described in the case of some viral and bacterial infections that use the autophagic machinery for their own survival, assembly, and proliferation. Preventing formation of autophagosomes under these conditions also has a beneficial effect in the infected cells by limiting the ability of the pathogen to survive and colonize the cell. As in the previous example, autophagy cannot be considered an active effector in cell death under these conditions, because it is the failure of the autophagic system to eliminate the pathogen that leads to colonization and cell death.

Despite these notes of caution when defining autophagic cell death, studies in different invertebrate systems, such as the fat body of the fly, have shown evidence in support of an active role of autophagy in cell death. In most of these instances, cell death is related to tissue differentiation, remodeling or embryogenesis.

Exacerbation of the autophagic pathway has also been shown to lead to cellular death, at least in cultured cells and invertebrates. The mechanisms linking excessive autophagy with cell death are still not clear, but the most intuitive explanation is that an imbalance in cell metabolism, in which autophagic cellular consumption exceeds the cellular capacity for synthesis, exhausts the cellular resources and eventually promotes cell death.

3.3. Molecular players of the autophagy–cell death cross-talk

The molecular mechanisms of autophagic cell death are, for the most part, still unknown. One of the first components proposed to regulate the cross-talk between autophagy and apoptosis has been the protein pair Beclin-1/Bcl-2. The initiation complex Beclin-1/Vps34 is negatively regulated by Bcl-2 in a nutrient-dependent manner. Bcl-2 family proteins are key regulators of apoptosis that are represented by anti-apoptotic proteins, such as Bcl-2, and proapoptotic proteins Bax and Bak, which regulate the efflux of proapoptotic molecules from mitochondria and possibly other organelles. Deficiency in Bax and Bak or expression of Bcl-2 results in marked resistance to many apoptotic stimuli. Binding of Bcl-2 to Beclin-1 prevents activation of autophagy, whereas knockdown of Bcl-2 or over-expression of Beclin-1 mutants unable to bind Bcl-2 results in unregulated massive autophagy and cell death. Thus the anti-oncogenic role of Bcl-2 may result not only from its ability to block apoptosis, but also from its ability to prevent unregulated (excessive) autophagy. The detrimental role of excessive autophagy has been recently confirmed in studies with Caenorhabditis elegans.

The autophagic protein Ser/Thr kinase Atg1 also appears to act as a convergence point for signals linking autophagic and apoptotic cell death. In Drosophila, over-expression of Atg1 is sufficient to induce high levels of autophagy that lead to caspase-dependent apoptotic cell death. The stimulatory effect of Atg1 on autophagy seems to depend on its ability to inhibit TOR signaling, although it is also possible that part of the autophagic upregulation is distinct from the TOR control. In fact, Vps34 has been recently shown to promote autophagy but not TOR signaling in Drosophila, although so far, in most mammalian cell types analyzed, the effect of Vps34 seems to be dependent on changes in TOR signaling. This may reflect a fundamental difference in signaling mechanisms between the fly and mammalian systems.

A second line of thought in the identification of the molecular regulators of autophagic cell death supports that excessive autophagy may degrade cytoprotective effectors. For example, removal of catalase by Jun kinase-regulated autophagy leads to cellular accumulation of reactive oxygen species (ROS) and lipid peroxidation products and eventually precipitates cell death. Finally, another appealing possibility is that particular autophagic gene products, when expressed at high levels or after post-translational modification, directly activate apoptosis in a manner independent of their effect on autophagy.

4. AUTOPHAGY, CELLULAR DEATH, AND CANCER

Of the various human disorders for which a connection with autophagy has been established, cancer is probably the one for which the relationship between autophagy and cell death has been most extensively explored. In this context, a dual anti-oncogenic and pro-oncogenic role of autophagy has also been described (Figure 7-6).

Downregulation of autophagy is a common feature of many cancer cells and has been shown to be necessary to maintain their oncogenic potential. In fact, expression of endogenous Beclin-1 protein is frequently low in human breast epithelial carcinoma cell lines, and restoration of normal levels of this protein or activation of autophagy by other means diminishes the tumorigenic capability of these cancer cells. Different mechanisms have been proposed to explain the antioncogenic effect of autophagy (summarized in Figure 7-6). For example, a switch from a catabolic to an anabolic status when autophagy is reduced will favor cellular growth and division and tumor progression. In addition, reduced autophagy could also stimulate oncogenesis by favoring a proinflammatory

different stressors induce autophagy are poorly understood. Accumulation of ROS, produced under many cellular responses to stress, can directly activate autophagy by inactivation of the cysteine protease Atg4. Blockage of this protease leads to accumulation of the Atg8phosphoethanolamine precursor required for the formation of autophagosomes. Current research efforts are focused on identifying this type of connections between stress and autophagy as they could become perfect targets of therapeutic approaches aimed to increase the susceptibility of cancer cells to anti-oncogenic treatments.

In summary, autophagy can have opposite functions (proor anti-oncogenic) in different steps of tumorigenesis and depending on the environmental conditions that surround the tumor.

5. CONCLUDING REMARKS AND PENDING QUESTIONS

If we have learned anything through recent studies of the role of autophagy in cell death, it is that the answer is never absolute. The better understanding of the autophagic process and its interplay with apoptosis and other forms of cell death is helping to reconcile the initially conflicting views of autophagy as a prosurvival or cell death mechanism. For example, the better definition of autophagy as the process leading to not only engulfment, but also to complete degradation of the sequestered cargo has replaced autophagy by “inefficient autophagy” as a cause of cell death, regaining thus a prosurvival role for autophagy in some of these conditions. However, even when the most strict criteria of what is understood by autophagy are applied, there are still clear conditions in which autophagy becomes an effector of cell death. In most of these cases, except for those related to embryogenesis and tissue remodeling, it is an excess in the rates of autophagy that leads to cell death, likely through consumption of cellular components essential for survival.

One aspect that has clearly increased the complexity of the role of autophagy in cell death is the fact that different cells exposed to the same cell death stimuli respond differently, and by the same token, the same cell type also responds in a different manner when exposed to different cell death stimuli. Are there specific autophagic responses for individual death stimuli? The answer to this question may become clear as we gain a better understanding about other types of autophagy and the cross-talk mechanisms among autophagic pathways and cellular systems. For example, in the same way that cells with impaired CMA can survive nutritional stress through compensatory activation of

macroautophagy, activation of CMA often detected as compensatory mechanism for impaired macroautophagy could also be beneficial in response to particular cell death stimuli. Thus recent studies have shown that exposure of mouse fibroblasts with compromised macroautophagy to Fas/tumor necrosis factor-α induces caspase-dependent apoptosis, whereas these cells become resistant to death from menadione and ultraviolet light because of upregulation of CMA.

Also unclear is the value of autophagic sequestration versus lysosomal degradation in the prosurvival effect of autophagy. Intuitively, sequestration – for example, of a leaky mitochondrion – even if its degradation cannot be completed, should be better than leaving the organelle free in the cytosol. However, the consequences of the accumulation of undegraded autophagic vacuoles in the cytosol (autophagic stress) should not be underestimated. Consequently, alterations in both the formation of autophagosomes or the degradation of autophagic vacuoles can lead to cell death, although the mechanisms are probably different. These findings force reevaluation of those interventions aimed to enhance cell survival by only upregulating autophagosome formation. Simultaneous upregulation of autophagosome formation and clearance should be the gold standard of any manipulation on the autophagic pathway with therapeutic purposes.

A current limitation of some of the current studies on the role of autophagy in cell death is that whereas we count on efficient genetic methods to inhibit autophagy through downregulation of autophagy genes, our means to upregulate autophagy, at least in mammals, are still very limited. In particular, good pharmacological regulators are unavailable, as all the compounds available today (such as mammalian TOR [mTOR], histone deacetylase [HDAC], or Akt inhibitors) also control other important processes in the cell. There is thus a pressing need to develop compounds that target selectively Atg proteins, which could then be used to directly address the role of different types of autophagy in cellular survival and death in response to particular stimuli.

SUGGESTED READINGS

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