TRP53 activates a global autophagy program to promote tumor suppression

Abstract

The mechanisms by which the TP53/TRP53 transcription factor acts as a tumor suppressor remain incompletely understood. To gain new insights into TP53/TRP53 biology, we used ChIP-seq and RNA-seq technologies to define global TRP53 transcriptional networks in primary cells subjected to DNA damage. Intriguingly, we identified a TRP53-regulated autophagy program, which can be coordinately regulated by the TRP53 family members TRP63 and TRP73 in certain settings. While autophagy is not involved in TRP53-dependent cell cycle arrest, it contributes to both TRP53-driven apoptosis in response to DNA damage and TRP53-mediated transformation suppression. Collectively, our genome-wide analyses reveal a profound role for TRP53 in regulating autophagy, through an extensive transcriptional network, and have demonstrated an important role for this program in promoting TRP53-mediated apoptosis and tumor suppression.

The TP53/TRP53 transcription factor is a critical tumor suppressor, as evidenced by both sporadic and inherited human cancers with TP53 mutations and the completely penetrant tumor predisposition of trp53 null mice. TP53/TRP53 becomes activated in response to a variety of stresses, including DNA damage, oncogenic signals, and nutrient starvation, and consequently drives apoptosis, cell cycle arrest, or cellular senescence to limit the expansion of damaged cells. While these classical TP53/TRP53 responses have long been thought to be responsible for tumor suppression, recent studies have questioned this notion. Thus, to gain new insight into TP53/TRP53 function, we used ChIP-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) technologies to perform global transcriptional profiling of TRP53 function in primary mouse embryonic fibroblasts (MEFs) subjected to DNA damage, to reveal novel TRP53-regulated genes and pathways.

Examination of the TRP53-bound genes from our TRP53 ChIP-seq analyses uncovered TRP53 binding to an array of genes encoding proteins involved in multiple steps of autophagy, including upstream regulation, autophagy core machinery function and lysosomal function (Fig. 1A). Furthermore, expression analysis revealed that these TRP53-bound genes are induced by DNA damage, albeit with varying TRP53-dependence. We validated induction of TRP53 autophagy target genes in response to nongenotoxic activation of TRP53 by Nutlin-3a, a compound that prevents TRP53 binding to its negative regulator MDM2, and by genetic activation of TRP53 expression, suggesting that the autophagy program is induced independently of DNA damage. TP53/TRP53 also activates the autophagy transcriptional program in diverse human and mouse cell types. Thus, TP53/TRP53 induces autophagy target gene expression in a variety of contexts.

Figure 1. TRP53-regulated autophagy program and its role in TRP53 responses. (A) List of genes belonging to the TRP53-regulated autophagy program, including genes encoding proteins involved in the upstream regulation of autophagy, autophagy core machinery function and lysosomal function. (B) Model showing the contribution of autophagy to TRP53 responses. TP53/TRP53 regulates target genes to induce cell cycle arrest/survival, apoptosis and autophagy, responses that are thought to contribute to TP53/TRP53-mediated tumor suppression. In some contexts, TRP63 and TRP73 can also contribute to TRP53 target gene regulation. X denotes additional known and unknown TP53/TRP53 target genes involved in these responses. TRP53-induced autophagy may contribute to tumor suppression through its role in TRP53-dependent apoptosis (red arrow), and may in addition directly contribute to tumor suppression independently of apoptosis [red arrow with (?)].

Intrigued by the observation that there was some TRP53-independent regulation of autophagy genes in response to DNA damage, we considered the possibility of compensation by the TRP53 family members TRP63 and TRP73, which can bind and regulate target genes through the same consensus site as TRP53. Notably, both TP63 and TP73 have been implicated previously in autophagy through transcriptional regulation of select autophagy genes, and, indeed, we found many of the TRP53-bound autophagy genes we identified to be bound by TP63 and TP73 in published genome-wide DNA-binding data sets. Interestingly, we found that knockdown of TRP63 and TRP73 in wild-type cells compromised induction of some autophagy genes by DNA damage, suggesting that the TP53/TRP53 family members contribute to the activation of the autophagy program. Moreover, induction of many autophagy genes by DNA damage is abolished upon knockdown of TRP63 and TRP73 in trp53^−/− MEFs, suggesting that physiological TRP63 and TRP73 can compensate for TRP53 deficiency. The shared role of the TP53/TRP53 family in inducing autophagy genes suggests that autophagy may represent an ancestral function common to all family members.

Further analyses revealed that induction of wild-type TRP53 correlates with increased levels of autophagy in various settings, including during both cell cycle arrest and apoptosis, indicating that autophagy is a core TRP53 response. We tested further whether expression of TRP53 mutants that lack wild-type TRP53 transcriptional activity by virtue of mutations in the transcriptional activation domains (TRP53^25,26,53,54) or DNA-binding domain (the tumor-derived mutants TRP53^R172H and TRP53^R270H), are able to induce autophagy. Indeed, we found that only wild-type, but not transcriptionally-deficient TRP53, is able to induce autophagy, supporting the importance of the TRP53-regulated autophagy transcriptional program for enhancing autophagic flux. To determine the functional relevance of the TRP53-induced autophagy program, we assessed the role of autophagy in classical TRP53 responses using primary Atg5 conditional knockout MEFs. TRP53-induced G₁ cell cycle arrest in response to DNA damage allows wild-type cells to repair DNA before progression through the cell cycle and to survive, while trp53^−/− cells exposed to DNA damage continue to proliferate, accumulate DNA damage and ultimately die due to mitotic catastrophy. Autophagy-deficient cells did not exhibit compromised TRP53-dependent G₁ arrest or survival. In contrast, in oncogene-expressing MEFs, which are sensitized to TRP53-dependent apoptosis, autophagy deficiency results in diminished DNA damage-induced apoptosis. Therefore, instead of mediating TRP53 function in cell cycle arrest and survival, autophagy contributes to TRP53-dependent apoptosis. Finally, we performed soft agar transformation assays, in which wild-type TRP53 restrains the growth of oncogene-expressing MEFs through the induction of apoptosis. Indeed, like TRP53 loss, autophagy deficiency increases colony number significantly relative to oncogene-expressing wild-type controls, suggesting that TRP53 suppresses transformation at least in part by triggering an autophagy program. This observation is consistent with a role for TRP53-induced autophagy in suppressing tumorigenesis (Fig. 1B).

In the future, it will be important to investigate the contexts in which TRP53-induced autophagy contributes to tumor suppression in vivo using mouse models, and its relative importance compared with the more well-established TP53/TRP53 responses. Recently, the dogma that the canonical TP53/TRP53 responses are required for tumor suppression has been challenged by the analysis of Trp53 knockin mice expressing mutants either compromised for cell cycle arrest and apoptosis in response to acute DNA damage (TRP53^25,26) or cell cycle arrest, senescence and apoptosis (TRP53^3KR), yet still able to function as tumor suppressors. It would be interesting to determine whether the TRP53^25,26 and TRP53^3KR mutants retain the capacity to induce the TRP53 autophagy program, which could provide further support for the importance of autophagy for TRP53-mediated tumor suppression. In addition, elaborating on the contribution of the TP53/TRP53 family members to the regulation of the autophagy program, and the mechanisms by which this cooperation occurs, is an exciting avenue for further investigation. Finally, it will also be critical to decipher the molecular mechanisms by which autophagy contributes to TRP53-dependent apoptosis and tumor suppression. Since autophagy can promote cancer cell survival and resistance to cancer therapies in some contexts, a deeper understanding of the factors dictating whether autophagy contributes to cell death or survival will be key for maximizing the benefits of modulating autophagy in cancer therapy.

Kenzelmann Broz D, Spano Mello S, Bieging KT, Jiang D, Dusek RL, Brady CA, Sidow A, Attardi LD. Global genomic profiling reveals an extensive p53-regulated autophagy program contributing to key p53 responses. Genes Dev. 2013;27:1016–31. doi: 10.1101/gad.212282.112.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.