Kaiso-deficient mice show resistance to intestinal cancer

Abstract

Kaiso is a BTB domain protein that associates with the signaling molecule p120-catenin and binds to the methylated sequence mCGmCG or the nonmethylated sequence CTGCNA to modulate transcription. In Xenopus laevis, xKaiso deficiency leads to embryonic death accompanied by premature gene activation in blastulae and upregulation of the xWnt11 gene. Kaiso has also been proposed to play an essential role in mammalian synapse-specific transcription. We disrupted the Kaiso gene in mice to assess its role in mammalian development. Kaiso-null mice were viable and fertile, with no detectable abnormalities of development or gene expression. However, when crossed with tumor-susceptible Apc(Min/+) mice, Kaiso-null mice showed a delayed onset of intestinal tumorigenesis. Kaiso was found to be upregulated in murine intestinal tumors and is expressed in human colon cancers. Our data suggest that Kaiso plays a role in intestinal cancer and may therefore represent a potential target for therapeutic intervention.

FIG. 1.

Generation of Kaiso-null mice. (A) Schematic representation of the genomic Kaiso locus, targeting vector, targeted locus, and deleted locus. Boxes represent Kaiso exons that are either translated (black) or untranslated (open). LoxP sites are shown as triangles. The position and direction of primers that were used to validate the targeting are depicted with arrows. (B) Validation of correct targeting by PCR. Primer pairs are indicated on the left. Genomic DNA from a correctly targeted ES clone was used as a template. PCR products were fractionated on 1% agarose gels. DNA fragment sizes are indicated on the right. (C) Northern blot hybridization with Kaiso cDNA. RNA was isolated from brain, kidney, liver, and spleen of wild-type (wt) and Kaiso-null (ko) animals. Kaiso mRNA corresponds to ca. 7 kb, and the hybridization signal is indicated by “kaiso.” Prior to the blotting, the gel was stained with ethidium bromide (EtBr) and photographed (bottom panel). (D) Western blot hybridization of liver nuclear extracts from wild-type (wt) and Kaiso-null (ko) animals. The Kaiso band (∼100 kDa) and products of Kaiso degradation (from 60 to 45 kDa) are indicated on the left. The bottom panel shows a Western blot of inhibitory κB-alpha protein as an internal control. The protein size markers are on the right. (E) Band-shift assays with nuclear extracts from wt or mutant (ko) liver. The labeled probe was either M+CG11 (methylated) or CG11 (unmethylated). The lower complex in wt M+CG11 lanes is the DNA methylation-specific Kaiso-DNA complex (KGB). This complex is absent in ko lanes. Lanes αKaiso contained anti-Kaiso antibody ZFH6 (33) that specifically supershifts the KGB complex.

FIG.2.

Kaiso-null cells show no defects in neural differentiation. (A) Wild-type or Kaiso-deficient ES cells expressing GFP from the Sox1 locus were induced toward neural differentiation for 3, 4, 5, 6, 7, or 8 days (D3, D4, etc.) and fluorescence-activated cell sorted for GFP expression. The experiment was performed in triplicate, with average values (± the standard error of the mean) plotted. (B) Kaiso gene expression was analyzed by RT-PCR in wt and null (clone A and B) sorted Sox1-positive cells. (C to F) Wild-type (C and E) and Kaiso-null (D and F) cultures were stained for β-tubulin (C and D) or Gfap (red) and DAPI (4′,6′-diamidino-2-phenylindole; blue) (E and F) after 12 days of monolayer differentiation. (G to N) Wild-type (G, I, K, and M) and Kaiso-null (H, J, L, and N) neural stem cells were stained for Nestin (G and H) and RC2 (I and J) or were induced to differentiate and stained for β-tubulin III (K and L) or Gfap (M and N). Cells were counterstained with DAPI (blue). Scale bars: 100 μm (C, D, E, F, M, and N) and 50 μm (G, H, I, J, K, and L).

FIG. 3.

No evidence for abnormal gene expression in Kaiso-null animals. (A) Total RNA from four different animals born in two independent families was isolated from wild-type (WT) and Kaiso-null (KO) strains. Sources of RNA were brain, liver, spleen, and muscle. The same blot was hybridized with S100A4 and Mta2 probes. Separate blots were prepared with muscle RNA and hybridized with a Rapsyn probe. Normalization of the amount of RNA loaded was performed by reprobing with β-actin (S100A4; Mta2) or S26 ribosomal protein (Rapsyn) probes. (B) In the left panel, chromatin immunoprecipitation was performed with M2 anti-FLAG monoclonal antibodies (M2 lanes) and chromatin was prepared from kidney of wild-type (WT) and Kaiso-null (KO) animals. PCR products amplified with IAP-specific primers from chromatin immunoprecipitated with or without (“no Ab”) the addition of antibodies are designated. Amplification without DNA (−) and with kidney genomic DNA (+) were used as negative and positive controls, respectively. PCR products were fractionated on 1% agarose gels. In the right panel is shown IAP expression analysis. RNA from liver of wild-type (WT) and Kaiso-null (KO) animals was either transcribed (+) or not transcribed (−) by reverse transcriptase (RT). Subsequent PCR amplification of IAP cDNA and control 18S cDNA produced DNA fragments that were resolved on agarose gels. Different dilutions of cDNA were used for PCR amplification as depicted. (C) Semiquantitative RT-PCR with serial dilutions of cDNA from WT or KO heart were amplified by using Wnt11-specific primers and compared to 18S rRNA-specific primers used to amplify the same samples. (D) Quantitative “real-time” PCR analysis of Wnt11 mRNA abundance in the hearts, livers, and testes of WT and KO mice. “Delta Ct” expresses the difference in cycle thresholds between Wnt11 and 18S amplification rates.

FIG. 4.

Kaiso deficiency decreases tumor size and increases life span of Apc^Min/+ mice. (A) Kaplan-Meier survival plot of Apc^Min/+ Kaiso^+/y (solid line) and Apc^Min/+ Kaiso^−/y (hashed line) mice. Apc^Min/+ Kaiso^−/y mice live significantly longer (median, 317 days; gray vertical line) than both Apc^Min/+ Kaiso^+/y (median, 217; black vertical line, P = 0.006 [log rank]). (B) Tumor number is not altered by Kaiso deficiency. Boxplots show numbers of adenomas per mouse at 180 days and at death. The horizontal boxed line represents the median. Gray boxes, Apc^Min/+ Kaiso^+/y; open boxes, Apc^Min/+ Kaiso^−/y. No significant differences were observed between Apc^Min/+ Kaiso^+/y and Apc^Min/+ Kaiso^−/y mice at either 180 days (P = 0.10 [Mann-Whitney], n ≥ 20) or at death (P = 0.62 [Mann-Whitney], n ≥ 20). (C) Tumor size, measured by area, is reduced in Kaiso-deficient mice at 180 days (P = 0.001 [Mann-Whitney], n ≥ 114) but not death (P = 0.55 [Mann-Whitney], n ≥ 239). Gray bars, Apc^Min/+ Kaiso^+/y; open bars, Apc^Min/+ Kaiso^−/y. Bars represent the standard error of the mean.

FIG. 5.

Kaiso expression is elevated in murine intestinal tumors. (A) Immunohistochemistry with an anti-Kaiso antibody 6F in colonic tumor and matched normal mucosa from Muc2^−/− mice at the indicated magnification factors. (B) Western blots performed with anti-Kaiso and antiactin antibodies in two pairs of Muc2^−/− intestinal tumors compared to matched normal mucosa.