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MiR-125b Increases Nasopharyngeal Carcinoma ... ... Small Molecule Therapeutics MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance by Targeting A20/NF-kB Signaling Pathway

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  • Small Molecule Therapeutics

    MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance by Targeting A20/NF-kB Signaling Pathway Li-Na Li1,2, Ta Xiao3, Hong-Mei Yi1, Zhen Zheng1, Jia-Quan Qu1,4,Wei Huang1, Xu Ye1, Hong Yi1, Shan-Shan Lu1, Xin-Hui Li1, and Zhi-Qiang Xiao1

    Abstract

    Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment, but little is known about how miRNA regulates this phenomenon. In this study, we investigated the function and mechanism of miR-125b in NPC radioresis- tance, one of upregulated miRNAs in the radioresistant NPC cells identified by our previous microarray analysis. We observed that miR-125b was frequently upregulated in the radioresistant NPCs, and its increment was significantly correlated with NPC radio- resistance, and was an independent predictor for poor patient survival. In vitro radioresponse assays showed that miR-125b inhibitor decreased, whereas miR-125b mimic increased NPC cell radioresistance. In a mouse model, therapeutic administra- tion of miR-125b antagomir dramatically sensitized NPC xeno- grafts to irradiation.Mechanistically, we confirmed that A20was a direct target of miR-125b and found that miR-125b regulated

    NPC cell radioresponse by targeting A20/NF-kB signaling. With a combination of loss-of-function and gain-of-function approaches, we further showed that A20 overexpression decreased while A20 knockdown increased NPC cell radioresis- tance both in vitro and in vivo. Moreover, A20 was significantly downregulated while p-p65 (RelA) significantly upregulated in the radioresistant NPCs relative to radiosensitive NPCs, and miR-125b expression level was negatively associated with A20 expression level, whereas positively associated with p-p65 (RelA) level. Our data demonstrate that miR-125b and A20 are critical regulators of NPC radioresponse, and high miR-125b expression enhances NPC radioresistance through targeting A20 and then activating the NF-kB signaling pathway, highlighting the thera- peutic potential of the miR-125b/A20/NF-kB axis in clinical NPC radiosensitization. Mol Cancer Ther; 16(10); 2094–106. �2017 AACR.

    Introduction Nasopharyngeal carcinoma (NPC) is the most frequent head

    and neck tumor in southern China and Southeast Asia, which poses one of the most serious public health problems in these areas (1). Radiotherapy is the major therapeutic modality used to treat NPC. Although NPC is sensitive to radiotherapy, a major impediment to achieve long-term survival is radioresis- tance that has been linked to an increased likelihood of recur- rence and a distant metastasis (2, 3). To realize the full poten- tial of radiotherapy, it is essential to understand the molecules and signaling pathways that mediate NPC radioresistance, a

    poorly characterized phenomenon, and identify druggable targets for radiosensitization.

    MicroRNAs (miRNA), a class of endogenous noncoding RNAs, act as negative gene regulators at posttranscriptional level. MiR- NAs are believed to play fundamental roles in the human cancers and have great potential in the diagnosis and treatment of cancers (4). Regulation of tumor radiosensitivity via miRNAs-associated mechanisms has attractedmuch attention in the recent years, and several miRNAs involving in tumor radioresistance have been identified (5–12). We previously used microarrays to compare the differences of miRNAs in the NPC cell lines with different radiosensitivity and found that miR-125b is one of upregulated miRNAs in the radioresistant NPC cells (NCBI-GSE48503). How- ever, the function and mechanism of miR-125b in NPC radio- resistance need to be elucidated.

    NF-kB is an important regulator of cell proliferation and survival (13). Activation of the NF-kB signaling pathway not only plays a crucial role in the development and progression of NPC (14–16), but also confers tumor resistance to radiotherapy (17–20). Ubiquitin modification, occurring at multiple steps within the NF-kB signaling cascades, serves as a regulator in NF-kB activation (21). A growing number of proteins, such as receptor-interacting protein kinase 1 (RIP-1), TNF receptor–asso- ciated factor 2 (TRAF2), and TRAF6, in the NF-kB signal pathway have been identified to bemodified by ubiquitin (21–24). Tumor necrosis factor alpha–induced protein 3 (TNFAIP3, also known as A20), functioning as an ubiquitin-editing enzyme (22), negative- ly regulates NF-kB signaling through dual mechanisms, i.e.,

    1Research Center of Carcinogenesis and Targeted Therapy, Xiangya Hospital, Central South University, Changsha, Hunan, China. 2Department of Pathology, Changzhi Medical College, Changzhi, Shanxi, China. 3Department of Dermatol- ogy, Xiangya Hospital, Central South University, Changsha, Hunan, China. 4Department of Oncology, Qianjiang Central Hospital of Chongqing, Jishou University, Hunan, China.

    Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

    L.-N. Li, T. Xiao, and H.-M. Yi contributed equally to this article.

    Corresponding Author: Z.-Q. Xiao, Xiangya Hospital, Central South University, 87# Xiangya Road, Changsha, Hunan 410008, China. Phone: 86-731-89753378; Fax: 86-731-84327332; E-mail: [email protected]

    doi: 10.1158/1535-7163.MCT-17-0385

    �2017 American Association for Cancer Research.

    Molecular Cancer Therapeutics

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    on January 24, 2020. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

    Published OnlineFirst July 11, 2017; DOI: 10.1158/1535-7163.MCT-17-0385

    on January 24, 2020. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

    Published OnlineFirst July 11, 2017; DOI: 10.1158/1535-7163.MCT-17-0385

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  • deconjugation of K63-linked polyubiquitin chains from RIP-1 and subsequent conjugation of RIP-1 with K48-linked polyubi- quitin chains for proteasomal degradation (23, 24). A20 can also catalyze the cleavage of K63-linked ubiquitin chains and the conjugation of K48-linked polyubiquitin chains, thereby target- ing TRAF2 and TRAF6 for proteasomal degradation (25, 26). Therefore, A20 serves as a negative regulator in the NF-kB signal pathway by proteasomal degradation of its upstream signaling transducers.

    Several studies have reported that A20 is a direct target of miR-125b, and miR-125b activates the NF-kB signaling pathway by inhibiting A20 expression (27–29). Numerous studies have demonstrated that A20 is involved in the pathogenesis of various types of human tumors. In some tumor types, A20 functions as a tumor suppressor due to its genetic or epigenetic inactivation, leading to A20 downregulation (30–32). In other tumors, A20 is upregulated and acts as oncogene (33–35). However, the roles of A20 in tumor radioresistance are unclear.Moreover, it is unknown whether NF-kB signaling mediates miR-125b/A20-regulating NPC radioresistance.

    In the present study,we investigatewhether andhowmiR-125b and A20 regulate NPC radioresistance. Here, we report that miR-125b is significantly upregulated, whereas A20 is significant- ly downregulated in the radioresistant NPCs relative to radiosen- sitive ones, and both are significantly correlated with poor patient survival; miR-125b increment confers NPC cell radioresistance both in vitro and in vivo by targeting the A20/NF-kB signaling pathway; A20 decrement also confers NPC cell radioresistance both in vitro and in vivo. These results can be extrapolated to clinical cases of NPC, as miR-125b level is significantly correlated with radioresistance and the levels of A20 and p-p65 (RelA) in NPC biopsies. Our findings demonstrate that bothmiR-125b and A20 are key molecules involved in NPC radioresistance, suggest- ing that NPC patients might benefit from radiosensitization therapies directed at miR-125b and A20.

    Materials and Methods Patients and tissue samples

    One hundred and eleven NPC patients without distant metas- tasis (M0 stage) at the time of diagnosis who were treated by radical radiotherapy alone in the Affiliated Cancer Hospital of Central South University, China, between January 2006 and December 2008 were recruited in this study. The radiotherapy was administered for a total dose of 60 to 70 Gy (2 Gy/fraction, 5 days a week). The neck received 60 Gy for lymph node–negative cases and 70 Gy for lymph node–positive cases. NPC tissue biopsies were obtained at the time of diagnosis before any therapy, fixed in 4% formalin, and embedded in paraffin. We also acquired 30 cases of formalin-fixed and paraffin-embedded normal nasopharyngeal mucosa (NNM) in the same period. On the basis of the 1978 WHO classification (36), all tumors were histopathologically diagnosed as poorly differentiated squamous cell carcinomas (WHO type III). The clinical stage of the patients was classified according to the 2008 NPC staging system of China (37).

    The radiotherapy response was evaluated clinically for primary lesions based on nasopharyngeal fiberscope and MRI 1 month after the initiation of radiotherapy according to the criteria as described previously by us (38). Based on the criteria, 111 NPC patients comprised 53 radioresistant and 58 radiosensitive ones.