Igf2 pathway dependency of the Trp53 developmental and tumour phenotypes

Abstract

Insulin-like growth factor 2 (IGF2) and the transformation related protein 53 (Trp53) are potent regulators of cell growth and metabolism in development and cancer. In vitro evidence suggests several mechanistic pathway interactions. Here, we tested whether loss of function of p53 leads to IGF2 ligand pathway dependency in vivo. Developmental lethality occurred in p53 homozygote null mice that lacked the paternal expressed allele of imprinted Igf2. Further lethality due to post-natal lung haemorrhage occurred in female progeny with Igf2 paternal null allele only if derived from double heterozygote null fathers, and was associated with a specific gene expression signature. Conditional deletion of Igf2(fl/fl) attenuated the rapid tumour onset promoted by homozygous deletion of p53(fl/fl) . Accelerated carcinoma and sarcoma tumour formation in p53(+/-) females with bi-allelic Igf2 expression was associated with reductions in p53 loss of heterozygosity and apoptosis. Igf2 genetic dependency of the p53 null phenotype during development and tumour formation suggests that targeting the IGF2 pathway may be useful in the prevention and treatment of human tumours with a disrupted Trp53 pathway.

Figure 1. Combination of Igf2 and p53 null alleles results in developmental lethality independent of embryo growth

1. Mean litter size at P0 was significantly reduced from Igf2^+m/−p, p53^+/− inter-cross (***p < 0.0001, n = 67 litters) relative to Igf2^+m/−p mated with WT (n = 11 litters), p53^+/− mated with WT (n = 15 litters) and Igf2^+m/−p mated with p53^+/− (n = 36 litters; one-way ANOVA, Tukey's multiple comparison).

2. Post-natal growth of progeny from a double heterozygote inter-cross (129, Igf2^+m/−p, p53^+/−). Mean weights of WT and Igf2^−m/+p mice were significantly greater (***p < 0.0001, one-way ANOVA, Tukey's multiple comparison) than Igf2^+m/−p and Igf2^−m/−p mice regardless of allelic dosage of p53.

3. Increased post-natal mortality by p30 in progeny null for both Igf2 and p53 (Igf2^+m/−p, p53^−/−, 2 of 7 and Igf2^−m/−p, p53^−/−, 5 of 9) relative to WT mice (1 of 15, p = 0.037, Fisher's exact test).

Figure 2. Lethality of female Igf2 ^+m/−p progeny derived from Igf2 ^+m/−p, p53 ^+/− fathers is associated with reduced lung growth

1. Male (n = 115) and female (n = 87) progeny at P10 from the 129 Igf2^+m/−p, p53^+/− inter-cross did not segregate according to the expected Mendelian distribution (see Table 1; p < 0.05) and were significantly different to expected numbers estimated from previous litter sizes (total expected numbers: males = 175, females = 175 based on mean litter size from earlier 129 breeding, p < 0.0001, χ²-test). Solid bars, observed numbers, grey unfilled bars, expected numbers (see also Table 2).

2. Litter of 13 pups delivered by caesarean section at E19.5 from a WT female mated with Igf2^+m/−p, p53^+/− male. One Igf2^+m/−p female (white arrow) became increasingly cyanotic and died 1hr post-delivery. The remaining four Igf2^+m/−p females (thick black arrows) were also cyanotic compared to their littermates (thin black arrow points to a representative Igf2^+m/−p male).

3. Lung weights from E18.5 Igf2^+m/−p female progeny derived from Igf2^+m/−p, p53^+/− fathers were significantly lower than those from Igf2^+m/−p male progeny (*p = 0.025), WT female progeny (***p < 0.0001) and Igf2^+m/−p, p53^+/− female progeny (*p = 0.049, two-tailed t-test, NS = not significant).

Figure 3. Paternal p53 genotype alters embryonic gene expression and Igf2^+m/−p female specific lethality

1. Schema of embryonic RNA expression analysis. Three parental genotype pairs were crossed to yield a number of female embryos of specific genotype as illustrated.

2. Comparison of gene expression in E9.5 female embryos derived from Igf2^+m/−p or WT fathers (WT and Igf2^+m/−p) with the expression in embryos from Igf2^+m/−p, p53^+/− fathers (WT, Igf2^+m/−p, p53^+/− and Igf2^+m/−p, p53^+/−). 1461 genes were significantly different in expression by SAM analysis and varied more than twofold, with most differentially expressed genes displayed in the heatmap.

3. Comparison of gene expression in Igf2^+m/−p female embryos derived from WT fathers with those Igf2^+m/−p and Igf2^+m/−p, p53^+/−embryos from Igf2^+m/−p, p53^+/− fathers. ‘Rescue’ signature of 23 genes shown in the heatmap.

4. H & E-stained lungs from E19.5 Igf2^+m/−p female embryos (low power above, bar 1 mm, high power 100 µm) were filled with blood in both bronchioles (br) alveolar air-spaces (alv) when compared to WT littermates. Confocal images showed there were no gross differences in lung structure labelled with E-cadherin, but fibronectin was predominantly present in blood within the airspaces (white arrow), and subjectively increased in small airway and alveolar regions (white arrow head). Bar = 100 µm.

Figure 4. Bi-allelic Igf2 expression accelerates tumour formation in p53^+/− female mice, irrespective of strain

1. Survival of B6 H19^−m/+p, p53^+/− mice was significantly reduced compared to p53^+/− mice (p = 0.0065, log-rank test). Due to accelerated tumour latency the humane end point was brought forward to 400 days.

2. Survival of 129 H19^−m/+p, p53^+/− mice was significantly reduced compared to Igf2^+/−p, p53^+/− (p = 0.0001), Igf2^+m/+p, p53^+/− (p = 0.008) and H19^+m/+p, p53^+/− mice (p = 0.036, log-rank test).

3. Survival of F2 hybrid H19^−m/+p, p53^+/− mice was significantly reduced compared to Igf2^+m/−p, p53^+/− (p = 0.0008), Igf2^+m/+p, p53^+/− (p = 0.0017) or H19^+m/+p, p53^+/− mice (p = 0.0018, log-rank test).

4. Tumour latency in B6 H19^−m/+p, p53^+/− female mice was significantly reduced compared to H19^−m/+p, p53^+/− males and p53^+/− females and male mice (p < 0.0007, log-rank test).

5. B6 H19^−m/+p, p53^+/− mice had significantly more solid tumours (carcinomas and sarcomas) and fewer lymphomas than B6 H19^+m/+p, p53^+/− mice (p = 0.037, Fisher's exact test).

Figure 5. p53 mutation, LOH and apoptosis in tumours from p53 ^+/− mice with bi-allelic Igf2 expression (H19 ^−m/+p)

1. Proportion of p53^+/− tumours with LOH/mutation was significantly reduced in tumours from mice bi-allelic for Igf2 (H19^−m/+p), compared to mice null for Igf2^+m/−p and WT (**p < 0.002, Fisher's exact test).

2. 100% of sarcomas and carcinomas from H19^−m/+p, p53^+/− retain an intact WT p53 allele (n = 9), compared to 100% of LOH in the littermate p53^+/− control mice (n = 5; **p = 0.0005, Fisher's exact test)

3. Sarcomas with intact WT p53 (n = 6) had significantly higher Igf2 mRNA expression than lymphomas and carcinomas, irrespective of p53 allelic status (p < *0.05–***0.01, one-way ANOVA, Tukey's multiple comparison). Sample annotation is as for genotypes in Fig 4A and B.

4. Solid tumours (n = 8) from H19^−m/+p, p53^+/− mice had significantly fewer apoptotic cells, assessed by staining for cleaved caspase-3 (right) than solid tumours from H19^+m/+p, p53^+/− mice (n = 4, *p = < 0.05, **p = < 0.01, ***p < 0.001, one-way ANOVA, Tukey's multiple comparison). Blue, male; Red, female.

Figure 6. Decreased tumour latency and altered tumour spectrum following homozygous conditional deletion of both Igf2 and p53

1. Progeny from the homozygous conditional cross of Igf2^fl/fl, p53^fl/fl females × Igf2^fl/fl, p53^fl/fl, R26^+/− males segregated according to the expected Mendelian distribution at P10, regardless of sex.

2. Following maternal IP injection of tamoxifen at E10.5, BW and musculoskeletal weights (MS) of carcasses, were significantly less for Igf2^Δ/Δ, p53^Δ/Δ, R26Cre^+/− mice (n = 6) compared to sham injected controls Igf2^fl/fl, p53^fl/fl, R26Cre^+/− (n = 3, *p = 0.05, unpaired t-test). No significant differences in the weight of viscera were detected; Br, brain, Int, small intestine; Liv, liver; Lu, lungs; Kid, kidney; Hrt, heart; Thy, thymus. Plot shows 95% of the range (whiskers), interquartile range (box), median (horizontal crossbar) and mean (cross).

3. Mice with homozygous conditional deletion of Igf2 and p53 (Igf2^Δ/Δ, p53^Δ/Δ, R26^+/−) developed tumours later and survived for longer (median survival = 325 days) than mice with an intact paternal allele of Igf2 (Igf2^Δm/+p, p53^Δ/Δ, R26^+/−, median survival = 206 days, p = 0.024, log-rank test, blue, males; red, females).

4. Igf2^Δ/Δ, p53^Δ/Δ, R26^+/− mice (right) developed more solid tumours than Igf2^Δm/+p, p53^Δ/Δ, R26^+/− mice (left, 4 and 1, respectively), and fewer lymphomas (3 and 10, respectively, p = 0.0474, Fisher's exact test).