RESUMEN
Previous studies suggest that the reduction of SMAD3 (mothers against decapentaplegic homolog 3) has a great impact on tumor development, but its exact pathological function remains unclear. In this study, we found that the protein level of SMAD3 was greatly reduced in human-grade IV glioblastoma tissues, in which LAMP2A (lysosome-associated membrane protein type 2A) was significantly up-regulated. LAMP2A is a key rate-limiting protein of chaperone-mediated autophagy (CMA), a lysosome pathway of protein degradation that is activated in glioma. We carefully analyzed the amino-acid sequence of SMAD3 and found that it contained a pentapeptide motif biochemically related to KFERQ, which has been proposed to be a targeting sequence for CMA. In vitro, we confirmed that SMAD3 was degraded in either serum-free or KFERQ motif deleted condition, which was regulated by LAMP2A and interacted with HSC70 (heat shock cognate 71 kDa protein). Using isolated lysosomes, amino-acid residues 75 and 128 of SMAD3 were found to be of importance for this process, which affected the CMA pathway in which SMAD3 was involved. Similarly, down-regulating SMAD3 or up-regulating LAMP2A in cultured glioma cells enhanced their proliferation and invasion. Taken together, these results suggest that excessive activation of CMA regulates glioma cell growth by promoting the degradation of SMAD3. Therefore, targeting the SMAD3-LAMP2A-mediated CMA-lysosome pathway may be a promising approach in anti-cancer therapy.
RESUMEN
Objective Investigation of biological characteristics of Cdc 20AAA/+APCmin/+ mouse embryonic fibroblast(MEFs) indicate the effect of Cdc20AAA/+on growth of mouse embryonic fibroblast and the possible mechanism . Methods MEFs of Cdc20AAA/+APCmin/+, Cdc20AAA/+, APCmin/+ and WT genotype were harvested from embryos for analysis.The growth characteristics of Cdc20AAA/+APCmin/+, Cdc20AAA/+,APCmin/+and WT mouse embryonic fibroblast were analyzed through growth curve analysis and foci formation assay .Separation of sister chromatid and the presence of aneuploid were detected by karyotype analysis .Results Cell proliferation assays showed that Cdc 20AAA/+APCmin/+cells grew at an accelerated rate compared with APC min/+MEFs(P<0.01).Foci formation assay showed that the clone forming ability was significantly increased .Cdc20AAA/+APCmin/+MEFs showed a significant increase in the frequency of aneuploid compared with WT MEFs , which had a karyotype of 38 and contained prematurely separated sister chromatids .Conclusion Cdc20 carrying a null allele (Cdc20AAA/+) may accelerate the growth and proliferation of APC min/+MEFs and present the growth characteristics of the tumor cells .The possible mechanism may be associated with chromosome instability .