Encoding gene RAB3B exists in linear chromosomal and circular extrachromosomal DNA and contributes to cisplatin resistance of hypopharyngeal squamous cell carcinoma via inducing autophagy

www.nature.com/cddis ARTICLE OPEN Encoding gene RAB3B exists in linear chromosomal and circular extrachromosomal DNA and contributes to cisplatin resistance of hypopharyngeal squamous cell carcinoma via inducing autophagy Changwei Lin 1,2 , Yifei Chen3, Fan Zhang1, Baiying Liu1, Canbin Xie1 and Yexun Song 4,5 ✉ 1234567890();,: © The Author(s) 2022 Cisplatin (DDP) resistance is an important factor that decreases the effect of chemotherapy, thus leading to local recurrence and lymph node metastasis of hypopharyngeal squamous cell carcinoma (HSCC). We aimed to explore the role and mechanism of extrachromosomal circular DNA (eccDNA) in the DDP resistance of HSCC. In our research, the HSCC cell line FaDu and the DDPresistant cell line FaDu/DDP were used as subjects. eccDNA sequencing and whole transcriptome sequencing were conducted, followed by a combined analysis of the two sequencing profiles. Outward PCR, inward PCR and Sanger sequencing were used to verify sequences of the eccDNAs. Bioinformatics analysis based on TCGA/GEO was performed in addition to plasmid transfection, RNA interference, qRT-PCR and Western blot experiments to verify the expression level of RAB3B amplified from eccDNA. mRFPGFP-LC3 adenoviral particle transfection and transmission electron microscopy were used to detect autophagic flux. Finally, we evaluated the role of RAB3B in FaDu/DDP cells and patient-derived organoids. Our results showed that we purified and sequenced more than 10 thousand eccDNAs from the two cell lines, and the size of the eccDNAs was distributed from 0.01 kb to 1000 kb. The combined analysis between eccDNA and transcript sequencing indicated that there were some highly expressed genes that were completely or partially transcribed from related sequences of eccDNAs and not from genome linear DNA. We further screened and verified the encoding gene RAB3B using full-length sequences that might be amplified from eccDNA [chr1circle 46219-52682 kb]. Finally, we confirmed that RAB3B could promote DDP resistance in HSCC by inducing autophagy. The eccDNA might play significant roles in DDP resistance in HSCC by amplifying related functional genes. Further study is needed to explore the novel mechanisms of eccDNA in the drug resistance of HSCC. Cell Death and Disease (2022)13:171 ; https://doi.org/10.1038/s41419-022-04627-w INTRODUCTION Extrachromosomal circles of DNA (eccDNA) are various sizes of circular DNA ranging from hundreds to thousands of base pairs (bp) that are found within a preparation of mammalian DNA [1] and have been proven to participate in physiological [2] or pathological [3] processes in a special way. Considering that oncogene amplification is one of the common types of cancer mutations, an increasing number of studies have focused on eccDNAs and found that they [4] play a similar role in oncogene amplification as chromosomes [5]. For example, Wu S et al. proved that oncogenes encoded on eccDNAs are among the most highly expressed genes in the transcriptome of tumors, thus linking increased copy numbers with high transcription levels [6]. Zhu Y et al. found that there are a number of eccDNAs in prostate cancer that can function as mobile transcriptional enhancers to promote tumor progression [7]. Furthermore, eccDNAs have also been frequently found in 29 cancers, including head and neck squamous cell carcinoma (HNSCC) [8]. Hypopharyngeal squamous cell carcinoma (HSCC) has the worst prognosis among HNSCCs and is rapidly rising in incidence [9]. Approximately 70–85% of HSCCs are diagnosed at stage III or IV, and the 5-year overall survival rate is less than 45% [10]. Cisplatin (DDP)-based chemotherapy is an important treatment to improve the prognosis of HNSCC [11]. However, the high incidence of drug resistance has become an important factor affecting the effect of chemotherapy, thus leading to local recurrence and consequent lymph node metastasis [12]. Therefore, identifying the cellular and molecular mechanisms of DDP resistance is helpful for developing 1 Department of Gastrointestinal Surgery, The Third Xiangya Hospital of Central South University, Changsha 410013 Hunan Province, China. 2School of Life Sciences, Central South University, Changsha 410078 Hunan Province, China. 3Department of Otolaryngology-Head Neck Surgery, The Fourth Hospital of Changsha (The Changsha Affiliated Hospital of Hunan Normal University), Hunan Normal University, Changsha 410006 Hunan Province, China. 4Department of Otolaryngology-Head Neck Surgery, The Third Xiangya Hospital of Central South University, Changsha 410013 Hunan Province, China. 5Department of Otolaryngology-Head Neck Surgery, The Xiangya Hospital of Central South University, Changsha 410008 Hunan Province, China. ✉email: Edited by Dr Angelo Peschiaroli Received: 28 August 2021 Revised: 23 January 2022 Accepted: 7 February 2022 Official journal of CDDpress C. Lin et al. 2 effective therapies. Mounting evidence shows that eccDNAs also play an important role in drug resistance. For example, D. H. Koo found that eccDNA-based amplification and transmission are key to obtaining herbicide resistance in Amaranthus palmeri [13]. In addition, eccDNA encompasses the pteridine reductase-1 gene (PTR1) responsible for MTX resistance [14]. These results suggest that oncogene amplification of eccDNAs is an important method by which tumor cells develop chemoresistance. However, the expression profiles of eccDNA in HSCC are still unclear, and whether there is a specific eccDNA devoted to DDP resistance in HSCC has not been well studied. Here, we first investigated the different expression profiles of eccDNAs between FaDu/DDP cells and FaDu cells. Then, we confirmed that eccDNAs are derived from every human chromosome with sequences from all known types of genomic structures, including genes and intergenic and repetitive regions, thus revealing that eccDNAs are common mutational elements in HSCC. Finally, we identified that eccDNA could promote FaDu/ DDP cell resistance to DDP by amplifying the RAB3B gene, which can enhance the autophagy of FaDu/DDP cells. Our discovery suggests that producing more eccDNAs is an important method used by FaDu cells to induce and promote DDP resistance. Understanding the tolerance mechanisms may provide new strategies for HSCC therapy. METHODS AND MATERIALS eccDNA enrichment for circle-Seq eccDNA enrichment, purification and sequencing were performed as described in a previous study [2]. Briefly, cells were suspended in L1 solution (Plasmid Mini AX; A&A Biotechnology) and supplemented with Proteinase K (Thermo Fisher) before incubation overnight at 50 °C with agitation. After lysis, the samples were treated with an alkaline solution, followed by the precipitation of proteins and separation of chromosomal DNA from circular DNA through an ion exchange membrane column (Plasmid Mini AX; A&A Biotechnology). Column-purified DNA was treated with FastDigest MssI (Thermo Scientific) to remove mitochondrial circular DNA a (...truncated)