?-Trcp-mediated Degradation Of FOXN2 Promotes Cell Proliferation And Radioresistance In Lung Cancer Cells

Objective:Lung cancer remains the highest morbidity and leading mortality among all cancer types worldwide,with an average 5-year survival of about 15%.Chemotherpy,radiotherapy,and targeted therapy are the main options for those advanced stage patients.Radioresistance is the important factor that due to treatment failure of lung cancer.It is of great significance to target new molecules and proteins.FOX family is a transcription factor involved in cell,tissue and organ development.Recently,it has been reported that FOX family also plays significant roles in tumorigenesis.?-Trcp(?-transducin repeat containing protein),which is substrate-recognition subunit of SCF(SKP1-Cullinl-F-box protein)E3 ligase complexes,plays various roles in different cancer types through ubiquitination and degradation of target proteins.Our preliminary data show that FOXN2 may interact with ?-Trcp.However,it remains largely unknown how ?-Trcp1 degrades FOXN2 and the biological function of FOXN2 in tumorigenesis.Methods:Tandem affinity purification(TAP)method was used to identify candidate?-Trcp-interacting protein.Co-immunoprecipitation(Co-IP)was used to validate the interaction between FOXN2 and ?-Trcp1.?-Trcp could ubiquitinate FOXN2 by using in vivo ubiquitination assay.Through site directed mutagenesis technique,we found ubiquitination site.In order to investigate the relevant biological function between ?-Trcp and FOXN2,we evaluated the effect of FOXN2 on lung cancer cell tumor growth and proliferation by inhibiting FOXN2(siRNA).Colony formation assay and y-H2AX foci experiment were conducted to assess the radio sensitivity.Moreover,gain of function was used to investigate the biological differences between FOXN2-WT and mutant form FOXN2-AA.Results:We demonstrate that FOXN2 interacts with ?-Trcp1,and the stability of FOXN2 was negatively controlled by ?-Trcpl.The protein level of FOXN2 was markedly increased after inhibition of(?-Trcp1.In addition,?-Trcpl ubiquitinates FOXN2 on the Ser365 and Ser369 site.Inhibition of FOXN2 in H1299 and A549 cells leads to cell growth and proliferation promotion.Consistent with this notion,the percentage of cells in the S phase was increased after FOXN2 depletion.Moreover,FOXN2 siliencing results in radioresistance in lung cancer cells.More importantly,mutant form of FOXN2(FOXN2-AA)exhibited more potent inhibition ability of cancer cell growth,proliferation and stronger radiosensitivity.Conclusions:We identify FOXN2 can bind to ?-Trcpl and is ubiquitinated by ?-Trcpl on the Ser365 and Ser369 site.FOXN2 can suppress cell growth and proliferation and enhance the radiosensitivity in lung cancer cells.P-Trcp-mediated degradation of FOXN2 promotes cell proliferation and radioresistance in lung cancer cells.Therefore,our findings suggest that FOXN2 may serve as a candidate tumor suppressor and a promising therapeutic target for lung cancer treatment,especially radiotherapy.