Circular RNA Circikbkb Promotes Breast Cancer Bone Metastasis
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Xu et al. Mol Cancer (2021) 20:98 https://doi.org/10.1186/s12943-021-01394-8 RESEARCH Open Access Circular RNA circIKBKB promotes breast cancer bone metastasis through sustaining NF-κB/bone remodeling factors signaling Yingru Xu1,2†, Shuxia Zhang1,2†, Xinyi Liao1,2, Man Li1,2, Suwen Chen1,2, Xincheng Li1,2, Xingui Wu1,2, Meisongzhu Yang1,2, Miaoling Tang1,2, Yameng Hu1,2, Ziwen Li1,2, Ruyuan Yu1,2, Mudan Huang1,2, Libing Song1,3 and Jun Li1,2* Abstract Background: Breast cancer (BC) has a marked tendency to spread to the bone, resulting in signifcant skeletal complications and mortality. Recently, circular RNAs (circRNAs) have been reported to contribute to cancer initiation and progression. However, the function and mechanism of circRNAs in BC bone metastasis (BC-BM) remain largely unknown. Methods: Bone-metastatic circRNAs were screened using circRNAs deep sequencing and validated using in situ hybridization in BC tissues with or without bone metastasis. The role of circIKBKB in inducing bone pre-metastatic niche formation and bone metastasis was determined using osteoclastogenesis, immunofuorescence and bone resorption pit assays. The mechanism underlying circIKBKB-mediated activation of NF-κB/bone remodeling factors signaling and EIF4A3-induced circIKBKB were investigated using RNA pull-down, luciferase reporter, chromatin isola- tion by RNA purifcation and enzyme-linked immunosorbent assays. Results: We identifed that a novel circRNA, circIKBKB, was upregulated signifcantly in bone-metastatic BC tissues. Overexpressing circIKBKB enhanced the capability of BC cells to induce formation of bone pre-metastatic niche dra- matically by promoting osteoclastogenesis in vivo and in vitro. Mechanically, circIKBKB activated NF-κB pathway via promoting IKKβ-mediated IκBα phosphorylation, inhibiting IκBα feedback loop and facilitating NF-κB to the promot- ers of multiple bone remodeling factors. Moreover, EIF4A3, acted acting as a pre-mRNA splicing factor, promoted cyclization of circIKBKB by directly binding to the circIKBKB fanking region. Importantly, treatment with inhibitor eIF4A3-IN-2 reduced circIKBKB expression and inhibited breast cancer bone metastasis efectively. Conclusion: We revealed a plausible mechanism for circIKBKB-mediated NF-κB hyperactivation in bone-metastatic BC, which might represent a potential strategy to treat breast cancer bone metastasis. Keywords: Bone metastasis, Breast cancer, circIKBKB, Bone remodeling factor, NF-κB Background Female breast cancer (BC) has become the leading cause *Correspondence: [email protected] of global cancer incidence worldwide [1]. Te majority †Yingru Xu and Shuxia Zhang contributed equally to this work. 1 Program of Cancer Research, Key Laboratory of Protein Modifcation of BC fatalities are due to complications from recurrent and Degradation and Guangzhou Institute of Oncology, Afliated or tumor metastases in distant organs [2, 3]. Tereinto, Guangzhou Women and Children’s Hospital, School of Basic Medical the most common site of BC metastasis is the bone, Sciences, Guangzhou Medical University, Guangzhou 510623, China Full list of author information is available at the end of the article occurring in 70% of patients with metastatic BC [4, 5]. © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Xu et al. Mol Cancer (2021) 20:98 Page 2 of 18 Currently, the clinical interventions for patients with BC- of multiple bone remodeling factors. Importantly, treat- bone metastasis (BC-BM) are bisphosphonates (BPs) or ment with an inhibitor of eukaryotic translation initiation denosumab (an anti-receptor activator of nuclear factor factor 4A3 (EIF4A3), the circIKBKB cyclization factor, kappa B ligand (RANKL) monoclonal antibody), which reduced circIKBKB expression and efectively inhibited disrupt the vicious cycle by targeting osteoclasts [6]. BC-BM. Taken together, our results uncover a plausible However, these drugs have no efect on the patient sur- mechanism underlying the intrinsic metastatic property vival rate and also cause severe side efects, even lead- of BC to bone and might represent an attractive thera- ing to BC metastasis to visceral organs [7–9]. Terefore, peutic target in the treatment of BC-BM. understanding the mechanisms of BC-BM is essential to develop innovative therapeutic strategies and improve of Methods patient outcomes. Patient information BC-BMs mostly are osteolytic metastases that are Tis study, which complied with all relevant ethical regu- characterized by aberrant bone destruction caused by lations for work with human participants, was conducted increased osteoclasts-mediated bone resorption in the on a total of 20 tumor-adjacent normal breast tissues and tumor-bone interface [10]. Accumulating evidence has 331 parafn-embedded BC samples, including 295 pri- proven that cancer-secreted factors play direct or indi- mary BC tissues (237 non-BM/BC and 58 BM/BC) and rect roles in the activation of osteoclasts, i.e., to absorb 36 bone-metastatic BC tissues (at bone), that were histo- the bone matrix in bone compartments, which estab- pathologically and clinically diagnosed at the Tird Afli- lishes a “bone pre-metastatic niche” to support cancer ated Hospital and Sun Yat-sen University Cancer Center bone metastasis [5, 9, 11–13]. In turn, the degraded bone from 2005 to 2018. Te study protocols were approved by matrix released various bone matrix-bound growth fac- the Institutional Research Ethics Committee of Sun Yat- tors that beneft the seeding and expansion of metastatic sen University for the use of these clinical materials for tumor cells in bone, which forming a “vicious cycle” [9]. research purposes. All Patients’ samples were obtained Hyperactivation of nuclear factor kappa B (NF-κB) according to the Declaration of Helsinki and each patient signaling has been reported to be involved in cancer signed a written informed consent for all the procedures. bone-metastasis via upregulation of multiple bone- remodeling factors, such as RANKL, parathyroid RNA sequencing of circRNA extracted from human BC hormone-related protein preproprotein (PTHrP), mac- tissues rophage colony stimulating factor (M-CSF) and granu- Te total RNA was extracted from six BC (without locyte–macrophage colony stimulating factor (GM-CSF) BM) and six BC (with BM) tissues using TRIzol reagent [14–18]. Systemically blocking NF-κB activity via IκB (Takara, Dalian, China) and further purifed by rRNA kinase (IKK) inhibitors or nondegradable NF-κB inhibi- depletion, followed by cDNA synthesis and RNA ampli- tor alpha (IκBα) dramatically inhibited osteolytic bone fcation, according to the manufacturer’s instructions. metastasis of BC in vivo [18]. However, systemic inhibi- Te RNA-seq libraries were constructed and sequenced tion of NF-κB activity might also promote tissue injury utilizing the Illumina HiSeq2500 platform (Illumina, San via inducing cell apoptosis and could even promote hepa- Diego, USA). tocarcinogenesis by inducing reactive oxygen species (ROS) accumulation and liver damage [19, 20]. Terefore, ASO‑mediated knockdown and ASO in vivo treatment investigating the mechanism of NF-κB hyperactivation in Te antisense oligonucleotides (ASOs) targeting circIK- BC would provide efective target for BC-BM treatment. BKB were obtained from RiboBio (Guangzhou, China). Circular RNAs (circRNAs) are a class of widespread Transfections with ASOs (50 nM) were performed with circular RNAs that are covalently closed single stranded Lipofectamine RNAiMAX according to the manufac- circular transcripts formed by precursor mRNA back- turer’s instructions. RNA and protein were harvested splicing or skipping events, which have no 5′ caps or 3′ for analysis 72 h after transfection. For in vivo treatment poly(A) tail [21]. Clinical evidence and animal studies experiment, 10 nmol ASOs were delivered into mouse have demonstrated that aberrant expression of circRNAs through tail vein injection twice a week. contribute to carcinogenesis and development of BC [22–24]. In the present study, we identifed a novel cir- Xenografted tumor models cRNA, circIKBKB (hsa_circ_0084100), derived from the All of the animal procedures were approved by the Sun IKBKB gene (encoding inhibitor of NF-κB kinase subu- Yat-sen University Animal Care Committee. Intracardiac 5 nit beta), which plays an important role in promotion of injections of luciferase-expressing BC cells (1 × 10 ) were osteoclastogenesis and BC-BM through inducing bone conducted in nu/nu nude mice (5 weeks old) for bone pre-metastatic