Abstract
In renal cell carcinoma, transglutaminase 2 (TGase 2) crosslinks p53 in autophagosomes, resulting in p53 depletion and the tumor's evasion of apoptosis. Inhibition of TGase 2 stabilizes p53 and induces tumor cells to enter apoptosis. This study explored the mechanism of TGase 2-dependent p53 degradation. We found that TGase 2 competes with human double minute 2 homolog (HDM2) for binding to p53; promotes autophagy-dependent p53 degradation in renal cell carcinoma (RCC) cell lines under starvation; and binds to p53 and p62 simultaneously without ubiquitin-dependent recognition of p62. The bound complex does not have crosslinking activity. A binding assay using a series of deletion mutants of p62, p53 and TGase 2 revealed that the PB1 (Phox and Bem1p-1) domain of p62 (residues 85–110) directly interacts with the β-barrel domains of TGase 2 (residues 592–687), whereas the HDM2-binding domain (transactivation domain, residues 15–26) of p53 interacts with the N terminus of TGase 2 (residues 1–139). In addition to the increase in p53 stability due to TGase 2 inhibition, the administration of a DNA-damaging anti-cancer drug such as doxorubicin-induced apoptosis in RCC cell lines and synergistically reduced tumor volume in a xenograft model. Combination therapy with a TGase 2 inhibitor and a DNA-damaging agent may represent an effective therapeutic approach for treating RCC.
Highlights
P53 Regulation by human double minute 2 homolog (HDM2) involves proteasomal degradation through ubiquitination, whereas TGase 2-mediated p53 regulation involves autophagosome degradation
This resembles HDM2 that binds to the N terminus of p53 and ubiquitinates multiple lysine residues including K370, 372, 373, 381, 382 and 386 in the C terminus of p53.16 We found that the TGase 2-binding domain and TGase 2-targeting sites in p53 are different because TGase 2 works as a chaperon of p53 to the autophagosome
TGase 2 is a possible therapeutic target for treating renal cell carcinoma (RCC), and this has been proved in a xenograft model.[3]
Summary
P53 is regulated competitively by TGase 2 and HDM2 in RCC under starvation. To assess the relative contributions of TGase 2 and HDM2 in p53 depletion in RCC cell lines, the two corresponding genes were silenced using small interfering RNAs (siRNAs; Figure 1). A similar pattern was observed in CAKI-1 cells, with a 1.8-fold and 2.0-fold increase, respectively (Figures 1c and d) This result suggests that p53 regulation depends on HDM2 and TGase 2 in RCC cells under starvation conditions. TGase 2 transports p53 to the autophagosome for degradation by autophagy through polymerization This idea was supported by immunoblotting of p53, which showed that high molecular weight polymers were only detected in the insoluble fraction after chloroquine treatment in RCC.[2] Here, we tested the binding of three molecules, namely, TGase 2, p53, and p62, in ACHN (Figure 3a) and CAKI-1 (Figure 3b) cells. To investigate which part of p62 binds to TGase 2, a series of FLAG-tagged deletion mutants of p62 was constructed, transfected into HEK293
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