87 Transcription factor RUNX1 activates OPN to promote tumor progression via MAPK signaling in head and neck cancer

BackgroundTumor progression and metastasis are still major burdens for head and neck squamous cell carcinoma (HNSCC) and are associated with eventual resistance to prevailing therapies. Complex molecular transcription and downstream signaling pathways have been implicated in the development, progression, invasion, metastasis, and treatment resistance of HNSCC. Runt-related transcription factor 1 (RUNX1) are involved in aggressive phenotypes in several cancers, while the molecular role of RUNX1 underlying cancer progression and metastasis of HNSCC remains largely unknown.MethodsRUNX1 expression levels in HNSCC cells and tissues were detected by quantitative real-time PCR (qPCR), Western blotting and immunohistochemistry (IHC). In vitro and in vivo assays were performed to investigate the function of RUNX1 in the metastatic phenotype and the tumorigenic capability of HNSCC cells. Luciferase reporter and chromatin immunoprecipitation (ChIP)-qPCR assays were performed to determine the underlying mechanism of RUNX1-mediated HNSCC aggressiveness.ResultsIn our study, RUNX1 expression was increased with disease progression in patients with HNSCC (figure 1). The silencing of RUNX1 significantly decelerated the malignant progression of HNSCC cells, reduced Osteopontin (OPN) expression in vitro, and weakened the tumorigenicity of HNSCC cells in vivo (figure 2). Moreover, we demonstrated that RUNX1 activated the MAPK signaling by directly binding to the promoter of OPN in tumor progression and metastasis of HNSCC (figure 3).Abstract 87 Figure 1RUNX1 expression in cancer progression of HNSCC. (A) Representative images of RUNX1 immunohistochemical staining between normal tissues and HNSCC tissues (scale bar 20μm). Insets (bottom) are lower magnification (15×) images of respective cores to show a more global view of individual samples. (B) The RUNX1 mRNA expression in tumor versus normal tissues from the TCGA database, which contains 31 normal samples and 91 HNSCC samples. (C) Immunoblotting analysis of RUNX1 expression in 3 pairs of HNSCC and non-tumoral laryngeal tissues. (D) Quantitative and statistical analysis of the immunoblotting analysis. *P<0.05, **P<0.01Abstract 87 Figure 2Effect of RUNX1 on progression and the interrelationship between RUNX1 and OPN in HNSCC. (A) The migration ability of FaDu and SCC-9 cells transfected as above were assessed by wound-healing assay. Representative images were obtained at 0h and 24h (upper, magnification 40×) and quantified (bottom). (B) The migration and invasion ability detected by transwell assays. Representative images of FaDu and SCC-9 cells from migration and invasion assays experiment were obtained at 24h (upper, magnification 12×) and quantified (bottom). (C) Correlation analysis was performed between RUNX1 expression and OPN expression in HNSCC tissues (n = 29) and (D) in TCGA HNSCC database (n = 91). All P values are shown in the graphs. (E) Levels of nucleus OPN mRNA and (F) protein in the FaDu cells transfected with lentiviral vector encoding shRUNX1 or scrambled control were determined by real-time RT-PCR and immunoblotting analysis. (G) The predicted OPN promoter sequence bound by RUNX1 and their ChIP-PCR primers. (H) The binding of RUNX1 to predicted OPN promoter binding region was confirmed in FaDu using ChIP-qPCR and ChIP-PCR. IgG was used as the control. (I) Relative OPN activity was detected by luciferase assay in 293T cells co-transfected with RUNX1 and luciferase reporter. **P<0.01, ****P<0.0001Abstract 87 Figure 3RUNX1-mediated HNSCC cell metastasis in MAPK pathway via stimulating OPN. (A) The migration ability of FaDu cells transfected as above were assessed by wound-healing assay. Representative images were obtained at 0h and 24h (magnification 40×). (B) The migration and invasion ability detected by transwell assays. Representative images of FaDu cells from migration and invasion assays experiment were obtained at 24h (magnification 12×). (C) Immunoblotting analysis for protein markers expression levels of the MAPK pathway in FaDu cells transfected as above. (D) The graph of tumor growth/volumes curve at the indicated time intervals (left). Tumor weights were quantified at the end of the experiment (right). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001ConclusionsOur results may provide new insight into the mechanisms underlying the role of RUNX1 in tumor progression and metastasis and reveal the potential therapeutic target in HNSCC.Ethics ApprovalThe study was approved by the Ethics Board of BenQ Medical Center, the Affiliated BenQ Hospital of Nanjing Medical University.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.