**5. Autophagy-Mediated TCTP Degradation**

The relationship between TCTP and autophagy has not received much investigative interest, and only a few studies have reported the regulation by and e ffect on TCTP by autophagy. The expression of TCTP is fine-tuned both by transcriptional and translational regulation by a variety of extracellular signals, but its degradation mechanism is poorly understood. Several studies reported that interaction with several proteins, including Hsp27 and Mcl-1, increase the protein stability of TCTP [78,79] and those studies suggested ubiquitin-proteasome mediated degradation as a TCTP protein regulation mechanism. However, recently, a new type of regulation called chaperone-mediated autophagy (CMA) was suggested as a possible mechanism for TCTP degradation [80]. Unlike in macroautophagy where the substrates are sequestered to the vesicles, which then fuse with lysosomes, in CMA, proteins are directly trapped inside vesicles by the invagination of the lysosomal membrane. Only proteins targeted to the lysosome can be taken up into the lumen of the lysosome [81]. Thus CMA works as a unique proteolytic system that degrades specific cytosolic proteins, which permits quality control of proteins. Thus regulation of CMA could be a promising strategy to combat cancer, as shown by several studies [82–84]. In mouse embryonic fibroblasts (MEFs), serum starvation decreased cellular TCTP expression and this was found to be independent of macroautophagy and the ubiquitin-proteasome pathway but was dependent on the 70 kDa heat shock cognate protein (Hsc70) and the lysosome-associated membrane protein type 2A (LAMP-2A) [80]. Hsc70 is a cytosolic chaperone that recognizes the KFERQ motif in the amino acid sequence of the substrate protein, which is mandatory for targeting to the lysosome membrane [85]. LAMP-2A works as a lysosomal receptor for the substrate protein–chaperone complex [86]. Moreover, the KFERQ-like motif in TCTP was generated by acetylation of Lys19, and this increased the binding of TCTP to Hsc70 [80].

### **6. The role of TCTP in the Regulation of Autophagy**

Since TCTP influences cell survival and proliferation, it has the potential to influence autophagy, most likely through its association with the mTORC1 signaling pathway. Being at the center of a signaling network, the mTORC1 pathway regulates many processes, which are important for cell growth. This pathway also negatively regulates autophagy by competing with adenosine monophosphate-activated protein kinase (AMPK). During glucose starvation, AMPK drives autophagy by phosphorylation of Ser317 and Ser777 in autophagy initiator, ULK1. When su fficient glucose is provided, mTORC1 phosphorylates Ser757 of ULK1, which prevents the interaction between ULK1 and AMPK, eventually blocking autophagy [87]. We have reported that TCTP is able to interfere with these processes at several points [88], and thereby negatively regulates autophagy (Figure 1). TCTP is translationally induced by growth stimulation through the mTORC1 pathway [89], and therefore regulation of cell growth by TCTP may be related to autophagy. Indeed, decreased TCTP expression promoted formation and maturation of autophagosomes, observed by LC3 puncta formation

and by co-localization of LC3 with the lysosomal marker, LAMP1 [88]. Further studies revealed that downregulation of TCTP in HeLa cells resulted in reduced levels of downstream effectors of the mTORC1 pathway, including p-EIF-4EBP1, p-RPS6KB, p-ULK1 (Ser757), but increased AMPK phosphorylation [88]. Interestingly, TCTP knockdown decreased the mTORC1 pathway synergistically with rapamycin, a well-known allosteric inhibitor of mTOR [90], suggesting that TCTP might be a potential target to antagonize rapamycin resistance in cancer therapy. TCTP also regulates autophagy in an AMPK-mTORC1-pathway-independent mode by modulating the formation of the Beclin-1 complex [88]. Beclin-1 is a mammalian orthologue of yeas<sup>t</sup> ATG-6 [91] and performs a central role as overall scaffold for the class III PI3K complex [92]. Additionally, Bcl-2 also blocks autophagy by binding to Beclin-1, disrupting the Beclin-1/PI3K complex [93]. TCTP knockdown reduced bcl-2 expression without affecting Beclin-1 expression and thereby promoted Beclin-1-mediated autophagosome formation [88]. Negatively regulated autophagy by TCTP was further confirmed by a mouse model, in which TCTP is haploinsufficiently expressed [88]. Although more detailed mechanistic studies need to be conducted, we can conclude from the present study that TCTP, because it is involved in regulating cellular autophagy, could be a potential target in cancer therapy.
