The RNA helicase p68 modulates expression and function of the Δ133 isoform(s) of p53, and is inversely associated with Δ133p53 expression in breast cancer

Abstract

The RNA helicase p68 is a potent co-activator of p53-dependent transcription in response to DNA damage. Previous independent studies have indicated that p68 and the Δ133p53 isoforms, which modulate the function of full-length p53, are aberrantly expressed in breast cancers. Here we identify a striking inverse association of p68 and Δ133p53 expression in primary breast cancers. Consistent with these findings, small interfering RNA depletion of p68 in cell lines results in a p53-dependant increase of Δ133p53 in response to DNA damage, suggesting that increased Δ133p53 expression could result from downregulation of p68 and provide a potential mechanistic explanation for our observations in breast cancer. Δ133p53α, which has been shown to negatively regulate the function of full-length p53, reciprocally inhibits the ability of p68 to stimulate p53-dependent transcription from the p21 promoter, suggesting that Δ133p53α may be competing with p68 to regulate p53 function. This hypothesis is underscored by our observations that p68 interacts with the C-terminal domain of p53, co-immunoprecipitates 133p53α from cell extracts and interacts only with p53 molecules that are able to form tetramers. These data suggest that p68, p53 and 133p53α may form part of a complex feedback mechanism to regulate the expression of Δ133p53, with consequent modification of p53-mediated transcription, and may modulate the function of p53 in breast and other cancers that harbour wild-type p53.

Figure 1. Short interfering RNA (siRNA)-mediated knockdown of p68 results in a striking increase of Δ133p53 mRNA expression in response to DNA damage

MCF-7 cells were transfected with an siRNA targeted against p68 or a non-silencing control (NS). Untransfected (UT) cells served as an additional control. 48hr after siRNA transfection, cells were treated with 100μM of the DNA damaging agent etoposide for 4 hr. qRT-PCR (TaqMan) was performed on RNA extracted from these cells to measure mRNA expression of A: p68, B: Δ133p53, C: p53 and D: p21. Expression was normalised to expression of TBP and graphs plotted as fold change from untransfected cells (UT) that had not been treated with etoposide. The average values from 3 independent experiments are shown ± standard error of the mean (s.e.m). E; Corresponding western blots showing expression of p68, p53, p21 and Δ133p53 from the p68 siRNA knockdown experiments. A longer exposure of the western blot with the p53-specific antibody (lower panel) to detect p53 isoforms with H1299 transfected with Δ133p53α for comparison. *This protein species is as yet unidentified.

Figure 2. Induction of Δ133p53 mRNA when p68 levels are depleted is p53-dependent

A: HCT116 p53−/− cells (which still express Δ133p53) and B: T47D cells (mutant p53) were treated with a siRNA targeted against p68 or a non-silencing control (NS). After 48hr, cells were treated with 100μM of etoposide for 4 hr and expression of p68 and Δ133p53 was examined by qRT-PCR. Untransfected cells and HCT116 p53+/+ cells served as additional controls. Expression of the gene of interest was normalised to TBP expression and graphs plotted as fold change from untransfected cells (UT) that had not been treated with etoposide. The average values from 3 independent experiments are shown ± s.e.m.

Figure 3. p68 depletion does not increase p53-mediated transcription from the intron 4 promoter in response to etoposide treatment

H1299 cells (p53 null) were treated with a p68 siRNA or a non-silencing control (NS). After 48hr, cells were co-transfected with the intron 4 promoter of p53 fused to the firefly luciferase reporter gene, the Renilla-luciferase control plasmid and increasing concentrations of p53 (0, 200, 400 and 800ng). p53 intron 4 promoter activity is shown A: in the absence of etoposide, and B: in the presence of etoposide (100μM for 4 hr). C and D show the corresponding western blots to confirm depletion of p68 and expression of p53. Reporter activity was calculated relative to Renilla luciferase activity to control for transfection efficiency. The average values from 2 independent experiments are shown ± s.e.m. * denotes a p53 cleavage product resulting from the overexpression.

Figure 4. Δ133p53 inhibits p68 co-activation of p53-dependent p21 induction

H1299 cells (p53 null) were cotransfected with the p21 promoter fused to the firefly luciferase reporter gene, the Renilla-luciferase control plasmid, 1μg p68, 1ng Δ133p53 and increasing concentrations of p53 (0, 0.2, 0.5 and 1ng). p21 promoter activity is shown A: in the absence of etoposide, and B: in the presence of etoposide (50μM for 8 hr). C and D show the corresponding western blots to confirm expression of p68, p53 and Δ133p53. Reporter activity was calculated relative to Renilla luciferase activity to control for transfection efficiency. The average values from 3 independent experiments are shown ± s.e.m.

Figure 5. p68 interacts with the C-terminal domain of p53 and co-immunoprecipitates with the Δ133p53α isoform; the ability of p53 to tetramerise is important for interaction with p68

GST-pulldowns were performed using GST-tagged p68 and A: in vitro-translated ³⁵S-labelled p53 isoforms and B: p53 deletion/mutation derivatives. In each case the top panel shows in vitro-translated/³⁵S-labelled proteins with arrows denoting the bands corresponding to specific p53 isoforms. * indicates non-specific bands. C: Co-immunoprecipitation from nuclear extracts of endogenous p68 with endogenous p53 (U2OS cells), or p53 isoforms in H1299 cells stably expressing either Δ133p53α, p53β or p53γ. D: Cartoon illustrating the different p53 species referred to in the figure.

Figure 6. Model illustrating the regulatory interactions between p68, p53 and Δ133p53

p53 transactivates expression of p21 and Δ133p53 (1). p68 alone does not influence expression of these genes but co-activates p53-dependent expression of p21 (2). p68 inhibits p53-dependent expression of Δ133p53 but independently of events at the intron 4 (Δ133p53) promoter (3). Δ133p53 in turn inhibits expression of p21, most likely through direct interaction with p53 and/or p68 (4). Variations in the p68 levels (as observed in tumours) will not only affect p21 expression directly but, additionally, through an indirect mechanism by regulating the levels of an inhibitor of p21 expression (i.e. Δ133p53). Notably, Δ133p53 itself does not appear to influence the levels of p68 (data not shown).