Haploinsufficiency of SGO1 results in deregulated centrosome dynamics, enhanced chromosomal instability and colon tumorigenesis

Abstract

Chromosome instability (CIN) is found in 85% of colorectal cancers. Defects in mitotic processes are implicated in high CIN and may be critical events in colorectal tumorigenesis. Shugoshin-1 (SGO1) aids in the maintenance of chromosome cohesion and prevents premature chromosome separation and CIN. In addition, integrity of the centrosome may be compromised due to the deficiency of Cohesin and Sgo1 through the disengagement of centrioles. We report here the generation and characterization of SGO1-mutant mice and show that haploinsufficiency of SGO1 leads to enhanced colonic tumorigenesis. Complete disruption of SGO1 results in embryonic lethality, whereas SGO1+/- mice are viable and fertile. Haploinsufficiency of SGO1 results in genomic instability manifested as missegregation of chromosomes and formation of extra centrosomal foci in both murine embryonic fibroblasts and adult bone marrow cells. Enhanced CIN observed in SGO1-deficient mice resulted in an increase in formation of aberrant crypt foci (ACF) and accelerated development of tumors after exposure to azoxymethane (AOM), a colon carcinogen. Together, these results suggest that haploinsufficiency of SGO1 causes enhanced CIN, colonic preneoplastic lesions and tumorigenesis in mice. SGO1 is essential for the suppression of CIN and tumor formation.

Figure 1

Generation of SGO1 mutant mice. (A) Schematic representation of knockout construct. Disruption of the SGO1 locus was obtained by a gene trap method. LTR, viral long-terminal repeat; SA, splice acceptor; NEO, neomycin; pA, poly adenylation sequence. (B) Structure of the wild-type and mutant SGO1 loci. *denotes the gene trap cassette insertion site. (C) Representative genotyping result by PCR. WT, wild type; HT, heterozygous. PCR products were run on 1% agarose gels. The WT allele generates a 423 bp PCR product, while the mutant allele generates a 280 bp product. (D) Relative mRNA level of SGO1 from mice tail tissue. mRNA levels were measured by quantitative-RT-PCR. The average tissue mRNA level of WT mice was arbitrarily set to 1, and the tissue mRNA level of HT mice was divided by the WT average level (n = 3/group). (E) Western blot analysis of SGO1 levels in tail tissue. (F) The signals shown in the western blot film were quantified by densitometry.

Figure 2

SGO^+/− MEFs exhibit enhanced chromosome missegregation. (A) Paired MEFs with indicated genotypes were stained with DAPI (blue) and antibody to α-tubulin (green). Arrows show the misaligned chromosome (middle part) or the lagging or missegregated chromosomes (lower part). (B) Paired MEFs with indicated genotypes were stained with antibodies to α-tubulin (green) and p-H3S10 (red). (C) Paired MEFs with indicated genotypes were stained with DAPI (blue) and antibody to α-tubulin (green). (D) Paired MEFs were subjected to chromosome spread analysis as described in Materials and Methods. Representative images are shown.

Figure 3

SGO^+/− MEFs exhibit extra centrosomal foci. (A) Paired MEFs were stained with the antibodies to γ-tubulin (green) and p-H3S10 (red). DNA was stained with DAPI (blue). (B) Paired MEFs were stained with the antibody to γ-tubulin (green). Centrosomal numbers in mitotic cells were counted for each type of MEFs (n = 50 for each). (C) Bone marrow cells from mice of each genotype were stained with the antibodies to γ-tubulin (green) and p-H3S10 (red). DNA was stained with DAPI (blue). Representative images are shown.

Figure 4

SGO1^+/− mice show enhanced acute colonic cell death with AOM treatments. (A) Schematic presentation of mouse tumorigenesis assay. At 7 weeks of age, mice were grouped, and starting at 8 weeks of age, they were given s.c. azoxymethane (AOM, 4 mg/kg body weight) two times weekly for 4 weeks. Asterisks indicate timing for FACS sample harvest to assess acute response to AOM on 1, 3 and 5 weeks after completion of AOM treatments. Twelve weeks after completion of AOM treatment, all remaining mice were sacrificed, and their colons were removed for FACS, immunoblotting and ACF/adenoma counting. (B) Colonic mucosa in SGO1^+/− mice show a higher acute cell death rate in response to AOM treatments. Cell death rate (i.e., percentage of cells with sub-G1 DNA) is estimated with FACS analysis of colonic mucosa in AOM-treated wild-type and SGO1^+/− mice. Cell death rate in untreated wild-type is less than 10%.

Figure 5

SGO1^+/− mice show enhanced colonic tumorigenesis with AOM treatments. (A) Examples of precancerous lesions (ACF): (i) Normal-looking colonic crypts (control), (ii) ACF with single crypt (upper right) and with two crypts (lower left), (iii) Large ACF with four crypts. (B) SGO1^−/+ mice are more susceptible to the development of precancerous legions (ACF) of various sizes. Size of ACF, indicated by number of crypts, is larger in SGO1^−/+ mice (green bars) than in wild type (blue bars). (C) (i) Methylene blue-stained colon from SGO1^+/−. Arrows indicate adenoma-like masses of cells, differentially stained with Methylene blue. (ii) Hematoxylin-stained colonic tissue sections from the adenoma. (D) Frequency of colon tumors is higher in Sgo1^+/− mice. Unit is incidence/animal. p-value is calculated with Student t-test.

Figure 6

Differential marker protein expressions in wild-type and SGO1^+/− mice. (A) Extracts of colonic mucosa from three wild-type and three SGO1^+/− mice were subjected to immunoblotting. All samples are from the endpoint (12 weeks after completion of AOM treatment). α-tubulin used as loading control. (B) Immunohistochemistry of endpoint samples for the overexpressed proteins (Bcl-2, left column; COX2, middle column; p53, right column) counterstained with Hematoxylin. Upper part: normal-looking colonic crypts from wild type. Lower part: normal-looking colonic crypts from SGO^+/− mice.