The Mechanism Of PHLDA3 In Regulating Lung Cancer Proliferation And Invasion Through Wnt Signaling Pathway

Lung cancer is the leading cause of cancer-related deaths worldwide.As the most common type of lung cancer,non–small cell lung cancer(NSCLC)accounts for 80–85% of cases.Surgical resection,platinum-based chemotherapy,and radiotherapy are still the primary treatments of NSCLC in the clinic.Nonetheless,the disease is usually in advanced stages when first detected in most patients,and the overall survival rates are low.Therefore,it is important to identify new oncogenes or tumor suppressor genes to prevent and treat NSCLC.Wnt/?-catenin alterations are prominent in human malignancies,and available data indicate that Wnt signaling substantially impacts NSCLC tumorigenesis,prognosis,and resistance to therapy.This signaling pathway is exquisitely regulated by a large and complex array of proteins,including stabilized cytoplasmic ?-catenin regulated by the destruction complex,which plays a key role in the signaling output of the canonical Wnt cascade.The PHLDA3 gene encodes a small 127 amino acid protein with a pleckstrin homology(PH)-only domain.In neuroendocrine tumors,PHLDA3 acts as a tumor suppressor by competitively binding PIP to inhibit Akt activity.In esophageal squamous cell carcinomas,low PHLDA3 expression is associated with poor prognosis,whereas PHLDA3 levels are significantly increased in head and neck squamous cell carcinoma.Nevertheless,the role of PHLDA3 in lung cancer biology has been poorly studied and remains to be fully elucidated.Objective:1.Examining the expression levels of PHLDA3 in lung cancer tissues,and analyzing the relationship between PHLDA3 expression and the prognoses,clinical pathological factors of lung cancer patients.2.Investigating the role and molecular mechanism of PHLDA3 in regulating Wnt signaling pathway and lung cancer proliferation and metastasis.Methods: We collected 206 NSCLC specimens from patients(average age was 58 years)who underwent complete surgical resection at the First Affiliated Hospital of China Medical University between 2012 and 2015.Some lung cancer samples(108 cases)were accompanied by corresponding normal lung tissue samples(> 5 cm distal to the primary tumor edge).We also collected 60 pairs of fresh primary lung cancer and matched normal lung tissue specimens.Samples were frozen in liquid nitrogen immediately after removal and stored at-80°C.Immunohistochemistry and immunoblotting analyses were used to assess PHLDA3 expression in lung cancer tissues and their correlations with clinicopathological factors in lung cancer.Plasmids encoding PHLDA3 and small interfering RNA against PHLDA3 were used to regulate the expression of PHLDA3 in lung cancer cells,and effects on proliferation and invasion were investigated using MTS proliferation assay,colony formation assay,matrigel invasion assay,and wound healing assay.We detected the protein levels of Wnt signaling pathway and EMT-related proteins using western blot and immunofluorescence after increasing or decreasing the expression of PHLDA3 in lung cancer cells.Co-immunoprecipitation analysis and inhibitors of both the Wnt signaling pathway and GSK3? were used to investigate regulatory mechanisms involving PHLDA3 in lung cancer cells.Results:1.The expression level of PHLDA3 was higher in lung cancers than in normal lung tissues.The positive expression of PHLDA3 was significantly correlated with sex,histological type,TNM stage,lymphatic metastasis,and tumor T stage in lung cancers.In60 pairs of fresh specimens,we found that the expression levels of PHLDA3 were significantly higher in lung cancer tissues than in normal lung samples by western blot analysis.2.After overexpressed PHLDA3 in H1299 and A549 cells by PHLDA3 gene transfection,or downregulated PHLDA3 with si PHLDA3,PHLDA3 overexpression promoted,and PHLDA3 knockdown inhibited cell proliferation,colony formation,matrigel invasion compared with that in control cells.3.After PHLDA3 gene transfection in H1299 and A549 cells,PHLDA3 overexpression promoted the protein levels of total ?-catenin,active ?-catenin,nuclear total ?-catenin,nuclear active ?-catenin,p-GSK3?,TCF4,c-Myc,Cyclin D1,and MMP7,whereas inhibited the GSK3? expression.Conversely,knockdown of PHLDA3 decreased the expression levels of total ?-catenin,active ?-catenin,nuclear total ?-catenin,nuclear active ?-catenin,p-GSK3?,TCF4,c-Myc,Cyclin D1,and MMP7,whereas that of GSK3? was increased in H1299 and A549 cells.4.Compared with that in control cells,the expression levels of N-cadherin,Snail,Twist,ZEB1,and MMP9,were significantly increased,whereas E-cadherin,ZO1,and Claudin1 were decreased in H1299 and A549 cells transfected with PHLDA3.In contrast,the expression of N-cadherin,Snail,Twist,ZEB1,and MMP9 were decreased,whereas the expression of E-cadherin,ZO1,and Claudin1 were increased in si PHLDA3-treated H1299 and A549 cells.Immunofluorescent examination also confirmed that PHLDA3 upregulated the expression of N-cadherin and vimentin,whereas it downregulated ZO1.5.PHLDA3 expression was positively correlated with active ?-catenin expression in8 pairs of lung cancer tissues and corresponding normal lung tissues.After ensuring a high transfection efficiency of PHLDA3(MYC-tag)in H1299 and A549 cells,we treated with the Wnt inhibitor XAV-939 and immunoblotting analyses revealed that XAV-939 reversed the upregulation of active ?-catenin,p-GSK3?,c-Myc,Cyclin D1,MMP7,N-cadherin and MMP9 induced by PHLDA3 overexpression,and reversed the downregulation of GSK3? and E-cadherin at the same time.Induction of proliferation,colony formation,and matrigel invasion of H1299 and A549 cells by PHLDA3 transfection was also partially reversed using XAV-939.6.We verified the interaction of PHLDA3 and GSK3? in H1299 and A549 cells using co-immunoprecipitation assays.In H1299 and A549 cells,transfection with si PHLDA3 and treatment with GSK3?/? inhibitor CHIR-99021 reversed the downregulation of active ?-catenin,p-GSK3?,c-Myc,Cyclin D1,MMP7,N-cadherin MMP9 induced by PHLDA3 knockdown,and reversed the upregulation of GSK3? and E-cadherin at the same time.The inhibitory effects of PHLDA3 knockdown on cell proliferation,colony formation,and matrigel invasion were also reversed by GSK3?/?inhibitor CHIR-99021,indicating that the function of PHLDA3 in the Wnt signaling pathway and lung cancer cells partially depends on the regulation of GSK3? activity.Conclusion:1.PHLDA3 overexpression was associated with poor prognosis of lung adenocarcinoma patients.2.PHLDA3 promoted lung cancer cell proliferation,invasion,and migration.3.PHLDA3 enhanced the proliferative and invasive ability of lung cancer cells via the Wnt signaling pathway.4.PHLDA3 may regulate Wnt signaling activity via GSK3? interactions modulated.