Bmi1 is required for tumorigenesis in a mouse model of intestinal cancer

Abstract

The epigenetic regulator BMI1 is upregulated progressively in a wide variety of human tumors including colorectal cancer. In this study, we assessed the requirement for Bmi1 in intestinal tumorigenesis using an autochthonous mouse model in which Apc was conditionally ablated in the intestinal epithelium. Germline mutation of Bmi1 significantly reduced both the number and size of small intestinal adenomas arising in this model, and it acted in a dose-dependent manner. Moreover, in contrast to wild-type controls, Bmi1(-/-) mice showed no increase in median tumor size, and a dramatic decrease in tumor number, between 3 and 4 months of age. Thus, Bmi1 is required for both progression and maintenance of small intestinal adenomas. Importantly, Bmi1 deficiency did not disrupt oncogenic events arising from Apc inactivation. Instead, the Arf tumor suppressor, a known target of Bmi1 epigenetic silencing, was upregulated in Bmi1 mutant tumors. This was accompanied by significant upregulation of p53, which was confirmed by sequencing to be wild-type, and also elevated apoptosis within the smallest Bmi1(-/-) adenomas. By crossing Arf into this cancer model, we showed that Arf is required for the induction of both p53 and apoptosis, and it is a key determinant of the ability of Bmi1 deficiency to suppress intestinal tumorigenesis. Finally, a conditional Bmi1 mutant strain was generated and used to determine the consequences of deleting Bmi1 specifically within the intestinal epithelium. Strikingly, intestinal-specific Bmi1 deletion suppressed small intestinal adenomas in a manner that was indistinguishable from germline Bmi1 deletion. Thus, we conclude that Bmi1 deficiency impairs the progression and maintenance of small intestinal tumors in a cell autonomous and highly Arf-dependent manner.

Figure 1. Bmi1 is required for small intestinal adenoma development, progression and maintenance

a Representative Haematoxylin & Eosin stained sections of small intestine from 120 day old Apc^fl/+;Vil-cre mice show adenomas (marked by arrowheads) in Bmi1^+/+ but not Bmi1^−/− context. Original magnification 4X. Scale bar represents 200 μm. b,c Quantification of (b) the number of visible (diameter > 1mm) adenomas, and (c) the cross-sectional area (CSA) of all adenomas, in 120-day old animals according to Bmi1 status and gender (n= 4–8). d,e Comparison of (d) the total number of small intestinal adenomas per histological cross-section (CS), and (e) adenoma cross-sectional area (CSA), in 90-day old versus 120-day old mice shows that Bmi1 is required for their maintenance and progression. Data were pooled for male and females, using equal numbers of each (n=8–10 for combined genders). For (a–e) mouse small intestine was dissected, fixed in formalin, coiled and subject to histological processing followed by paraffin embedding. Sections were examined using a Nikon Eclipse E600 microscope with a SPOT RT digital camera. SPOT Basic imaging software was utilized for capture and area/length measurements. Data are presented as box plots with marked median values and whiskers spanning the 5^th to the 95^th percentiles. For statistical analyses (b,c) ANOVA was performed with a Tukey post-test for paired comparisons or (d,e) a student's 2-sided t-test was performed for paired comparisons.

Figure 2. Bmi1 loss promotes Arf-dependent, p53 stabilization and apoptosis within small intestinal adenomas

a Western blot of lysates from small intestinal adenomas demonstrates higher p19^Arf expression in Bmi1^+/− versus Bmi1^+/+ adenomas. Lysates were prepared from small intestinal adenomas that were minced, digested for 1 hour at 37°C in dispase (150 u/ml final; Gibco, Japan), triturated and lysed in RIPA buffer (200 mM Tris pH 7.4, 130 mM NaCl, 10% glycerol, 0.1% SDS, 0.5% DOC, 1% Triton) containing protease inhibitors (Roche, Germany). Protein was separated by SDS-PAGE and western blot was performed with anti-p19^Arf (top panel; BD Biosciences, San Jose CA, NB200-106) and Histone H3 (lower panel; Cell Signaling, Danvers MA, 4620) antibodies, followed by incubation with a 1:5000 dilution of HRP-conjugated secondary antibodies (Cell Signaling, Danvers, MA). Relative levels of p19^Arf were quantified and normalized to levels of Histone H3 after band density measurements using ImageJ software. b Bmi1^+/− and Bmi1^−/− adenomas are more proliferative than Bmi1^+/+ adenomas in 90-day old mice, as determined by quantification of the average percentage of adenoma nuclei staining positive for incorporated BrdU. Data were stratified by adenoma cross-sectional area. BrdU (10 μg/g body weight; Sigma, St. Louis MO) was injected I.P. two hours prior to euthanasia. c Analysis of average number of cleaved caspase-3 positive adenoma cells, normalized to adenoma cross-sectional area, shows that Bmi1 deficiency elevated apoptosis in the smallest adenomas in an Arf-dependent manner. d Adenomas of 90 day old Bmi1^−/−mice have an average high percentage of cells with p53 stabilization, which is lost in the absence of Arf. Original magnification was 40X. For b–d: Adenomas from at least 5 mice of each genotype consisting of both males and females were assayed. Immunohistochemistry on intestinal sections was performed after antigen retrieval in 10 mM citric acid pH 6.0 using a decloaking chamber, followed by standard DAB protocol and haematoxylin counterstain. Antibodies were: anti-BrdU (1:100; Beckton Dickson, Franklin Lakes NJ, 347580), p53 (1:500, Santa Cruz, Santa Cruz CA, FL-393) and cleaved caspase-3 (1:1000, Cell Signaling, Danvers MA, 9661). Tissue processing, image capture/measurements and statistical analysis were performed as described for Figure 1b,c and error bars represent standard deviation.

Figure 3. The tumor suppressive effects of Bmi1 deficiency are Arf-dependent

a Representative Haematoxylin & Eosin stained sections of small intestine from 90 day old Apc^fl/+;Vil-cre mice show adenomas (marked by arrowheads) in a Bmi1^+/+, Bmi1^+/+;Arf^−/−, Bmi1^−/−;Arf^−/− but not Bmi1^−/− context. Original magnification 4X. Scale bar represents 200 μm. b,c Quantification of the effect of Bmi1 and Arf status on (b) the total number of adenomas per cross-section (CS) and (c) adenoma size, as measured by cross-sectional area (CSA), in 90 day old Apc^fl/+;Vil-cre mice (n≥5 for each genotype consisting of both males and females) shows that Arf loss suppresses Bmi1^+/+ tumors while promoting Bmi1^−/− tumors. Tissue processing and image capture/measurements were performed as described for Figure 1. For statistical analysis, ANOVA was performed, followed by an Unpaired Fisher's LSD test for paired comparisons.

Figure 4. The oncogenic effect of Bmi1 in small intestinal tumorigenesis reflects its action within the intestinal epithelium

a–d Comparison of (a) the number of visible adenomas (diameter>1 mm), (b,c) the total number of adenomas per cross-section (CS) and (d) adenoma size, as measured by CSA, shows a similar degree of tumor suppression in the small intestines of 120 day Apc^fl/+;Vil-cre mice that are Bmi1^fl/fl versus Bmi1^−/− (both genders; n=8 or greater). For (b), original magnification was 4X and the scale bar represents 200 μm. e,f The relative (e) number and (f) size of small intestinal adenomas in Bmi1^−/− or Bmi1^fl/fl Apc^fl/+;Vil-cre mice at 90 days versus 120 days (both genders; n=5–10) phenocopies those of Bmi1^−/−;Apc^fl/+;Vil-cre mice. The Bmi1 conditional allele (Bmi1^fl) was generated using standard gene targeting to insert loxP sites in introns 3 and 8 and validated as shown in Supplemental Figure 6. Tissue processing, image capture/measurements and statistical analyses were performed as described for Figure 1.