Erika Nakatsuka

Clinical relevance of circulating cell-free microRNAs in ovarian cancer

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.

Exosomes Promote Ovarian Cancer Cell Invasion through Transfer of CD44 to Peritoneal Mesothelial Cells

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.

Interleukin 6 Receptor Is an Independent Prognostic Factor and a Potential Therapeutic Target of Ovarian Cancer

Ovarian cancer remains the most lethal gynecologic cancer and new targeted molecular therapies against this miserable disease continue to be challenging. In this study, we analyzed the expressional patterns of Interleukin-6 (IL-6) and its receptor (IL-6R) expression in ovarian cancer tissues, evaluated the impact of these expressions on clinical outcomes of patients, and found that a high-level of IL-6R expression but not IL-6 expression in cancer cells is an independent prognostic factor. In in vitro analyses using ovarian cell lines, while six (RMUG-S, RMG-1, OVISE, A2780, SKOV3ip1 and OVCAR-3) of seven overexpressed IL-6R compared with a primary normal ovarian surface epithelium, only two (RMG-1, OVISE) of seven cell lines overexpressed IL-6, suggesting that IL-6/IL-6R signaling exerts in a paracrine manner in certain types of ovarian cancer cells. Ovarian cancer ascites were collected from patients, and we found that primary CD11b+CD14+ cells, which were predominantly M2-polarized macrophages, are the major source of IL-6 production in an ovarian cancer microenvironment. When CD11b+CD14+ cells were co-cultured with cancer cells, both the invasion and the proliferation of cancer cells were robustly promoted and these promotions were almost completely inhibited by pretreatment with anti-IL-6R antibody (tocilizumab). The data presented herein suggest a rationale for anti-IL-6/IL-6R therapy to suppress the peritoneal spread of ovarian cancer, and represent evidence of the therapeutic potential of anti-IL-6R therapy for ovarian cancer treatment.

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

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.

Abstract 5060: Exosome transfer from ovarian cancer cells to mesothelial cells promotes cell invasion by upregulating MMP-9 secretion and increasing clearance of mesothelial cells

Ovarian cancer cells metastasize to organs in the abdominal cavity, such as the omentum, which are covered by a single layer of mesothelial cells. Thus, the cell-cell connection between ovarian cancer cells and mesothelial cells is the crucial step of metastasis. Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions through transfer of proteins, mRNAs and microRNAs. However, whether exosome-mediated transfer plays any role in ovarian cancer cell invasion remains poorly understood. Thus, the aim of this study is to identify the functional role of ovarian cancer-derived exosomes during the process of ovarian cancer metastasis. Methods: Exosomes were isolated from two ovarian cancer cell lines (HeyA8 and TYK-NU) and immortalized normal ovarian epithelial cell line (IOSE) using differential centrifugation. Human peritoneal mesothelial cells (HPMCs) were isolated from normal omentum of patients undergoing gynecologic surgery. The isolation of exosomes was confirmed by electron microscope and the transfer of exosomes into HPMCs was confirmed by fluorescent-labeling exosomes. The effects of exosome transfer from ovarian cancer cells to mesothelial cells were analyzed in vitro 3D culture model, morphological assessment, Western Blotting and gelatin zymography. We found that CD44 was enriched in cancer derived exosome. Thus, gain or loss of function of CD44 was analyzed. CD44 expression of ovarian cancer omental metastasis and surrounding organs in clinical samples was assessed by immunohistochemistry. Results: Fluorescent-labeled exosomes were evidently transferred into HPMCs. Exosome-treated HPMCs changed in cellular morphology to spindle phenotype. Ovarian cancer invasion was significantly promoted in the presence of exosome-treated HPMCs. In exosome-treated HPMCs, MMP-9 secretion was up-regulated and E-cadherin expression was down-regulated. The clearance of mesothelial barrier was increased in exosome-treated HPMC monolayer. By Western Blotting, we confirmed CD44 was enriched in exosomes and exosome-treated HPMCs display high-level of CD44. When CD44 expression was knocked down by siRNA in ovarian cancer cells, these effects to HPMCs were significantly attenuated. In contrast, the enforced expression of CD44 into HPMCs promoted cancer invasion by secreting MMP-9 and increasing mesothelial clearance. In human omentum with microscopic metastasis of ovarian cancer, positive CD44 expression was confirmed in a mesothelial cell layer when cancer cells are attaching onto it, while CD44 expression was generally negative in normal mesothelial cells. Conclusion: Herein, we revealed that ovarian cancer-derived exosomes transfer CD44 to HPMCs, which can facilitate ovarian cancer invasion by up-regulating of MMP-9 secretion and increasing clearance of HPMCs. Citation Format: Koji Nakamura, Kenjiro Sawada, Yasuto Kinose, Akihiko Yoshimura, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Exosome transfer from ovarian cancer cells to mesothelial cells promotes cell invasion by upregulating MMP-9 secretion and increasing clearance of mesothelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5060. doi:10.1158/1538-7445.AM2015-5060

Exosomal miR-1290 is a potential biomarker of high-grade serous ovarian carcinoma and can discriminate patients from those with malignancies of other histological types

BackgroundmicroRNAs (miRNAs) stably exist in circulating blood encapsulated in extracellular vesicles such as exosomes; therefore, serum miRNAs have the potential to serve as novel cancer biomarkers. New diagnostic markers to detect high grade serous ovarian cancer (HGSOC) are urgently needed. The aim of this study was to identify miRNAs specific to HGSOC and analyze whether serum miRNA can discriminate HGSOC patients from healthy controls or patients with ovarian malignancies of other histological types.MethodsExosomes from ovarian cancer cell lines were collected and exosomal miRNAs extracted. miRNA microarray analysis revealed several elevated miRNAs specific to HGSOC. Among these, we focused on miR-1290. Sera from 70 ovarian cancer patients and 13 healthy controls were gathered and its expression levels detected by quantitative real-time polymerase chain reaction.ResultsIn HGSOC patients, serum miR-1290 was significantly overexpressed compared to in healthy controls (3.52 fold; P = 0.03), unlike in patients with ovarian cancers of other histological types. The relative expression of miR-1290 was higher in advanced stages of HGSOC than in early stages (4.23 vs. 1.58; P = 0.23). Its expression significantly decreased after operation (5.87 to 1.17; P < 0.01), indicating that this miRNA reflects tumor burden. A receiver operating characteristic curve analysis showed that at the cut-off of 1.20, the sensitivity and specificity were 63% and 85% respectively for discriminating patients with HGSOC (area under the curve [AUC] = 0.71) from healthy controls, and at the cut-off of 1.55, the sensitivity and specificity were 47% and 85% respectively for discriminating patients with HGSOC (AUC = 0.76) from those with malignancies of other histological types.ConclusionsSerum miR-1290 is significantly elevated in patients with HGSOC and can be used to discriminate these patients from those with malignancies of other histological types; it is a new potential diagnostic biomarker for HGSOC.

Effects of minodronate in postmenopausal women with osteoporosis who received prior treatment with raloxifene

Background In clinical practice, patients with postmenopausal osteoporosis have often shown a poor response to treatment with an antiresorptive agent for several years. The purpose of this study was to investigate the efficacy of switching raloxifene with minodronate in patients who responded poorly to the treatment of postmenopausal osteoporosis with raloxifene. Patients and methods This observational study was conducted based on a single-arm, non-randomized, open-label design and was approved by the institute’s institutional review board. Postmenopausal women with osteoporosis who became unresponsive in terms of bone mineral density (BMD) after being administered raloxifene for two or more years were enrolled. Patients were treated with 1 mg minodronate daily or 50 mg minodronate monthly. Changes in BMD and serum bone turnover markers were monitored at baseline, 6, 12, and 24 months after switching treatment. Results Twenty-seven patients were enrolled. Two discontinued treatment because of adverse events related to the study drug. Among the remaining 25 patients, lumbar BMD significantly increased by 3.67%, 5.08%, and 6.97% at 6, 12, and 24 months, respectively, and femoral neck BMD increased by 1.63%, 2.18%, and 3.85% at 6, 12, and 24 months, respectively. Serum bone-specific alkaline phosphatase showed a significant reduction of 30.35% from the baseline (p<0.0001) within the first 6 months, suggesting a stronger antiresorptive effect of minodronate. Serum N-terminal telopeptide of type I collagen showed a tendency to decrease. Conclusion Switching raloxifene with minodronate is effective in poor responders of osteoporosis treatment and should be considered as one of the treatment options for osteoporosis.

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

Objective Aberrant 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. 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.

Abstract 1579: Exosomes promote ovarian cancer invasion through CD-44 transfer to mesothelial cells

Purpose: The peritoneum and organs in the peritoneal cavity are covered by a single layer of mesothelial cells. Therefore, ovarian cancer cells, which metastasize within the peritoneal cavity, directly encounter mesothelial cells as the initial step of metastasis. This contact has been found to involve cell-cell communication that affects cancer progression. Possible actors in this cell-cell communication are exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, and microRNAs. Here, we aim to identify the functional roles of ovarian cancer-derived exosomes in this metastatic process. Methods: Exosomes were isolated from two ovarian cancer cell lines (HeyA8 and TYK-NU) and immortalized normal ovarian epithelial cell line (IOSE) using differential centrifugation. Human peritoneal mesothelial cells (HPMCs) were isolated from normal omentum of patients undergoing gynecologic surgery. The isolation of exosomes was confirmed by electron microscope, nanoparticle tracking analysis, Western blotting and electrophoresis of RNA. The transfer of exosomes into HPMCs was confirmed by fluorescent-labeled exosomes. The effects of exosome transfer from ovarian cancer cells to mesothelial cells in cancer invasion were analyzed in vitro 3D culture model, morphological assessment, Western blotting and gelatin zymography. CD-44 was enriched in cancer derived exosome. Thus, gain or loss of function of CD-44 was analyzed. CD-44 expression in ovarian cancer omental metastasis and surrounding organs was assessed by immunohistochemistry using clinical samples. Results: Fluorescent-labeled exosomes were evidently transferred into HPMCs. Exosome-treated HPMCs changed in cellular morphology to spindle phenotype. Ovarian cancer invasion was significantly promoted in the presence of exosome-treated HPMCs. In exosome-treated HPMCs, MMP-9 secretion was up-regulated and E-cadherin expression down-regulated. The clearance of mesothelial barrier was increased in exosome-treated HPMC monolayer. CD-44 was enriched in cancer-derived exosomes and exosome-treated HPMCs display high-level of CD-44. When CD-44 expression was knocked down by siRNA in ovarian cancer cells, these effects to HPMCs were significantly attenuated. In contrast, the enforced expression of CD-44 into HPMCs promoted cancer invasion. In human omentum with microscopic metastasis of ovarian cancer, positive CD-44 expression was confirmed in a mesothelial cell layer when cancer cells are attaching onto it, while CD-44 expression was generally negative in normal mesothelial cells. Conclusion: Ovarian cancer-derived exosomes transfer CD-44 to HPMCs, which can facilitate ovarian cancer invasion by up-regulating of MMP-9 secretion and increasing mesothelial clearance. Citation Format: Koji Nakamura, Kenjiro Sawada, Yasuto Kinose, Akihiko Yoshimura, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Exosomes promote ovarian cancer invasion through CD-44 transfer to mesothelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1579.

Targeting Inhibitor of κB Kinase β 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 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.