Autophagy and senescence, converging roles in pathophysiology as seen through mouse models.

Both senescence and autophagy have been strongly linked to aging and also cancer development. Numerous molecular, cellular, and physiological changes are known to correlate with an increasing age, yet our understanding of what underlies these changes or how they combine to give rise to the various pathologies associated with aging is still unclear. Levels of autophagy activity are known to decrease with advancing age, in a variety of organisms including mammals. Whereas senescent cells are known to accumulate in our bodies with age. Herein we review evidence from some elegant genetic mouse models linking senescence and also autophagy to aging and cancer. It is especially interesting to note the convergence in the pathological phenotypes of these two processes, senescence and autophagy, in these mouse models.

Temporal inhibition of autophagy reveals segmental reversal of ageing with increased cancer risk.

Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially.

Cell-based screen for altered nuclear phenotypes reveals senescence progression in polyploid cells after Aurora kinase B inhibition.

Cellular senescence is a widespread stress response and is widely considered to be an alternative cancer therapeutic goal. Unlike apoptosis, senescence is composed of a diverse set of subphenotypes, depending on which of its associated effector programs are engaged. Here we establish a simple and sensitive cell-based prosenescence screen with detailed validation assays. We characterize the screen using a focused tool compound kinase inhibitor library. We identify a series of compounds that induce different types of senescence, including a unique phenotype associated with irregularly shaped nuclei and the progressive accumulation of G1 tetraploidy in human diploid fibroblasts. Downstream analyses show that all of the compounds that induce tetraploid senescence inhibit Aurora kinase B (AURKB). AURKB is the catalytic component of the chromosome passenger complex, which is involved in correct chromosome alignment and segregation, the spindle assembly checkpoint, and cytokinesis. Although aberrant mitosis and senescence have been linked, a specific characterization of AURKB in the context of senescence is still required. This proof-of-principle study suggests that our protocol is capable of amplifying tetraploid senescence, which can be observed in only a small population of oncogenic RAS-induced senescence, and provides additional justification for AURKB as a cancer therapeutic target.

Histone H3.3 and its proteolytically processed form drive a cellular senescence programme.

The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Here, using models of oncogene-induced and replicative senescence, we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first 21 amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence.

Inside and out: the activities of senescence in cancer.

The core aspect of the senescent phenotype is a stable state of cell cycle arrest. However, this is a disguise that conceals a highly active metabolic cell state with diverse functionality. Both the cell-autonomous and the non-cell-autonomous activities of senescent cells create spatiotemporally dynamic and context-dependent tissue reactions. For example, the senescence-associated secretory phenotype (SASP) provokes not only tumour-suppressive but also tumour-promoting responses. Senescence is now increasingly considered to be an integrated and widespread component that is potentially important for tumour development, tumour suppression and the response to therapy.

Redistribution of the Lamin B1 genomic binding profile affects rearrangement of heterochromatic domains and SAHF formation during senescence.

Senescence is a stress-responsive form of stable cell cycle exit. Senescent cells have a distinct gene expression profile, which is often accompanied by the spatial redistribution of heterochromatin into senescence-associated heterochromatic foci (SAHFs). Studying a key component of the nuclear lamina lamin B1 (LMNB1), we report dynamic alterations in its genomic profile and their implications for SAHF formation and gene regulation during senescence. Genome-wide mapping reveals that LMNB1 is depleted during senescence, preferentially from the central regions of lamina-associated domains (LADs), which are enriched for Lys9 trimethylation on histone H3 (H3K9me3). LMNB1 knockdown facilitates the spatial relocalization of perinuclear H3K9me3-positive heterochromatin, thus promoting SAHF formation, which could be inhibited by ectopic LMNB1 expression. Furthermore, despite the global reduction in LMNB1 protein levels, LMNB1 binding increases during senescence in a small subset of gene-rich regions where H3K27me3 also increases and gene expression becomes repressed. These results suggest that LMNB1 may contribute to senescence in at least two ways due to its uneven genome-wide redistribution: first, through the spatial reorganization of chromatin and, second, through gene repression.

Cell senescence as both a dynamic and a static phenotype.

It has been 50 years since cellular senescence was first described in human diploid fibroblasts (HDFs), yet its mechanism as well as its physiological and clinical implications are still not fully appreciated. Recent progress suggests that cellular senescence is a collective phenotype, composed of complex networks of effector programs. The balance and quality within the effector network varies depending on the cell type, the nature of the stress as well as the context. Therefore, understanding each of these effectors in the context of the whole network will be necessary in order to fully understand senescence as a whole. Furthermore, searching for new effector programs of senescence will help to define this heterogeneous and complex phenotype according to cellular contexts.

Autophagy in cancer: having your cake and eating it.

Autophagy, one of two major intracellular degradation pathways, plays a critical role in energy homeostasis and the quality control of macromolecules and intracellular organelles. Previous work has demonstrated the importance of autophagy in maintaining cellular fitness, both in healthy and stressful conditions, revealing the complex interplay between autophagy and other stress-responsive phenotypes. The complex outcomes of stress-responsive autophagy confer on it both pro- and anti-tumourigenic roles, depending on the cellular and environmental context. Furthermore, recent findings that functionally link autophagy to the tumour suppressor mechanism, cellular senescence, have revealed a new role of autophagy in cancer biology. In this review we summarise the current evidence on the relationship between autophagy and cancer, with a focus on its role in senescence.

Spatio-temporal association between mTOR and autophagy during cellular senescence.

Evidence for a connection between lysosomes and mTOR is emerging. Seminal work from the Sabatini laboratory has shown that mTOR can be recruited to the lysosomal surface in response to amino acids, in a Rag GTPase-dependent manner, to become activated by Rheb. However the biological significance of this is not fully understood. Recent work from our laboratory has shown that lysosomes spatially link mTOR and autophagy forming a cytoplasmic compartment in close proximity to the Golgi apparatus (GA) during oncogenic Ras-induced senescence. The TOR-autophagy spatial coupling compartment (TASCC) is enriched for autolysosomes, but largely excludes autophagosomes. Our data suggest that mTOR, which is a positive regulator of protein synthesis, is recruited, in part, by the amino acid-rich environment surrounding the autolysosomes. This then facilitates protein synthesis at the nearby rER-GA system, reinforcing lysosome and autophagy biogenesis. Proper TASCC formation contributes to the production of secretory proteins, which also utilizes the rER-GA system. Since mTOR inhibits autophagy during the initial stages of autophagosome formation, TASCC formation is likely to facilitate autophagy by sequestering mTOR, suggesting that the TASCC is a self-enhancing structure.

Spatial coupling of mTOR and autophagy augments secretory phenotypes.

Protein synthesis and autophagic degradation are regulated in an opposite manner by mammalian target of rapamycin (mTOR), whereas under certain conditions it would be beneficial if they occurred in unison to handle rapid protein turnover. We observed a distinct cellular compartment at the trans side of the Golgi apparatus, the TOR-autophagy spatial coupling compartment (TASCC), where (auto)lysosomes and mTOR accumulated during Ras-induced senescence. mTOR recruitment to the TASCC was amino acid- and Rag guanosine triphosphatase-dependent, and disruption of mTOR localization to the TASCC suppressed interleukin-6/8 synthesis. TASCC formation was observed during macrophage differentiation and in glomerular podocytes; both displayed increased protein secretion. The spatial coupling of cells' catabolic and anabolic machinery could augment their respective functions and facilitate the mass synthesis of secretory proteins.

Autophagy facilitates oncogene-induced senescence.

Oncogenic stress triggers a range of intracellular protective responses including DNA damage checkpoints, senescence and apoptosis, depending on the cell type and the severity of the particular stress. Senescent cells are metabolically viable but are stably arrested. Senescence is a collective phenotype, however, that is comprised of various signaling pathways and effector mechanisms. Thus, to understand and manipulate the senescence phenotype, it is critical to find its effector mechanisms and determine the relationships between them. We have recently found that autophagy is activated upon acute induction of senescence and facilitates another effector mechanism: the senescence associated secretory phenotype (SASP).

Autophagy mediates the mitotic senescence transition.

As a stress response, senescence is a dynamic process involving multiple effector mechanisms whose combination determines the phenotypic quality. Here we identify autophagy as a new effector mechanism of senescence. Autophagy is activated during senescence and its activation is correlated with negative feedback in the PI3K-mammalian target of rapamycin (mTOR) pathway. A subset of autophagy-related genes are up-regulated during senescence: Overexpression of one of those genes, ULK3, induces autophagy and senescence. Furthermore, inhibition of autophagy delays the senescence phenotype, including senescence-associated secretion. Our data suggest that autophagy, and its consequent protein turnover, mediate the acquisition of the senescence phenotype.

Atg9 trafficking in Mammalian cells.

The molecular mechanisms of autophagy have been best characterized in the yeast Saccharomyces cerevisiae, where a number of proteins have been identified to be essential for this degradative pathway. ATG (autophagy-related) proteins(1) localize to a unique compartment, the pre-autophagosomal structure (PAS). Isolation membranes are suggested to originate from the PAS, enwrapping cytoplasmic components to form a double membrane autophagosome, which then fuses with the vacuole. Although many Atg proteins have been identified, the source of the PAS membrane in yeast is unknown. Identification of the source of the PAS in yeast has been hindered due to the transient association of Atg proteins with forming autophagosomes.(2) Likewise, in mammalian cells, it is not known if a PAS equivalent exists or if the formation of autophagosomes occurs from numerous membrane sources. The identification of stably associated markers would allow us to address this question further. Thus, characterization of the only transmembrane autophagy protein so far identified, Atg9, may aid in the search for the source of the PAS. Recent data from our lab suggests that mammalian Atg9 (mAtg9) traffics between the Golgi and endosomes, and suggests an involvement of the Golgi complex in the autophagic pathway.(3) Here we address the implications of our model with regard to membrane trafficking events in mammalian cells after starvation.

Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and endosomes.

Autophagy, fundamentally a lysosomal degradation pathway, functions in cells during normal growth and certain pathological conditions, including starvation, to maintain homeostasis. Autophagosomes are formed through a mechanism that is not well understood, despite the identification of many genes required for autophagy. We have studied the mammalian homologue of Atg9p, a multi-spanning transmembrane protein essential in yeast for autophagy, to gain a better understanding of the function of this ubiquitious protein. We show that both the N- and C-termini of mammalian Atg9 (mAtg9) are cytosolic, and predict that mAtg9 spans the membrane six times. We find that mAtg9 is located in the trans-Golgi network and late endosomes and colocalizes with TGN46, the cation-independent mannose-6-phosphate receptor, Rab7 and Rab9. Amino acid starvation or rapamycin treatment, which upregulates autophagy, causes a redistribution of mAtg9 from the TGN to peripheral, endosomal membranes, which are positive for the autophagosomal marker GFP-LC3. siRNA-mediated depletion of the putative mammalian homologue of Atg1p, ULK1, inhibits this starvation-induced redistribution. The redistribution of mAtg9 also requires PI 3-kinase activity, and is reversed after restoration of amino acids. We speculate that starvation-induced autophagy, which requires mAtg9, may rely on an alteration of the steady-state trafficking of mAtg9, in a Atg1-dependent manner.