Dorothy Hallberg, Gordon Mills, Tan Ince, Victor Velculescu, Fiona Simpkins, Ronny Drapkin. COMPREHENSIVE GENOMIC, PROTEOMIC, AND EXPERIMENTAL CHARACTERIZATION OF OVARIAN CLEAR CELL CARCINOMA CELL LINES FOR IMPROVED DRUG DEVELOPMENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-036.

Purpose: Ovarian cancer is heterogeneous with multiple histological subtypes and a wide range of genetic aberrations. While high-grade serous ovarian carcinoma (HGSOC) is the most common, clear cell ovarian carcinoma (CCOC) is the most challenging to treat and exhibits low response rates to standard therapies. However, efforts to understand CCOC and develop new therapies have been limited because it represents a minority of ovarian cancers in the U.S. and Europe. In contrast, CCOC accounts for approximately 30% of all ovarian cancer in Japan. To improve the survival of patients with CCOC, a deeper understanding of the molecular features that define available model systems is needed. Our goal is to comprehensively characterize a panel of CCOC lines using next generation sequencing and functional in vitro and in vivo experiments to define the lines that are most faithful to CCOC and are tractable for subsequent in vivo drug discovery. Method: We obtained 9 CCOC cells from ATCC, Riken Cell Bank, and University of Miami (ES-2, TOV21G, OVTOKO, OVMANA, OCI-C5x, JHOC-5, JHOC-7, JHOC-9, and OVISE). Genomic DNA, RNA, and protein were isolated and subjected to whole exome DNA-seq, RNA-seq, and reverse phase protein array (RPPA), respectively. We performed in vitro MTT assays to test the sensitivity of these lines to chemotherapies. Tumorigenicity was evaluated by injecting 5 million cells of luciferized CCOC lines into NSG female mice using both the subcutaneous route and the intraperitoneal route. Imaging was performed weekly using the In Vivo Imaging System. Results: ARID1A mutation, the most prevalent mutation in CCOC, is present in 8 of the 9 cell lines (TOV21G, OVTOKO, OVMANA, OCI-C5x, JHOC-5, JHOC-7, JHOC-9, and OVISE), whereas the second common mutation in CCOC (PIK3CA) is detected in 5 lines (TOV21G, OVMANA, OCI-C5x, JHOC-7, and OVISE). The ES-2 cell line has both TP53 and BRAF mutations and its genomic profile is not typical of CCOC. Principal component analysis of RPPA showed distinct groups between the 9 CCOC lines and the 6 HGSOC lines. Interestingly, we also observed two distinct clusters within the CCOC lines. Consistent with our genomic analysis, the ES-2 cell line correlated more closely with the HGSOC lines based on RPPA data. In our in vitro drug studies, OVTOKO and OCI-C5x exhibited resistance to Carboplatin/Paclitaxel. In xenograft study, 4 cell lines (ES-2, TOV21G, OVTOKO, and OCI-C5x) formed measurable tumor within a month. In contrast, OVMANA, JHOC-7, JHOC-9, and OVISE took over 100 days to form tumors. Conclusion: Our data suggests that there may exist two functionally distinct groups within CCOC that warrants further study. In vitro and in vivo studies identified 4 cell lines that represent tractable models for rigorous therapeutic studies: ES-2, TOV21G, OVTOKO, and OCI-C5x. However, ES-2 appears to cluster more closely with HGSOC and may not represent the CCOC histotype. Citation Format: Yasuto Kinose, Dorothy Hallberg, Kai Doberstein, Gordon Mills, Tan Ince, Victor Velculescu, Fiona Simpkins, Ronny Drapkin. Comprehensive molecular and experimental characterization of ovarian clear cell carcinoma cell lines for in vivo drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1065.

Ovarian cancer is the leading cause of death among gynecologic malignancies. Since ovarian cancer develops asymptomatically, it is often diagnosed at an advanced and incurable stage. Despite many years of research, there is still a lack of reliable diagnostic markers and methods for early detection and screening. Recently, it was discovered that cell-free microRNAs (miRNAs) circulate in the body fluids of healthy and diseased patients, suggesting that they may serve as a novel diagnostic marker. This review summarizes the current knowledge regarding the potential clinical relevance of circulating cell-free miRNA for ovarian cancer diagnosis, prognosis, and therapeutics. Despite the high levels of ribonucleases in many types of body fluids, most of the circulating miRNAs are packaged in microvesicles, exosomes, or apoptotic bodies, are binding to RNA-binding protein such as argonaute 2 or lipoprotein complexes, and are thus highly stable. Cell-free miRNA signatures are known to be parallel to those from the originating tumor cells, indicating that circulating miRNA profiles accurately reflect the tumor profiles. Since it is well established that the dysregulation of miRNAs is involved in the tumorigenesis of ovarian cancer, cell-free miRNAs circulating in body fluids such as serum, plasma, whole blood, and urine may reflect not only the existence of ovarian cancer but also tumor histology, stage, and prognoses of the patients. Several groups have successfully demonstrated that serum or plasma miRNAs are able to discriminate patients with ovarian cancer patients from healthy controls, suggesting that the addition of these miRNAs to current testing regimens may improve diagnosis accuracies for ovarian cancer. Furthermore, recent studies have revealed that changes in levels of cell-free circulating miRNAs are associated with the condition of cancer patients. Discrepancies between the results across studies due to the lack of an established endogenous miRNA control to normalize for circulating miRNA levels, as well as differing extraction and quantification methods, are the pitfalls to be resolved before clinical application. There is still a long way, however, before this can be achieved, and further evidence would make it possible to apply circulating cell-free miRNAs not only as biomarkers but also as potential therapeutic targets for ovarian cancer in the future.

High grade serous ovarian cancers (HGSOC) have defective homologous recombination (HR) genes in 50% of cases, while a distinct 20% demonstrate CCNE1 amplification (CCNE1amp). HR-deficient HGSOC are initially sensitive to Poly(ADP-ribose) polymerase inhibitors (PARPi) but drug resistance ultimately emerges. CCNE1amp HGSOC show resistance to PARPi and platinum treatments. Here, we investigated the anti-tumor activity of the potent, highly selective, orally bioavailable small molecule inhibitor of Checkpoint kinase 1 (Chk1), SRA737, in both acquired PARPi-resistant and CCNE1amp HGSOC models. HR-deficiencies and CCNE1amp are known to increase replication stress, leading to increased reliance on Chk1, a key regulator of cell cycle progression and the replication stress response. We hypothesized that Chk1 inhibition by SRA737 will result in increased replication stress, inducing subsequent cell death and tumor regression in both PARPi-resistant and CCNE1amp ovarian cancer models. In colony formation assays, SRA737 monotherapy decreased cell survival in HR-deficient, PARPi-resistant and CCNE1amp cells. Additionally, the combination of SRA737 with PARPi was synergistic in decreasing colony formation in HR-deficient (PEO1, best coefficient of drug interaction (CDI)=0.53; JHOS4, CDI=0.45) and PARPi-resistant cell models (PEO1-PR, CDI=0.11; PEO4, CDI=0.08). SRA737 treatment led to a dose-dependent increase in the replication stress marker pCHK1 (S345), confirming an on-target drug effect in PARPi-resistant (PEO1-PR) and CCNE1amp (OVCAR3) cells. Furthermore, treatment with SRA737 induced gH2AX (indicator of DNA damage) which increased modestly in combination with PARPi. SRA737 was also evaluated in a PARPi-resistant PDX model as well as in CCNE1amp in vivo mouse models. Preliminary evidence in a PARPi resistant PDX model demonstrated tumor growth inhibitory activity of SRA737 in combination with PARPi. Consistent with in vitro activity, SRA737 inhibited tumor growth in an OVCAR3 xenograft model. Lastly, in an orthotopic PDX model established from a platinum-resistant CCNE1amp ovarian cancer patient, SRA737 monotherapy caused significant tumor regression, similar to SRA737 in combination with PARPi. Strategies to optimize treatments for PARPi-resistant HGSOC, as well as for platinum-resistant CCNE1amp HGSOC, are needed. In PARPi-resistant models, SRA737 is active as a monotherapy, and the combination of SRA737 with PARPi demonstrated synergy. In CCNE1amp tumors, SRA737 showed profound activity as a monotherapy in this PARPi-resistant model. SRA737 is a new potent and selective Chk1 inhibitor that demonstrated activity in acquired PARPi-resistant as well as CCNE1amp preclinical cancer models, warranting further development in these HGSOC subgroups. Citation Format: Haineng Xu, Sergey Medvedev, Ashka Pandya, Hyoung Kim, Yasuto Kinose, Eric Brown, Ryan J. Hansen, Bryan Strouse, Snezana Milutinovic, Christian Hassig, Fiona Simpkins. The novel oral Chk1 inhibitor, SRA737, is active in both PARP inhibitor resistant and CCNE1 amplified high grade serous ovarian cancers [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 LB-265.

Preterm delivery (PTD) remains a serious challenge in perinatology. Intrauterine infection and/or inflammation, followed by increased inflammatory cytokines, represented by IL-6, are involved in this pathology. Our aim was to identify IL-6-producing cells in the placenta and to analyze the potential of targeting IκB kinase β (IKKβ) signaling to suppress IL-6 production for the treatment of PTD. Immunohistochemical analyses using placentas complicated with severe chorioamnionitis revealed that IL-6 is mainly expressed in human amniotic mesenchymal stromal cells (hAMSCs). Primary hAMSCs were collected, and strong IL-6 expression was confirmed. In hAMSCs, the treatment of tumor necrosis factor-α or IL-1β drastically induced IL-6 production, followed by the phosphorylation of IKKs. A novel IKKβ inhibitor, IMD-0560, almost completely inhibited IL-6 production from hAMSCs. Using an experimental lipopolysaccharide-induced PTD mouse model, the therapeutic potential of IMD-0560 was examined. IMD-0560 was delivered vaginally 4 hours before lipopolysaccharide administration. Mice in the IMD-0560 (30 mg/kg, twice a day) group had a significantly lower rate of PTD [10 of 22 (45%)] without any apparent adverse events on the mice and their pups. In uteri collected from mice, IMD-0560 inhibited not only IL-6 production but also production of related cytokines, such as keratinocyte-derived protein chemokine/CXCL1, macrophage inflammatory protein-2/CXCL2, and monocyte chemoattractant protein-1/chemokine ligand 2. Targeting IKKβ signaling shows promising effects through the suppression of these cytokines and can be explored as a future option for the prevention of PTD.

Purpose: Ovarian cancer is the most lethal gynecologic malignancy. Platinum-paclitaxel combination chemotherapy is the current standard postoperative care for patients with advanced ovarian cancer. Despite developments in current chemotherapy regimens in the last decades, overall 5-year survival rate for patients with advanced disease remains approximately 30%, mainly due to primary or acquired drug resistance. Therefore, overcoming chemoresistance remains the great challenge in ovarian cancer management. However, mechanisms underlying chemoresistance to paclitaxel have not been fully understood. The aim of this study is to identify key miRNAs which regulate paclitaxel resistance in ovarian cancer and to pursue those potential as therapeutic targets. Methods: Using two serous ovarian cancer cell lines, SKVO3ip1 and HeyA8, paclitaxel resistant ovarian cancer cell lines were established by a continuous exposure of paclitaxel. MiRNA PCR arrays were performed and miR-194 was found to be one of down-regulated miRNAs in paclitaxel-resistant cell lines. The effect of miR-194 on paclitaxel resistance was assessed by transducing the precursor miRNA into ovarian cancer cells. In silico analysis revealed MDM-2 is one possible putative target of miR-194 and it was assessed using luciferase reporter assay. Since MDM-2 is one of key regulator of P53 ubiquitination, the effect of miR-194 on cell cycle in ovarian cancer cell was assessed. To analyze the impact of miR-194 and MDM-2 expression on patient survival, Kaplan-Meier and log-rank methods were used using The Cancer Genome Atlas (TCGA) dataset. Results: While IC50 values of SKOV3ip1 and HeyA8 were 5.0 nM and 3.5 nM respectively, those of paclitaxel resistant cell lines, named as SKOV3ip1-TR and HeyA8-TR, were both over 300 nM. In both resistant cell lines, miR-194 was found to be down-regulated compared with their parental cell lines. Up-regulation of miR-194 sensitized resistant cells to paclitaxel. Conversely, its downregulation induced paclitaxel resistance in parental cells. Luciferase reporter assay revealed that miR-194 directly suppressed MDM-2 transcriptional activity. In resistant cell lines, MDM-2 was up-regulated compared with their parental cell lines. MiR-194 induced P21 upregulation and G1 phase arrest in resistant cell lines through the downregulation of MDM-2. In TCGA dataset, high MDM-2 expression was associated with shorter progression-free survival of ovarian cancer patients (concrete data, p=0.046). Conclusion: miR-194 acts as a tumor suppressor miRNA through the sensitization to paclitaxel and can be considered as a therapeutic option to overcome paclitaxel-resistant in ovarian cancer patients. Citation Format: Koji Nakamura, Kenjiro Sawada, Akihiko Yoshimura, Erica Nakatsuka, Yasuto Kinose, Seiji Mabuchi, Tadashi Kimura. MiR-194 modulates paclitaxel resistance in ovarian cancer cells through the regulation of MDM-2 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3436. doi:10.1158/1538-7445.AM2017-3436

Objective: There is a critical need for improved diagnostic markers to detect ovarian high grade serous cancer (HGSC). microRNAs (miRNAs) stably exist in circulating blood, reflecting tissue or organ conditions and present in circulating microvesicles such as exosomes. We aimed to identify which miRNAs are highly produced from HGSCs and analyze whether serum miRNA can discriminate patients with HGSC from healthy controls. Methods: Secreted exosomes from ovarian cancer cell lines were collected and exosomal miRNAs extracted. miRNA microarray was performed and several elevated miRNAs specific to ovarian cancer cells were picked up. Among these, we focused on miR-99a. Sera were collected from 15 patients with HGSC (stage III and IV) and 10 healthy controls. Expression level of miR-99a was determined by miRNA RT-qPCR. Results: miRNA microarray revealed several miRNAs were highly expressed in exosomes in patients with HGSC. In patients, serum miR-99a levels were significantly increased (4.8 fold) compared with those of healthy controls. ROC analysis showed that at the cut-off of 1.4, the sensitivity and specificity were 73 % and 80 % respectively for detecting HGSC (AUC = 0.85). Conclusion: Exosomal miRNAs can be detected in sera of patients with HGSC and have the potential to predict ovarian cancer. Citation Format: Akihiko Yoshimura, Kenjiro Sawada, Koji Nakamura, Yasuto Kinose, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Elevated level of serum miR-99a is correlated with serous epithelial ovarian cancer and can be a potential biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4655. doi:10.1158/1538-7445.AM2017-4655