Estrogen induces genomic instability in high-risk HPV-infected cervix and promotes the carcinogenesis of cervical adenocarcinoma.

High-risk human papillomavirus (HPV) infection is the major cause of cervical cancer. However, the factors that modulate the process from infection to carcinogenesis are poorly understood. Although cervical cancer is clinically considered an estrogen-independent tumor, the role of estrogen in cervical cancer, particularly cervical adenocarcinoma, remains controversial. In this study, we showed that estrogen/GPR30 signaling induced genomic instability, which leads to carcinogenesis in high-risk HPV-infected endocervical columnar cell lines. The expression of estrogen receptors in a normal cervix was confirmed through immunohistochemical analysis which showed that G protein-coupled receptor 30 (GPR30) was predominantly expressed in endocervical glands and estrogen receptor-α (ERα) was expressed at higher levels in the squamous epithelium than in the cervical gland. E2 increased the proliferation of cervical cell lines, particularly normal endocervical columnar and adenocarcinoma cells via GPR30 rather than ERα, and increased the accumulation of DNA double-strand breaks (DSBs) in high-risk HPV-E6-expressing cells. The increase in DSBs was caused by the impairment of Rad 51 and accumulation of topoisomerase-2-DNA complexes under HPV-E6 expression. In addition, chromosomal aberrations increased in cells with E2-induced DSB accumulation. Collectively, we conclude that E2 exposure in high-risk HPV-infected cervical cells increases DSBs, leading to genomic instability and thus carcinogenesis via GPR30.

Clinical trial assessing the safety of edoxaban with concomitant chemotherapy in patients with gynecological cancer-associated thrombosis (EGCAT study).

BACKGROUND: Gynecological cancer is one of the highest risk factors for cancer-associated thrombosis (CAT). Although low-molecular-weight heparin (LMWH) is recommended as an anticoagulant for treating CAT, recent studies have shown that direct oral anticoagulants (DOACs) are an acceptable alternative. Patients with cancer require a series of chemotherapies concomitantly with DOAC administration; however, the extent to which these drugs influence DOAC blood concentrations is unknown. In this study, we measured the plasma concentration of edoxaban during chemotherapy for gynecological cancers to determine its safety. METHODS: Patients histologically diagnosed with ovarian or uterine corpus cancer and CAT were recruited after primary surgery and before the initiation of postoperative adjuvant chemotherapy, including paclitaxel. Patients were administered edoxaban (30 or 60 mg) orally for CAT. The plasma concentrations of edoxaban and active factor Xa were determined and their percentage change before and after chemotherapy was calculated. Additionally, blood coagulation tests were analyzed. RESULTS: Sixteen patients with gynecological cancer (12 with ovarian cancer and 4 with uterine corpus cancer) were enrolled. Among these, 15 samples were collected one day after chemotherapy initiation. During chemotherapy, the trough concentration of edoxaban changed from 17.6 ± 10.6 to 20.0 ± 15.6 ng/ml, and the mean percentage change in edoxaban concentration was 14.5%. Therefore, the trough concentrations of edoxaban, which represent excretion capacity, were not significantly increased by chemotherapy with paclitaxel. The area under the plasma edoxaban concentration-time curve and the active factor Xa concentration were also unaffected. CONCLUSION: Patients with CAT and ovarian or uterine corpus cancer administered edoxaban orally showed no significant increase in the trough concentration of edoxaban while undergoing chemotherapy. This suggests the safety of edoxaban use during the treatment of gynecological cancers. TRIAL REGISTRATION: EGCAT study; Japan Registry of Clinical Trials, jRCTs051190024.

A single institution's experience with minimally invasive surgery for ovarian cancer, and a systematic meta-analysis of the literature.

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.

Safety and efficacy of Ninjin'yoeito along with iron supplementation therapy for preoperative anemia, fatigue, and anxiety in patients with gynecological disease: an open-label, single-center, randomized phase-II trial.

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 < 11.0 g/dL Hemoglobin [Hb]) were randomly assigned (1:1) into the iron supplementation and NYT groups. Patients of the iron supplementation group and the NYT group received 100 mg/day iron supplementation with and without NYT (7.5 g/day) for at least 10 days before surgery. The primary endpoint was improvement in Hb levels before and after treatment, and Cancer Fatigue Scale (CFS) and Visual Analogue Scale for Anxiety (VAS-A) scores between groups. Statistical analyses were performed with Wilcoxon signed rank test, Wilcoxon rank sum test, and Fisher's exact test as appropriate. RESULTS: Forty 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 fatigue and anxiety in addition to the recovery from anemia. TRIAL REGISTRATION: jRCT1051190012 (28/April/2019, retrospectively registered).

Postoperative lymphocyst formation after pelvic lymphadenectomy for gynecologic cancers: comparison between laparoscopy and laparotomy.

PURPOSE: The goal of this study was to evaluate, using definitive diagnostic criteria, the incidence of lymphocyst formation following pelvic lymphadenectomy for gynecological cancer, and to compare rates between the approaches of laparoscopy and laparotomy. METHODS: We retrospectively reviewed the medical records of all patients who underwent pelvic lymphadenectomy for cervical or endometrial cancer between March of 2010 and March of 2016. We defined a lymphocyst as a circumscribed collection of fluid within the pelvic cavity, with a diameter of 2 cm or more, as diagnosed with ultrasound or computed tomography. RESULTS: During the six-year observational period, a pelvic lymphadenectomy was conducted in 196 women with clinical stage I uterine cancer; 90 cases underwent laparoscopy, 106 underwent laparotomy. The minimally invasive laparoscopic group had a lower estimated blood loss (p < 0.01), shorter hospital stay (p < 0.01). Lymphocysts were observed in 14.4% (13/90) of the laparoscopy cases, and in 15.1% (16/106) of the laparotomy cases which means no significant difference of lymphocyst (p = 1.00). The median size of symptomatic lymphocyst was significantly larger in laparotomy group than in laparoscopy group (4.8 cm v.s. 2.8 cm, median) (p = 0.04). Symptomatic lymphocysts were more common in laparotomy [7/90 (7.8%) vs 14/106 (13.2%) (p = 0.253)]. CONCLUSIONS: In a retrospective analysis with a strict diagnostic criteria, we could find no statistical difference in lymphocyst occurrence between laparoscopy and laparotomy. The median size of the lymphocyst was bigger and lymphocyst was likely to be symptomatic in the laparotomy group.

Ubiquitin specific peptidase 32 acts as an oncogene in epithelial ovarian cancer by deubiquitylating farnesyl-diphosphate farnesyltransferase 1.

Epithelial ovarian cancer (EOC) is the seventh most common cancer worldwide and the deadliest gynecological malignancy because of its aggressiveness and high recurrence rate. To discover new therapeutic targets for EOC, we combined public EOC microarray datasets with our previous in vivo shRNA screening dataset. The top-ranked gene ubiquitin specific peptidase 32 (USP32), coding a deubiquitinating enzyme, is a component of the ubiquitin proteasome system. Clinically, USP32 is expressed in primary ovarian cancer, especially in metastatic peritoneal tumors, and negatively impacts the survival outcome. USP32 regulates proliferative and epithelial mesenchymal transition capacities that are associated with EOC progression. Proteomic analysis identified farnesyl-diphosphate farnesyltransferase 1 (FDFT1) as a novel substrate of USP32 that is an enzyme in the mevalonate pathway, essentially associated with cell proliferation and stemness. USP32 and FDFT1 expression was higher in tumor spheres than in adherent cells. Inhibition of USP32, FDFT1, or mevalonate pathway considerably suppressed tumor sphere formation, which was restored by adding squalene, a downstream product of FDFT1. These findings suggested that USP32-FDFT1 axis contributes to EOC progression, and could be novel therapeutic targets for EOC treatment.

Exploring the potential of engineered exosomes as delivery systems for tumor-suppressor microRNA replacement therapy in ovarian cancer.

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.

The role of myeloid-derived suppressor cells in increasing cancer stem-like cells and promoting PD-L1 expression in epithelial ovarian cancer.

The aim of this study was to investigate the role of myeloid-derived suppressor cells (MDSC) in the induction of cancer stem-like cells (CSC) and programmed death ligand 1 (PD-L1) expression in ovarian cancer. CSC were defined as tumor cells expressing high levels of aldehyde dehydrogenase 1 (ALDH 1). We inoculated G-CSF-expressing or Mock-expressing ovarian cancer cells into mice, and the frequencies of MDSC and CSC in tumors of these models were compared by flow cytometry. To directly demonstrate the role of MDSC in the induction of CSC and the increase in PD-L1 expression, we performed in vitro co-culture. MDSC and CSC (ALDH-high cells) were more frequently observed in G-CSF-expressing cell-derived tumors than in Mock-expressing cell-derived tumors. Co-culture experiments revealed that MDSC increased the number of CSC via the production of PGE2. Moreover, PGE2 produced by MDSC increased tumor PD-L1 expression via the mammalian target of rapamycin (mTOR) pathway in ovarian cancer cells. In an in vitro experiment in which ovarian cancer cells were co-cultured with MDSC, higher expression of PD-L1 was observed in CSC than in non-CSC (ALDH-low cells). Furthermore, by immunofluorescence staining, we found that PD-L1 was co-expressed with ALDH1 in in vivo mouse models. In conclusion, PGE2 produced by MDSC increases the stem cell-like properties and tumor PD-L1 expression in epithelial ovarian cancer. Depleting MDSC may be therapeutically effective against ovarian cancer by reducing the number of CSC and tumor PD-L1 expression.

In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a deadly cancer, and its prognosis has not been changed significantly during several decades. To seek new therapeutic targets for EOC, we performed an in vivo dropout screen in human tumor xenografts using a pooled shRNA library targeting thousands of druggable genes. Then, in follow-up studies, we performed a second screen using a genome-wide CRISPR/Cas9 library. These screens identified 10 high-confidence drug targets that included well-known oncogenes such as ERBB2 and RAF1, and novel oncogenes, notably KPNB1, which we investigated further. Genetic and pharmacological inhibition showed that KPNB1 exerts its antitumor effects through multiphase cell cycle arrest and apoptosis induction. Mechanistically, proteomic studies revealed that KPNB1 acts as a master regulator of cell cycle-related proteins, including p21, p27, and APC/C. Clinically, EOC patients with higher expression levels of KPNB1 showed earlier recurrence and worse prognosis than those with lower expression levels of KPNB1. Interestingly, ivermectin, a Food and Drug Administration-approved antiparasitic drug, showed KPNB1-dependent antitumor effects on EOC, serving as an alternative therapeutic toward EOC patients through drug repositioning. Last, we found that the combination of ivermectin and paclitaxel produces a stronger antitumor effect on EOC both in vitro and in vivo than either drug alone. Our studies have thus identified a combinatorial therapy for EOC, in addition to a plethora of potential drug targets.

Lurbinectedin (PM01183), a selective inhibitor of active transcription, effectively eliminates both cancer cells and cancer stem cells in preclinical models of uterine cervical cancer.

Objective The objective of this study was to evaluate the antitumor effects of lurbinectedin on cervical cancer with a special focus on its effects on cancer stem cells (CSCs). Methods Using two cervical cell lines (ME180 and CaSki cells), the antitumor effects of lurbinectedin were assessed in vitro using the MTS assay and colony formation assay. The growth inhibitory effects of paclitaxel and cisplatin were also evaluated as controls. By employing ALDH1 activity as a marker of CSCs, the antitumor effects of lurbinectedin on cervical CSCs and non-CSCs were individually evaluated. Finally, we investigated the mechanisms by which lurbinectedin eliminated cervical CSCs. Results Lurbinectedin had significant antitumor activity toward cervical cancer cells at low nanomolar concentrations in vitro. Mouse xenografts of cervical cancer revealed that lurbinectedin significantly inhibits tumor growth. The growth-inhibitory effect of lurbinectedin was greater than that of cisplatin and paclitaxel. ALDH-high CSCs were observed in both cervical cancer cell lines (4.4% and 2.4% in ME180 and CaSki cells, respectively). Lurbinectedin downregulated stem cell-related gene expression (Oct4, Nanog, and SOX2), inhibited HDAC1 activity, and effectively eliminated ALDH-high CSCs. Conclusions Lurbinectedin is highly effective on uterine cervical cancer because it eliminates CSCs, and lurbinectedin is a promising agent to overcome platinum resistance in cervical cancer.

Exosomal miR-99a-5p is elevated in sera of ovarian cancer patients and promotes cancer cell invasion by increasing fibronectin and vitronectin expression in neighboring peritoneal mesothelial cells.

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.

[A Case of Retroperitoneal Metastasis of Breast Cancer with Effective Insertion of Plural Stents].

A 61-year-old woman was referred to our hospital because of epigastric pain during chemotherapy for breast cancer recurrence. She was diagnosed with left breast cancer and underwent mastectomy with axillary lymph node dissection 13 years previously. The postoperative pathological examination result was luminal invasive lobular carcinoma. Hydronephrosis appeared after 1 month, which we diagnosed as ureter stenosis caused by radiation therapy for the lumbar metastasis and thus inserted an ureteralstent. After 1 month, computed tomography demonstrated expansion of the tumor into the stomach and duodenum. Upper gastrointestinalendoscopy demonstrated stenosis of the duodenum with intact mucosa. We diagnosed the duodenalstenosis due to the retroperitonealmetastasis of breast cancer and inserted duodenal, biliary, and pancreatic duct stents. The plural stent insertion was effective, and chemotherapy was administered with enforcement possibility for 7 months afterward.

Targeting Inhibitor of κB Kinase ß Prevents Inflammation-Induced Preterm Delivery by Inhibiting IL-6 Production from Amniotic Cells.

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 ß (IKKß) 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-1ß drastically induced IL-6 production, followed by the phosphorylation of IKKs. A novel IKKß 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 IKKß signaling shows promising effects through the suppression of these cytokines and can be explored as a future option for the prevention of PTD.

Predictors for recurrent/persistent high-grade intraepithelial lesions and cervical stenosis after therapeutic conization: a retrospective analysis of 522 cases.

OBJECTIVE: The objective of the study is to identify the risk factors associated with recurrent/persistent disease and cervical stenosis after conization. METHODS: Five hundred twenty-two (522) cases of high-grade intraepithelial lesions treated by conization were retrospectively reviewed. Risk factors associated with recurrent/persistent disease were analyzed by univariate and multivariate analysis using a Cox hazard regression model. Factors that could potentially affect the risk of cervical stenosis were examined by univariate and by multivariate analysis using the χ 2 test and logistic regression, respectively. RESULTS: Recurrent/persistent diseases and cervical stenosis occurred in 4.8% and 5.4% of the patients, respectively. Age ≥46 years [hazard ratio (HR) 3.6, 95% CI 1.36-10.3, p = 0.0092] and surgical margin involvement (HR 13.44, 95% CI 5.07-46.37, p < 0.001) were independent predictors for recurrent/persistent diseases. Age ≥46 years [odds ratio (OR) 4.27, 95% CI 1.88-10.07, p < 0.001] and shortened interval after childbirth to conization (within 12 months) (OR 5.42, 95% CI 1.42-17.41, p = 0.016) were independent risk factors for cervical stenosis. CONCLUSION: Elderly patients (aged ≥46 years) are at high risk of recurrence and cervical stenosis, which may lead to unsatisfactory follow-up. Subsequent hysterectomy is beneficial to patients aged 46 or older with surgical margin involvement. Clinicians should recognize the possibility of cervical stenosis after conization during the breastfeeding period, leading to secondary infertility or hematometra.

The Novel IκB Kinase ß Inhibitor, IMD-0560, Has Potent Therapeutic Efficacy in Ovarian Cancer Xenograft Model Mice.

OBJECTIVE: Aberrant activation of nuclear factor-kappa ß (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. METHODS: NF-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. RESULTS: The 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. CONCLUSIONS: IMD-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.

Analysis of acquired resistance to metronomic oral topotecan chemotherapy plus pazopanib after prolonged preclinical potent responsiveness in advanced ovarian cancer.

An alternative or follow-up adjunct to conventional maximum tolerated dose (MTD) chemotherapy now in advanced phase III clinical trial assessment is metronomic chemotherapy--the close regular administration of low doses of drug with no prolonged breaks. A number of preclinical studies have shown metronomic chemotherapy can cause long term survival of mice with advanced cancer, including metastatic disease, in the absence of overt toxicity, especially when combined with targeted antiangiogenic drugs. However, similar to MTD chemotherapy acquired resistance eventually develops, the basis of which is unknown. Using a preclinical model of advanced human ovarian (SKOV-3-13) cancer in SCID mice, we show that acquired resistance can develop after terminating prolonged (over 3 months) successful therapy utilizing daily oral metronomic topotecan plus pazopanib, an oral antiangiogenic tyrosine kinase inhibitor (TKI). Two resistant sublines were isolated from a single mouse, one from a solid tumor (called KH092-7SD, referred to as 7SD) and another from ascites tumor cells (called KH092-7AS, referred to as 7AS). Using these sublines we show acquired resistance to the combination treatment is due to tumor cell alterations that confer relative refractoriness to topotecan. The resistant phenotype is heritable, associated with reduced cellular uptake of topotecan and could not be reversed by switching to MTD topotecan or to another topoisomerase-1 inhibitor, CPT-11, given either in a metronomic or MTD manner nor switching to another antiangiogenic drug, e.g. the anti-VEGFR-2 antibody, DC101, or another TKI, sunitinib. Thus, in this case cross resistance seems to exist between MTD and metronomic topotecan, the basis of which is unknown. However, gene expression profiling revealed several potential genes that are stably upregulated in the resistant lines, that previously have been implicated in resistance to various chemotherapy drugs, and which, therefore, may contribute to the drug resistant phenotype.

miR-92a inhibits peritoneal dissemination of ovarian cancer cells by inhibiting integrin α5 expression.

Ovarian cancer is characterized by widespread peritoneal dissemination and ascites and has a cure rate of only 30%. As has been previously reported, integrin α5 plays a key role in the peritoneal dissemination of ovarian cancer. Our aim was to identify a new miRNA that regulates integrin α5 expression and analyze the therapeutic potential of targeting this miRNA. By using an IHC analysis, we proved that high integrin α5 expression correlates with a poor prognosis in Japanese patients with International Federation of Gynecology and Obstetrics stage III ovarian cancer. Based on an miRNA algorithm search, we identified hsa-mir-92a (miR-92a) as a candidate. The level of miR-92a expression was significantly inversely correlated with ITGA5 expression in various cancer cells. Transfection of precursor miR-92a reduced integrin α5 expression in ovarian cancer cells, which was accompanied by the inhibition of cancer cell adhesion, invasion, and proliferation. miR-92a overexpression reduced the luciferase activity of the ITGA5 3'-untranslated region, suggesting that ITGA5 mRNA is a direct target of miR-92a. In in vivo ovarian cancer xenografts, the enforced expression of miR-92a in HeyA-8 cells suppressed peritoneal dissemination. Although we still have a long way to go before an effective and nontoxic miRNA-based cancer therapy can be introduced into the clinic, the inhibition of integrin α5 expression by targeting miR-92a needs to be explored further for future applications in ovarian cancer treatment.

Patient-Derived Exosomes as siRNA Carriers in Ovarian Cancer Treatment.

RNA interference is a powerful gene-silencing tool with potential clinical applications. However, its therapeutic use is challenging because suitable carriers are unavailable. Exosomes are stable small endogenous vesicles that can transport functional molecules to target cells, making them ideal small interfering RNA (siRNA) carriers. Herein, we elucidated the therapeutic potential of patient-derived exosomes as an siRNA carrier for ovarian cancer (OC) treatment. The exosomes were extracted from the culture medium of primary fibroblasts collected from the omentum of patients with OC during surgery. MET proto-oncogene, receptor tyrosine kinase (MET) was selected for gene silencing, c-Met siRNAs were synthesized and loaded into the exosomes (Met-siExosomes) via electroporation, and the treatment effect of the Met-siExosomes was assessed in vitro and in vivo. The Met-siExosomes downregulated the c-Met protein levels and inhibited OC cell proliferation, migration, and invasion. In xenograft experiments using SKOV3-13 and ES-2 cells, Met-siExosomes were selectively extracted from peritoneally disseminated tumors. Intraperitoneal treatment suppressed the c-Met downstream targets in cancer cells and prolonged mouse survival. The synthesized siRNAs were successfully and selectively delivered via the exosomes to intraperitoneally disseminated tumors. As patients with OC routinely undergo omentectomy and abundant fibroblasts can be easily collected from the omentum, patient-derived exosomes may represent a promising therapeutic siRNA carrier to treat OC.

Continuous Administration of Anti-VEGFA Antibody Upregulates PAI-1 Secretion from Ovarian Cancer Cells via miR-143-3p Downregulation.

Although bevacizumab (BEV) plays a key role in ovarian cancer treatment, BEV resistance is often observed in clinical settings. This study aimed to identify the genes responsible for BEV resistance. C57BL/6 mice inoculated with ID-8 murine ovarian cancer cells were treated with anti-VEGFA antibody or IgG (control) twice weekly for 4 weeks. The mice were sacrificed, then, RNA was extracted from the disseminated tumors. qRT-PCR assays were performed to identify angiogenesis-related genes and miRNAs that were altered by anti-VEGFA treatment. SERPINE1/PAI-1 was found to be upregulated during BEV treatment. Therefore, we focused on miRNAs to elucidate the mechanism underlying the upregulation of PAI-1 during BEV treatment. Kaplan-Meier plotter analysis revealed that higher expression levels of SERPINE1/PAI-1 were associated with poor prognoses among BEV-treated patients, suggesting that SERPINE1/PAI may be involved in the acquisition of BEV resistance. miRNA microarray analysis followed by in silico and functional assays revealed that miR-143-3p targeted SERPINE1 and negatively regulated PAI-1 expression. The transfection of miR-143-3p suppressed PAI-1 secretion from ovarian cancer cells and inhibited in vitro angiogenesis in HUVECs. Next, miR-143-3p-overexpressing ES2 cells were intraperitoneally injected into BALB/c nude mice. ES2-miR-143-3p cells downregulated PAI-1 production, attenuated angiogenesis, and significantly inhibited intraperitoneal tumor growth following treatment with anti-VEGFA antibody. Continuous anti-VEGFA treatment downregulated miR-143-3p expression, which upregulated PAI-1 and activated an alternative angiogenic pathway in ovarian cancer. In conclusion, the substitution of this miRNA during BEV treatment may help overcome BEV resistance, and this may be used as a novel treatment strategy in clinical settings. IMPLICATIONS: Continuous administration of VEGFA antibody upregulates SERPINE1/PAI-1 expression via the downregulation of miR-143-3p, which contributes to acquiring bevacizumab resistance in ovarian cancer.

Benefits of Laparoscopic Surgery for Bleeding Events in Patients with Implantable Left Ventricular Assist Devices during Antithrombotic Therapy.

Hemorrhagic ovarian cysts (HOCs), a common gynecological disease causing intraabdominal bleeding, can be life threatening in patients undergoing antithrombotic therapy, especially those with left ventricular assist device (LVAD) implantation under strong antithrombotic therapy. We encountered three postLVAD implantation cases with intraabdominal bleeding due to suspected HOCs, which required surgery for hemostasis. Such patients are not only at a higher risk of bleeding but also have restrictions in available surgical incision sites to avoid damaging the LVAD driveline located underneath the abdominal wall. Laparoscopic surgery, which can be performed through minute incisions with flexible site selection, may benefit intraabdominal hemorrhage patients with LVADs.