Tumor Suppressor Spred2 Interaction With LC3 Promotes Autophagosome Maturation And Induces Autophagy-dependent Cell Death

Background:Autophagy is an evolutionarily conserved self-protective mechanism and a common process in eukaryotes maintaining cellular homeostasis.It is an important manner of cell death.The initiation of autophagy includes the formation of the phagophore,the initial sequestering compartment,which expands into an autophagosome.These autophagosomes mature by fusing with the endocytic compartments(e.g.early and late endosomes,multivesicular bodies)and then fusing with the lysosomal compartment to form autolysosomes,in which the cargo is degraded by acidic lysosomal hydrolases.The process is tightly regulated by a set of core autophagy-related(ATG)genes.More than 30 Atg genes have been identified in yeast so far.During autophagy,the microtubule-associated protein 1 light chain 3(LC3),which is the mammalian homologue of yeast ATG8,is converted to lipidated LC3 II and then associates with the autophagic membrane.The accumulation of LC3 II and its localization to the autophagosome(punctate dot formation)are generally used as markers for autophagy.During late autophagy,autophagosome matured by fusing with the lysosomal compartment to form autolysosomes,further to degrade its contents.The degradation of the substrate protein of autophagy p62(also known as SQSTM1)can be used as a hallmark of autophagy flux.Autophagy is mainly regulated by m TOR,class PI3 Kand classΙΙΙ PI3 K singaling pathways.Basically,autophagy is activated by the inhibition of m TOR and class Ι PI3 K,while the class ΙΙΙ PI3 K pathway has the opposing effect.Signaling molecules such as AMPK,p53 and Bcl-2 are also involved in autophagy.Autophagy is an important biology process playing significant roles during cell growth and differentiation,the dysfunction of autophagy always leads to various diseases including tumorigenesis.It can play both anti-and pro-tumorigenic roles depending on tumor type,stage,and cellular context.Based on the dual character of autophagy,autophagy could be used as a new therapeutic target for cancer.Sprouty-related EVH1 domain(Spreds)proteins are a new Sprouty-related membrane protein family,containing Spred1,Spred2,Spred3 three members in mammals.Several researches have shown that Spreds play a critical role in cell growth and differentiation,influence angiogenesis and lymphangion genesis also skeletogeny and histokinesis.Spreds are closely associated with tumorigenesis and EMT.Accumulating evidence indicates that expression of Spred1 and/or Spred2 was frequently reduced in hepatocellular carcinoma and prostate cancer,while decreased Spred levels were associated with increased tumor invasion and metastasis and may be a marker protein of tumorigenesis and aggressiveness.In this regard,to investigate the molecular mechanism of Spred may provide a new therapeutic target in tumor therapy.Tumor suppressor Spred2 functions as a negative regulator of ERK/MAPK signaling pathway,inducing cell death and differentiation.However,the underlying mechanism remains to be elucidated.In the current study we show that Spred2 modulates autophagosome maturation in tumor cells and induces autophagy-dependent cell death.We demonstrate that Spred2 knockdown induces accumulation of lapidated LC3,whereas Spred2 overexpression increases autophagosome formation and autophagosome-lysosome fusion.Mechanistically,Spred2 co-localizes and interacts with LC3 via the LC3-interacting region(LIR)motifs in its SPR domain.Mutations in the LIR motifs impair Spred2-mediated autophagosome maturation.In addition,Spred2 interacts and co-localizes with p62/SQSTM1 through its SPR domain,indicating that p62 may be involved in Spred2-mediated autophagosome maturation.Functionally,mutations in the LIR motifs impaired Spred2-mediated tumor cell death,indicating that the functional LIR is required for Spred2 to trigger cell death.Additionally,silencing the expression of autophagy-related genes ATG5,LC3 or p62 in He La cells reduced Spred2-mediated cell death.Pharmacological inhibition of autophagy with lysosomal inhibitors chloroquine gave similar results,suggesting that autophagy is required for Spred2-induced cell death.Collectively,these data indicate that Spred2 induces tumor cell death in an autophagy-dependent way.Method: 1.Molecular subcloning and PCR-based site-directed mutagenesis of LIR motif in Spred2 2.Cell culture and transfection 3.Adenovirus infecte cell induce the expression of aim proteins 4.Immunecoprecipitation and GST-Pull-down in vitro to test the interaction between proteins 5.Immunofluorescence assay explore the co-localization 6.Colony formation assay,Trypan blue exclusion assay and Flow cytometry analysis of apoptosisResults: 1.Overexpress Spred2 induce tumor cell growth 2.Spred2 interact and co-localization with LC3 and p62 3.Spred2 is involved in autophagy and promotes autophagosome maturation 4.Spred2 induces autophagy-dependent cell death Conclusion: 1.Tumor suppressor Spred2 promotes autophagosome maturation 2.Tumor suppressor Spred2 promotes autophagosome maturation induce autophagy-dependent cell death in tumor cell...