A platform for interrogating cancer-associated p53 alleles

Abstract

p53 is the most frequently mutated gene in human cancer. Compelling evidence argues that full transformation involves loss of growth suppression encoded by wild-type p53 together with poorly understood oncogenic activity encoded by missense mutations. Furthermore, distinguishing disease alleles from natural polymorphisms is an important clinical challenge. To interrogate the genetic activity of human p53 variants, we leveraged the Drosophila model as an in vivo platform. We engineered strains that replace the fly p53 gene with human alleles, producing a collection of stocks that are, in effect, 'humanized' for p53 variants. Like the fly counterpart, human p53 transcriptionally activated a biosensor and induced apoptosis after DNA damage. However, all humanized strains representing common alleles found in cancer patients failed to complement in these assays. Surprisingly, stimulus-dependent activation of hp53 occurred without stabilization, demonstrating that these two processes can be uncoupled. Like its fly counterpart, hp53 formed prominent nuclear foci in germline cells but cancer-associated p53 variants did not. Moreover, these same mutant alleles disrupted hp53 foci and inhibited biosensor activity, suggesting that these properties are functionally linked. Together these findings establish a functional platform for interrogating human p53 alleles and suggest that simple phenotypes could be used to stratify disease variants.

Figure 1. Human p53 functionally complements the Drosophila counterpart

(a) The Dp53⁺ rescue and the humanized p53 fly set (HPFS) were generated by integrating a BAC containing a 20kb genomic fragment of the Dp53 locus (CH322-115D03) into an attP landing site on the X chromosome (PBac{y⁺-attP-9A}VK00006) via phiC31-driven recombination(42). Exons 1-8 of the Dp53 locus code for the predominant gene product and Exons A&B are alternative exons used in a less abundant isoform. To humanize p53, exons 1-8 of Dp53 were replaced with wild-type hp53 cDNA (NM_000546.5) via recombineering. To generate hp53 mutant lines, we engineered five of the most prevalent point mutations in human cancer into the cDNA using site-directed mutagenesis. The relative prevalence of the five mutations are indicated by height of the red bars. Orange: Drosophila exons, gray: UTRs, blue: human sequence, dark blue: hp53 DNA binding domain. Translation start sites are shown as black arrows. (b) Western blot for hp53 levels in HPFS ovary lysates using the hp53 DO-1 monoclonal antibody. Note that wild-type hp53 lines (hp53¹ or hp53²) and hp53 mutants (R175H, G45S, R248Q, R273C, R273H) express hp53 (black arrow) at similar levels. Human bronchial epithelial cell (HBEC) lysate was included as a positive control for full-length hp53. * and ** are non-specific cross-reacting bands. ** serves as a loading control. (c-d) Acridine Orange (AO) staining of wing discs (c) and embryos (d) mock treated (-IR) or irradiated at 40 gray (+IR) as in Akdemir et al(20). Note that dp53^−/− embryos and wing discs do not elicit an apoptotic response after irradiation. This response is restored by presence of the Dp53⁺ rescue fragment and partially restored by hp53. (e) Confocal micrographs of immunofluorescence on whole mount ovarioles from HPFS flies carrying a biosensor (p53R-GFPcyt) for p53 activity. Flies were irradiated at 40 gray (+IR), their ovaries dissected, fixed, stained, mounted and imaged as in Lu et al(18). An ovary is made up of many ovarioles, which function as egg factories. Insets focus on the germanium, the region of the ovariole where stem cells (white arrowheads) reside. Note that the Dp53⁺ rescue can restore biosensor activation in the stem cells (a, inset) and that hp53 (hp53¹ or hp53²) activates the biosensor throughout the ovariole. Antibodies: mouse anti-hp53 DO-1 (Santa Cruz, 1:1000), rabbit anti-GFP (Life Technologies, IF:1:500). Genotypes: in (b) WT is yw and dp53^−/− is dp53^{5A-1-4/5A-1-4}, in (c-e) all flies are in a p53R-GFPcyt,dp53^NS/TM3,Sb background except for WT which is w;p53R-GFPcyt. The TM3,Sb balancer chromosome contains a breakpoint in the p53 locus(43) and behaves like a p53 mutant in our hands.

Figure 2. Cancer-associated hp53 alleles are defective for in vivo activation of a p53 biosensor

(a) Confocal micrographs of immunofluorescence on whole mount ovarioles from HPFS flies carrying a biosensor (p53R-GFPcyt) for p53 activity. Flies were treated and their ovaries stained as in Figure 1E. Note that wild-type (hp53¹) but not mutant (R175H, G45S, R248Q, R273C, R273H) hp53 can activate the biosensor throughout the ovariole. (b) Western blots of lysates from HPFS ovaries from (a) mock treated (−) or irradiated at 40 gray (+). Note that wild-type hp53 flies show radiation induced upregulation of the p53 biosensor (anti-GFP) without increased stabilization of hp53. Furthermore, all five oncogenic mutants fail to activate the biosensor. (c) Embryos of the indicated genotypes were collected for 2 hours, aged for 2.5 hours, mock treated or irradiated at 40 gray, allowed to recover for 2.5 hours and then processed for RNA as in Akdemir et al(20). WT embryos show induction of selected radiation-induced p53-dependent (RIPD) genes(20) after irradiation. This response is lost in dp53^−/− embryos and rescued by the Dp53⁺ genomic fragment. Hp53 shows potent rescue of Xrp1 induction and modest rescue of hid, ku80 and egr induction but fails to rescue rpr and skl induction. Levels were normalized to rp49. Note log scale to accommodate Dp53⁺ induction of Xrp1. Ovaries were dissected, fixed, stained, mounted and imaged as in Figure 1e. Antibodies: rabbit anti-GFP (Life Technologies, 1:500 (IF), 1:1000 (WB)), mouse anti-hp53 DO-1 (Santa Cruz, 1:1000), mouse anti-tubulin E7 (1:5000 Developmental Studies Hybridoma Bank). Genotypes: HPFS flies in (a) are in a p53R-GFPcyt,dp53^NS/TM3,Sb background and those in (b) in a p53R-GFPcyt,dp53^NS/NS background, and in (c) WT is yw, and dp53^−/− is dp53^{5A-1-4/5A-1-4}.

Figure 3. Normal but not mutant hp53 is recruited to SUMO-associated nuclear foci

Confocal micrographs of immunofluorescence on whole mount ovaries. (a) Representative germaria of indicated HPFS genotypes stained with anti-hp53 (see Supplementary Figure 3 for the other cancer alleles). White arrowheads indicate representative foci. Note lack of foci in the two representative mutants shown (see Supplementary Figure 3b for the complete HPFS panel). (b-c) Quantification of nuclear foci from confocal micrographs of HPFS germaria (see Supplementary Figure 3c) using Imaris (Bitplane).(b) Average volume of nuclear foci within a ~50um Z-stack (in um³). (c) Total number of nuclear foci present within a ~50um Z-stack. Dotted blue line in (b) and (c) represent level of background. Data represents two germaria. Error bars represent SD. (d) Germarium showing hp53 (red) and Dp53 (green) colocalization. White arrowheads indicate representative colocalization of hp53 foci and Dp53 foci. (e) Germarium showing examples of hp53 (red) and dSUMO (green) colocalization. White arrowheads indicate representative colocalization of hp53 foci and dSUMO staining. Note that with both Dp53 and dSUMO, extensive but not complete colocalization is seen. All images represent collapsed Z-stacks (~50um). Ovaries were dissected, fixed, stained, mounted and imaged as in Figure 1e. Antibodies: rabbit monoclonal anti-hp53 7F5 (Cell Signaling, 1:500), anti-lamin Dm0 (Developmental Studies Hybridoma Bank, 1:100), mouse anti-Dp53 25F4 (Developmental Studies Hybridoma Bank, 1:500), rabbit anti-hp53 FL-393 (Santa Cruz, 1:500), mouse anti-hp53 DO-1 (Santa Cruz, 1:500), rabbit anti-dSUMO (kind gift from Anne Dejean, 1:300). Genotypes: all HPFS flies are in a dp53^{5A-1-4/5A-1-4} background except for (e), in which Dp53^+/− is +/TM3,Sb.

Figure 4. Cancer-associated hp53 proteins disrupt wild-type hp53 foci and biosensor activation

Confocal micrographs of immunofluorescence on whole mount ovarioles from HPFS flies carrying a biosensor (p53R-GFPcyt) for p53 activity. (a) Hp53¹ was put in trans to either a wild-type chromosome (hp53¹/+) or to mutant hp53 (hp53¹/mutant) and then ovarioles were stained for hp53. Note that hp53¹ can form foci but these do not occur when cancer-associated hp53 proteins are present. White arrowheads indicate representative foci. (b) The same ovarioles in (a) stained with anti-GFP to detect biosensor activation. Note that disruption of hp53 foci by hp53 variants results in decreased biosensor activation. (c) Quantification of foci present within confocal micrographs in (a). In (c) confocal images were processed using Imaris (Bitplane). Background subtraction was applied, surfaces were created using the hp53 channel to isolate foci between 0.05-0.2 um³, and then values for the total number of foci per Z-stack were plotted. Data represents four germaria. Error bars represent SD. Ovaries were dissected, fixed, stained, mounted and imaged as in Figure 1e. Antibodies: mouse anti-hp53 DO-1 (Santa Cruz, 1:500), rabbit anti-GFP (Life Technologies, 1:500). Genotypes: HPFS flies are in a p53R-GFPcyt,dp53^NS/5A-1-4 background. All flies carry two copies of the p53 biosensor.