P70S6K Promotes IL-6 Induced EMT And Metastasis Of Head And Neck Squamous Carcinoma

Head and neck cancer is one common sort of malignant tumors,of which more than 95% are squamous cell carcinomas.Most patients are diagnosed at an advanced stage and accompanied by metastasis,and lymph node metastasis is a major factor affecting the prognosis of HNSCC.Therefore,the identification of key molecules associated with metastasis could guide effective diagnosis and treatment of HNSCC.Interleukin-6(IL-6)plays a central role in regulating various tumour behaviours including angiogenesis,proliferation,migration,and invasion of tumour cells.Elevated serum IL-6 levels are known as an indicator of poor prognosis in most malignancies.IL-6 gene transcription is induced by many stimuli,such as RNA or DNA virus infection,bacterial endotoxin,lipopolysaccharide and serum,the inflammatory cytokines,tumor necrosis factor(TNF),IL-1,platelet-derived growth factor,and the interferons(IFNs).In recent years,investigations of IL-6 as the representative of cytokines in oncology have attracted great attention.IL-6 promotes tumor development by regulating a number of signaling pathways,but to its role and mechanisms in the metastasis of HNSCC are still not elucidated.In this study,we first detected the expression of IL-6 mRNA and EMT markers in paired poorly-highly metastatic HNSCC cell lines,and it showed that the expression of IL-6 mRNA was increased in high metastatic HNSCC cells,686LN-M4 e,compared with low metastatic HNSCC cells,686 LN.And 686LN-M4 e cells exhibited more EMT characters as well as elevated total-and phospho-p70S6 K and S6 levels than 686 LN cells.Then we found that IL-6 promoted the EMT and the migration of low metastatic 686 LN cells,and IL-6 short-term treatment significantly increased phosphorylation protein levels of p70S6 K and S6.It indicates that p70S6 K was involved in IL-6 induced EMT and migration of HNSCC cells.Meanwhile,phosphorylation protein levels of AKT,ERK1/2,and STAT3 were also significantly increased with short-term IL-6 treatment,which was consistent with previous reports that IL-6 activated PI3K/AKT,JAK/STAT3,Ras/MAPKs signaling pathways.To determine the role of p70S6 K in the metastasis of HNSCCs,we detected the effects of p70S6 K overexpression on EMT and migration of low metastatic HNSCCs.The results showed that transfection of plasmids which encodes p70S6 K induced EMT and promted migration of HNSCCs just as IL-6 did.Using RNAi to downregulate p70S6 K expression inhibited IL-6 induced EMT and migration of HNSCCs.Then we explored which pathway mediated IL-6 induced phosphorylation of p70S6 K.We found activation of PI3K/AKT/mTOR,STAT3,MAPK/ERK signaling pathways when low metastasis HNSCC cells were treated with IL-6.We pretreated cells with PI3 K inhibitors LY294002 or wortmannin,mTORC1 inhibitor rapamycin,or MEK1/2 inhibitor U0126,then stimulated the cells with IL-6,we found that all these inhibitors reduced p-p70S6 K levels.STAT3 is the most important downstream signals of IL-6,we detected elevated total-and phospho-STAT3 levels in 686LN-M4 e cells compared to 686 LN cells.Moreover,STAT3 siRNAs reduced basal p-p70S6 K levels,but not IL-6 induced p-STAT3 and p-p70S6 K.Using STAT3 inhibitor niclosamide decreased p-p70S6 K levels as well as IL-6-induced p-p70S6 K.However,p70S6 K siRNA transfection decreased p-STAT3 levels but not IL-6-induced p-STAT3 levels.These results suggested that IL-6 induced p70S6 K activation located downstream of multiple signaling pathways,including PI3K/AKT/mTOR,STAT3,and MAPK/ERK pathways.In summary,our findings suggest that p70S6 K plays an important role in IL-6 induced EMT and metastasis of head and neck cancer.This study investigated the role of IL-6 in the metastasis of HNSCC and the possible mechanism of activating p70S6 K signaling pathway.Our findings will further clarify the mechanisms of HNSCC metastasis and provide new diagnostic metastasis markers and therapeutic targets for HNSCC.