TRAIL-Receptor 2 as a novel regulator of tumor suppressor protein p53
TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) receptor 2 (TRAIL-R2) has been shown to induce cell death preferentially in tumor cells upon binding of its ligand TRAIL. Nevertheless, TRAIL-R2 can also induce various pro-inflammatory pathways which promote invasion, migration and proliferation of apoptosis resistant malignant cells. Moreover, high intracellular levels of TRAIL-R2 are a characteristic feature for various tumors, suggesting a pro-tumoral function. Indeed, nuclear TRAIL-R2 (nTRAIL-R2) was shown to execute tumor promoting functions by regulating the maturation of microRNAs (miRNAs). Consistently, a high intracellular abundance of TRAIL-R2 was correlated with worse patient prognosis. In the present study a novel mechanism was uncovered how nTRAIL-R2 promotes tumor progression. TRAIL-R2 co-localizes and interacts with tumor suppressor protein p53 in the nucleus. Silencing of TRAIL-R2 in wild type p53 expressing HCT116 and A549 cells, elevates the protein level of p53 as well as the expression of its targets p21, BAX (Bcl-2 associated x protein) and MDM2 (mouse double minute 2 homolog). Vice versa exogenous overexpression of TRAIL-R2 isoforms decreased the protein levels of p53, p21 and MDM2 in A549 cells. Thereby, TRAIL-R2 acts independent of TRAIL and its death inducing function as an activator of caspases. Gene expression and protein half-life analyses indicated that TRAIL-R2 does not impact p53 transcription, but destabilizes p53 protein. Furthermore, this effect is dependent on the conventional p53 degradation pathway by the ubiquitin-proteasome-system. Blocking of the proteasomal degradation pathway by inhibition of the 26S proteasome via MG132 as well as blocking MDM2-mediated p53 ubiquitination by Nutlin 3a, abolished the TRAIL-R2-mediated destabilization of p53. Consequently, TRAIL-R2 might promote MDM2-mediated p53 ubiquitination. Immunoprecipitation experiments revealed no TRAIL-R2-MDM2 interaction, but immunofluorescence stainings indicated a presence of TRAIL-R2 in protein complexes with both p53 and MDM2. Thereby, p53 and MDM2 associate independent of the TRAIL-R2 status in A549 cells. Therefore, besides MDM2, a co-factor might participate in the TRAIL-R2-p53 regulatory axis to modulate p53 protein stability. On the track of identifying potential co-players, an involvement of ubiquitin peptidases, especially USP10 (ubiquitin specific peptidase 10), as well as HMGA2 (high mobility group AT-hook 2), which was shown to promote MDM2-mediated p53 ubiquitination in colorectal cancer, was excluded. Interestingly, TRAIL-R2 was identified as a novel potential interaction partner of promyelocytic leukemia (PML) protein. Moreover, PML knockdown led to an increased p53 protein level in TRAIL-R2-expressing cells to similar extent as the knockout of TRAIL-R2 itself, whereas no alteration of p53 level was seen by PML knockdown in TRAIL-R2 knockout cells. PML is the key organizer of PML nuclear bodies that can sequester, modify, or degrade partner proteins including p53 and MDM2. Accordingly, PML might assist TRAIL-R2 in destabilizing p53. In summary, in the present study a potential regulatory feedback signaling loop between nTRAIL-R2 and p53 was identified in wild type p53 expressing cancer cells. Activated p53 transcriptionally upregulates TRAIL-R2 expression, vice versa nTRAIL-R2 destabilizes p53 and thereby inhibits its transcriptional output. The detailed mechanism how TRAIL-R2 impacts p53 stability will be examined in future studies.
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