Here we show that deletion, depletion or inhibition of p53 induces autophagy in human, mouse and nematode cells. Moreover, various inducers of autophagy cause p53 degradation, whereas inhibition of p53 degradation prevents autophagy, suggesting that p53 inhibition is essential for induction of autophagy. These findings have implications for the spatial regulation of the p53 system, the connections between p53 and autophagy/survival pathways and the role of p53 as a tumour suppressor protein.
At present, the exact mechanisms by which autophagy-inducing stimuli, such as ER stress, cause the cytoplasmic translocation and subsequent degradation of p53 remain unknown. However, this process involves sequential phosphorylation of p53 on Ser 315 and Ser 376, its nuclear export, ubiquitination by HDM2 and proteasome-mediated degradation30,31
. As shown here, inhibiting HDM2 or the proteasome prevents degradation of p53 induced by various autophagy triggers, and inhibits autophagy. The present study provides some insight into the mechanisms by which p53 depletion de-represses autophagy. We have shown that p53 inhibits autophagy through a cytoplasmic (non-nuclear) effect, as indicated by the finding that pharmacological inhibition of p53 can trigger autophagy in cytoplasts. Moreover, cytoplasm-targeted, but not nucleus-targeted, variants of p53 suppress autophagy. Importantly, either a point mutation that changes the conformation of p53 (R175H) or the removal of the πPP abolished the autophagy-inhibitory action of p53. Another cytoplasmic function of p53, namely the induction of mitochondrial membrane permeabilization and apoptosis, is abolished by the R175H and ΔPP mutations as well27,36,37
. Factors that interact with p53 to induce apoptosis belong to the Bcl-2 family of proteins27,36,38
, which also regulate autophagy39
. As with pro-apoptotic BH3-only proteins, p53 inhibits the anti-apoptotic effect of Bcl-2 homologues36
and activates Bax38
, which are Bcl-2 proteins that promote apoptosis. However, BH3-only proteins are autophagy inducers, not inhibitors41
, indicating that the modulatory effect of p53 in autophagy is different from that of BH3-only proteins. Hence, the exact molecular pathway through which cytoplasmic p53 inhibits autophagy remains elusive.
Irrespective of the molecular details, induction of autophagy by p53 has been linked to inhibition of mTOR10,42
, whereas autophagy inhibition by p53 correlates with enhanced mTOR activity. Moreover, it appears that induction of autophagy by p53 depends on transactivation of genes such as DRAM14
, whereas inhibition is a cytoplasmic effect. Nucleo-cytoplasmic shuttling of p53 is regulated by post-translational modifications of nuclear export signals43
and has a major role in regulation of autophagy.
p53 is activated by various stressors, including agents that affect DNA structure (for example, ultraviolet light, ionizing irradiation and chemotherapeutics) and conditions that induce reactive oxygen species. The activation of p53 by stress involves multiple mechanisms that often lead to stabilization of the p53 protein44
. Once activated, p53 mediates several effects ranging from stimulation of DNA repair after transient cell-cycle arrest to irreversible cell-cycle arrest (senescence) or induction of apoptosis. Most of these effects depend on the transactivation of p53 target genes9,45
. Determining whether p53 induces adaptive responses (DNA repair) or cellular demise has been linked to distinct transcriptional programmes, correlating with the particular pattern of post-transcriptional modifications of p53 (such as phosphorylation of Ser 46, which is pro-apoptotic). Although DNA-damaging and pro-oxidant conditions activate p53, some cellular stressors inactivate p53, as demonstrated for ER stressors30,31
and starvation (as shown in this study). These findings demonstrate that subtle differences in the nature of the stressors can either induce or inhibit p53.
As mentioned earlier, p53 may potently induce autophagy through transcriptional activation of the autophagy-inducing protein DRAM14
, and also through inactivation of the mTOR pathway10
. This p53-mediated activation of autophagy has been linked to cell death in a positive fashion (because DRAM is also required for apoptosis induction via p53) or in a negative fashion (because autophagy inhibition can enhance p53-mediated apoptosis). In addition, we show here that physiological levels of p53 repress autophagy. Thus, any perturbation of p53 system — either activation or inhibition — may induce autophagy. It is possible that any kind of cellular stress, caused by either inactivation or overactivation of p53, may induce autophagy, perhaps in the context of genotoxic stress or failure to handle such stress. Autophagy induced by p53 overactivation can either be cytocidal or cytoprotective, as discussed above. In contrast, it seems plausible that autophagy linked to p53 inhibition is cytoprotective, as illustrated by the finding that autophagy is required for the enhanced resistance of p53-/-
cells to metabolic stress.
Together, these findings indicate that p53 may be either activated or inhibited by different stressors, that p53 can inhibit or enhance autophagy and that autophagy can increase or reduce cell survival, in a series of binary decisions that are probably determined by cellular events, as well as by the intensity of the signals. For example, reduction of glucose concentration can activate p53 through AMPK-mediated phosphorylation42,46
, but the removal of all nutrients from the medium induces p53 degradation, as shown here. Moreover, although p53 can stimulate autophagy by mTOR inhibition10
or DRAM transactivation14
in the context of DNA damage or p53 overexpression, we show here that baseline p53 levels can inhibit autophagy in other cellular contexts.
Undoubtedly, p53 is one of the tumour suppressors most frequently inactivated in cancer. What could be the advantage, in teleological terms, of increasing autophagy in tumour cells? Pharmacological stimulation of autophagy increases the resistance of cells to apoptosis, presumably due to the removal of pro-apoptotic mitochondria47
; both resistance to apoptosis and reduction of oxidative phosphorylation are hallmarks of cancer48,49
. Autophagy is a process by which cells struggle to survive under conditions of reduced intracellular metabolite concentrations, caused by loss of growth factor signalling that governs the uptake of nutrients24
. As shown here, cells lacking p53 are particularly resistant to ATP depletion and cell death induced by metabolic stress caused by hypoxia and nutrient depletion, and this resistance is lost when autophagy is inhibited. Oxygen and glucose supply to cancer cells is often low, especially in non-vascularized areas of tumour nodules. In this tumour microenvironment, an enhanced level of baseline autophagy may improve the fitness of malignant cells and constitute an initial advantage for those cancer cells that lose paracrine or contact-dependent growth signals as they infiltrate normal tissue or metastasize.
Although enhanced autophagy may confer an advantage, at least for stressed cells, it is plausible (yet remains to be proven) that a constant increase in self-cannibalism may reduce cell proliferation rates. In this context, the partial inhibition of autophagy either by loss of one beclin 1 allele (and perhaps other, yet-to-be-discovered genetic or epigenetic modifications) or constitutive activation of the PI-3K/Akt axis may function as a `corrective' measure to dampen autophagy. It will be important to determine in which order p53 and Beclin-1 are inactivated (or the PI-3K/Akt pathway is activated) during the natural history of breast, ovary and prostate cancers and how this correlates with the enhanced or reduced autophagic capacity of premalignant and malignant cells.