Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan

Abstract

The BubR1 gene encodes for a mitotic regulator that ensures accurate segregation of chromosomes through its role in the mitotic checkpoint and the establishment of proper microtubule-kinetochore attachments. Germline mutations that reduce BubR1 abundance cause aneuploidy, shorten lifespan and induce premature ageing phenotypes and cancer in both humans and mice. A reduced BubR1 expression level is also a feature of chronological ageing, but whether this age-related decline has biological consequences is unknown. Using a transgenic approach in mice, we show that sustained high-level expression of BubR1 preserves genomic integrity and reduces tumorigenesis, even in the presence of genetic alterations that strongly promote aneuplodization and cancer, such as oncogenic Ras. We find that BubR1 overabundance exerts its protective effect by correcting mitotic checkpoint impairment and microtubule-kinetochore attachment defects. Furthermore, sustained high-level expression of BubR1 extends lifespan and delays age-related deterioration and aneuploidy in several tissues. Collectively, these data uncover a generalized function for BubR1 in counteracting defects that cause whole-chromosome instability and suggest that modulating BubR1 provides a unique opportunity to extend healthy lifespan.

Fig. 1

Transgenic mouse strains with moderate and high levels of Flag-BubR1 are chromosomally stable. (a) Flag-mBubR1 transgenic vector design. pCAGGS = promoter consisting of the CMV immediate enhancer and the chicken beta-actin promoter. (b) Western blots of tissue and MEF extracts from wildtype and Flag-BubR1 transgenic mice (strains T7 and T23). Tubulin was used as a loading control. (c) Both Flag-BubR1 transgenes correct the growth retardation and aging-associated phenotypes of BubR1^H/H mice. (d) High BubR1 expression does not induce aneuploidy. Chromosome counts were done on splenocytes from 5-month-old mice and passage (P) 5 MEFs (n = 3 samples per genotype). Fifty spreads were counted per sample (150 total). Values represent means ± SD. (e) High BubR1 levels do not induce chromosome missegregation. (f) The mitotic checkpoint is not hyperactive at supranormal BubR1 levels. Three independent MEF lines per genotype were used in e and f. Values represent means ± SD in f.

Fig. 2

Resistance to tumorigenesis and chromosomal instability in BubR1 transgenic mice. (a) Tumor incidence and multiplicity in animals treated with DMBA on dorsal skin on post-natal day 5 and biopsied at 5 months of age. Values represent means ± SEM. (b) Resistance of BubR1 transgenic mice to lung tumors induced by oncogenic Kras (G12D). Cohorts of Kras^LA1 and T23;Kras^LA1 mice were killed at 6 weeks of age and lung tumors counted under a dissection microscope. Values represent means ± SD. (c) Western blots of cell extracts from wildtype and T23 MEFs with and without retrovirally expressed oncogenic Hras (G12V). Actin was used as a loading control. (d and e) Oncogenic Hras-induced aneuploidy (d) and chromosome missegregation (e) rates of MEF lines with and without BubR1 overexpression (n = 3 independent MEF lines each). Values represent means ± SD in d and means ± SEM in e. (f) Aneuploidy rates in lungs of wildtype and Kras^LA1 mice with and without BubR1 overexpression. n = 3 mice for WT and T23, and n = 4 for Ras and T23;Ras. Values represent means ± SEM. (g) Nocodazole-challenge assay showing that elevated BubR1 expression restores normal mitotic checkpoint activity in MEFs with low amounts of Rae1. Values represent means ± SD. Three independent MEF lines per genotype were used. (h) Aneuploidy rates of mutant MEF lines with and without BubR1 overexpression (n = 5 MEF lines each). Values represent means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. n indicates the number of mice (mixed gender) used per genotype in a and b.

Fig. 3

Increased BubR1 expression protects against spontaneous tumors and extends lifespan. (a) Survival curves of wildtype, T-GFP, and T23 mice dying of cancer. Only mice with malignant lymphomas, sarcomas and carcinomas are included. Statistical analysis of the survival curves is represented by the asterisks (log-rank test). (b) Overall survival curves of wildtype, T-GFP, and T23 mice. We note that maximum lifespan of T23 mice was also significantly extended compared to both wildtype and T-GFP control mice (p = 0.05 and 0.0056), respectively; one-sided Wang/Allison test referring to the proportion of mice alive at the 90^th percentile survival point. Furthermore, we note that the median lifespan of our wildtype cohort is similar to that of an earlier, independent study performed at the same site. *P < 0.05, **P < 0.01, ***P < 0.001. n indicates the number of mice (mixed gender) per genotype.

Fig. 4

Increased BubR1 expression delays select age-related pathologies. (a) Sustained BubR1 expression attenuates muscle fiber atrophy. Gastrocnemius muscle fiber diameter declines with age in all genotypes but T23. (b) Quantitative RT-PCR analysis of p16^Ink4a expression in gastrocnemius muscles 3 and 24-month-old transgenic and control mice relative to 3-month-old wildtype muscles. (c) Same as (b) but for p19^Arf. (d–f) Exercise ability is enhanced in aged T23 mice. Time (d), distance (e), and work performed (f) are all improved in 18-month-old T23 animals compared to WT and T-GFP. (g) Hematoxylin-eosin stained kidney sections of 24-month-old mice. Dashed area depicts interstitial inflammation; arrows denote glomerular hypercellularity of WT mice (above) and normal glomeruli of T23 mice (below). Scale bar, 100 μm. (h) Percentage of sclerotic glomeruli from 24-month-old kidney sections. 40 glomeruli were scored for each animal. (i) DNA damage, as measured by γ-H2AX staining on cryosections of kidney tissue, is increased in BubR1^H/H mice at a young age and reduced in transgenic mice at advanced age. (j) Cardiac stress tolerance correlates with BubR1 level of expression. (k) Ptah stained heart sections of 24-month-old wildtype and T23 mice. Interstitial fibrosis (pink area) is significantly reduced in T23 animals. Scale bar, 50 μm. For all analyses in a–k, n = 5 males per genotype per age group. Error bars represent SD. (l) Quantification of the percentage of stem cells isolated from 3 and 24-month-old wildtype and T23 mouse skeletal muscle (satellite cells) and heart (cardiac stem cells). n = 3 males per genotype per tissue. Values represent means ± SEM. We note that there are no significant differences between wildtype and T23. *P < 0.05, **P < 0.01, ***P < 0.001.