Molecular chaperones in mechanism of gain-of-function activity of p53 mutants

Magdalena Pruszko1,2, Maciej ylicz1, Alicja ylicz1

1International 2Institute

Institute of Molecular and Cell Biology in Warsaw of Biochemistry and Biophysics PAS, Warsaw

p53 mutants in cancer

The TP53 gene is a powerful tumor suppressor which is commonly mutated in human cancers. Most of the mutations occur in a DNA-binding domain of a polipetide chain. This mutations result in a p53 protein which is transcriptionally inactive because of reduced DNA-binding specificity. However, it was observed in vitro and in vivo that some TP53 mutants support cancer development and metastasis, unlike the TP53-null controls (Dittmer et al, 1993; Liu et al, 2000). This observation has led to a hypothesis that some p53 mutants are not fully inactive. They enhance or inhibit an expression of many genes by interactions with other transcription factors. The acquisition of pro-oncogenic activity by p53 mutants is called gain-of-function.

Molecular chaperones and p53

Molecular chaperones are necessary for normal cells to survive under stress conditions. Interestingly, they are also essential for tumorigenesis induced by mutations in RAS orTP53 (Dai et al, 2007). The level of Hsp70 molecular chaperone is elevated in many cancers and correlates with a poor prognosis for patient survival and response to therapy. Our laboratory demonstrated that heat shock proteins Hsp90 and Hsp70 form different complexes with wild type and mutant p53 and are required for p53 tumor suppressor activity (King et al, 2001; Walerych et al, 2009). These observations suggest that heat shock proteins can be involved in mutant p53 gain-of-function mechanism.
Transactivation Proline domain rich region 1 61 64

DNA-binding domain
Hsp90 Hsp70

Oligomerisation Cterminal domain domain 292 325 356 393

Hsp90 and Hsp70 bind to DNA-binding domain of the p53

The TP53 mutations are grouped in two categories: Contact - directly compromising binding of the protein to DNA e.g. R273H, R248Q, R248W Conformational - causing global conformational distortion of the protein e.g. R175H, R249S, Y220C

Aim of the project

We are studying the mechanisms by which different proto-oncogenes such as GRO1 or ID4 are regulated by common p53 gain-of-function mutants: R175H and R273H. Our main interest is a role of heat shock proteins in these processes.

Joerger AC, Fersht AR; 2010

Preliminary results
p53 R175H augments ID4 but not GRO1 expression in H1299 cells
Fig.1 The H1299 cells (p53-/-, Non-Small Cell Lung Cancer) were stably transfected with vector encoding TP53 R175H under inducible promoter. The expression is induced by Ponasteron A [A]. Upon TP53 R175H induction, the expression of ID4 is significantly enhanced [B]. No significant changes is observed in GRO1 expression [C].

p53 R175H augments GRO1 expression in SK-BR-3 cells

Fig.2 The expression of TP53 R175H in SK-BR-3 cells (breast cancer cells with endogenous p53 R175H) was silenced using shRNA [A]. The silencing of TP53 R175H reduces expression of GRO1 [B]

Questions to answer
1. Do Hsp90 and Hsp70 influence a transcriptional activity of p53 gain-of-function mutants? 2. What is the mechanism? 3. How do p53 gain-of-function mutants and ID4 collaborate on a metastasis induction?

Hsp70

? ?
p53
mutant

ID4
augments expression

?
stabilizes mRNA

EGFR integrins Receptors in lipids rafts

ID4 p53
mutant

Metastasis

Hsp90

changes

Lipids metabolism

The " Studies of nucleic acids and proteins - from basic to applied research" project is realized within International PhD Projects Programme of Foundation for Polish Science. The project is cofinanced from European Union - Regional Development Fund