Disabled-2 Down-regulates LIG4 To Increase Radiosensitivity Of Lung Cancer Via Classical Wnt Pathway

Objective: Disabled-2(Dab2)is a multifunctional binding protein that can negatively regulate Wnt signaling pathway.Previous studies have found that the down-regulation of Dab2 expression in lung cancer is positively correlated with poor progression and prognosis of lung cancer.Overexpression Dab2 can significantly inhibit the activity of Wnt pathway in lung cancer cells.DNA ligase IV(LIG4)mainly participated in the repair of non-homologous terminal junction(NHEJ)and mediated radioresistance induced by Wnt pathway.Therefore,we speculated Dab2 could downregulate the expression of LIG4 through Wnt/?-catenin signaling pathway and inhibit the repair ability of DNA double strand break(DSB)in lung cancer cells,which induce the increased radiosensitivity of lung cancercells.Methods: Immunohistochemical staining was used to detect the expression of Dab2 and LIG4 in 103 lung cancer specimens.The relationship between the expression of Dab2 and LIG4 and clinicopathological factors and prognosis was analyzed.The wild type Dab2 and mutant Dab2 plasmids(Dab2-PTB and Dab2-?PTB)were constructed.Dab2 siRNA,Wnt pathway inhibitor iCRT-14 and LIG4 inhibitor SCR7 were used to bi-directionally regulate the expression of Dab2 and the activity of Wnt pathway.Western blot was used to detect the expression of Dab2,Wnt pathway factor,LIG4 and phosphorylated ?-H2 AX.Immunofluorescence was used to observe the number of focal points of phosphorylated ?-H2 AX to judge the repair of DSB after X-ray.Colony formation test and matrigel invasion test were used to determine the radiosensitization of lung cancer cells after irradiation.Results: Immunohistochemistry showed that low expression of Dab2(high expression of LIG4)was positively correlated with poor differentiation,high TNM stage,lymph node metastasis and poor prognosis.Western Blot showed that overexpression of Dab2 and Dab2-PTB decreased the expression of key molecules in Wnt pathway,cyclin D1,MMP-7 and LIG4(P<0.05),and increased the expression of phosphorylated ?-H2AX(P<0.05),while transfection of Dab2 ?PTB did not show the same effect.After down-regulating the expression of Dab2 in A549 by siRNA,the expression of ?-catenin,cyclin D1,MMP-7 and LIG4 was significantly up-regulated,while the expression of phosphorylated ?-H2 AX was down-regulated(P<0.05).This change was significantly reversed in combination with Wnt pathway inhibitor iCRT-14(P<0.05).Clonalformation and Transwell invasion experiment showed that wild-type Dab2 and mutant Dab2-PTB transfection significantly decreased the clonal formation rate and invasive cells number in H1299 cells,while transfection of Dab2-PTB showed no significant change.After siRNA and X-ray treatment,the clonal formation rate and invasive cells number of A549 did not decrease significantly,but after the combined treatment with iCRT14 or SCR7,the clonal formation rate and invasive cells number decreased significantly.Conclusion: The low expression of Dab2(high expression of LIG4)is positively correlated with the progression and poor prognosis of lung cancer patients.Dab2 inhibits the ability of DNA damage repair and enhances the sensitivity of lung cancer radiotherapy by down-regulating the expression of LIG4 via Wnt pathway.Dab2 gene might be a potential target for clinically enhancing the radiosensitivity of lung cancer.