Published OnlineFirst January 4, 2019; DOI: 10.1158/1078-0432.CCR-18-2363 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Exosome-Transmitted PSMA3 and PSMA3-AS1 Promote Proteasome Inhibitor Resistance in Multiple Myeloma Hongxia Xu1,2, Huiying Han1, Sha Song1, Nengjun Yi3, Chen'ao Qian4, Yingchun Qiu1, Wenqi Zhou1, Yating Hong5, Wenyue Zhuang6, Zhengyi Li7, Bingzong Li5, and Wenzhuo Zhuang1 Abstract Purpose: How exosomal RNAs released within the bone Results: We identified that PSMA3 and PSMA3-AS1 in MSCs marrow microenvironment affect proteasome inhibitors' (PI) could be packaged into exosomes and transferred to myeloma sensitivity of multiple myeloma is currently unknown. This cells, thus promoting PI resistance. PSMA3-AS1 could form an study aims to evaluate which exosomal RNAs are involved and RNA duplex with pre-PSMA3, which transcriptionally promot- by which molecular mechanisms they exert this function. ed PSMA3 expression by increasing its stability. In xenograft Experimental Design: Exosomes were characterized by models, intravenously administered siPSMA3-AS1 was found dynamic light scattering, transmission electron microscopy, and to be effective in increasing carfilzomib sensitivity. Moreover, Western blot analysis. Coculture experiments were performed to plasma circulating exosomal PSMA3 and PSMA3-AS1 derived assess exosomal RNAs transferring from mesenchymal stem from patients with multiple myeloma were significantly asso- cells (MSC) to multiple myeloma cells. The role of PSMA3-AS1 ciated with PFS and OS. in PI sensitivity was further evaluated in vivo. To determine the Conclusions: This study suggested a unique role of exoso- prognostic significance of circulating exosomal PSMA3 and mal PSMA3 and PSMA3-AS1 in transmitting PI resistance from PSMA3-AS1, a cohort of patients with newly diagnosed multiple MSCs to multiple myeloma cells, through a novel exosomal myeloma was enrolled to study. Cox regression models and PSMA3-AS1/PSMA3 signaling pathway. Exosomal PSMA3 and Kaplan–Meier curves were used to analyze progression-free PSMA3-AS1 might act as promising therapeutic targets for PI survival (PFS) and overall survival (OS). resistance and prognostic predictors for clinical response. mechanisms underlying proteasome inhibitor (PI) resistance Introduction have been studied: genetic mutations, gene expression signatures, copy number abnormalities, and bone marrow microenviron- Multiple myeloma is a malignancy of the plasma cell charac- ment (4). Nevertheless, multiple myeloma is a multifaceted terized by proliferation of plasma cell clones (1). The proteasome disease related to genetic, epigenetic, and chromosomal altera- inhibitor bortezomib has shown promise in the treatment of tions, and the mechanisms underlying PI resistance in multiple multiple myeloma (2). However, its therapeutic activity was myeloma remain elusive. severely impeded by bortezomib resistance (3). Multifactorial The proteasome is a 26S enzyme complex that consists of a 19S regulatory complex and a core 20S catalytic complex. The 20S complex is composed of two rings of seven a subunits and two 1 Department of Cell Biology, School of Biology & Basic Medical Sciences, rings of seven b subunits (5). PIs could slightly inhibit the peptidyl Soochow University, Suzhou, China. 2Xiangyang No.1 People's Hospital, Hubei 3 glutamyl–like activity at the b1 subunit (PSMB6) and mainly University of Medicine, Xiangyang, China. Department of Biostatistics, Univer- b sity of Alabama at Birmingham, Birmingham, Alabama. 4Department of Bioin- target the chymotrypsin-like (ChT-L) activity of 5 subunit formatics, School of Biology & Basic Medical Sciences, Soochow University, (PSMB5; refs. 6, 7). The mutations in b5 subunit or the increased Suzhou, China. 5Department of Haematology, the Second Affiliated Hospital of expression of b5 subunit was detected in bortezomib-resistant Soochow University, Suzhou, China. 6Department of Molecular Biology, School hematologic tumor cell lines (8). However, the b5 mutations and 7 of Laboratory Medicine of Beihua University, Jilin, China. Department of Clinical overexpression are absent in clinical samples derived from borte- Examination Basis, Laboratory Academy of Jilin Medical College, Jilin, China. zomib-resistant patients (9, 10). Therefore, the b5 subunit dysre- Note: Supplementary data for this article are available at Clinical Cancer gulation may not be uniquely responsible for PI resistance. It is Research Online (http://clincancerres.aacrjournals.org/). conceivable that other mechanisms could be associated with PI H. Xu and H. Han contributed equally to this article. resistance. Corresponding Authors: Wenzhuo Zhuang, Soochow University, Ren Ai Road The bone marrow microenvironment activates many pathways 199, Suzhou 215123, China. Phone: 8651-2627-23369; Fax: 8651-2658-80103; leading to disease progression. MSCs support tumor cell growth, E-mail: [email protected]; and Bingzong Li, The Second Affiliated metastasis, and evasion of the immune system (11). The inter- Hospital of Soochow University, San Xiang Road 1055, Suzhou 215006, China. action between MSCs and myeloma cells performs a critical role in Phone: 8651-2677-84069; E-mail: [email protected] multiple myeloma pathogenesis, progress, and chemotherapy doi: 10.1158/1078-0432.CCR-18-2363 resistance (12, 13). These findings indicate a need for developing Ó2019 American Association for Cancer Research. novel drugs to counteract these cancer–stroma interactions. www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst January 4, 2019; DOI: 10.1158/1078-0432.CCR-18-2363 Xu et al. Translational Relevance Materials and Methods Patient samples PI resistance is a major challenge for multiple myeloma. Samples were taken from patients with newly diagnosed mul- The bone marrow microenvironment promotes myeloma cell tiple myeloma who were enrolled to the study. The study was survival and proliferation through the interaction between conducted in accordance with the Declaration of Helsinki. the MSCs and multiple myeloma cells. The bone marrow Informed written consent was obtained from each subject or each microenvironment promotes the interactions between the subject's guardian. The human investigations were performed MSCs and multiple myeloma cells that permit multiple mye- after approval by an institutional review board and in accordance loma to survive and proliferate. Here we discovered that with an assurance filed with. The detailed clinical features of these exosome-mediated transfer of PSMA3 (encodes proteasome patients are listed in Supplementary Table S1. Progression-free subunit a7) and lncPSMA3-AS1 from MSCs to multiple mye- survival (PFS) was defined as the time between initiation of loma cells contributed to PI resistance. PSMA3-AS1, which bortezomib therapy and the date of first evidence of progressive. arises from the antisense strand of PSMA3, was highly Patients who were progression-free at the time of analysis were expressed in myeloma cells (r-MM) and MSCs (r-MSCs) censored using the time between initial treatment and last follow- derived from bortezomib-resistant patients. As a pair of pro- up. More details of the patient samples are available in Supple- tein-coding/noncoding antisense transcripts, PSMA3 and mentary Data. PSMA3-AS1 were disordered concurrently and correlated pos- itively in multiple myeloma cells, driving PI sensitivity in multiple myeloma. These results provided in vitro and in vivo Plasmid construction evidence that interference with exosomal RNAs served as a Full-length cDNA (PMSA3 or PSMA3-AS1) was synthesized Eco Bam promising approach to overcome PI resistance in multiple and subcloned into the RI and HI sites of Lenti-CMV-puro fi myeloma. Moreover, our data indicated that circulating exo- vector. The constructs were veri ed by DNA sequencing. somal PSMA3 and PSMA3-AS1 could develop the prognostic fi stratification of patients with multiple myeloma, in addition Exosome puri cation to the international staging system. Exosomes secreted by cultured cell lines and MSCs were iso- lated using ExoQuick solution (System Biosciences). Exosomes in the plasma were isolated using ExoQuick-LP solution (System Biosciences). The detailed procedures of exosome isolation are Exosomes act as key communicators between tumor microen- available in the Supplementary Data. The exosome pellet was vironment and cancer cells (14, 15). The interaction between isolated and the protein content of the exosome suspension was MSCs and multiple myeloma cells plays a crucial role in multiple analyzed by BCA Protein Assay Kit (Beyotime Biotechnology). myeloma pathogenesis and drug resistance by exosomes (16, 17). Recently, lncRNAs have been reported to exist in exosomes Dynamic light scattering analysis (18, 19). LncRNAs are transcribed from thousands of loci in Dynamic light scattering (DLS) analysis was performed to mammalian genomes and function in a wide range of biological characterize and quantify the particle size distribution of exo- processes (20, 21). In cancers, lncRNAs are emerging important somes (Zetasizer Nano S90). regulators in oncogenic pathways (22, 23). However, the role of lncRNAs in the multiple myeloma pathogenesis and progression In vivo xenograft studies has not been fully elucidated. In particular, how exosomal The animal experiments were performed after acquired per- lncRNAs derived from the bone marrow microenvironment