Inhibitory Effect And Mechanisms Of PDCD4on Tumor And Macrophage Autophagy

Objective:PDCD4(programmed cell death4), an important tumor suppressor, inhibits carcinogenesis, progression and invasion via suppressing gene transcription and translation. It has been reported that Pdcd4transgenic mice show significant resistance to tumor induction whereas pdcd4-/-mice develop spontaneously lymphomas. In addition, loss or reduction of PDCD4expression has been observed in multiple types of human tumors, such as glioma, lung, ovarian, liver tumor, implicating development and progression of these human tumors. Further, PDCD4suppresses the malignant phenotype and enhances the chemosensitivity of tumors. Overall, these are indications that PDCD4is a tumor suppressor. However, the mechanisms by which PDCD4inhibits tumors remain unclear and conflict in different tumors. Some researchers have demonstrated that PDCD4promotes tumor cell apoptosis in glioma, hepatocellular, ovarian, breast and gastric tumor. However, others have reported that PDCD4plays an antiapoptotic role in HeLa cells or has no impact on apoptosis in colon carcinoma. Furthermore, PDCD4plays inhibitory functions or no effect on cell cycles. These suggest that roles of PDCD4in apoptosis and cell cycle may be limited to certain cell types, which cannot explain the suppression of PDCD4on multiple tumors.(Macro)autophagy, a conserved catabolic process whereby cellular proteins and organelles are engulfed by autophagosomes, digested in lysosomes, and recycled to sustain cellular homeostasis, has dual roles in tumor. Some data support the idea that autophagy is classified as an antioncogenic mechanism; However, accumulating evidences strongly support that autophagy enhances tumorigenesis and protects tumor cells from death. In addition, autophagy also plays an important role in regulation of macrophage functions such as lipid metabolism, which is involved in the initiation and development of atherosclerosis. The process of autophagosome formation is regulated by several autophagy-related genes(ATG), such as ATG5, ATG6(known as BECN1), ATG8(microtubule-associated protein1light chain3, LC3) and ATG12. ATG5, a protein involved at the early stage of autophagosome formation, contributes to elongation and closure of the autophagosomes. BECN1, acting upstream of autophagosome formation, governs the autophagy process by the subsequent recruitment of additional ATG proteins for initiating autophagosome formation.In our current study, we will investigate these questions from the following several aspects:(1) Effect of PDCD4on tumor autophagy and its related functions. PDCD4suppresses autophagy in multiple cell types and PDCD4-attenuated autophagy is required for its inhibition of tumor cell growth.(2) Effect of Pdcd4on macrophage autophagy and its related functions. Pdcd4suppresses macrophage autophagy and then cholesterol efflux, resulting in formation of foam cell, contributing to the initiation and development of atherosclerosis.(3) Molecular mechanisms by which PDCD4suppresses autophagy. PDCD4suppresses autophagy dependent on inhibition of PDCD4on ATG5protein via PDCD4-EIF4A-ATG5axis but neither BECN1nor ATG12.Methods1. Effect of PDCD4on tumor autophagy and its related functions(1) After plasmid transfection or siRNA interference, autophagy related markers LC3B-Ⅱ, SQSTMl/p62were detected by western blot. IF was carried out to demonstrate the formation of autophagosome.(2) Pepstatin A plus E64d were applied to block autophagic flux.(3) Xenograft animal model was established, treated with N.S, Mock and PDCD4plasmid. IHC and IF were applied to indicate PDCD4expression and autophagosome formation; western blot analysis to show LC3B-Ⅱ and SQSTM1expression.(4) After plasmid or siRNA transfection, cells were treated with3-MA or siATG5to inhibit autophagy or z-VAD to inhibit apoptosis, cell viability was detected by CCK8.2. Effect of Pdcd4on macrophage autophagy and its related functions(1) Western blot and IF were used to show autophagy level in wide type and pdcd4-/-mice primary peritoneal macrophages, BMDM, RAW264.7cells and lymphocytes after treated by EBSS, LPS, rapamycin and ox-LDL. (2) Oil Red "O" staining was used to show foam cell formation.(3) IF was used to demonstrate co-location of cholesterol, lysosome and autophagosome. MDC staining to show activated lysosome in macrophage(4) Real time PCR was used to determine the expression of Lxr-a, Fas, Ppar-y, Leptin, C/EBPa-R,Abcal and Abcgl.(5) Western blot was used to show the expression of Lc3b and Sqstml in vessel protein of apoE-/-and pdcd4-/-apoE-/-AS model mice.(6) IF was used to show co-location of cholesterol, autophagosome and macrophage, Sqstml expression in apoE-/-and pdcd4-/-apoE-/-AS model mice aortic root local plaque. After bone marrow transplantation assay, above experiments were repeated.(7) IHC was applied to demonstrate PDCD4expression and IF to show autophagosome in human plaque.3. Molecular mechanisms by which PDCD4suppresses autophagy(1) After plasmid transfection or siRNA interference, cells were subjected to EBSS, and PDCD4, BECN1, ATG5, ATG12and LC3B were analyzed by western blot.(2) Samples from human liver, ovarian cancers, glioma and xenograft tumors were stained with an anti-PDCD4and ATG5antibody for IHC analysis.(3) After plasmid transfection, HEK293cells were subjected to western blot to show PDCD4, ATG5expression; real time PCR to show mRNA levels of PDCD4, ATG5; RNA-IP to show binding between PDCD4protein and ATG5or ATG12mRNA.(4) After transfection with a mutant plasmid, cells was subjected to western blot to demonstrate BECN1, ATG5, ATG12, LC3; IF to show the formation of autophagosome; CCK8to demonstrate cell viability.(5) HEK293cells were cotransfected with PDCD4and ATG5plasmid. PDCD4, ATG5, and LC3B-I/LC3B-II were analyzed by western blot analysis.ResultsⅠ. Effect of PDCD4on tumor autophagy and its related functions1. PDCD4suppresses autophagy in multiple cell types in vitro(1) Forced PDCD4suppresses autophagy Overexpressed PDCD4in Skov3and U87cells led to decreases in the amount of LC3B-Ⅱ and increases in the amount of SQSTM1in absence or presence of Pep A and E64d detected by western blot, and a decrease in the number of autophagosomes detected by IF.(2) Silencing of PDCD4expression promotes autophagy Silencing of PDCD4 expression in HepG2and HeLa cells led to increases in the amount of LC3B-Ⅱ and decreases in the amount of SQSTM1in absence or presence of Pep A and E64d detected by western blot, and activated autophagosomes formation detected by IF.2. PDCD4suppresses autophagy in murine xenograft tumorsMurine xenograft tumors were established and treated with Mock and PDCD4plasmid. Western blot revealed LC3B-Ⅱ expression was decreased and SQSTM1was increased in the PDCD4group, as confirmed by IF:a decrease in the number of autophagosomes in the PDCD4group.3. PDCD4-attenuated autophagy required for its inhibition of proliferation(1)3-MA blockage assay indicates PDCD4-attenuated autophagy is required for its inhibition to tumor growth Skov3-Mock cells grew significantly faster than Skov3-PDCD4cells, retarded by3-MA mediated blockage of autophagy. Knockdown of PDCD4expression caused an increase of cell viability in HepG2, which was inhibited by3-MA-mediated blockage of autophagy.(2) Si-ATG5assay demonstrates that PDCD4-attenuated autophagy is required for tumor inhibition Si-ATG5caused depletion of autophagy both in Skov3and HepG2cells, which was enhanced by overexpression of PDCD4in Skov3cells and retarded by silencing of PDCD4in HepG2cells, and the cell viability after treatment with siATG5was similar to that of cells treated with3-MA.(3) Blockage assay of apoptosis by Z-VAD confirms that PDCD4inhibits cell viability independent on apoptosis Blockage of apoptosis by z-VAD caused cells with a good status, cell proliferation was still suppressed by PDCD4, whereas blockage of autophagy by3-MA led to cell fragmentation, nuclear condensation, suggesting PDCD4inhibits cell viability independent on apoptosis.Ⅱ. Effect of Pdcd4on macrophage autophagy and its related functions1. Pdcd4inhibits macrophage autophagy induced by various stimuli(1) Pdcd4deficiency upregulates immunocytes autophagy induced by various stimuli Western blot and IF revealed Pdcd4deficiency caused upregulated autophagy level in primary peritoneal macrophages, BMDM, mouse RAW264.7and even in spleen lymphocytes after treated by EBSS, LPS, rapamycin.(2) Pdcd4deficiency upregulates autophagy induced by ox-LDL IF showed that Pdcd4deficiency led to an increase in the number of autophagosomes, but no obvious difference in fluorescence intensity of Sqstm1induced by ox-LDL.2. Effects of Pdcd4-attenuated autophagy on macrophage-derived foam cell formation(1) Pdcd4deficiency attenuates macrophage-derived foam cell formation dependent on autophagy Oil Red "0" staining was used in wide type and pdcd4-/-mouse primary macrophage, demonstrating that Pdcd4deficiency caused a decrease in lipid accumulation and attenuation of foam cell formation, enhanced by EBSS and rapamycin, and restored by WM,3-MA and Pep A+E64d.(2) Pdcd4controls autophagy dependent cholesterol efflux Uptake tests demonstrated no differences between wide type and pdcd4-/-primary macrophage. A decrease in cholesterol accumulation and an increase in number of autophagosomes, and activated lysosome were observed in pdcd4-/-mouse primary macrophage. Lysosome was colocalized with autophagosome and cholesterol. Real time PCR showed Pdcd4deficiency led to an increase in amount of Abcal, Abcgl and Lxra.3. Effects of Pdcd4-attenuated macrophage autophagy on atherosclerosis(1) Pdcd4deficiency promotes macrophage autophagy and lipid accumulation in atherosclerotic plaque in apoE-/-mice Western blot and IF showed Pdcd4deficiency led to an increase in amount of Lc3and Sqstm1, and a decrease in cholesterol accumulation and an increase in number of autophagosomes, localizing in macrophage in atherosclerotic plaque.(2) Pdcd4-attenuated macrophage autophagy may play an important role in AS via bone marrow transplantation Pdcd4deficiency led to a decrease in cholesterol accumulation and an increase in number of autophagosome, colocalizing with macrophage in local plaque after bone marrow transplantation.(3) Relationship between PDCD4expression and autophagy in human atherosclerotic plaque PDCD4expression were detected by IHC and autophagosomes were analyzed by IF. The correlationship analysis demonstrated a negative relation between PDCD4expression and autophagy level in human plaque.Ⅲ.Molecular mechanisms by which PDCD4suppresses autophagy1. PDCD4inhibits autophagy-related gene ATG5protein expression(1) PDCD4inhibits ATG5protein expression in vitro Western blot demonstrated in both tumor cells and macrophages BECN1was not affected by PDCD4under starvation, LPS or rapamycin. Higher PDCD4expression remarkably inhibited ATG5expression and formation of the ATG12-ATG5complex.(2) PDCD4inhibits ATG5protein expression in vivo ATG5expression in human liver, ovarian cancers, gliomas and murine xenograft with negative or low PDCD4expression was significantly higher than that with higher PDCD4expression.2. PDCD4inhibits ATG5expression and autophagy via its ma3domains(1) PDCD4protein binds ATG5mRNA ATG5protein was suppressed significantly, while ATG5mRNA was unaffected by PDCD4expression, suggesting the inhibition of PDCD4on ATG5occurs translationally. A RIP assay was applied to demonstrate an association between ATG5mRNA and PDCD4protein.(2) PDCD4inhibits ATG5-mediated autophagy via its ma3domains PDCD4mutation analysis demonstrated only wild-type PDCD4but not mutant PDCD4led to ATG5downregulation, and a decrease in the number of autophagosomes, suppressed cell viability and an association with ATG5mRNA, suggesting PDCD4inhibited ATG5by binding of PDCD4-ma3with EIF4A-ATG5mRNA.Conclusions1. PDCD4inhibits activation of autophagy and formation of autophagosome in tumor cells and immunocytes. PDCD4-attenuated autophagy is required for inhibition of tumor cell growth.2. Pdcd4promotes macrophage-derived foam cell formation via suppressing autophagy and cholesterol efflux, at last promotes the initiation and development of atherosclerosis.3. Mechanically, PDCD4inhibits ATG5protein expression and further inhibits ATG12-ATG5complex formation in vitro and in vivo. The inhibition of PDCD4on ATG5occurs translationally, and RIP assay demonstrates ATG5but not ATG12mRNA, binds with PDCD4protein. Once PDCD4fails to binding with ATG5, it loses the ability to suppress ATG5expression, autophagy and cell viability.Originality and Significance1. For the first time, we demonstrate PDCD4inhibits autophagy and formation of autophagosome in multiple cells including tumor and immune cells, an universal phenomenon. We report here that inhibition of PDCD4on autophagy contributes to its tumor suppressor activity and effect of Pdcd4on lipid metabolism and atherosclerosis.2. Importantly, PDCD4inhibits the expression of an essential autophagy related gene, ATG5and the formation of ATG12-ATG5complex, and its ma3domains are required for PDCD4-mediated inhibition of autophagy. Our findings indicate that PDCD4negatively regulates autophagy by targeting ATG5, which provides a novel mechanism of tumor suppression.