Overexpression Of TAGLN2 Promotes Metastasis Of Esophageal Squamous Cell Carcinoma Via Inhibition Of Hippo Signaling Pathway By Rac1 Activation

BackgroundChina is one of the highest areas for esophageal cancer(EC),which is one of the most common gastrointestinal cancers worldwide,and the mortality of EC ranks the fourth leading cause of cancer-related death in China.EC is characterized by its geographic distribution in terms of its incidence and mortality and northern China in Taihang Mountain areas is one the highest incidence areas for EC.The two most common histological subtypes of EC comprise esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC),of which ESCC accounts for nearly 90% of ECs in Asia.With regards to the diagnosis and treatment of ESCC,there are lack of high specificity and sensitivity of diagnostic markers and effective treatments at present.Therefore,the 5-year overall survival rate of ESCC is only10%.Currently,surgery is the prodominant treatment of choice for ESCC but the majority of ESCC are diagnosed at middle-and late-stages,resulting in limited treatment option.Although multimodality treatment regimen comprising radiotherapy,chemotherapy and biotherapy,the survival time of patients at middle-and late-stages has improved little yet.Thus,identification of biomarkers and potential therapeutic targets with high specificity and sensitivity is of critical importance.TAGLN2(Transgelin-2,actin-binding protein 2),an important actin-binding protein,has a molecular weight of 23 kDa.It locates at chromosome 1q2l-q25,and is widely expressed in various tissues and organs in human body.Via regulation of the three-dimensional network structure of the cytoskeleton after binding to actin,it is directly involved in modulation of a variety of biological functions.Recently,many studies documented that TAGLN2 was overexpressed in colorectal cancer,lung cancer,ovarian cancer,stomach cancer,cervical cancer,and head and neck cancer,whereas low level of TAGLN2 expression was found in breast cancer,hepatocellular carcinoma and esophageal adenocarcinoma.At sub-cellular level,TAGLN2 is localized at cell membrane,in cytoplasm or in nucleus under different physiological or pathological conditions.Therefore,deregulation of TAGLN2 is correlated with the development of a variety of malignant diseases,suggesting that TAGLN2 may be a specific tumor marker and a potential therapeutic target.The Hippo signaling pathway plays an important role in organ size control,tissue regeneration and tumorigenesis.Numerous studies indicate that YAP/TAZ is a transcription co-activator downstream of the Hippo signaling pathways with oncogenic function.Deregulation of the Hippo signaling pathway can stimulate the activity of YAP/TAZ which transacts their target genes,contributing to tumorigenesis.Literature reveals that overexpression of YAP is observed in liver cancer,colon cancer,ovarian cancer,lung cancer,prostate cancer,etc.Our previous studies revealed that the overexpression of TAGLN2 was positively correlated with TNM stages of ESCC and negatively correlated with postoperative the 5-year overall survival rate of ESCC.In addition,TAGLN2 was one of independent risk factors for postoperative overall survival rate of ESCC.In the present study,the roles of TAGLN2 in cell proliferation,migration,invasion and its underlying molecular mechanisms were determined after respective manipulation of TAGLN2 in ESCC cells or immortalized esophageal epithelial cells mediated by lentiviral vectors.AimsUsing the ESCC cells with TAGLN2 overexpression and the immortalized esophageal epithelial cells with low TAGLN2 expression,we aim to examine the effects of TAGLN2 on cell proliferation,migration,invasion,and to define the underlying mechanisms via TAGLN2 knockdown in ESCC cells and TAGLN2 overexpression in immortalized cells,respectively.MethodsThe ESCC cells EC9706 and NE6-T with overexpression of TAGLN2 were transfected with shTAGLN2 lentiviral vector,and the immortalized esophageal epithelial cells NE6 with low expression of TAGLN2 were transfected with the lentiviral vector containing TAGLN2 cDNA.Subclones with stable knockdown of TAGLN2 and overexpression of TAGLN2,respectively,were established through puromycin selection.Western blot analyses were used to verify the knockdown effects of TAGLN2 and TAGLN2 overexpression.The effects of TAGLN2 on the proliferation,migration and invasion of EC9706,NE6-T and NE6 were evaluated by cell proliferation assay,migration assay,wound-healing assay and invasion assay in vitro and mouse model of tail vein lung metastasis in vivo.Western blot,gelatin zymography,RT-PCR,and immunofluorescence(IF)were used to investigate the changes of EMT-relatedmolecules and MMPs after TAGLN2 deletion in ESCC cells EC9706 and NE6-T,and TAGLN2 overexpression in immortalized esophageal epithelial cells NE6.The roles of Hippo signaling pathway in the progression of ESCC associated with TAGLN2 were explored by Western blot and IF as well.The effects of Rac1-GTP on the proliferation,migration and invasion of NE6 TAGLN2 with Rac1-GTP overexpression and interactions between Rac1 and the Hippo signaling pathway were evaluated by Rac1 inhibitor of NSC23766.Results1 The abilities of migration and invasion of ESCC cells EC9706 and NE6-T with TAGLN2 overexpression were abolished after ablation of TAGLN2 mediated by shTAGLN2 lentiviral vector,whilst the capacities of proliferation,migration and invasion of immortalized esophageal epithelial cells NE6 with low expression of TAGLN2 were significantly increased after TAGLN2 overexpression.2 TAGLN2 knockdown in ESCC cells EC9706 and NE6-T enhanced expression of E-cadherin and reduced expression of N-cadherin,Vimentin,MMP2,MMP7 and MMP9,whereas TAGLN2 overexpression in immortalized esophageal epithelial cells NE6 resulted in suppressed expression of E-cadherin and increased levles of N-cadherin,Vimentin,MMP2,MMP7 and MMP9.3 Hippo signaling pathway was activated by TAGLN2 knockdown in EC9706 and NE6-T cells.TAGLN2 knockdown induced phosphorylation of LATS1 and YAP,and decreased expression of YAP/TAZ and CTGF.In contrast,TAGLN2 overexpression in immortalized esophageal epithelial cell NE6 suppressed the Hippo signaling pathway with reversed expression pattern of p-LATS1,p-YAP,YAP/TAZ and CTGF.4 Activated levels of Rac1,PAK1 and Merlin were suppressed by TAGLN2 knockdown in EC9706 and NE6-T cells.In contrast,TAGLN2 overexpression in immortalized esophageal epithelial cell NE6 activated Rac1 with reversed expression of p-PAK1 and p-Merlin.NSC23766 suppressed the abilities of proliferation,migration and invasion of NE6 TAGLN2 with overexpression of Rac1-GTP and activated the Hippo signaling pathway.The low expression Rac1-GTP induced phosphorylation of YAP,and decreased expression of YAP/TAZ and CTGF.Conclusions1 High level of TAGLN2 induces EMT,MMP7 upregulation and enhanced activities of MMP2 and MMP9,which promotes the abilities of invasion and metastasis of ESCC;2 The overexpression of TAGLN2 in ESCC suppresses the activity of Hippo signaling pathway and subsequently increase the transcriptional activities of YAP/TAZ,which increase expression of YAP/TAZ target genes such as CTGF;3 The deletion of TAGLN2 in ESCC suppresses the activation of Rac1-GTP,which activates Hippo pathway activity and then contribute to the reduced abilities of proliferation,invasion and metastasis of ESCC;4 Our findings indicate that TAGLN2 is one of key biomarkers implicated in development and progression of ESCC,and that TAGLN2 and key components of Hippo signaling cascades are potential molecular targets applicable for ESCC management.