Different phenotypic consequences of simultaneous versus stepwise Apc loss

Abstract

APC is considered a gatekeeper for colorectal cancer (CRC). Cells with heterozygous APC mutations have altered expression profiles suggesting that the first APC hit may help set the stage for subsequent transformation. Therefore, we measured transformation efficiency following what we have designated as 'simultaneous' versus 'stepwise' Apc loss. We combined a conditional Apc allele (Apc(CKO)) with a Cre reporter gene and an out-of-frame Cre allele (Pms2(cre)) that stochastically becomes functional by a frameshift mutation in single cells. Loss of one Apc allele (Apc(CKO/+)) had little consequence, whereas simultaneous loss of both Apc alleles (Apc(CKO/CKO)) resulted in increased clonal expansion (crypt fission), consistent with the gatekeeper function of Apc. Interestingly, our analyses showed that most of the Apc-deficient crypts in Apc(CKO/CKO) mice appeared normal, with morphological transformation, including β-catenin deregulation, occurring in only 17% of such crypts. To determine whether transformation efficiency was different following stepwise Apc loss, we combined Apc(CKO) with a germline mutant allele, either Apc(Min) or Apc(1638N). Transformation efficiency following stepwise Apc loss (Apc(Min/CKO) or Apc(1638N/CKO)) was increased five-fold and essentially all of the Apc-deficient cells were dysplastic. In summary, our data suggest that the gatekeeper function of Apc consists of two roles, clonal expansion and morphological transformation, because simultaneous Apc loss frequently leads to occult clonal expansion without morphological transformation, whereas stepwise Apc loss more often results in visible neoplasia. Finally, that Apc-deficient cells in certain scenarios can retain a normal phenotype is unexpected and may have clinical implications for surveillance strategies to prevent CRC.

Figure 1. Increased crypt fission in Apc^CKO/CKO mice

β-gal⁺ foci counted in whole mount (a, b), or sections of the proximal small intestine (c, d). (a) Note, there was no significant difference in the total number of β-gal⁺ foci between the three genotypes. (b) However, there was a significant increase in the number of “larger” β-gal⁺ foci, those involving more than three villi in Apc^CKO/CKO mice (p=0.02). (c) In sections of the proximal small intestine, there was no significant difference in the percentage of β-gal⁺ foci. (d) In contrast, we did observe an increase in the percentage of β-gal⁺ foci involving more than one crypt in Apc^CKO/CKO mice (p=0.001).

Figure 2. Increased number of larger β-gal⁺ foci in Apc^CKO/CKO mice

(a–d) Whole mount images of proximal small intestine of Apc^+/+ and Apc^CKO/CKO mice. Adenomas denoted with circles and larger normal β-gal⁺ foci with arrows. (b, d) Images showing examples of larger normal β-gal⁺ foci in Apc^CKO/CKO mice. (e) Distribution of β-gal⁺ foci sizes in the proximal small intestine of Apc^+/+, Apc^CKO/+ and Apc^CKO/CKO mice. Apc^CKO/+ and Apc^CKO/CKO mice show a significant increase in β-gal⁺ foci involving 3 villi (p=0.01). Apc^CKO/CKO show a significant increase in β-gal⁺ foci involving 6 and 10 or more villi (p=0.02 and p=0.03, respectively).

Figure 3. Distribution of normal and adenomatous β-gal⁺ foci

(a) Images of different representative β-gal⁺ foci illustrating a single β-gal⁺ cell, single β-gal⁺ crypt, multiple β-gal⁺ crypts, β-gal⁺ microadenoma and β-gal⁺ adenoma. (b) Percentage of the β-gal⁺ foci illustrated in (a).

Figure 4. Apc is efficiently recombined in Pms2^cre/cre; Apc^CKO/CKO mice

Images of (a) β-gal⁻ crypts, (b) β-gal⁺ crypts and (c) adenomas from the proximal small intestine isolated on LCM CAPs. (d) Gel of PCR reactions for the different isolated cell types. CKO is the unrecombined allele and Δ is the recombined allele. Each gel was normalized to the signal obtained from an Apc^Δ/CKO sample, which has a 1:1 ratio of recombined:unrecombined Apc^CKO alleles. (e) Graph of normalized recombined:unrecombined ratios that illustrates that 76% (16/21) of normal appearing β-gal⁺ samples had a 6–10 fold increase in the recombined:unrecombined ratio over β-gal⁻ cells (p<0.001), similar to the increase observed for the adenoma samples (p=0.85).

Figure 5. Characteristics of Cre-reporter⁺ foci

Characterization of Cre-Reporter⁺ tumor (a–c, g–i) and normal (d–f, j–l) tissue. (a, d) Show deregulated expression of β-catenin in GFP⁺ tumor tissue (single arrowheads), but normal β-catenin expression in normal GFP⁺ tissue (single arrowheads) (β-catenin:blue, mTomato:red[unrecombined], mGFP:green[recombined]). Double arrowheads point out unrecombined, epithelial tissue. (b, e) Show increased apoptosis in β-gal⁺ tumor tissue, but normal apoptosis levels in normal, β-gal⁺ tissue (β-gal⁺:blue, TUNEL:brown). Arrows denote TUNEL positive cells. (c, f) Loss of alkaline phosphatase staining in β-gal⁺ tumor tissue, but continued expression of alkaline phosphatase in normal, β-gal⁺ tissue (β-gal⁺:blue, alkaline phosphatase:purple). In c) arrows show demarcation of alkaline phosphatase and X-gal staining. In f) purple and blue lines illustrate the overlap of alkaline phosphatase and β-gal⁺ cells. (g, j) Merged images of immunofluorescence for β-catenin (red), β-gal (green) and DAPI (blue). Arrows point to the same β-gal⁺ regions. (h, k) Shown are images of only the β-gal (green) channel. (i, l) Images of only the β-catenin (red) channel. Note, deregulated β-catenin expression n the tumor, but normal β-catenin pattern in the normal tissue.

Figure 6. Increased tumor formation in Apc^Min/CKO and Apc^1638N/CKO mice

(a) Survival curve illustrating that Apc^Min/CKO (red squares) and Apc^1638N/CKO (green triangles) mice become anemic at an earlier age than Apc^CKO/CKO (blue diamonds) mice (p<0.001). (b) Increased number of small intestinal adenomas in Apc^Min/CKO and Apc^1638N/CKO mice compared to Apc^CKO/CKO mice (p=0.01).

Figure 7. Percentage of β-gal⁺ cells in proximal small intestine adenomas

(a) Whole mount image of β-gal⁺ adenomas in Apc^CKO/CKO mice. Single-headed arrow shows a tumor scored as 100% β-gal⁺. Double-headed arrow shows a tumor scored as 50% β-gal⁺. (b) Classification of adenomas into % β-gal⁺ in the proximal small intestine.