Yasuto Kinose

The Role of MicroRNAs in Ovarian Cancer

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.

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.

Targeting interleukin-6 receptor inhibits preterm delivery induced by inflammation

Intrauterine infection is still a common trigger of preterm delivery (PTD) and also a determinant risk factor for the subsequent development of neurodevelopmental abnormalities in neonates. In this study, we examined the expressional pattern of various inflammatory cytokines such as interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in placentae complicated with severe chorioamnionitis (CAM) and found that IL-6 is mainly expressed in macrophages in villous mesenchyme by immunohistochemical analysis with anti-CD-68 antibody. Using an experimental lipopolysaccharide (LPS)-induced PTD model, the therapeutic potential of targeting this cytokine was investigated. Anti-IL-6 receptor antibody (MR16-1) was delivered 6 h before LPS treatment. Mice in the MR16-1 group had a significantly lower rate of PTD (17%) than in the controls (53%, P = 0.026). As a result, MR16-1 treatment significantly prolonged the gestational period (control; 18.4 ± 1.7d, MR16-1; 19.8 ± 1.5d, P = 0.007) without any apparent adverse events on the mice and their pups. In primary human amniotic epithelial cells, pretreatment with a humanized anti-human IL-6 receptor antibody, tocilizumab, significantly inhibited the production of prostaglandin E2 induced by IL-6. In conclusion, IL-6 was strongly expressed mainly in macrophages in villous mesenchyme in placentae complicated with CAM. Anti-IL-6R antibody significantly decreased the rate of PTD in LPS-induced inflammatory model in mice, and inhibited PGE2 production from human primary amniotic epithelial cells. Targeting IL-6 signaling could be a promising option for the prevention of PTD and needs to be further explored for future clinical application.

Comparison of the prognoses of FIGO stage I to stage II adenosquamous carcinoma and adenocarcinoma of the uterine cervix treated with radical hysterectomy.

Objectives To evaluate the significance of adenosquamous carcinoma (ASC) compared with adenocarcinoma (AC) in the survival of surgically treated early-stage cervical cancer. Methods We retrospectively reviewed the medical records of 163 patients with International Federation of Gynecology and Obstetrics stage IA2 to stage IIB cervical cancer who had been treated with radical hysterectomy with or without adjuvant radiotherapy between January 1998 and December 2008. The patients were classified according to the following: (1) histological subtype (ASC group or AC group) and (2) pathological risk factors (low-risk or intermediate/high-risk group). Survival was evaluated using the Kaplan-Meier method and compared using the log-rank test. Multivariate analysis of progression-free survival (PFS) was performed using the Cox proportional hazards regression model to investigate the prognostic significance of histological subtype. Results Clinicopathological characteristics were similar between the ASC and AC histology groups. Patients with the ASC histology displayed a PFS rate similar to that of the patients with the AC histology in both the low-risk and intermediate/high-risk groups. Neither the recurrence rate nor the pattern of recurrence differed between the ASC group and the AC group. Univariate analysis revealed that patients with pelvic lymph node metastasis and parametrial invasion achieved significantly shorter PFS than those without these risk factors. Conclusions Characteristics of the patients and the tumors as well as survival outcomes of ASC were comparable to adenocarcinoma of early-stage uterine cervix treated with radical hysterectomy. Our results in part support that the management of ASC could be the same as the one of AC of the uterine cervix.

IKKβ Regulates VEGF Expression and Is a Potential Therapeutic Target for Ovarian Cancer as an Antiangiogenic Treatment.

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.

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

Abstract 4386: Identification of microRNA which regulates paclitaxel resistance of ovarian cancer cells - a potential of miR-194 by attenuating paclitaxel resistance through the down-regulation of oncogene BMI-1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Ovarian cancer remains a deadly disease. Despite developments in current chemotherapy, overall survival rate for patients with advanced disease remains approximately 30%, mainly due to primary or acquired drug resistance. Therefore, overcoming chemoresistance is one of the greatest challenges in ovarian cancer management. However, mechanisms underlying chemoresistance to paclitaxel, a key drug, have not been fully understood. MicroRNAs (miRNAs) are non-coding small RNAs and more than 50% of miRNA genes are located in cancer-associated genomic regions, suggesting that miRNAs are deeply involved in the pathogenesis of cancers, including acquiring chemoresistance. In this study, we attempted to identify miRNAs that regulate paclitaxel resistance and to analyze the therapeutic potential of targeting these. Methods: Two ovarian cancer cell lines, SKOV3ip1 and HeyA-8, were continuously exposed by gradually increasing concentrations of paclitaxel and finally, paclitaxel resistant cell lines, SKOV3ip1-TR and HeyA8-TR were established. MicroRNA PCR arrays were performed and miRNA expressional patterns were compared with those of parental cells. miR-194 was found to be one of down-regulated miRNAs in both resistant cell lines. The effect of miR-194 was assessed by the enforced expression of precursor miRNA. In silico analyses found that Bmi-1 is a possible target gene. miR-194 expression was examined by quantitative RT-PCR and BMI expression by Western blotting. In order to investigate whether miR-194 directly targets the 3′-UTR of Bmi-1 mRNA, a luciferase reporter assay was performed. The stably miR-194 expressing HeyA8-TR cells were constructed and injected subcutaneously onto female nude mice. Two weeks after the inoculation, paclitaxel was treated to mice and the effect of paclitaxel was assessed by measuring tumor volumes. Results: IC50 values of parentally paclitaxel sensitive cell lines, SKOV3ip1 and HeyA-8, were 2nM and 19nM, respectively, while those of SKOV3ip1-TR and HeyA8-TR were both over 300nM. The enforced expression of miR-194 attenuated paclitaxel resistance significantly. While miR-194 expression was down-regulated in SKOV3ip1-TR and HeyA8-TR cells compared parental cells, BMI-1 expression was highly expressed in these cell lines. Transfection of precursor miR-194 into resistant cells reduced BMI-1 expression. In a luciferase reporter assay, miR-194 directly suppressed BMI-1 transcriptional activity. In a xenograft model, the transduction of miR-194 enhanced the effect of paclitaxel and significantly shrunk the tumor volumes. Conclusion: MiR-194 attenuated paclitaxel resistance by inhibiting BMI-1 expression. The overcome of chemoresistace is a critical issue for ovarian cancer treatment and herein we suggest that miR-194 is a potential therapeutic target. Citation Format: Koji Nakamura, Kenjiro Sawada, Yasuto Kinose, Kae Hashimoto, Seiji Mabuchi, Tadashi Kimura. Identification of microRNA which regulates paclitaxel resistance of ovarian cancer cells - a potential of miR-194 by attenuating paclitaxel resistance through the down-regulation of oncogene BMI-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4386. doi:10.1158/1538-7445.AM2014-4386