During malignancy, proteins are usually uncontrollably over-expressed or structurally affected due to genetic mutations, resulting in changes in activity and interaction of these proteins in cancer cells. It remains, however, largely unexplored whether aggregation of tumor suppressor/oncogenes could contribute to the induction or progression of malignancy. The tumor suppressor p53 is a key regulator of the cell cycle and gets mutated in over 50% of reported human tumor cases, making it a major target for anti-cancer therapy. Previously, it has been shown that the DNA-binding domain of p53 is conformationally unstable and that the majority of hot-spot disease mutants such as R175H, R282W, R248Q and R249S further destabilize the DNA-binding domain. Consequently, a proportion of these mutants are at least partially unfolded and therefore inactive. Hence, these mutants, present in about 30% of reported clinical cases, are usually referred to as ‘structural’ mutants. We found that the dominant-negative activity and gain-of-function effects of structurally destabilized p53 mutants results from their increased aggregation propensity. Upon its aggregation, mutant p53 not only induced misfolding and co-aggregation of wild-type p53, but also of its paralogues p63 and p73 into cellular inclusions, causing inefficient transcription of target genes that are important for cell growth control and apoptosis.
We assessed the oligomerization state of p53 in SaOS-2 cells by Blue-Native PAGE (BN-PAGE) and Western Blot analysis. Transiently over-expressed wild type p53 appeared as monomers, tetramers and octamers on Western blot and an identical pattern was also observed for the DNA-contact mutants R248W and R273H. However, over-expression of the aggregating mutants R175H, R282W, R248Q, R249S, P250L, E258V, R110L and R110P caused a shift in molecular mass ranging from 800kDa up to the fractionation limit of the gel (10,000kDa), consistent with the formation of large multimeric assemblies.
The crystal structures of the DNA-binding domains of p53, p63 and p73 show high homology, with aggregating sequences in the same structural motif (marked in red). The TANGO analysis combining sequence alignment revealed that the aggregating sequences of p53 (251-257), p63 (321-327) and p73 (271-277) sit in a highly conserved region of DNA-binding domain.
Mutant p53 R110P was coexpressed with p63 in SaOS-2 cells, and three-dimensional confocal microscopy revealed the morphology of large aggregates formed by mutant p53 and p63 in the perinuclear regions. The ability of mutant p53 to coaggregate with p63/p73 explains its gain of oncogenic function.
In two large-scale studies on prognosis of breast and colon cancers, the end point survival of patients with non-aggregating (DNA-contact) mutations was significantly higher than those carrying aggregating (structural) mutations.
