The MEKK3-mediated Autophagy And Its Function In Tumor Cells

Background:Autophagy is a cellular process that degrades and recycles organelles,macromolecular complexes and proteins in eukaryotic cells.Autophagy plays an important role in regulation of cell metabolism,stress response and survival.Abnormal autophagy is directly related to many diseases.Autophagy is involved in tumorigenesis,progression,therapeutic resistance and functions in both tumor cell survival and death.The kinase MEKK3(mitogen-activated protein kinase kinase kinase 3)is known in regulation of cell proliferation,cycle progression,differentiation,migration,apoptosis and inflammation.MEKK3 is also involved in tumorigenesis and progression.However,the role of MEKK3 in regulation of autophagy has not been defined and the cellular function of MEKK3 in lung and prostate cancer remains elusive.Objective:This thesis is to determine the role of MEKK3 in regulation of autophagy,proliferation and migration of tumor cells,and the role of autophagy in drug resistance.It is expected that this study is going to lay a foundation for improving the efficiency of cancer targeted and chemo therapies.Methods:1.Cell culture and treatment: Human embryonic kidney cell line HEK293 T,the non-small cell lung cancer cell line A549 and the prostate cancer cell line DU145 were cultured in DMEM complete medium(containing 10% FBS,1%penicillin/streptomycin mixture)at 37°C with 5% CO2.The treatment was performed by directly adding chemicals or drugs into the medium.2.The expression systems for sh RNAs and c DNAs.The lentiviral expression vector(p LKO.1 or p LKO.1-Tet-on)was used to construct the MEKK3 sh RNA constructs p LKO.1-MEKK3-sh RNA and p LKO.1-Tet-on-MEKK3-sh RNA.To produce the MEKK3 sh RNA cell lines in lung cancer A549 and prostate cancer DU145 cells,the virus infected cells were selected with 2.5 ?g/ml puromycin.For expression of the MEKK3 c DNA,the lentiviral expression vector TETO-FUW was used to construct the MEKK3-WT and MEKK3-KD expression plasmids.For expression of ATG5 c DNA,the lentiviral expression vector FUW-M2 rt TA was used to construct the HA-tagged ATG5 expression plasmid.3.The cell counting assay under a microscope with a hemocytometer was used for detecting the proliferation rate of tumor cells.4.The wound healing assay was used for detecting the tumor cell migration.5.The Western blot assay was used for detecting the protein level.6.The immunofluorescence staining assay was used for detecting intracellular localization of proteins and autophagic activity.7.The GST-fusion protein pulldown and co-immunoprecipitation assays were used for detecting the interaction and interactive sites of proteins.8.The GST-UBA pulldown assay was used for detecting ubiquitination of proteins.9.The FITC-Annexin V staining assay was used for detecting cellular apoptosis.Results:1.Knockdown of MEKK3 significantly inhibits proliferation of lung cancer A549 and prostate cancer DU145 cells.Overexpression of MEKK3-WT significantly promotes the proliferation of A549 cells,while overexpression of MEKK3-KD has no effect.2.Knockdown of MEKK3 in A549 cells significantly inhibits cell migration.Overexpression of MEKK3-WT significantly promotes cell migration,while overexpression of MEKK3-KD has no effect.Knockdown of MEKK3 in DU145 cells has no effect on cell migration while knockdown of MEKK3 in the ATG5-expressed DU145 cells inhibits cell migration.3.Overexpression of MEKK3-WT in A549 cells increase the LC3-? level and decreases the SQSTM1 level,while overexpression of the kinase dead mutant MEKK3 has no effect on the LC3-? level.Knockdown of MEKK3 in A549 cells results in a decrease in the LC3-? level and an increase in the SQSTM1,the phospho-m TOR and the phospho-S6 K levels.4.The results of GST-pulldown and co-immunoprecipitation showed that MEKK3 interacts with WWP1.The PPGY motif of MEKK3 and the WW3 domain of WWP1 are the interactive sites for the interaction.5.Immunofluorescence staining experiments showed that MEKK3 is co-localized with WWP1 in A549 cells.6.The GST-UBA-pulldown assay has demonstrated that MEKK3 is not a substrate of WWP1.However,MEKK3 enhances the WWP1-catalyzed ubiquitination of SQSTM1.7.Ectopic expression of ATG5 in DU145 cells results in formation of the autophagosomal protein LC3-?,reduction of SQSTM1,and enhancement of cell proliferation and migration.8.The cell counting and the FITC-Annexin V staining assays indicate that ectopic expression of ATG5 in DU145 cells resumes the autophagy and enhances the sensitivity of the cells to the cytotoxicity of chemotherapeutic drugs.Overexpression of MEKK3 in A549 cells promotes autophagic activity and enhances the sensitivity of the cells to the cytotoxicity of chemotherapeutic drugs.Conclusions:1.MEKK3 promotes autophagy in lung cancer A549 cells possibly by inhibiting the phosphorylation of m TOR.2.MEKK3 promotes proliferation of tumor cells.3.The promoting effect of MEKK3 on tumor cell migration may be dependent on the autophagic activity.4.The interaction of MEKK3 with WWP1 may promote the WWP1-catalyzed ubiquitination of SQSTM1.5.Increasing the autophagic activity may enhance sensitivity of tumor cells to the cytotoxicity of chemotherapeutic drugs.