G of the regional and distant spread of this disease. In fact, most treatment failure

G of the regional and distant spread of this disease. In fact, most treatment failure in cases of HNSCC is due to locoregional recurrence or distant metastatic disease [4, 5]. Thus, it is still urgent to advance our understanding of the mechanisms of the progression and metastasis of HNSCC in order to improve the survival outcome for patients with HNSCC. The transcription factor AP-1 (activator protein 1), one of the major effectors activating gene transcription, is a heterodimeric protein composed of Fos family (Fos, FosB, FosL1 and FosL2), Jun family (Jun, JunB, and JunD), Atf (activating transcription factor) and Maf (musculoaponeurotic fibrosarcoma) proteins [6, 7]. Phosphorylation of Fos and Jun or extracellular stimuli such as cytokines, stress, infection, and growth factors inducing the expression of Fos and Jun, the main AP-1 proteins in mammalian cells, can activate the AP-1 pathway. The activated AP-1 complex then binds to a consensus DNA sequence in the promoter region to regulate AP-1 target genes expression thus playing an important role in a number of cellular processes, including proliferation, differentiation, apoptosis, cell migration, and transformation [8]. While some AP-1 proteins have been reported to have tumor suppressor activity, AP-1 is well known to have oncogenic activity [6, 9, 10]. In fact, AP-1 activation in epithelial cells has been reported to be required for SCC transformation in a transgenic mouse model [11], and to promote metastasis in SCC [12]. Among the AP-1 family, Jun and FosL1 have also been reported to promote invasion via epithelial-to-mesenchymal transition (EMT) [9]. Thus, the activation of AP-1 has been reported to play a critical role in the invasive growth and metastasis of human cancers, although the significance of AP-1 in metastasis in HNSCC is not yet fully understood. In this study, we initially characterized the distant metastatic potential in vivo using 26 different HNSCC cell lines in an experimental lung metastatic mouse model with tail vein injection of HNSCC. A whole gene microarray was performed with 8 selected HNSCC cell lines, and upstream and key node AZD3759 site analysis was then used to investigate the upstream key molecules involved in the mechanisms of distant metastasis in HNSCC. The AP-1 family was identified as the key molecules regulating the pathways related to distant metastasis in HNSCC. We therefore hypothesize that the AP-1 family plays a crucialrole in inducing cell invasion, migration and distant metastasis in HNSCC. In the present study, we show that the small interfering RNA (siRNA)-mediated knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (cas9) system (CRISPR/Cas9 [13, 14])-mediated knockout of JunB in HNSCC cells significantly inhibited both invasion and migration in vitro, as well as lung metastasis in vivo.MethodsCell linesInformation on and appropriate growth media for the 26 HNSCC cell lines are shown in Additional file 1: Table S1. All cells were authenticated by short tandem repeat genotyping as described previously [15, 16]. Adherent monolayer cultures were maintained on plastic and incubated at 37 and 5 CO2.Animals and maintenanceAthymic nude mice, aged 7? weeks, were purchased from PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/25432023 the animal production area of the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD) and Oriental Yeast (Tokyo, Japan). The mice were housed and maintained in laminar flow cabin.