JWA Regulates The Molecular Mechanism And Clinical Significance Of HER2-positive Gastric Cancer Cell Migration

Background and Purpose:Tumor metastasis,as an important biological hallmark of malignancies,is a complex multi-step process,including tumor cells spreading out from the primary tumor,invasion in situ,intravasation,evasion from immune surveillance,extravasation,micro-metastasis and colonization.In this process,the migratory ability of tumor cells is essential throughout all process of tumor metastasis,which is the rate-limiting step of tumor metastasis.Migration is divided into directional and non-directional migration.Directional migration,referred as chemotaxis,plays an important role in metastasis and invasion of tumor cells.Human epidermal growth factor receptor 2(human epidermal growth factor receptor-2,HER2),is one member of the epidermal growth factor receptor family,HER2-positive(overexpression or amplification)of the breast cancer represents aggressive,highly metastatic potential and poor prognosis.However,targeted antibody trastuzumab significantly increased the overall survival and disease-free survival of the HER2-positive breast cancer patients.In addition,HER2-positive also occurs in 10-30% of gastric cancer,which is closely associated with metastatic phenotype.Although patients with HER2-overexpressing gastric cancer can also benefit from trastuzumab,but its response rate was only about half of patients compared with breast cancer,which may be due to its unique molecular genetic profile of gastric cancer and related regulatory networks.Therefore,the dissection of mechanism underlying metastasis in HER2-positive gastric cancer will contribute to the development of anti-tumor drugs for gastric cancer.JWA,as a microtubule-associated protein(MAP),is implicated in the process of apoptosis by directly binding to tubulin to adjust its dynamic stability.In addition,recent studies have shown JWA may be a tumor suppressor involved in several steps of metastasis.For JWA-deficiency melanoma cells,the abilities of migration,adhesion,invasion,angiogenesis exhibited significantly enhanced.Furthermore,our previous studies have demonstrated that JWA is involved in arsenic trioxide(As2O3)-and phorbol ester(PMA)-related cellular migration and cytoskeletal regulation in cervical cancer.However,the biological effects of JWA on tumor cell migration behavior,mechanism and clinical value is not fully elucidated,especially in HER2-positive gastric cancer.The study aims to explore its effects of JWA on EGF-induced tumor cell chemotaxis and its mechanism in HER2-positive gastric cancer.Moreover,the clinical value of JWA in HER2-positive gastric cancer is also evaluated.Methods:In this study,NCI-N87 and HGC-27 cells were regarded as HER2-positive gastric cancer cell models to overexpress and downregulate JWA expression,with a variety of molecular biology,as follows:1.EGF-induced chemotaxis assay JWA of ectopic overexpressing and silencing gastric cancer cells according to actin staining immunofluorescence assay after EGF stimulation JWA actin distribution and aggregation of muscle.2.Using Ed U assay to assess the impact of JWA expression on cell proliferation.3.The effect of JWA expression on total and phosphorylated protein,m RNA levels of HER2,EGFR and ERBB3 were determined by real-time quantitative PCR and Western Blotting.The downstream PI3 K / AKT signal and MEK / ERK signal activation were analyzed using Western Blotting with antibodies targetingphosphorylated Akt,ERK,PAK.And G-LISA assay was conducted to quantatively measure Rac1 and Cdc42 activation.Genetic and pharmatical inhibition of HER2 expression or activity was followed by Transwell,immunofluorescence,G-LISA assays,Western Blotting to detect the alterations of HER2 protein,its downstream pathways and cell migration ability in JWA-deficiency cells.4.The real-time quantitative PCR was done to screen the potential affected m RNA levels of upstream transcription factor of HER2 gene by JWA.Western Blotting,nuclear/cytosol fractionation were used to validate total protein and nucleoprotein levels of PEA3 modulated by JWA.Luciferase reporter gene assay and gel mobility shift assay(EMSA)were performed to measure the binding capacity of PEA3 to HER2 promoter as well as HER2 promoter transcriptional activity.With si RNA silencing PEA3 combined with Western Blotting,Transwell and G-LISA assays,the essential role of PEA3 in JWA-modulated migration and HER2 expression was.5.Luciferase reporter gene assay and Western Blotting combined with the MEK inhibitor and PI3 K inhibitor were applied to investigate how JWA regulates PEA3 activation.6.In the HER2-negative BGC-823 cells,Western Blotting,Transwell for determining whether JWA regulates EGF-induced cell migration and HER2,PEA3 expression in the absence of HER2.7.To evaluate the correlation between HER2 and JWA,Western Blotting was performed with 10 pairs of fresh gastric cancer and adjacent normal tissues.8.Immunohistochemistry was conducted using the tissue microarray of 128 patients with advanced gastric cancer to further clarify the relationship between JWA andHER2 expression,their expression and clinicopathological features and their prognostic significance.Result:This study has confirmed the following findings:1)JWA inhibits EGF-induced cell migration and actin cytoskeletal rearrangement.When overexpressing JWA,EGF-induced migration and stretch lamellipodia and filopodia were significantly reduced and actin cytoskeletal rearrangement was weaken,but in the absence of EGF,inhibition of migration by JWA is not obvious.2)Ed U assays showed that JWA has no significant effect on proliferation,which excludes the impact of proliferation on the migration.3)Inhibition of EGF-induced cell migration by JWA is dependent on HER2 and downstream PI3 K / AKT signal,thus weakening Rac1 / Cdc42 activation of PAK1,affecting cell movement.However,EGFR and ERBB3 showed no significant regulatory role.4)HER2 downregulation by JWA is mediated by HER2 negative transcription factor PEA3 activation.Compared with the vehicle control group,JWA-overexpressing cells has an increased PEA3 m RNA,nuclear protein levels and its binding capacity to HER2 proximal promoter along with a reduced HER2 promoter activity.Although the mutation of the PEA3 binding sites decreased HER2 promoter activity,JWA overexpression showed little influence on HER2 promoter activity.PEA3 silencing could partly relieve migration repression due to JWA and almost completely restore HER2 expression.5)JWA-regulated PEA3 / HER2 axis requires ERK activation.JWA can activate MEK / ERK pathway,thus maintaining PEA3 protein expression and activation ofits functions.Use MEK inhibitor-treated cells after JWA overexpression may significantly impede PEA3 expression,partially recovered HER2 promoter activity.JWA overexpression mitigates AKT phosphorylation,which could be enhanced by MEK inhibitor;and PI3 K inhibitor significantly reduced ERK phosphorylation,indicating,JWA-regulated cell motility may be PEA3 / HER2-dependent or-independent.6)In the HER2-negative BGC-823 cells,JWA cannot regulate HER2 expression.Even though the JWA still affects migration,but expression and activation of HER2 showed little difference between JWA-overexpressing or vector cells.Nevertheless,when exogenous HER2 was overexpressed,JWA could decrease the HER2 protein level in a HER2-dependent pattern,indicating that HER2 modulation by JWA is cell-specific.It may depend on the level of cellular HER2.7)Western Blotting with 10 pairs of fresh gastric cancer and their adjacent tissues found that HER2 was negatively correlated with JWA.8)Tissue microarray of 128 patients with advanced gastric cancer demonstrated that there was a negative correlation between JWA and HER2 expression.Both JWA and HER2 expression were associated with histological type,lymph node metastasis,and liver metastasis.JWA low expression is an independent adverse prognostic factor for advanced gastric cancer,but in the general population studied,HER2 overexpression is not assioated with prognosis.According to JWA and HER2 staining score,patients can be divided into three groups: both are high or low expression,JWA high and low expression of HER2,low JWA and high HER2,patients with low JWA expression and higher HER2 expression exhibited worse prognosis than the other two groups.After adjustment for other risk factors,the combination of JWA and HER2 remains a strong prognostic marker for advancedgastric cancer.When stratified by JWA expression level(JWA low expression or high expression),HER2-positivity was significantly associated with poor prognosis only in the low JWA expression group.Nevertheless,in patients with high expression of JWA,overall survival time between HER2-positive and negative groups showed no significant difference.Conclusion:In summary,JWA could inhibit HER2 expression through activation of MEK / ERK/ PEA3 signals in HER2-positive gastric cancer cells,to regulate cell motility and actin cytoskeletal remodeling.In addition,JWA could also be a useful prognostic marker for advanced gastric cancer to help stratify HER2-positive subgroup to better identify high-risk candidates for timely individualized treatment.This study illustrates a new upstream regulatory factor of HER2,its role in HER2-induced migration behavior and the mechanism of the regulation.More importantly,we found the prognostic value of JWA in patients with advanced HER2-positive gastric cancer,which would provide a new target and predictor for anti-metastatic drugs.