FULL PAPER British Journal of Cancer (2018) 118, 233–247 | doi: 10.1038/bjc.2017.385 Keywords: nasopharyngeal carcinoma; BPIFB1; LPLUNC1; VTN; VIM; metastasis BPIFB1 (LPLUNC1) inhibits migration and invasion of nasopharyngeal carcinoma by interacting with VTN and VIM Fang Wei1,2,3, Yingfen Wu2, Le Tang2,YiHe2,4, Lei Shi2, Fang Xiong1, Zhaojian Gong2, Can Guo2, Xiayu Li2,3, Qianjin Liao2,4, Wenling Zhang2, Ming Zhou1,2,3, Bo Xiang1,2,3, Xiaoling Li1,2,3, Yong Li2,5, Guiyuan Li1,2,3, Wei Xiong*,1,2,3 and Zhaoyang Zeng*,1,2,3 1The Key Laboratory of Carcinogenesis of the Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China; 2The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute, Central South University, Changsha, Hunan 410078, China; 3Hunan Key Laboratory of Nonresolving Inflammation and Cancer, Disease Genome Research Center, the Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, China; 4Hunan Key Laboratory of Translational Radiation Oncology, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410006, China and 5Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA Background: Bactericidal/Permeability-increasing-fold-containing family B member 1 (BPIFB1, previously termed LPLUNC1) is highly expressed in the nasopharynx, significantly downregulated in nasopharyngeal carcinoma (NPC), and associated with prognosis in NPC patients. Because metastasis represents the primary cause of NPC-related death, we explored the role of BPIFB1 in NPC migration and invasion. Methods: The role of BPIFB1 in NPC metastasis was investigated in vitro and in vivo. A co-immunoprecipitation assay coupled with mass spectrometry was used to identify BPIFB1-binding proteins. Additionally, western blotting, immunofluorescence, and immunohistochemistry allowed assessment of the molecular mechanisms associated with BPIFB1-specific metastatic inhibition via vitronectin (VTN) and vimentin (VIM) interactions. Results: Our results showed that BPIFB1 expression markedly inhibited NPC cell migration, invasion, and lung-metastatic abilities. Additionally, identification of two BPIFB1-interacting proteins, VTN and VIM, showed that BPIFB1 reduced VTN expression and the formation of a VTN-integrin aV complex in NPC cells, leading to inhibition of the FAK/Src/ERK signalling pathway. Moreover, BPIFB1 attenuated NPC cell migration and invasion by inhibiting VTN- or VIM-induced epithelial–mesenchymal transition. Conclusions: This study represents the first demonstration of BPIFB1 function in NPC migration, invasion, and lung metastasis. Our findings indicate that re-expression of BPIFB1 might represent a useful strategy for preventing and treating NPC. Nasopharyngeal carcinoma (NPC) constitutes a squamous cell tumourigenesis comprises a complex, multi-step, and multi- carcinoma of the nasopharynx that exhibits significant ethnic and factorial process involving abnormal activation of certain onco- regional differences and is often found to occur in Southeast Asia genes, as well as the inactivation of several tumour-suppressor and southern China (Lo et al, 2004; Chua et al, 2016; Song et al, genes (Xiong et al, 2004; Zeng et al, 2011, 2014, 2015; Gong et al, 2016; Tu et al, 2017). Previous studies showed that NPC 2016; Tang et al, 2017). NPC shows a tendency towards early *Correspondence: Professor W Xiong; E-mail: [email protected] or Professor Z Zeng; E-mail: [email protected] Received 24 June 2017; revised 21 September 2017; accepted 4 October 2017; published online 9 November 2017 r The Author(s) named above Published by Springer Nature on behalf of Cancer Research UK. 233 BRITISH JOURNAL OF CANCER BPIFB1 inhibits NPC metastasis via VTN and VIM lymphatic spread and a relatively high incidence of lymph node were supplemented with 200 ml of media containing 10% Matrigel. metastasis among head and neck cancers (Lu et al, 2013; Bo et al, After 4 days, 400 ml of media was exchanged, and images depicting 2015; He et al, 2016). Radiotherapy is the primary effective method cell morphology were obtained for 10 consecutive days. The utilised for NPC treatment and has significantly improved its local invasive degree of spheroids was classified into two types: regional control (Razak et al, 2010). However, radiation resistance, noninvasive spheroids exhibiting smooth edges with no visible local recurrence, and distant metastasis of NPC comprise the major cellular protrusions or only occasional scattered protrusions; or reasons for treatment failure (Tian et al, 2013). Therefore, a better invasive spheroids exhibiting fully scattered protrusions. understanding of the metastatic mechanisms associated with NPC Clinical NPC samples. A total of 20 NPC samples and 11 paired, would allow the development of more effective anticancer adjacent, non-tumour nasopharyngeal epithelium (NPE) tissues therapies. were collected from newly diagnosed patients with NPC at the Bactericidal/Permeability-increasing (BPI)-fold-containing Second Xiangya Hospital of Central South University (Changsha, family B member 1 (BPIFB1), also known as long-palate lung China). The study was approved by the Joint Ethics Committee of and nasal epithelium clone 1 (LPLUNC1), belongs to the BPI-fold- the Central South University Health Authority and informed containing family (Weston et al, 1999; Bingle and Craven, 2003; consent was obtained from each participant. The diagnoses of all Bingle et al, 2011). Our previous studies showed that BPIFB1 is specimens were confirmed by histopathological examination. tissue-specifically expressed in nasopharyngeal epithelia and downregulated in NPC tissues (Zhang et al, 2003). Its expression RNA extraction and quantitative real-time polymerase chain also negatively correlates with the clinical stages of NPC, reaction (qPCR). Total RNAs were extracted using TRIzol reagent suggesting that the reduction in BPIFB1 expression constitutes a (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s novel adverse prognostic factor in NPC (Liao et al, 2013). BPIFB1 protocol. The cDNA was prepared from total RNA using 5 Â All- inhibits NPC growth by downregulating mitogen-activated protein In-One RT master mix (Applied Biologic Materials, Richmond, (MAP) kinases and the cyclin D1/E2F pathways (Yang et al, 2013) Canada), after which real-time qPCR was conducted using a Mini and can also significantly suppress interleukin (IL)-6-induced Option system (Bio-Rad, Hercules, CA, USA) with SYBR Green inflammation and NPC cell proliferation by inhibiting signal (Applied Biologic Materials). The amount of each target gene was transducer and activator of transcription 3 (STAT3) activity (Liao quantified by the comparative CT method using glyceraldehyde 3- et al, 2013). However, its role in NPC metastasis has not been phosphate dehydrogenase (GAPDH) as the normalisation control. clarified. The following primers were synthesised by Life Technologies: In this study, BPIFB1 was re-expressed in NPC cell lines to BPIFB1 forward primer (50-ATC GGA TCC AGC TGA TGA AC- demonstrate its function in NPC migration and invasion in vitro 30) and reverse primer (50-AGG AGG CTG GAG TAA GCA CA- and in vivo. Additionally, a co-immunoprecipitation (co-IP) assay 30); GAPDH forward primer (50-CAA CGG ATT TGG TCG TAT combined with mass spectrometry (MS) analysis was performed to TGG-30) and reverse primer (50-TGA CGG TGC CAT GGA ATT identify BPIFB1-interacting proteins. Among the potential inter- T-30). acting proteins, roles of the cell adhesion molecule vitronectin Western blotting. Whole-cell lysates were extracted using radio- (VTN) and the intermediate filament protein vimentin (VIM) were immunoprecipitation assay buffer, and protein concentration was confirmed. Our results suggest that BPIFB1 might reduce NPC determined using the BCA protein assay kit (Pierce, Grand Island, migration and invasion by interacting with VTN or VIM and their NY, USA). Equal levels of protein from the samples were separated associated signalling pathways. by 10% sodium dodecyl sulphate polyacrylamide gel electrophor- esis (SDS–PAGE), and the separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) MATERIALS AND METHODS and blocked with Tris-buffered saline-Tween-20 with 5% dry skimmed milk for 1 h at room temperature. To assess protein Cell lines and transfection constructs. NPC cell lines 5-8F, expression, the blots were incubated with the following primary HNE2, and HONE1 were maintained in our laboratory and grown antibodies at 4 1C overnight: rabbit antibodies against His-tag, in RPMI-1640 medium (Life Technologies, Grand Island, NY, E-cadherin, ZO-1, b-catenin, N-cadherin, VIM, Snail, and Slug USA) supplemented with 10% foetal bovine serum (FBS; Life (Cell Signaling Technology, Danvers, MA, USA); and mouse Technologies) and 1% penicillin-streptomycin (Life Technologies). antibodies against Flag (Sigma-Aldrich, St Louis, MO, USA), Cells were incubated at 37 1C in a humidified atmosphere with 5% BPIFB1 (Abnova, Taipei, Taiwan); VTN and integrin aV (BD CO2. Biosciences). After washing, the blots were incubated with To generate the vector overexpressing BPIFB1, the full-length horseradish peroxidase-labelled secondary antibodies (Cell Signal- BPIFB1-coding sequence was amplified, tagged with Flag, cloned ing Technology)