Suppression Of The Growth And Invasion Of Human Head And Neck Squamous Cell Carcinomas Via STAT3 And MiR-21/β-catenin Axis With HJC0152

Head and neck squamous cell carcinoma（HNSCC）is the sixth most common type of human cancer.More than 70% of patients with HNSCC suffer from relapsed or metastatic disease.There is an urgent need,therefore,for effective therapeutic regimens to treat HNSCC.Signal transducer and activator of transcription 3（STAT3）is validatedtobewidely expressed in various human tissues.STAT3 is implicated in cancer proliferation,invasion,and metastasis in human cancers of epithelial origin,including HNSCC.However,only a few of them have progressed into early-phase clinical trials.There is an urgent need,therefore,for effective therapeutic regimens to treat HNSCC.HJC0152 was designed to be a novel potent STAT3 signaling inhibitor.To identify the anticancer effect of HJC0152 in HNSCC and underlying mechanism,we hold following four distinct chapters:Method:Chapter1: First,we explorerd STAT3 and p-STAT3（Tyr705）in six distinct HNSCC cell lines.MTT assay was employed to assess IC₅₀ value of HJC0152 i.Western blot was employed to assessthe expression levelof STAT3 and p-STAT3.In addition,immunofluorescence was performed to show the locational changes of certain protein.Chapter2: MTT assay and plate clone formation assay valued tumor proliferation capacity.Flow cytometry analysis measured cell apoptosis and cell cycle distribution.We also measured tumor invasion and migration capacity in HNSCC via transwell and wound healing assays.Gelatin zymography was used to test secreted MMP2/9.Immunofluorescence staining showed locational differences of EMT biomarkers.Finally,we detected the invasion andproliferation related protein levels in HNSCC.Chapter3: After 24 hours of HJC0152 treatment,we used real-time PCR to detect the expression level of mi R-21.Western blot analysis was used to determine VHL and β-catenin.Immunofluorescence staining show locational differences of β-catenin.Restoration analysis further confirmed the critical role of VHL.Chapter4: HNSCC orthotropic tumor model was established inordertoevaluatethe anti-cancer capacityof HJC0152 invivo.Fluorescence images were captured toassess tumor volume.TUNEL assay was used to investigate active cell death.IHC was used to examine the relative protein expression.Results:Chapter1: SCC25 and CAL27 cell lines showed higher levels of p-STAT3 than did the Hep-2,TSCCA,Tb3.1,and UM1 cell lines.In HJC0152-treated CAL27 and SCC25 cells,p-STAT3 expression was significantly lower than that in untreated control cells.Moreover,HJC0152 and si-STAT3 both dramatically inhibited tumor cell proliferation,migration and invasion in vitro.In contrast to attenuation of phosphorylation of STAT3,phosphorylation of AKT and MAPK（Erk1/2）were not inhibited in both HNSCC cell lines tested.Immunofluorescence staining showed that p-STAT3 levels were lower in HJC0152-treated cells,with higher p-STAT3 accumulation in the cytoplasm than in the nucleus.We also found that HJC0152 inhibited p-STAT3 in HNSCC cells in a dose-and time-dependent manner.Chapter2:Transwell assay results suggested that HJC0152 inhibited SCC25（P < 0.05）and CAL27（P < 0.05）cell migration and invasion.Scratch test demonstrated that HJC0152 delayed wound healing in both HNSCC cell lines（P < 0.05）.In both cell lines tested,HJC0152 treatment significantly inhibited expression and secretion of MMP2/9.The subsequent immunofluorescence staining indicated the gain of E-cadherin and loss of N-cadherin followed by HJC0152 administration,similar to the western blot findings.As shown by the clonogenicity assay,after HJC0152 treatment,clones were reduced.We observed an increase in the accumulation of cells in the G1 phase in SCC25（P< 0.05）and CAL27（P< 0.05）cell lines treated with HJC0152.Cyclin-D1 was remarkably suppressed,while p21 was upregulated.HJC0152 significantly induced apoptosis in both SCC25（P< 0.05）and CAL27（P< 0.05）cell lines.BCL-2,a regulator of apoptosis,was decreased,while BAX was increased.Expression levels of cleaved caspase-3 also rose with increasing drug concentrations.Chapter3: q RT-PCR showed that in both cells that were transfected with si-STAT3 and those that were exposed to HJC0152,mi R-21 was attenuated.Subsequently,SCC25 and CAL27 cells were transfected with mi R-21 mimics for 3 days.VHL expression was decreased,and β-catenin levels were increased.mi R-21 levels were remarkably decreased after HJC0152 treatment in both cell lines（P< 0.05）.We also found that VHL expression was upregulated and β-catenin attenuated by HJC0152 in dose-and time-dependent manners.HJC0152 also dramatically blocked the nuclear translocation of β-catenin.TOP/FOP luciferase assay was used to detect the transcriptional activity of β-catenin.VHL abrogation significantly reversed HJC0152’s anticancer effects on cell proliferation and migration in vitro.Chapter4: HJC0152-treated tumors showed significantlylower tumor volume（P< 0.05）and tumor weight（P< 0.05）than did DMSO-treated tumors.TUNEL assay results demonstrated that more HJC0152-treated cells had apoptotic nuclei than did DMSO-treated cells（P< 0.05）.Furthermore,we found that Ki-67,cyclin D1,and BCL-2 expression were all dramatically attenuated and that cleaved caspase-3 was upregulated in HJC0152-treated tumors in vivo.HJC0152-treated mice had lower levels of local invasion and lymph node metastasis than did DMSO-treated mice.MMP2/9 expression was also attenuated in HJC0152-treated tumors.IHC staining showed that HJC0152 treatment significantly inhibited p-STAT3 expression,elevated VHL expression,and decreased β-catenin expression in SCC25 orthotropic tumors.Conclusion1、HJC0152 specifically inhibits phosphorylation and nuclear accumulation of STAT3 in HNSCC cells in vitro.2、HJC0152 reduces cell motility and cell proliferation in HNSCC cell lines in vitro.3、HJC0152 regulates the activity of the STAT3/mi R-21/β-catenin axis.4、HJC0152 reduces HNSCC tumor growth and invasion in vivo.