Yutaka Nezu

Clinical significance of circulating miR-25-3p as a novel diagnostic and prognostic biomarker in osteosarcoma

Background Emerging evidence has suggested that circulating microRNAs (miRNAs) in body fluids have novel diagnostic and prognostic significance for patients with malignant diseases. The lack of useful biomarkers is a crucial problem of bone and soft tissue sarcomas; therefore, we investigated the circulating miRNA signature and its clinical relevance in osteosarcoma. Methods Global miRNA profiling was performed using patient serum collected from a discovery cohort of osteosarcoma patients and controls and cell culture media. The secretion of the detected miRNAs from osteosarcoma cells and clinical relevance of serum miRNA levels were evaluated using in vitro and in vivo models and a validation patient cohort. Results Discovery screening identified 236 serum miRNAs that were highly expressed in osteosarcoma patients compared with controls, and eight among these were also identified in the cell culture media. Upregulated expression levels of miR-17-5p and miR-25-3p were identified in osteosarcoma cells, and these were abundantly secreted into the culture media in tumor-derived exosomes. Serum miR-25-3p levels were significantly higher in osteosarcoma patients than in control individuals in the validation cohort, with favorable sensitivity and specificity compared with serum alkaline phosphatase. Furthermore, serum miR-25-3p levels at diagnosis were correlated with patient prognosis and reflected tumor burden in both in vivo models and patients; these associations were more sensitive than those of serum alkaline phosphatase. Conclusions Serum-based circulating miR-25-3p may serve as a non-invasive blood-based biomarker for tumor monitoring and prognostic prediction in osteosarcoma patients.

Circulating MicroRNA-92b-3p as a Novel Biomarker for Monitoring of Synovial Sarcoma

The lack of useful biomarkers is a crucial problem for patients with soft tissue sarcomas (STSs). Emerging evidence has suggested that circulating microRNAs (miRNAs) in body fluids have novel impact as biomarkers for patients with malignant diseases, but their significance in synovial sarcoma (SS) patients remains unknown. Initial global miRNA screening using SS patient serum and SS cell culture media identified a signature of four upregulated miRNAs. Among these candidates, miR-92b-3p secretion from SS cells was confirmed, which was embedded within tumour-derived exosomes rather than argonaute-2. Animal experiments revealed a close correlation between serum miR-92b-3p levels and tumour dynamics. Clinical relevance was validated in two independent clinical cohorts, and we subsequently identified that serum miR-92b-3p levels were significantly higher in SS patients in comparison to that in healthy individuals. Moreover, serum miR-92b-3p was robust in discriminating patients with SS from the other STS patients and reflected tumour burden in SS patients. Overall, liquid biopsy using serum miR-92b-3p expression levels may represent a novel approach for monitoring tumour dynamics of SS.

miR-135b, a key regulator of malignancy, is linked to poor prognosis in human myxoid liposarcoma.

Myxoid/round cell (RC) liposarcomas (MLS) were originally classified into two distinct populations based on histological differences; a myxoid component and a RC component. It is notable that, depending on an increase of the RC component, the prognosis significantly differs. Hence, the RC component is associated with metastasis and poor prognosis. However, the molecular mechanisms that contribute to the malignancy of the RC component still remain largely unknown. Here, we report microRNA-135b (miR-135b), a key regulator of the malignancy, highly expressed in the RC component and promoting MLS cell invasion in vitro and metastasis in vivo through the direct suppression of thrombospondin 2 (THBS2). Decreased THBS2 expression by miR-135b increases the total amount of matrix metalloproteinase 2 (MMP2) and influences cellular density and an extracellular matrix structure, thereby resulting in morphological change in tumor. The expression levels of miR-135b and THBS2 significantly correlated with a poor prognosis in MLS patients. Overall, our study reveals that the miR-135b/THBS2/MMP2 axis is tightly related to MLS pathophysiology and has an important clinical implication. This work provides noteworthy evidence for overcoming metastasis and improving patient outcomes, and sheds light on miR-135b and THBS2 as novel molecular targets for diagnosis and therapy in MLS.

RPN2 gene confers osteosarcoma cell malignant phenotypes and determines clinical prognosis

Drug resistance and metastasis are lethal characteristics of tumors. We previously demonstrated that silencing of ribophorin II (RPN2), which is part of the N-oligosaccharyl transferase complex, efficiently induced apoptosis and reduced resistance to docetaxel in human breast cancer cells. Here, we report the clinical and functional correlations of RPN2 expression in osteosarcoma. Immunohistochemical evaluation of 35 osteosarcoma patient biopsies revealed that RPN2 was moderately to highly expressed in all specimens, and higher RPN2 mRNA expression was significantly correlated with poor prognosis. To investigate whether lethal phenotypes of osteosarcoma could be reduced by regulating the expression of RPN2, we conducted a study of RNAi-induced RPN2 knockdown in highly metastatic human osteosarcoma cells. The results indicated that RPN2 silencing reduced cell proliferation, sphere formation, cell invasion, and sensitized drug response in vitro. Mice bearing RPN2-silenced highly metastatic osteosarcoma xenografts showed reduced tumor growth and lung metastasis, and survived longer than mice bearing control tumor xenografts. Taken together, our data suggest that RPN2 silencing contributes to regulation of lethal osteosarcoma phenotypes and could be a novel target for RNAi-based therapeutics against osteosarcoma.

Circulating MicroRNAs in Sarcoma: Potential Biomarkers for Diagnosisand Targets for Therapy

The importance of microRNAs (miRNAs) in tumor biology has been recognized over the past several years. Recently, evidence of circulating miRNAs in both healthy and unhealthy individuals has been accumulated, and is accelerating their potential to transform clinical diagnostics and therapeutics. Since there is a lack of useful biomarkers for bone and soft tissue sarcomas, the discovery of novel biomarkers that can be used at early disease stages to detect tumors or predict tumor response to chemotherapy or the chance of survival is one of the most important challenges in sarcoma management. Further more highly, sensitive and specific biomarkers might help diagnostic classification, since some cases are unclassifiable using modern diagnostic modalities. In this review, we summarize the emerging evidence of circulating miRNAs in sarcoma and discuss their potential as novel biomarkers and therapeutics.

A tissue microRNA signature that predicts the prognosis of breast cancer in young women

Since breast cancers in young women are generally aggressive, young patients tend to be intensively treated with anti-cancer drugs. To optimize the strategy for treatment, particularly in young women, prognostic biomarkers are urgently required. The objective of this study was to identify a tissue microRNA (miRNA) signature that predicts prognosis in young breast cancer patients. Total RNA from 45 breast cancer patients aged <35 years was extracted from formalin-fixed paraffin-embedded (FFPE) tissues and analyzed using miRNA microarrays. Patients were categorized into two groups according to recurrence status within the 5 year period after surgery: recurrence (n = 11) and non-recurrent (n = 34). Histological parameters of hormone receptors and Ki-67 were statistically compared between the two groups. Differentially expressed miRNAs were identified, and their associations with overall survival (OS) were evaluated by log-rank test. The median observation period was 5.8 years for the recurrent group, and 9.1 years for the non-recurrent group. Nine miRNAs were significantly differentially expressed between the recurrent and non-recurrent groups. Receiver Operating Characteristic curve analysis was performed to evaluate the prediction accuracy of the identified miRNAs, and the resultant area under the curve was >0.7. Five of the miRNAs were validated by qRT-PCR, and the expression levels of three of those five (miR-183-5p, miR-194-5p, and miR-1285-5p), both alone and in combination, were associated with OS. In conclusion, we identified three candidate miRNAs that could be used separately or in combination as prognostic biomarkers in young breast cancer patients. This miRNA signature may enable selection of better treatment choices for young women with this disease.

Abstract 456: Identification of circulating tumor-derived microRNA signatures in osteosarcoma

Introduction: The lack of useful biomarkers is one of the most important clinical problems of bone and soft tissue sarcomas, although early detection of recurrent or metastatic disease and early decision making according to tumor response to chemotherapy is important to improve patient prognosis. The recent discovery of circulating cell-free microRNAs (miRNAs) in human blood has represented a new approach for the diagnostic screening for malignant diseases. In this study, we investigated whether the serum miRNA levels could be used as novel biomarkers for osteosarcoma (OS) patients. Methods: Global miRNA profiling was performed using the serum samples collected from OS patients, age-matched non-OS patients and healthy volunteers. The expression levels of extracted miRNA were confirmed in human OS cell lines (SaOS2, U2OS, HOS, 143B), human mesenchymal stem cell (hMSC), and their culture media. The miRNA candidates were evaluated using the serum samples of the validation set. Results: The miRNA expression profile of the serum samples of 10 OS patients, 10 age-matched non-OS patients, and 10 healthy controls identified 236 serum miRNAs to be highly expressed in the OS compared to the non-OS patients and healthy controls. Among these miRNAs, 8 miRNAs were overlapped with the secretory miRNAs in the culture medium of 7 osteosarcoma cell lines. Quantitative RT-PCR revealed that two candidates were significantly upregulated in the OS cells and culture medium, compared to MSCs. The expression of these miRNAs in the culture media from all OS cell lines increased along with culture time and tumor cell numbers, indicating that it is an important secretory miRNA derived from OS cells. The serum concentration of these miRNAs in OS patients was significantly higher than those in healthy volunteers and non-OS patients. Our ROC analyses revealed that an AUC value based on the miRNA expression was 0.868 (95% confidence interval = 0.743 to 0.993), which was was higher than that of alkaline phosphatase (ALP). Finally, the detected serum miRNA expression levels markedly decreased at the postoperative status in operative cases, while gradually decreased during neoadjuvant chemotherapy, which revealed that serum miRNA expression levels correlated with the tumor burden and drug sensitivity. Conclusions: We identified circulating tumor-derived miRNA signatures in the serum obtained from the OS patients. Furthermore, the definitive miRNA reflected tumor burden in OS patients. Evaluating the serum miRNA expression may thus have important clinical implications for risk stratification and the planning of post-therapeutic surveillance. Citation Format: Tomohiro Fujiwara, Koji Uotani, Aki Yoshida, Takuya Morita, Tadashi Komatsubara, Kazuhisa Sugiu, Toshinori Omori, Ken Takeda, Toshiyuki Kunisada, Yutaka Nezu, Akira Kawai, Hirotaka Kanzaki, Takahiro Ochiya, Toshifumi Ozaki. Identification of circulating tumor-derived microRNA signatures in osteosarcoma. [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 456.

The Biological Role and Clinical Implication of MicroRNAs in Osteosarcoma

The main causes of death in osteosarcoma (OS) patients are the development of distant metastasis and resistance to chemotherapy. Clarification of the pathophysiological molec‐ ular mechanisms that contribute to the malignant phenotype in OS and identification of a molecular target, such as a diagnostic marker, prognostic predictor, or chemosensitivity sensor, are strongly desired to develop therapeutics for OS patients. Accumulating evi‐ dence has demonstrated that microRNAs (miRNAs), small endogenous single‐stranded noncoding RNAs, play critical roles not only in biological but also pathological processes such as cancer. miRNAs can function as oncogenes or tumor‐suppressive genes depend‐ ing on the mRNA they target. They are strongly associated with OS invasion, metastasis, and chemoresistance as well as OS cancer stemness. Furthermore, miRNAs are associ‐ ated with commonly altered genes, such as TP53 and RB1. Additionally, recent global microRNA expression analyses have identified specific miRNAs correlated with the clinical stage and the response to chemotherapy. In this chapter, we summarize the cur‐ rent understanding of the pathological roles of miRNAs as well as their potential utility as OS biomarkers.

MicroRNAs in Bone and Soft Tissue Sarcomas and Their Value as Biomarkers

Abstract Bone and soft tissue sarcomas are malignant neoplasms that are histologically and genetically heterogeneous, and various subtypes of them have been identified. Evidence of microRNA (miRNA) dysregulation in bone and soft tissue sarcomas has been recently described. miRNA dysregulation that is associated with genetic abnormalities unique to the specific subtypes of sarcomas, functionally important, or correlated with clinical prognosis has been gradually identified. Furthermore, the discovery of circulating miRNAs in patient blood has accelerated interest in their potential to transform clinical applications. Considering the lack of useful bone and soft tissue sarcoma biomarkers, the discovery of miRNA dysregulation may provide a diagnostic method that can be used at early disease stages to detect tumors, predict tumor response to chemotherapy, or identify prognoses. Such interventions would address some of the most important challenges in sarcoma management. In this review, we summarize the emerging evidence of miRNA dysregulation in bone and soft tissue sarcomas and discuss their potential as novel biomarkers and therapeutics.

Abstract 455: Identification of exosomal surface marker derived from Ewing sarcoma cells

Introduction: Exosomes are small vesicles that are secreted by a multitude cell types. Recent studies have identified that circulating exosomes play an important role in tumor develompment and provide perspectives in novel diagnostic and therapeutic challenge. In this study, we investigated the surface protein on the tumor-derived exosomes by proteome analysis to utilize for a new biomarker of Ewing sarcoma. Methods: Exosomes was collected from the culture medium of two Ewing sarcoma cells (SKES1, RDES) and human mesenchymal stem cells (hMSC) by gel filtration (EVsecond®) and ultracentrifugation. Exosomes extracted from each cell line were checked by transmission electron microscopy (TEM) and Nanosight®, a nanoparticle-tracking system. Global analysis of exosomal surface proteins were performed by liquid chromatography and mass spectrometry (LC/MS), then ES specific protein candidates were validated by immunoblot and ExoScreen for exosomes extracted by ultracentrifugation, and enzyme-linked immunosorbent assay (ELISA) for exosomes extracted by EVsecond®. Results: Extracellular vesicles from ES cells were obtained by ultracentrifugation and EVSecond®. TEM and Nanosight® showed that the size of extracted exosomes derived from ES cells were 40-100 nm in diameter. Proteomic analysis detected 287 proteins from SKES-derived exosomes, 256 proteins from RDES-derived exosomes, and 133 proteins from hMSC-derived exosomes by LC/MS. We identified CD99 as entrapped protein on the exosomes derived from ES cells. For validation study, we performed immunoblot analysis that CD9, CD63, CD81 and CD99 were detected in ES-derived exosomes. Furthermore, immunogold TEM detected CD81 and CD99 on the exosomal surface. Next, we performed ELISA to detect the CD9 and CD99 on the surface of ES-derived exosomes purified by EVsecond®. Finally, we identified that ExoScreen, that has been clinically useful, was able to detect ES-derived exosomes, which expressed CD9, CD63 and CD99 on their surface. Discussion and conclusion: Although the EWS-FLI1 oncogenic activity is increased by CD99 in ES, no study, to date, has reported that CD99 is on the tumor-derived exosomes and circulating in the patients’ serum. This study demonstrates that exosomes derived from ES cells can be detected by the CD99 surface marker, providing the possibility of the exosome as a novel biomarker for liquid biopsy. Citation Format: Aki Yoshida, Tomohiro Fujiwara, Koji Uotani, Yusuke Yoshioka, Koji Ueda, Takuya Morita, Ken Takeda, Toshiyuki Kunisada, Yutaka Nezu, Hiroyuki Kawashima, Takahiro Ochiya, Toshifumi Ozaki. Identification of exosomal surface marker derived from Ewing sarcoma 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 455.