Autophagy is a lysosomal degradation pathway for the breakdown of intracellular proteins and organelles Despite various efforts have been made in the past few years in dissecting the differential functions and the related underlying regulations of autophagy in different cellular situations; our understanding on the molecular regulatory mechanisms of autophagy is still far from complete Survivin is a member of the inhibitors-of-apoptosis proteins family It is widely demonstrated that survivin is mainly expressed in tumors but not in the differentiated normal tissues The expression of survivin can be transcriptionally and post-translationally regulated in cells At the post-translational level survivin protein can be stabilized by Heat shock protein 90 (Hsp90) through physical interactions Dissociation of Hsp90 promotes survivin ubiquitination and subsequent protein degradation by the 26S proteasome It is widely believed that survivin exhibits two important pro-tumorigenic functions First it promotes mitosis by forming the chromosomal passenger complex (CPC) and second it inhibits apoptosis through direct and indirect caspases inhibition In this study we found that survivin is a novel negative regulator of autophagy Fluorescence microscopic analysis and Western blot analysis revealed that overexpression of survivin decreased acidic vesicular organelles (AVOs) formation and inhibited autophagic flux as indicated by the decreased amount of MDC labeled fluorescence puncta present and the increased accumulation of p62/SQSTM1 in breast cancer cells respectively Western blot analysis also revealed that overexpression of survivin negatively regulated both the expression of Atg7 Atg5 monomer Atg12 monomer and the formation of Atg5-Atg12 conjugate which are important molecules and protein complex for the formation of autophagosome in the cells Interestingly the same treatment also decreased the expression and activity of cathepsin L and the truncated Atg5 which is a molecule recently discovered to play a role in the positive regulation of caspase-3 activation These results indicate that survivin may negatively regulate autophagy through down-regulation of the autophagosome formation and indirectly inhibit caspase-3 activation through reduction of the amount of truncated Atg5 present in breast cancer cells Interestingly we also found that survivin is an autophagic substrate protein Inhibiting autophagy by either pharmacological inhibitors or siRNA against LC3 and Atg7 increased survivin expression in breast cancer cells Importantly Western blot analysis and immunoprecipitation assay showed that survivin bound to p62/SQSTM1 in MDA-MB-231 cells In addition inhibition of autophagy by either chloroquine or LC3 siRNA increased the protein stability of survivin in breast cancer cells In contrast inducing autophagy by serum starvation tamoxifen and rapamycin decreased survivin expression in breast cancer cells Furthermore mechanistic study revealed YM155 a survivin inhibitor that was originally developed to inhibit survivin gene transcription also down-regulated survivin expression through autophagic protein degradation Taken together findings of this study provide new mechanistic insights into both molecular functions and regulations of survivin and the molecular mechanism of actions of YM155 a drug currently undergoes clinical trials in cancer cells
Investigation of the novel inter-regulatory relationship between survivin and autophagy in cancer cells
琇涵, 詹. (Author). 2015 Aug 17
Student thesis: Master's Thesis