Heterozygosity for hypoxia inducible factor 1alpha decreases the incidence of thymic lymphomas in a p53 mutant mouse model

Abstract

Hypoxia inducible factors (HIF) are critical mediators of the cellular response to decreased oxygen tension and are overexpressed in a number of tumors. Although HIF1alpha and HIF2alpha share a high degree of sequence homology, recent work has shown that the two alpha subunits can have contrasting and tissue-specific effects on tumor growth. To directly compare the role of each HIFalpha subunit in spontaneous tumorigenesis, we bred a mouse model of expanded HIF2alpha expression and Hif1alpha(+/-) mice to homozygotes for the R270H mutation in p53. Here, we report that p53(R270H/R270H) mice, which have not been previously described, develop a unique tumor spectrum relative to p53(R270H/-) mice, including a high incidence of thymic lymphomas. Heterozygosity for Hif1alpha significantly reduced the incidence of thymic lymphomas observed in this model. Moreover, reduced Hif1alpha levels correlated with decreased stabilization of activated Notch1 and expression of the Notch target genes, Dtx1 and Nrarp. These observations uncover a novel role for HIF1alpha in Notch pathway activation during T-cell lymphomagenesis.

Figure 1. Breeding scheme and characterization of HIF2α expression in adult mouse tissues

A. Breeding scheme to generate Hif1α^KI/+ p53^H/H, Hif1α^+/−p53^H/H, and Hif1α^+/+ p53^H/H mice. B. Representative genotyping PCR results for each genotype. C. Graph showing Hif2α and Hif1α mRNA expression in normal duodenum and skeletal muscle of Hif1α^+/+ (black), Hif1α^+/− (gray), and Hif1α^KI/+(white) mice. *p<0.05

Figure 2. Dosage of p53^R270H allele influences tumor spectrum

A. Graph comparing tumor types arising in p53^H/H (n=23, black) and p53^H/− mice (n=19, gray). * p<0.035; ** p<0.01 B. Age at onset of thymic lymphomas in p53^H/H and p53^H/− mice. C. Immunohistochemistry for p53 shows accumulation of mutant protein (brown) in lymphomas from both p53^H/H and p53^H/− mice. No p53 protein can be detected in tumors from p53^−/− mice. Nuclei were counterstained with hematoxylin (blue).

Figure 3. Characterization of survival, tumor spectrum, and tumor burden in p53^H/H mice

A. Kaplan-Meier curve of Hif1α^+/+p53^H/H (n=23, green), Hifα1^+/−p53^H/H (n=20, black), and Hif1α^KI/+p53^H/H (n=24, red) mice. Significance was calculated using a Log-rank test over all three genotypes. When comparing the Hif1α^KI/+p53^H/H and Hif1α^+/+p53^H/H groups, p=0.024. B. Average age at sacrifice of Hif1α^+/+p53^H/H, Hif1α^+/−p53^H/H, and Hif1α^KI/+p53^H/H mice. C. Tumor burden for Hif1α^+/+p53^H/H, Hif1α^+/−p53^H/H, and Hif1α^KI/+p53^H/H mice. D. Tumor spectrum in Hif1α^+/+p53^H/H, Hifα1^+/−p53^H/H, and Hif1α^KI/+p53^H/H mice. * p = 0.055, # p = 0.073 (student’s two-tailed t test for pair-wise comparisons).

Figure 4. Thymic lymphomas were the predominant tumor type observed in Hif1α^+/+ p53^H/H mice

A. Gross image of a thymic lymphoma (*) in a dissected mouse. B. Histological section of a thymic lymphoma stained with Hematoxylin and Eosin (H&E). C. H&E of a liver with infiltrating malignant T-cells in a mouse with a thymic lymphoma. D. H&E of a kidney infiltrated with malignant T-cells in a mouse with a thymic lymphoma.

Figure 5. Molecular and phenotypic differences between thymic lymphomas

A. Average age (days) at presentation with thymic lymphoma in Hif1α^+/+p53^H/H (black), Hif1α^+/−p53^H/H (gray), Hif1α^KI/+p53^H/H (white) mice. * p < 0.05, ** p = 0.01. B. Mitotic cells were identified by immunohistochemistry for phospho-histone H3 (Ser10). C. TUNEL staining of apoptotic cells. Hif1α^+/− * p = 0.01, Hif1α^KI/+ p = 0.14 D. QRT-PCR analysis of HIFα and HIF target gene expression in thymic lymphomas from Hif1α^+/+p53^H/H, Hif1α^+/−p53^H/H, Hif1α^KI/+p53^H/H. * p < 0.05.

Figure 6. Differential activation of the Notch pathway in thymic lymphomas arising in Hif1α^+/+p53^H/H, Hifα1^+/−p53^H/H, and Hif1α^KI/+p53^H/H mice

A. QRT-PCR for IL-2 receptor-α (CD25), Dtx1 and Nrarp, two Notch target genes, in thymic lymphoma tissue derived from Hif1α^+/+p53^H/H (n =9, black), Hif1α^+/−p53^H/H (n = 7, gray), and Hif1α^KI/+p53^H/H (n = 5, white) mice. * p < 0.05. B. Western blot for cleaved Notch (Val1744) using protein lysates derived from primary tumors. β–tubulin serves as the loading control. Differences in protein migration likely reflect changes in Notch1 phosphorylation. C. Model depicting the correlation between Notch activity and CD25 expression during T-cell development. D. Model depicting potential crosstalk between HIF1α levels and Notch activity in thymic lymphomas arising in p53^H/H mice.