The role of p53/p73 family members in the cytotoxic response
Programme Leader: Gerry Melino
Summary of Research Interests
Several chemicals exert toxic or carcinogenic effects by damaging DNA and unrepaired DNA damage may cause uncontrolled cell proliferation or apoptosis. A major DNA repair mechanism involves the tumour suppressor gene p53 (Figure 1). The two homologues of p53, p63 and p73 are also involved in the decisions of survival or death upon DNA damage (Figures 1, 2). Like p53, both p63 and p73 possess an N-terminal transactivation domain (TAD), a DNA-binding domain (DBD), and a C-terminal oligomerization domain (OD) and share 60% similarity with the p53 DBD. This similarity allows them to transactivate same target genes and to induce cell cycle arrest and apoptosis. However, knockout mice studies clearly demonstrated that both proteins also have distinct functions. Whereas p53 null mice develop normally, p63 and p73 knockout mice show severe developmental defects. p63-null mice fail to develop limbs, skin and epithelial tissues; and p73-null mice exhibit neurodevelopmental and inflammatory defects.
The “Apoptosis & Cancer” Group is focused on understanding the role of p73 in response to toxic insults, and subsequently in tumorigenesis and development. In general, we investigate both p73 interactors and p73 degradation pathways.
1. Identification of p73 Target Genes
We generated TET-inducible p53, p63 and p73 expressing cell lines and analysed differentially expressed genes using Affymetrix DNA chip technology. We have extensively validated and characterised p63/p73 target genes and extended our findings to cancer. p73 protein levels and the ratio TAp73/DNp73 are extremely important in cancer, and we demonstrated that in head and neck cancers and hepatomas, p73 protein expression is a prognostic factor that predicts chemotherapy sensitivity. We also identified that p73 mediates cell death, inducing pro-apoptotic genes like CD95, TRAIL, caspase 8, FLIP, Scotin, Bax, Puma, Noxa.
2. Control of p73 Protein Stability
Abnormalities in regulation of protein stability result in impairment of many cellular processes and, therefore, it is important to understand how the balance between synthesis and degradation is maintained.
We demonstrated that the HECT-type E3 ubiquitin ligase Itch regulates p73 and p63 stability inducing their proteosomal degradation. p73 and p63 protein levels are maintained low through Itch-mediated ubiquitylation, but they accumulate in tumor cells upon gamma-irradiation or chemotherapeutic drugs treatment, concomitantly with DNA damage-caused Itch downregulation (Figure 3). p73 protein stability is also controlled by caspases. Indeed, we showed that, following caspases activation, the trans-inhibitory domain of TAp63 is cleaved, generating a highly apoptotic TAp63 protein. Likewise, we identified that p73 and its caspase-cleaved fragments localize to mitochondria, enhancing apoptotic signals.
Calcium-mediated activation of the proteases Calpain leads to degradation of many proteins among which p73. Indeed, we proved that, in-vitro, Calpain I cleaves p73, while overexpression of calpastatin, a selective calpain inhibitor, increases p73 level, suggesting that calpains regulate p73 protein stability. Previously, we showed that DNp73 is less stable than TAp73 in cells treated with DNA damaging drugs and recently we identified PIR2, as an E3 ubiquitin ligase that preferentially targets DNp73. The stability of p73 also requires post-translational modifications (i.e. phosphorylations and acetylations) and our group unveiled a role for the PML protein in this regulation.
In summary, p73 function is regulated via complex mechanisms at transcriptional level, via specific protein-protein interactions. We characterized the interaction of p73 with several partners, including Sumo1, DNp73, Bub1, Bub3, BubR1, c-Abl. Similarly, we identified several p73 transcriptional targets (Bax, CD95, TRAIL, Noxa, Puma, p21, loricrin) (Figure 4). As Itch is a major regulator of p73 stability, this E3 ligase is a potential therapeutic target. Indeed, in collaboration with the MRC-Technology, we filed a patent and are currently developing Itch inhibitors as potential anti-cancer drugs.
3. Transgenic p73 Mice
To understand the roles of p73 isoforms in-vivo, we developed selective TAp73 and DNp73 null mice. Cells from TAp73-/-mice exhibit genomic instability and aneuploidy, a characteristic of most solid tumors, often associated with poor prognosis. Moreover, decreased TAp73 expression correlates with increased expression of kinetochore-related proteins in lung cancer patients. We also discovered that ectopic TAp73alpha induces aneuploidy, due to its interaction with kinetochore-related proteins Bub1, Bub3, and BubR1 and impaired mitotic checkpoint activity. Cells from DeltaNp73(-/-) mice are sensitized to DNA-damaging agents and show increased p53-dependent apoptosis. In this way, we found that DeltaNp73 localizes to the site of DNA damage and inhibits ATM activation and subsequent p53 phosphorylation/activation. We are actively working on these mice to fully elucidate p73 contribution to development and tumor formation.
In summary, the “Apoptosis & Cancer” Group is focused on the biology of p73 and its role in responding to toxic insults (Figure 5).