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A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo - PubMed

. 2007 Apr;117(4):1008-18.

doi: 10.1172/JCI28920. Epub 2007 Mar 8.

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A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo

Helen S Bell et al. J Clin Invest. 2007 Apr.

Abstract

The tumor suppressor p53 is a potent inducer of tumor cell death, and strategies exist to exploit p53 for therapeutic gain. However, because about half of human cancers contain mutant p53, application of these strategies is restricted. p53 family members, in particular p73, are in many ways functional paralogs of p53, but are rarely mutated in cancer. Methods for specific activation of p73, however, remain to be elucidated. We describe here a minimal p53-derived apoptotic peptide that induced death in multiple cell types regardless of p53 status. While unable to activate gene expression directly, this peptide retained the capacity to bind iASPP - a common negative regulator of p53 family members. Concordantly, in p53-null cells, this peptide derepressed p73, causing p73-mediated gene activation and death. Moreover, systemic nanoparticle delivery of a transgene expressing this peptide caused tumor regression in vivo via p73. This study therefore heralds what we believe to be the first strategy to directly and selectively activate p73 therapeutically and may lead to the development of broadly applicable agents for the treatment of malignant disease.

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Figures

Figure 1
Figure 1. The p53-derived peptide 37AA induces cell death without direct activation of p53 target genes.

(A) Representation of the p53 mutants used in this study. Full-length p53 is shown in green with 5 evolutionarily conserved regions (boxes) shown in yellow. Two truncations, tr105 (aa 1–105) and tr210 (aa 1–210), are shown as well as the composition of the 37AA peptide, with aa from conserved box II (residues 118–142) in red and those from conserved box III (residues 171–181) in blue. (B–E) Cells were transiently transfected with the indicated plasmids and subsequently analyzed 36 hours (C and E) and 48 hours later (D) for changes in cell cycle and cell death by flow cytometry. Ap, apoptosis. (B) Cells were transiently transfected with the indicated plasmids, and transgene expression was determined by Western blotting. The additional lower band seen following expression of 37AA represents expression from an alternate internal methionine. Mutation of this methionine to alanine resulted in expression of the larger protein species only and produced identical results (not shown). (F) No direct activation of p53 target genes by 37AA was observed. Saos-2 cells were transfected with luciferase reporter plasmids for the p53 target genes p21, Bax, and PIG3 as well as with the indicated expression constructs. After 24 hours, cells were assayed for luciferase activity, and the data were normalized against transfected β-gal activity. Values represent fold activation relative to activity of GFP alone, which was assigned as 1.

Figure 2
Figure 2. 37AA causes selective killing of tumor cells.

Tumor cell lines LoVo (A and B) and HeLa (C and D) as well as primary cell cultures MEF (E and F), HFF (G and H), and RPE (I and J) were transfected or infected with the indicated transgenes. After 48 hours (transfection, A–D) or 72 hours (infection, E–K), transgene expression was assessed by Western blotting (A, C, E, G, and I), and the effects on cell death were determined by flow cytometry (B, D, F, H, J, and K).

Figure 3
Figure 3. 37AA binds to iASPP and disrupts its interaction with p73.

(A) 37AA binds iASPP. Saos-2 cells were infected with adenoviruses expressing GFP, wtp53, tr105, tr210, and 37AA following transfection with Flag-iASPP. Cells were lysed and split into 2 and used for either Western analysis (GFP blot) or IP (Flag-iASPP pulldown and GFP blot). WB, Western blot. (B) 37AA does not affect iASPP protein levels. Cells were transfected with iASPP and either GFP, 37AA, or empty vector. Protein levels were measured by Western blotting. (C and D) 37AA affects iASPP localization and dissociates iASPP from p73. Cells were transfected with iASPP, GFP, 37AA, or p73. (C) iASPP and GFP localization was determined by immunofluorescence. (D) iASPP binding to p73 was determined by IP.

Figure 4
Figure 4. Expression of iASPP or interference with p73 inhibits 37AA-mediated cell death.

(A) Saos-2 cells were transfected with the indicated plasmids and then infected with an adenovirus (Ad) expressing ΔN-p73α or empty virus control (Con). After 48 hours, cells were analyzed for cell death by flow cytometry or for expression of transfected plasmids by Western blotting. (B) Saos-2 cells that had been infected with a retrovirus expressing either an shRNA directed against TA-p73 (pRS-p73) or a scrambled control (pRS-Scr) were transfected with the indicated plasmids. After 48 hours, cells were analyzed for cell death by flow cytometry or for expression of transfected plasmids by Western blotting. (C) Saos-2 cells were transiently cotransfected with the indicated amounts of 37AA and iASPP. After 48 hours, cells were analyzed for cell death by flow cytometry and for expression of transfected plasmids by Western blotting. (D) The effects of iASPP knockdown by RNA interference in p53-null cells were determined. Saos-2 cells were transfected with plasmids expressing 2 different shRNAs that target iASPP or a scrambled shRNA control. The effects of the shRNAs on transfected iASPP expression were determined by Western blotting after 24 hours, and the effects on long-term survival were determined by assessing the clonogenic capacity of the cells. G418 con, control for drug activity; cells were mock-transfected and selected with G418. Original magnification, ×1.

Figure 5
Figure 5. 37AA does not transactivate directly, but causes secondary activation through p73.

(A) Saos-2 cells were transiently transfected with 5 μg PUMA luciferase plasmid and 200 ng each of the indicated expression constructs. After 72 hours, cells were assayed for luciferase activity, and the data were normalized against transfected β-gal activity. Values represent fold activation relative to the activity of GFP alone, which was assigned as 1. (B) Saos-2 cells were infected as indicated with adenoviruses expressing tr105, 37AA, ΔN-p73 adenovirus, or control empty adenovirus. At the indicated time points, RNA was isolated from the cells and subjected to RT-PCR for PUMA and GAPDH. (C) At the same time cells were harvested and assessed for changes in cell death by flow cytometry. (D) Cells were infected with the indicated adenoviruses for 48 hours. The mRNA levels for PUMA, DR5, Bax, and p21 were determined by qPCR. Samples were normalized to the levels of 18S ribosomal RNA. (E) Schematic of the mode of action of 37AA. Introduction of 37AA into p53-null cells sequesters iASPP and thereby derepresses p73. TA-p73 subsequently activates apoptotic target genes such as PUMA to bring about programmed cell death. In line with this model, cell death from 37AA can be inhibited by decreasing p73 activity or increasing iASPP.

Figure 6
Figure 6. 37AA causes tumor regression in vivo.

Cultures of LoVo (A), LoVo-E6 (B), LoVo-E6–pRS-Scr (C), LoVo-E6–pRS-p73 (D), and LoVo-E6–iASPP cells (E) were injected into the flanks of athymic mice. Upon tumor formation (day 0), mice were treated by intravenous tail vein injection on days 0, 2, 4, 6, and 8 with DNA-dendrimer complexes containing 50 μg of 37AA, wtp53, or tr105. Tumor volume was measured daily, and growth was plotted as relative to tumor size on day 0.

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