The role of mannose-6-phosphate receptor and autophagy in influencing the outcome of combination therapy

Combining two different treatment modalities for targeting malignancies is gaining importance, with preclinical/clinical results indicating higher success rates in eradicating tumors or having longer remission periods. A better understanding of the synergy between the treatments helps in optimizing the dose and time of administration. We found that chemotherapy enhanced the levels of insulin-like growth factor 2 receptor/cation-independent mannose-6-phosphate receptor (IGF2R) on the surface of tumor cells, which leads to better tumor targeting by cytotoxic T cells (CTLs). Early evidence indicates that upregulation of IGF2R involves the autophagy pathway.     

Recognizing the cooperative and independent mitochondrial functions of Parkin and PINK1

Comment on: Johnson BN, et al. Proc Natl Acad Sci USA 2012; 109:6283-8.     

Starvation-induced autophagy promotes the invasion and migration of human bladder cancer cells via TGF-β1/Smad3-mediated epithelial-mesenchymal transition activation

The biological characteristics of bladder cancer include enhanced invasion and migration, which are the main causes of death in patients. Starvation is a typical feature of the bladder cancer microenvironment and can induce autophagy. Autophagy has an important relationship with the invasion and migration of tumors. However, the role of autophagy in the invasion and migration of bladder cancer cells remains unclear. Hence, the aim of the current study was to clarify this role and underlying mechanism. In this study, we found that starvation enhanced the epithelial-mesenchymal transition (EMT)-mediated invasion and migration of T24 and 5637 cells while inducing autophagy. The inhibition of autophagy with chloroquine (CQ) or 3-methyladenine (3MA) decreased EMT-mediated invasion and migration. In addition, the expression of transforming growth factor 1 (TGF-β1) and phosphorylated Smad3 (p-Smad3) increased after starvation. The inhibition of autophagy with CQ or 3MA also decreased the expression of TGF-β1 and p-Smad3. The inhibitor of TGF-β receptor sb431542 also inhibited the invasion, migration, and EMT of T24 and 5637 cells during starvation. Furthermore, recombinant TGF-β1 induced autophagy and inhibition of the TGF-β/Smad signaling pathway with sb431542 suppressed autophagy. In summary, our results suggested that autophagy promotes the invasion and migration of bladder cancer cells by inducing EMT through the TGF-β1/Smad3 signaling pathway. Moreover, autophagy and TGF-β1 can form a positive feedback loop to synergistically promote invasion and migration. Thus, our findings may provide a theoretical basis for the prevention of invasion and migration in bladder cancer.     

Commentary: ATG5: A distinct role in the nucleus

Both apoptotic and autophagic pathways are activated in cells during anticancer treatment using DNA-damaging agents. Thus, the outcome is balanced between apoptotic cell death and enhanced autophagy, with the possibility of prolonged cell survival. It seems intuitively obvious that this survival mechanism might interfere with the desired tumor cell killing. We addressed this question by tipping the balance in favor of autophagy, using etoposide or cisplatin at low, sublethal doses. Over 4 days, only a little apoptosis was observed, but both drugs sharply increased autophagic flux. Surprisingly, cells underwent a cell cycle arrest at G₂/M, followed later by mitotic catastrophe with formation of multipolar spindles, missegregated chromosomes, or enlarged, irregular, sometimes multiple nuclei. Why? The answer is that even a low level of DNA damage not only upregulates autophagy, but also provokes the recruitment of an autophagy-related protein, ATG5, to the nucleus, where it binds BIRC5/survivin, thereby interfering with correct assembly of the chromosome passenger complex needed for cytokinesis.     

Modulation of apoptosis sensitivity through the interplay with autophagic and proteasomal degradation pathways

Autophagic and proteasomal degradation constitute the major cellular proteolysis pathways. Their physiological and pathophysiological adaptation and perturbation modulates the relative abundance of apoptosis-transducing proteins and thereby can positively or negatively adjust cell death susceptibility. In addition to balancing protein expression amounts, components of the autophagic and proteasomal degradation machineries directly interact with and co-regulate apoptosis signal transduction. The influence of autophagic and proteasomal activity on apoptosis susceptibility is now rapidly gaining more attention as a significant modulator of cell death signalling in the context of human health and disease. Here we present a concise and critical overview of the latest knowledge on the molecular interplay between apoptosis signalling, autophagy and proteasomal protein degradation. We highlight that these three pathways constitute an intricate signalling triangle that can govern and modulate cell fate decisions between death and survival. Owing to rapid research progress in recent years, it is now possible to provide detailed insight into the mechanisms of pathway crosstalk, common signalling nodes and the role of multi-functional proteins in co-regulating both protein degradation and cell death.     

Transient Receptor Potential Cation Channel V1 (TRPV1) Is Degraded by Starvation- and Glucocorticoid-Mediated Autophagy

A mammalian cell renovates itself by autophagy, a process through which cellular components are recycled to produce energy and maintain homeostasis. Recently, the abundance of gap junction proteins was shown to be regulated by autophagy during starvation conditions, suggesting that transmembrane proteins are also regulated by autophagy. Transient receptor potential vanilloid type 1 (TRPV1), an ion channel localized to the plasma membrane and endoplasmic reticulum (ER), is a sensory transducer that is activated by a wide variety of exogenous and endogenous physical and chemical stimuli. Intriguingly, the abundance of cellular TRPV1 can change dynamically under pathological conditions. However, the mechanisms by which the protein levels of TRPV1 are regulated have not yet been explored. Therefore, we investigated the mechanisms of TRPV1 recycling using HeLa cells constitutively expressing TRPV1. Endogenous TRPV1 was degraded in starvation conditions; this degradation was blocked by chloroquine (CLQ), 3MA, or downregulation of Atg7. Interestingly, a glucocorticoid (cortisol) was capable of inducing autophagy in HeLa cells. Cortisol increased cellular conversion of LC3-I to LC-3II, leading autophagy and resulting in TRPV1 degradation, which was similarly inhibited by treatment with CLQ, 3MA, or downregulation of Atg7. Furthermore, cortisol treatment induced the colocalization of GFP-LC3 with endogenous TRPV1. Cumulatively, these observations provide evidence that degradation of TRPV1 is mediated by autophagy, and that this pathway can be enhanced by cortisol.     

Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation.     

PIASy-mediated Tip60 sumoylation regulates p53-induced autophagy

Posttranslational modifications of p53 integrate diverse stress signals and regulate its activity, but their combinatorial contribution to overall p53 function is not clear. We investigated the roles of lysine (K) acetylation and sumoylation on p53 and their relation to apoptosis and autophagy. Here we describe the collaborative role of the SUMO E3 ligase PIASy and the lysine acetyltransferase Tip60 in p53-mediated autophagy. PIASy binding to p53 and PIASy-activated Tip60 lead to K386 sumoylation and K120 acetylation of p53, respectively. Even though these two modifications are not dependent on each other, together they act as a “binary death signal” to promote cytoplasmic accumulation of p53 and execution of PUMA-independent autophagy. PIASy-induced Tip60 sumoylation augments p53 K120 acetylation and apoptosis. In addition to p14^ARF inactivation, impairment in this intricate signaling may explain why p53 mutations are not found in nearly 50% of malignancies.     

EMT impairs breast carcinoma cell susceptibility to CTL-mediated lysis through autophagy induction

Epithelial to mesenchymal transition (EMT) has become one of the most exciting fields in cancer biology. While its role in cancer cell invasion, metastasis and drug resistance is well established, the molecular basis of EMT-induced immune escape remains unknown. We recently reported that EMT coordinately regulates target cell recognition and sensitivity to specific lysis. In addition to the well-characterized role for EMT in tumor phenotypic change including a tumor-initiating cell phenotype, we provided evidence indicating that EMT-induced tumor cell resistance to cytotoxic T-lymphocytes (CTLs) also correlates with autophagy induction. Silencing of BECN1 in target cells that have gone through the EMT restored CTL susceptibility to CTL-induced lysis. Although EMT may represent a critical target for the development of novel immunotherapy approaches, a more detailed understanding of the inter-relationship between EMT and autophagy and their reciprocal regulation will be a key determinant in the rational approach to future tumor immunotherapy design.     

Autophagy in osteoporosis: Relation to oxidative stress

Impaired autophagy and oxidative stress are implicated in the development of many diseases. This study aimed to investigate the involvement of autophagy represented by autophagy-related gene 7 (Atg7) and oxidative stress represented by superoxide dismutase 2 (SOD2) gene expression and enzyme activity in the pathogenesis of osteoporosis. Atg7 and SOD2 gene relative expression were evaluated by SYBR green quantitative real-time-polymerase chain reaction in the osteoporotic group (n = 26) versus the osteoporosis free group (n = 14). SOD2 enzyme activity was evaluated by colorimetric method in both study groups. Both Atg7 and SOD2 relative expression showed highly significant decrease (P < 0.01) between both groups. However, SOD2 enzyme activity showed no significant difference between the two groups. There was a significant direct correlation between Atg7 and SOD2 gene expression in both study groups. Atg7 relative expression showed significant ( P < 0.01) direct correlation with vitamin D serum levels and body mass index in osteoporotic group. In conclusion, both genes are involved in the pathogenesis of osteoporosis and this could be amenable to future therapeutic intervention.