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Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice
(2015)
Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1-null (col6a1(-/-)) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1(-/-) mice. Systemic administration of spermidine in col6a1(-/-) mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.
The extracellular matrix is rapidly emerging as a prominent contributor to various fundamental processes of tumorigenesis. In particular, decorin, a member of the small leucine-rich proteoglycan gene family, is assuming a central role as a potent soluble tumor repressor. Decorin binds and antagonizes various receptor tyrosine kinases and inhibits downstream oncogenic signaling in several solid tumors. Among other functions, decorin evokes cell cycle arrest, apoptosis, and antimetastatic, and antiangiogenic programs. Recent work has revealed a paradigmatic shift in our understanding of the molecular mechanisms underlying its tumoricidal properties. Decorin adversely compromises the genetic signature of the tumor microenvironment and induces endothelial cell autophagy downstream of VEGFR2. Moreover, decorin selectively evokes destruction of tumor cell mitochondria downstream of Met through mitophagy. Acting as a partial agonist, decorin signals via proautophagic receptors and triggers procatabolic processes that parallel the classical tumoricidal properties of this multifaceted proteoglycan.
Autophagy can act either as a tumor suppressor or as a survival mechanism for established tumors. To understand how autophagy plays this dual role in cancer, in vivo models are required. By using a highly heterogeneous C. elegans germline tumor, we show that autophagy-related proteins are expressed in a specific subset of tumor cells, neurons. Inhibition of autophagy impairs neuronal differentiation and increases tumor cell number, resulting in a shorter life span of animals with tumors, while induction of autophagy extends their life span by impairing tumor proliferation. Fasting of animals with fully developed tumors leads to a doubling of their life span, which depends on modular changes in transcription including switches in transcription factor networks and mitochondrial metabolism. Hence, our results suggest that metabolic restructuring, cell-type specific regulation of autophagy and neuronal differentiation constitute central pathways preventing growth of heterogeneous tumors.
As current classical Hodgkin lymphoma (cHL) treatment strategies have pronounced side-effects, specific inhibition of signaling pathways may offer novel strategies in cHL therapy. Basal autophagy, a regulated catabolic pathway to degrade cell's own components, is in cancer linked with both, tumor suppression or promotion. The finding that basal autophagy enhances tumor cell survival would thus lead to immediately testable strategies for novel therapies. Thus, we studied its contribution in cHL.We found constitutive activation of autophagy in cHL cell lines and primary tissue. The expression of key autophagy-relevant proteins (e.g. Beclin-1, ULK1) and LC3 processing was increased in cHL cells, even in lymphoma cases. Consistently, cHL cells exhibited elevated numbers of autophagic vacuoles and intact autophagic flux. Autophagy inhibition with chloroquine or inactivation of ATG5 induced apoptosis and reduced proliferation of cHL cells. Chloroquine-mediated inhibition of basal autophagy significantly impaired HL growth in-vivo in NOD SCID γc-/- (NSG) mice. We found that basal autophagy plays a pivotal role in sustaining mitochondrial function.We conclude that cHL cells require basal autophagy for growth, survival and sustained metabolism making them sensitive to autophagy inhibition. This suggests basal autophagy as useful target for new strategies in cHL treatment.
Recently, the conserved intracellular digestion mechanism ‘autophagy’ has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas.
Autophagy in cancer therapy
(2017)
Autophagy represents a catabolic program involved in the degradation of cellular components via lysosomes. It serves to mitigate cellular stress and to provide metabolic precursors especially upon starvation. Thereby, autophagy can support the survival of cancer cells. In addition, there is now convincing evidence showing that under certain conditions autophagy can also foster cell death. This dual function of autophagy is also relevant upon anticancer treatment, as many chemotherapeutic agents engage autophagy. A better understanding of the molecular mechanisms that are critical for mediating autophagic cell death in cancer cells will be instrumental to selectively interfere with this cellular program in order to increase the cancer cell’s response to cytotoxic drugs. This review illustrates how anticancer drug-induced autophagy is involved in mediating cell death.
Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.
Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson’s disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.
Autophagy is a cytosolic quality control process that recognizes substrates through receptor‐mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR‐Cas9 or knockout‐mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER‐resident lectin chaperone Calnexin (CANX) and the ER‐phagy receptor FAM134B are required for autophagy‐mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co‐receptor that recognizes ER luminal misfolded procollagens and interacts with the ER‐phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane‐associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome‐resistant misfolded clients from the ER.
Protein aggregates and cytoplasmic vacuolization are major hallmarks of multisystem proteinopathies (MSPs) that lead to muscle weakness. Here, we identify METTL21C as a skeletal muscle-specific lysine methyltransferase. Insertion of a β-galactosidase cassette into the Mettl21c mouse locus revealed that METTL21C is specifically expressed in MYH7-positive skeletal muscle fibers. Ablation of the Mettl21c gene reduced endurance capacity and led to age-dependent accumulation of autophagic vacuoles in skeletal muscle. Denervation-induced muscle atrophy highlighted further impairments of autophagy-related proteins, including LC3, p62, and cathepsins, in Mettl21c−/− muscles. In addition, we demonstrate that METTL21C interacts with the ATPase p97 (VCP), which is mutated in various human MSP conditions. We reveal that METTL21C trimethylates p97 on the Lys315 residue and found that loss of this modification reduced p97 hexamer formation and ATPase activity in vivo. We conclude that the methyltransferase METTL21C is an important modulator of protein degradation in skeletal muscle under both normal and enhanced protein breakdown conditions.