IRE1α-XBP1 inhibitors exerted anti-tumor activities in Ewing's sarcoma

// Yu Tanabe 1 , Yoshiyuki Suehara 1 , Shinji Kohsaka 2 , Takuo Hayashi 3 , Keisuke Akaike 1 , Kenta Mukaihara 1 , Taisei Kurihara 1 , Youngji Kim 1 , Taketo Okubo 1 , Midori Ishii 1 , Saiko Kazuno 4 , Kazuo Kaneko 1 and Tsuyoshi Saito 3 1 Department of Orthopedic Surgery, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan 2 Department of Medical Genomics, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan 3 Department of Human Pathology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan 4 Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan Correspondence to: Yoshiyuki Suehara, email: ysuehara@juntendo.ac.jp Keywords: Ewing’s sarcoma (ES); proteomics; EWS/FLI1; XBP1; unfolded protein response (UPR) Received: September 04, 2017     Accepted: February 03, 2018     Epub: February 12, 2018     Published: March 06, 2018 ABSTRACT Ewing’s sarcoma (ES) is the second-most frequent pediatric bone tumor. Chromosomal translocation t(11;22)(q24:q12) results in the formation of EWS/FLI1 gene fusion, which is detected in approximately 90% of tumors of the Ewing family. Several transcriptome studies have provided lists of genes associated with EWS/FLI1 expression. However, the protein expression profiles associated with EWS/FLI1 have yet to be elucidated. In this study, to identify the regulated proteins associated with EWS/FLI1 and therapeutic targets in ES, we conducted proteomic studies using EWS/FLI1 knockdown in four Ewing’s sarcoma cell lines and human mesenchymal stem cells (hMSCs) expressing EWS/FLI1. Isobaric tags for relative and absolute quantitation (i-TRAQ) analyses identified more than 2,000 proteins regulated by the EWS/FLI1 fusion. In addition, the network analyses identified several critical pathways, including XBP1, which was ranked the highest. XBP1 is a protein well known to play an important role in the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress through the IRE1α-XBP1 pathway. We confirmed the high mRNA expression of XBP1 (spliced XBP1 and unspliced XBPl) in surgical samples and cell lines in ES. The silencing of XBP1 significantly suppressed the cell viabilities in ES cell lines. In the inhibitor assays using IRE1α-XBP1 inhibitors, including toyocamycin, we confirmed that these agents significantly suppressed the cell viabilities, leading to apoptosis in ES cells both in vitro and in vivo . Our findings suggested that IRE1α-XBP1 inhibitors might be useful for developing novel therapeutic strategies in ES.