Joe Hasei

Dual Programmed Cell Death Pathways Induced by p53 Transactivation Overcome Resistance to Oncolytic Adenovirus in Human Osteosarcoma Cells

Tumor suppressor p53 is a multifunctional transcription factor that regulates diverse cell fates, including apoptosis and autophagy in tumor biology. p53 overexpression enhances the antitumor activity of oncolytic adenoviruses; however, the molecular mechanism of this occurrence remains unclear. We previously developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301, that kills human osteosarcoma cells, but some human osteosarcoma cells were OBP-301–resistant. In this study, we investigated the antitumor activity of a p53-expressing oncolytic adenovirus, OBP-702, and the molecular mechanism of the p53-mediated cell death pathway in OBP-301–resistant human osteosarcoma cells. The cytopathic activity of OBP-702 was examined in OBP-301–sensitive (U2OS and HOS) and OBP-301–resistant (SaOS-2 and MNNG/HOS) human osteosarcoma cells. The molecular mechanism in the OBP-702–mediated induction of two cell death pathways, apoptosis and autophagy, was investigated in OBP-301–resistant osteosarcoma cells. The antitumor effect of OBP-702 was further assessed using an orthotopic OBP-301–resistant MNNG/HOS osteosarcoma xenograft tumor model. OBP-702 suppressed the viability of OBP-301–sensitive and -resistant osteosarcoma cells more efficiently than OBP-301 or a replication-deficient p53-expressing adenovirus (Ad-p53). OBP-702 induced more profound apoptosis and autophagy when compared with OBP-301 or Ad-p53. E1A-mediated miR-93/106b upregulation induced p21 suppression, leading to p53-mediated apoptosis and autophagy in OBP-702–infected cells. p53 overexpression enhanced adenovirus-mediated autophagy through activation of damage-regulated autophagy modulator (DRAM). Moreover, OBP-702 suppressed tumor growth in an orthotopic OBP-301–resistant MNNG/HOS xenograft tumor model. These results suggest that OBP-702–mediated p53 transactivation is a promising antitumor strategy to induce dual apoptotic and autophagic cell death pathways via regulation of miRNA and DRAM in human osteosarcoma cells. Mol Cancer Ther; 12(3); 314–25. ©2012 AACR.

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.

Ablation of MCL1 expression by virally induced microRNA-29 reverses chemoresistance in human osteosarcomas

Osteosarcoma is a rare disease diagnosed as malignant bone tumor. It is generally refractory to chemotherapy, which contributes to its poor prognosis. The reversal of chemoresistance is a major clinical challenge to improve the prognostic outcome of osteosarcoma patients. We developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301 (telomelysin) and assessed its synergistic effects with chemotherapeutic agents (cisplatin and doxorubicin) using human osteosarcoma cell lines and a xenograft tumor model. The molecular mechanism underlying the chemosensitizing effect of OBP-301 was evaluated in aspects of apoptosis induction. OBP-301 inhibits anti-apoptotic myeloid cell leukemia 1 (MCL1) expression, which in turn leads to chemosensitization in human osteosarcoma cells. The siRNA-mediated knockdown of MCL1 expression sensitized human osteosarcoma cells to common chemotherapeutic agents. We also found that upregulation of microRNA-29 targeting MCL1 via virally induced transcriptional factor E2F-1 activation was critical for the enhancement of chemotherapy-induced apoptosis in osteosarcoma cells. Telomerase-specific oncolytic adenovirus synergistically suppressed the viability of human osteosarcoma cells in combination with chemotherapeutic agents. The combination treatment also significantly inhibited tumor growth, as compared to monotherapy, in an osteosarcoma xenograft tumor model. Our data suggest that replicative virus-mediated tumor-specific MCL1 ablation may be a promising strategy to attenuate chemoresistance in osteosarcoma patients.

Favorable outcome after complete resection in elderly soft tissue sarcoma patients: Japanese Musculoskeletal Oncology Group study.

BACKGROUND The surgical management of soft tissue sarcoma (STS) in elderly patients has only been addressed in a few studies. The objective of the current study was to assess surgical outcomes in patients with STS aged 70 years and older and the association of older age with the survival after complete resection. METHODS A retrospective analysis was conducted in 158 elderly patients with localized STS who visited 11 institutions participating in Japanese Musculoskeletal Oncology Group between 1995 and 2006 and were treated by surgical resection. Univariate and multivariate analyses were performed to identify prognostic factors. RESULTS Median follow-up period was 38 months. Histologically high-grade tumors were detected in 71% of the patients. Wide resection with adequate margins was performed in 66% of the cases. Systemic chemotherapy was performed in only 5 patients. Univariate analysis identified histological grade and gender as statistically significant prognostic factors for sarcoma-specific survival. Multivariate analysis did not identify significant prognostic factors for sarcoma-specific survival, although high grade sarcoma emerged as a potentially significant prognostic factor (P = 0.050). Local recurrence was detected in 19% of the patients. Multivariate analysis of local recurrence-free survival showed that tumor site and surgical margins were statistically significant prognostic factors. CONCLUSIONS Older age was not identified as a prognostic factor for sarcoma-specific survival, which is not consistent with the findings of previous studies showing that older age was associated with decreased sarcoma-specific survival. Complete resection should be indicated and can lead to optimal treatment outcome for properly selected elderly patients.

A simple detection system for adenovirus receptor expression using a telomerase-specific replication-competent adenovirus

Adenovirus serotype 5 (Ad5) is frequently used as an effective vector for induction of therapeutic transgenes in cancer gene therapy or of tumor cell lysis in oncolytic virotherapy. Ad5 can infect target cells through binding with the coxsackie and adenovirus receptor (CAR). Thus, the infectious ability of Ad5-based vectors depends on the CAR expression level in target cells. There are conventional methods to evaluate the CAR expression level in human target cells, including flow cytometry, western blotting and immunohistochemistry. Here, we show a simple system for detection and assessment of functional CAR expression in human tumor cells, using the green fluorescent protein (GFP)-expressing telomerase-specific replication-competent adenovirus OBP-401. OBP-401 infection induced detectable GFP expression in CAR-expressing tumor cells, but not in CAR-negative tumor cells, nor in CAR-positive normal fibroblasts, 24 h after infection. OBP-401-mediated GFP expression was significantly associated with CAR expression in tumor cells. OBP-401 infection detected tumor cells with low CAR expression more efficiently than conventional methods. OBP-401 also distinguished CAR-positive tumor tissues from CAR-negative tumor and normal tissues in biopsy samples. These results suggest that GFP-expressing telomerase-specific replication-competent adenovirus is a very potent diagnostic tool for assessment of functional CAR expression in tumor cells for Ad5-based antitumor therapy.

Impact of autophagy in oncolytic adenoviral therapy for cancer

Oncolytic virotherapy has recently emerged as a promising strategy for inducing tumor-specific cell death. Adenoviruses are widely and frequently used in oncolytic virotherapy. The mechanism of oncolytic adenovirus-mediated tumor suppression involves virus-induced activation of the autophagic machinery in tumor cells. Autophagy is a cytoprotective process that produces energy via lysosomal degradation of intracellular components as a physiologic response to various stresses, including hypoxia, nutrient deprivation, and disruption of growth signaling. However, infection with oncolytic adenoviruses induces autophagy and subsequent death of tumor cells rather than enhancing their survival. In this review, we summarize the beneficial role of autophagy in oncolytic adenoviral therapy, including the roles of infection, replication, and cell lysis. Numerous factors are involved in the promotion and inhibition of oncolytic adenovirus-mediated autophagy. Furthermore, recent evidence has shown that oncolytic adenoviruses induce autophagy-related immunogenic cell death (ICD), which enhances the antitumor immune response by inducing the activation of danger signal molecules and thus represents a novel cancer immunotherapy. Understanding the precise role of oncolytic adenovirus-induced autophagy and ICD could enhance the therapeutic potential of oncolytic adenoviral therapy for treating various cancers.

Inflammation-induced miRNA-155 inhibits self-renewal of neural stem cells via suppression of CCAAT/enhancer binding protein β (C/EBPβ) expression

Intracerebral inflammation resulting from injury or disease is implicated in disruption of neural regeneration and may lead to irreversible neuronal dysfunction. Analysis of inflammation-related microRNA profiles in various tissues, including the brain, has identified miR-155 among the most prominent miRNAs linked to inflammation. Here, we hypothesize that miR-155 mediates inflammation-induced suppression of neural stem cell (NSC) self-renewal. Using primary mouse NSCs and human NSCs derived from induced pluripotent stem (iPS) cells, we demonstrate that three important genes involved in NSC self-renewal (Msi1, Hes1 and Bmi1) are suppressed by miR-155. We also demonstrate that suppression of self-renewal genes is mediated by the common transcription factor C/EBPβ, which is a direct target of miR-155. Our study describes an axis linking inflammation and miR-155 to expression of genes related to NSC self-renewal, suggesting that regulation of miR-155 may hold potential as a novel therapeutic strategy for treating neuroinflammatory diseases.

TWIST1 induces MMP3 expression through up-regulating DNA hydroxymethylation and promotes catabolic responses in human chondrocytes

The objective was to investigate the levels of TWIST1 in normal and OA cartilage and examine its role in regulating gene expression in chondrocytes. Human cartilage tissues and chondrocytes were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee arthroplasty. TWIST1 expression was increased in human OA knee cartilage compared to normal knee cartilage. TWIST1 induced matrix metalloproteinase 3 (MMP3) expression without direct binding to MMP3 promoter and increased the 5-hydroxymethylcytosine (5hmC) level at the MMP3 promoter. The effect of TWIST1 on expression of TET family (TET1, 2 and 3) was measured in stable TWIST1 transfected TC28 cells, and TET1 expression was up-regulated. TWIST1 dependent upregulation of Mmp3 expression was suppressed in Tet triple KO fibroblast derived from mouse ES cells. Increased TWIST1 expression is a feature of OA-affected cartilage. We identified a novel mechanism of catabolic reaction where TWIST1 up-regulates MMP3 expression by enriching 5hmC levels at the MMP3 promoter via TET1 induction. These findings implicate TWIST1 as an important factor regulating OA related gene expression. Clarifying epigenetic mechanisms of 5hmC induced by TWIST1 is a critical molecule to understanding OA pathogenesis.

Role of zoledronic acid in oncolytic virotherapy: promotion of antitumor effect and prevention of bone destruction

Osteosarcoma is an aggressive malignant bone tumor that causes bone destruction. Although tumor‐specific replicating oncolytic adenovirus OBP‐301 induces an antitumor effect in an osteosarcoma tumor, it cannot prevent bone destruction. Zoledronic acid (ZOL) is a clinically available agent that inhibits bone destruction. In this study, we investigated the potential of combination therapy with OBP‐301 and ZOL against osteosarcomas with bone destruction. The antitumor activity of OBP‐301 and ZOL in monotherapy or combination therapy was assessed using three human osteosarcoma cell lines (143B, MNNG/HOS, SaOS‐2). The cytotoxic effect of OBP‐301 and/or ZOL was measured by assay of cell apoptosis. The effect of OBP‐301 and ZOL on osteoclast activation was investigated. The potential of combination therapy against tumor growth and bone destruction was analyzed using an orthotopic 143B osteosarcoma xenograft tumor model. OBP‐301 and ZOL decreased the viability of human osteosarcoma cells. Combination therapy with OBP‐301 and ZOL displayed a synergistic antitumor effect, in which OBP‐301 promoted apoptosis through suppression of anti‐apoptotic myeloid cell leukemia 1 (MCL1). Combination therapy significantly inhibited tumor‐mediated osteoclast activation, tumor growth and bone destruction compared to monotherapy. These results suggest that combination therapy of OBP‐301 and ZOL suppresses osteosarcoma progression via suppression of MCL1 and osteoclast activation.

Abstract 5188: Molecular radiosensitization in soft tissue sarcomas by telomerase-specific oncolytic adenovirus

INTRODUCTION:Treatment options for soft tissue sarcoma (STS) include surgical resection and adjuvant chemotherapy and radiotherapy. Despite the development of combined modality treatments in recent years, a significant proportion of patients with sarcomas respond poorly to adjuvant therapy, leading to local recurrence or distant metastasis. Therefore, there is an urgent need to develop novel therapeutic strategies for improvement of patient prognoses. We previously reported the therapeutic potential of OBP-301, a telomerase-specific oncolytic adenoviruses, and OBP-702, an armed OBP-301 expressing the wild-type p53 tumor suppressor gene against bone and soft tissue sarcoma cells. Furthermore, the synergistic anti-tumor effect of OBP-301 in combination with radiation has been observed. While OBP-702 suppressed the viability of OBP-301-sensitive and -resistant osteosarcoma cells more efficiently, the combination effect of OBP-702 and radiotherapy has been unknown. In this study, we investigated the radiosensitizing effect of OBP-702 against human STS cells. METHODS:We used four human STS cell lines, HT1080 (fibrosarcoma), NMS-2 (malignant peripheral nerve sheath tumor), and SYO-1 (synovial sarcoma). Cells were irradiated 24 h after infection with OBP-301 and OBP-702, and cell viability was assessed by XTT assay 4 days after irradiation. Combined effect of radiation with OBP-301 and OBP-702 was analyzed with the CalcuSyn software (BioSoft). These cells were also analyzed for apoptosis and DNA damage using Western blot analysis. RESULTS:While OBP-301 and OBP-702 showed anti-tumor effect for STS cell lines respectively, HT1080 and NMS-2 were highly resistant to radiation. When combined with radiation, not only OBP-301 but also OBP-702 enhanced the inhibitory anti-tumor effect in all STS cell lines. The calculation of combination index demonstrated additive or synergistic anti-tumor effect in combination therapy. Further analysis revealed that OBP-301 and OBP-702 increased radiation-induced apoptosis in STS cells. Notably, the radio-sensitizing effect of OBP-702 was associated with increase of p53 expression and interruption of anti-apoptotic myeloid cell leukemia 1 (MCL1) expression. DISCUSSION:Our study demonstrated that OBP-702 had much stronger anti-tumor effect compared to OBP-301, and sensitized radiotherapy to various types of osteosarcoma cell lines. Currently, the clinical trial of OBP-301 is performed, and preclinically OBP-702 accumulates good therapeutic results in various tumors. Thus, OBP-702 may provide a novel treatment strategy for STSs and wide application of radiotherapy for localized as well as advanced STSs. Citation Format: Tadashi Komatsubara, Toshinori Omori, Hiroshi Tazawa, Kazuhisa Sugiu, Yusuke Mochizuki, Yasuaki Yamakawa, Shuhei Osaki, Joe Hasei, Tomohiro Fujiwara, Toshiyuki Kunisada, Yasuo Urata, Tsoshifumi Ozaki, Toshiyoshi Fujiwara. Molecular radiosensitization in soft tissue sarcomas by telomerase-specific oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5188. doi:10.1158/1538-7445.AM2017-5188