Origin of Somatic Mutations in β-Catenin vs. APC in Colon Cancer

Powell, S. M., Zilz, N., Beazer-Barclay, Y., Bryan, T. M., Hamilton, S. R., Thibodeau, S. N., Vogelstein, B., and. Kinzler, K. W. (1992) APC mutations...
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Origin of Somatic Mutations in β‑Catenin versus Adenomatous Polyposis Coli in Colon Cancer: Random Mutagenesis in Animal Models versus Nonrandom Mutagenesis in Humans Da Yang, Min Zhang, and Barry Gold* Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, United States ABSTRACT: Wnt signaling is compromised early in the development of human colorectal cancer (CRC) due to truncating nonsense mutations in adenomatous polyposis coli (APC). CRC induced by chemical carcinogens, such as heterocyclic aromatic amines and azoxymethane, in mice also involves dysregulation of Wnt signaling but via activating missense mutations in the βcatenin oncogene despite the fact that genetically modified mice harboring an inactive APC allele efficiently develop CRC. In contrast, activating mutations in β-catenin are rarely observed in human CRC. Dysregulation of the Wnt signaling pathway by the two distinct mechanisms reveals insights into the etiology of human CRC. On the basis of calculations related to DNA adduct levels produced in mouse CRC models using mutagens, and the number of stem cells in the mouse colon, we show that two nonsense mutations required for biallelic disruption of APC are statistically unlikely to produce CRC in experiments using small numbers of mice. We calculate that an activating mutation in one allele near the critical GSK3β phosphorylation site on β-catenin is >105-times more likely to produce CRC by random mutagenesis due to chemicals than inactivating two alleles in APC, yet it does not occur in humans. Therefore, the mutagenesis mechanism in human CRC cannot be random. We explain that nonsense APC mutations predominate in human CRC because of deamination at 5-methylcytosine at CGA and CAG codons, coupled with the number of human colonic stem cells and lifespan. Our analyses, including a comparison of mutation type and age at CRC diagnosis in U.S. and Chinese patients, also indicate that APC mutations in CRC are not due to environmental mutagens that randomly damage DNA.

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CONTENTS

1. Introduction 2. Animal Models of CRC 2.1. Azoxymethane-Induced CRC 2.2. PhIP-Induced CRC 3. Age at Diagnosis and APC Mutation Pattern 4. Deamination and CRC in Humans 5. Discussion 6. Methods 6.1. Patients and Mutation Data 7. Statistical Analysis Author Information Corresponding Author ORCID Funding Notes Biographies Abbreviations References

most cancers, the etiology of the disease remains obscure, although several dietary risk factors have been suggested based on epidemiological studies2−7 coupled with chronic bioassays of identified mutagens in animal models.8,9 In addition, CRC is attributable to distinct germ line mutations: 3% to hereditary nonpolyposis colorectal cancer (HNPCC) Lynch Syndrome)10 and 1% to familial adenomatous polyposis (FAP).11 In human CRC, the incidence of mutations in the tumor suppressor adenomatous polyposis coli (APC) reaches 75%12 and is thought to be the earliest driver mutation in CRC.13,14 Most of these mutations (65%) are nonsense mutations that afford a truncated APC gene product.12 The remaining mutations are frameshift (27%), splice site (5%), and missense (3%). The nonsense mutations are not random with a high incidence at CGA Arg and CAG Gln codons.15 In humans, the second APC allele is inactivated by promotor methylation, chromosomal loss, or by another truncating nonsense, frame shift, or splice site mutation,12 while in rodents, chromosomal loss is most frequently observed.16 The result of biallelic loss APC or activating mutations of β-catenin causes increased nuclear β-catenin levels due a decrease in GSK-3β mediated

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1. INTRODUCTION Colorectal cancer (CRC) is the third most common cancer in men and women, affecting more than a 130 000 people yearly in the U.S. with almost 50 000 deaths per year.1 As is the case in © 2017 American Chemical Society

Received: April 5, 2017 Published: June 4, 2017 1369

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phosphorylation of β-catenin, which leads to adenomas and adenocarcinomas.17,18 There are a number of rodent models of CRC that use chemical carcinogens (e.g., azoxymethane, AOM, or 2-amino-1methyl-6-phenylimidazo(4,5-b)pyridine, PhIP), that closely mimic the physiology of the disease in humans.8,9 However, APC mutations are rarely observed in the colon tumors that develop in wild-type rodents treated with these mutagens.19−21 Instead, the Wnt signaling pathway is activated by point mutations at key residues in β-catenin that prevent its phosphorylation at Ser,33 Ser,37 Thr,41 and Ser45 by GSK3β, which normally leads to β-catenin degradation.17,18 The mutated β-catenin protein is not degraded, and its levels build up in the nucleus. Accordingly, Wnt signaling dysregulation is achieved but by mutation of two distinct proteins, one a tumor suppressor and one an oncogene. The observation of APC mutations in humans and β-catenin mutations in rodents persists even when other promoting factors such as inflammation are involved.19,20 An exception to this pattern is observed in people defective in mismatch repair (HNPCC) that develop CRC with mutant β-catenin at an early age.22 What do the different mutation signatures in the different genes (APC tumor suppressor and β-catenin oncogene) imply about the etiology of CRC in humans? To address this question, we analyzed the reported adduct levels and mutation patterns in chemical carcinogenesis models of CRC relative to the mutation pattern in human CRC tissue. The results suggest that (i) despite what is observed in humans, random mutagenesis will create an activating mutation in the β-catenin oncogene >105-fold more efficiently than it will a biallelic loss of the tumor suppressor APC; (ii) mutagenic agents that randomly react with DNA, including those that are present in the western diet and can induce CRC in animal models, appear to be relatively ineffective in the induction of CRC in humans, at least at levels of normal environmental exposure; and (iii) hydrolytic deamination of 5-methylcytosine (5-mC) at CpG and CpApG sites coupled with random polymerase errors are the major sources of inactivating APC mutations observed in human CRC.

Table 1. Comparison of Physical Parameters of Human versus Mouse Colon length (cm) diameter (cm) surface (cm2) crypts/colon stem cells/crypt stem cells/colon

160.0 7.0 3.3 × 103 1.4 × 107 15 2.2 × 108

8.2 0.3 7.7 1.1 × 105 5 5.4 × 104

would be 4.0 × 1011 6-mG generated in colonic stem cells per mouse: (6 weekly 10 mg/kg AOM) × (1.2 × 105 6‐mG/stem cell /10 mg/kg/AOM treatment) × (5.5 × 105stem cells/mouse colon) = 4.0 × 1011

Of these DNA lesions, only ∼1% (4.0 × 109) would be converted into G → A transitional mutations after the AOM treatments because of efficient DNA repair by methylguanineDNA methyltransferase (MGMT), correct base insertion of C opposite the lesion, and cell death.25,27,32 What are the odds that one of these G → A mutations produced in colonic stem cells by AOM will occur at a base that will lead to a nonsense mutation in a tumor suppressor gene (i.e., APC) versus the odds for an activating missense mutation in an oncogene (i.e., β-catenin)? For APC, nonsense mutations derived from a G•C → A•T transition caused by 6-mG can occur at the following codons (including on the complementary strand): Arg (CGA → TGA), Gln (CAA → TAA and CAG → TAG), and Trp (TGG → TAG or TGA). In the APC gene, there are 27 Arg, 149 Gln, and 13 Trp codons that can yield a G•C → A•T nonsense mutation, respectively.15 This translates to 378 Gs on both strands in the two APC alleles. Since there are 1.7 × 109 G per genome, statistically the chance of a 6-mG induced transitional mutation occurring at one of the critical Gs in Arg, Gln, and Trp codons is 378 Gs/1.7 × 109 Gs per stem cell = 2.2 × 10−7

When 2.2 × 10−7 is multiplied by the number of G•C → A•T mutations in the stem cells in a mouse colon (4.0 × 109 stem cell mutations/colon), it predicts that 880 stem cells would harbor a G → A mutation at a site that could lead to a nonsense mutation in one APC allele. Therefore, there is a predictable inactivation of one APC allele as an outcome of the AOM dosing. However, inactivation of APC requires that both alleles be dysfunctional via a truncating mutation or loss of expression. The statistical chance of the AOM treatments generating a second inactivating hit at a critical site in the remaining WT allele is

2. ANIMAL MODELS OF CRC 2.1. Azoxymethane-Induced CRC. A frequently used animal model of CRC involves repetitive i.p. or s.c. administration of azoxymethane (AOM) or related 1,2dimethylhydrazine (DMH).8,9 Both compounds are metabolized to a common DNA methylating agent (methanediazonium ion) that yields a complex mixture of DNA lesions.23,24 The adduct that appears to be most important in carcinogenesis is O6-methylguanine (6-mG), which is both toxic and miscoding.25−27 The latter activity leads to an increase in G•C → A•T transitional mutations.25−27 Unfortunately, studies in DNA repair mutant cells and animals do not dissect out toxicity effects from mutagenicity since both go up and down simultaneously with unrepaired adduct levels. A 10 mg/kg dose of AOM, which is commonly used in 6 weekly treatments to induce CRC, yields 1.2 × 105 6-mG per cell based on measured adduct levels of 70 pmol 6-mG/μmol G in the distal colon28 and 1.7 × 109 Gs per genome.29 Assuming the target cells for CRC are in the stem cell niche, that there are 5.5 × 105 stem cells/mouse colon (Table 1),30,31 and the DNA in all cells undergoes the same chemical reactions with the reactive electrophilic metabolites derived from AOM, there

(880 stem cells with a nonsense mutation in one APC allele) × (378 critical Gs/1.7 × 109 Gs/stem cell) = 1.9 × 10−4

Therefore, only one mouse in >5000 treated with six exposures to AOM would develop CRC due to loss of both APC alleles, which would not observable in typical experiments with small numbers of animals. We used estimates of the number of colonic stem cells and conversion efficiency of 6-mG to a G → A mutations in the above analysis. However, even if the number 1370

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accentuated by treatment with DSS in the drinking water, βcatenin activation is observed.20,21 Similar to AOM-induced CRC, nonsense mutations in APC are not found. G•C → T•A transversions (at the underlined base) could result in a nonsense mutation at Glu (GAA and GAG), Gly (GGA), Ser (TCA and TCG), Tyr (TAC and TAT), and Cys (TGC) codons in APC. There are 426 of these codons in APC. If heterocyclic aromatic amines (HAA), such as PhIP, were a source of the APC nonsense mutations in human CRC, mutations at these sites should be elevated above that predicted by random errors. However, the incidence of nonsense mutations at these codons in human CRC is lower than that predicted based on random mutations.15 Overall, 41% of the APC codons that can potentially be converted into a stop codon by a single base substitution involving G•C → T•A transversions (Table 2), but only 22.2% of the APC mutations in human CRC are G•C → T•A mutations.

of nonsense APC mutations was ,000-fold higher, it would still require >50 treated mice to observe a single biallelic APC mutation. What does a similar calculation show for an activating missense mutation of β-catenin by a random G alkylating agent? For β-catenin, only one allele needs to be activated but the target size for an activating missense mutation in β-catenin is smaller than that for an inactivating nonsense mutation in an APC allele. On the basis of human and animal studies, activating mutations of β-catenin are confined to a small region around the GSKβ3 phosphorylation site. Only six amino acids are mutated in human endometrial cancers (Figure 1), which

Figure 1. Sequence of β-catenin mutation hotspots (underlined sequences) observed in chemically-induced CRC. Mutations in this region of β-catenin affect its phosphorylation by GSK3β, which targets the protein for degradation.

Table 2. Potential G•C → T•A Nonsense Mutations per APC Allele amino acid

show a high (31%) incidence of β-catenin mutations.12 In human CRC, β-catenin mutations are rare (8 in 233 tumors), and no site is mutated more than once.12 The AOM-induced transitional G•C → A•T mutations that can occur in the codons for these amino acids are D32N (GAC → AAT), S33F (TCT → TTT), G34R or G34E (GGA → AGA or GAA), S37F (TCT → TTT), T41I (ACC → ATC or ATT), and S45F (TCT → TTT). Therefore, the target for 6-mG derived mutations is limited to 16 bases for both alleles, although only one needs to undergo an activating missense mutation. Accordingly, the odds of hitting one of these 16 G bases with a transitional mutation derived from a 6-mG lesion are 16/1.7 × 109 Gs or 9.4 × 10−9. The product of this value and the number of mutations that will be generated by AOM (4.0 × 109 stem cell mutations/colon) indicates that 37.6 activating mutations of β-catenin in stem cells are statistically predicted to occur as a result of the 6 AOM treatments. Clearly, the mutation frequency of 37.6 versus the 2.0 × 10−4 for biallelic truncation of APC is consistent with the mouse data on AOMinduced CRC having only β-catenin mutations. Some mouse strains, for example, C57Bl/6, are more resistant to AOM-induced CRC. However, AOM followed by DSS in these mice efficiently produces CRC, but the mutation pattern is unchanged: tumors bear activating mutations in βcatenin and not truncating mutations in APC.32 This is expected since DSS inflammation alone does not produce colonic stem cell mutations in mice. 33 An alternative explanation for the lack of APC mutations in animal models is that biallelic deletion of APC activity by any agent may not be a viable transformation pathway in mice. However, APCMin mice, which carry a truncating nonsense mutation in one allele, rapidly develop GI tumors due to loss of the remaining wildtype allele.34 2.2. PhIP-Induced CRC. Another mouse model for CRC involves PhIP, which is the most abundant of the many mutagenic HAA produced during the grilling of meat.5−7 The major DNA lesion produced by PhIP, after N-oxidation, is 2amino-9-(2R,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-(1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridin-2-yl-amino)-1H-purin-6(9H)one (dG-C8-PhiP),35 which causes mainly G → T transversions in vitro and in vivo.36−39 In the PhIP model of CRC, which can be

Glu Gly Ser Tyr Cys total a

codon

stop codon

no. codons in APC

% of total codons

% of mutations observed

GAA GAG GGA TCA TCG TAC TGC

TAA TAG TGA TAA TAG TAA TGA

161 49 69 114 7 16 10 426

15.6 6.0 6.7 11.1 0.7 0 1.0 41.0

11.4 1.2a 0.6a 7.2 0.6 0 1.2 22.2

Observed significantly less than predicted (p < 0.05).

Low exposure of PhIP (5 μg/day) in human studies indicated that the yield of dG-C8-PhIP is 2 × 105-fold lower than that observed in a noncarcinogenic dose of PhIP in rats.40 The efficiency for conversion of PhIP DNA lesions into mutations is not known, but the dG-C8-PhIP adduct would have to be >1000-fold more mutagenic than 6-mG to inactivate two APC alleles. The measured levels of aromatic amines in food products, the adducts levels in “control” human colon tissue, and the mutation pattern are not consistent with these environmental molecules posing a significant burden for CRC by APC inactivation. As with AOM-induced CRC, we suggest that truncation of APC via nonsense mutations is not an observable driver mutation in PhIP-induced CRC in rodents because insufficient adducts are formed to cause nonsense mutations in both APC alleles. Additional evidence that aromatic amines are not a significant cause of CRC is that in addition to G → T transversions, PhIP produces frameshift deletions.41 Analysis of the 38 frameshift deletions that occur in APC in human CRC indicates a random pattern with no hotspots: only three sites have >1 hit.12 The majority (76%) of the “random” frameshifts involve deletion of A•T bp’s and are associated with poly dA runs of ≥3. The fact that HAA, such as PhIP, selectively react at Gs, and PhIPinduced CRC show G•C deletion mutations at 5′-GGGA,41 reinforces the suggestion that is unlikely that aromatic amines play a significant role in human CRC. A comparison between the APC mutation patterns in CRC in the U.S.12 versus China42 provides additional insight into the role of diet and HAA in human CRC. While the diets in China and the U.S. are probably becoming more similar, this was not 1371

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Table 3. APC Nonsense Mutations in Human CRC in China versus USA amino acid Glu (E) Lys (K) Ser (S)

Gly (G) Arg (R) Leu (L)

Tyr (Y)

codon GAA GAG AAA AAG TCA TCG GGA CGA AGA TTG TTA TAC TAT

Trp (W)

TGG

Gln (Q)

CAG CAA TGT TGC

Cys (C) a

stop codon TAA TAG TAA TAG TAA TGA TAG TAA TGA TGA TAG TGA TAA TAG TAA TAA TAG TAG TGA TAG TAA TGA TGA

no. of codons in APC

% of total codons

no. mutations observed (China)42

% observed in China

% observed in U.S.15

G•C→T•A G•C→T•A A•T→T•A A•T→T•A G•C→T•A G•C→C•G G•C→T•A G•C→T•A G•C→A•T A•T→T•A A•T→T•A A•T→C•G A•T→T•A G•C→C•G G•C→T•A A•T→T•A A•T→C•G G•C→A•T

161 49 133 62 114

15.6 4.8 12.9 6.0 11.1

7 69 27 71 39 49

0.7 6.7 2.6 6.9 3.8 4.8

13

1.6 0 3.3 0 1.9

11.8 3.2 3.2a 0a 2.2a 3.2 0 3.2 45.2b 4.3 0 3.2 1.1 0 0 0 0 2.4

11.4 1.2 2.4a 1.2a 7.2 0.6 0.6 0.6a 43.4b 1.2a 0 1.8

16

11 3 3 0 2 3 0 3 42 4 0 3 1 0 0 0 0 2

G•C→A•T G•C→A•T A•T→T•A G•C→T•A

81 68 28 10

7.7 6.7 2.8 1.0

10 3 1 2

10.8 3.2 1.1 2.2

19.9b 4.2 0.6 1.2

mutation

34

1.2 0 1.2 0 0

Observed significantly less than predicted (p < 0.05). bObserved significantly more than predicted (p < 0.05).

the case when the population that is currently at risk for CRC was young. The age-standardized rate of CRC in China is 0.017% compared to 0.040% in Asian and Pacific Islanders in the U.S. population.1,43 However, the APC mutation patterns in both countries are virtually identical (Table 3): nonsense mutations account for 64% of the CRC mutations, and C → T mutations at CGA Arg and CAG Gln codons account for 63% of the APC nonsense mutations in both China42 and the U.S.12 The only difference observed is the lower incidence of C → T nonsense mutations at CAG Gln codons (Table 3), but this may be a function of the smaller number of tumors analyzed. The similar mutation profile implies that the different incidences of CRC in the U.S. and China cannot be directly related to exposure to different endogenous and environmental mutagens.

Figure 2. Relationship between age at diagnosis of CRC and the nonsense mutations in APC observed in the CRC tumor: (A) in U.S.44 and (B) China.43

3. AGE AT DIAGNOSIS AND APC MUTATION PATTERN Against the backdrop of random mutations, exposure to environmental factors, such as HAA, would increase the incidence and decrease the latency period for CRC. Therefore, the nature of the APC point mutations in the The Cancer Genome Atlas (TCGA) CRC samples was analyzed as a function of age at diagnosis (Figure 2A).44 While age at diagnosis reflects the latency for initiation as well as tumor growth and medical surveillance, it is reasonable to assume that it provides a rough relationship to disease onset. In comparing the potential base substitutions in APC that can afford a nonsense mutation, no significant difference was observed for the different point mutations: C → T and G → T mutations had mean ages at diagnoses of 66.4 and 65.8, respectively (p = 0.66, t test). A similar analysis was done using the Chinese (ICGC) database.42 As in the U.S. population, there is no difference (p = 0.13, t test) between age at diagnosis and

between the two largest groups of different APC nonsense mutations (Figure 2B). Moreover, there is no difference between the age at diagnosis between the two populations (Figure 2). Thus, the age-related incidence of G → T mutations to afford stop codons at Glu (GAA and GAG) and Gly (GGA), which would be accelerated if HAA in the diet were a cause of the mutations, was not observed.

4. DEAMINATION AND CRC IN HUMANS The much higher probability of a random DNA damaging event generating a missense mutation in β-catenin versus truncating mutations in both APC alleles raises the question of why the ∼105 more efficient route to transformation is rarely observed in human CRC.12 We suggest that (i) exposure of humans to DNA damaging agents that randomly react with DNA is too low to account for a significant amount of human 1372

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5. DISCUSSION The major differences between humans and rodents related to their susceptibility to CRC are the physical dimension of the colon and the number of stem cells, and lifetime. We estimate the surface area of the colon in humans is 400-fold larger than the mouse, and that there are three-fold more stem cells/crypt relative to the mouse (Table 1).30,31 In addition, humans live almost 40-times longer than mice. Therefore, the probability of acquiring a truncating APC mutation via hydrolytic deamination is almost 1.6 × 104-fold higher in humans than in mice since the rate of formation of G−T mismatches is a function of the deamination rate and the number of 5-mC in APC at CGA and CAG codons in colonic stem cells. The increase in the number of stem cells and longer lifespan make a biallelic truncation of APC statistically possible due to deamination, a process that is also biased against the generation of missense mutations in β-catenin because of the lack of CpG and CpApG sequences at the region that affords an oncogenic mutation. Only in HPNCC individuals with high mutation rates are activating β-catenin mutations observed.22 The analysis of the data also suggests that it is unlikely that environmental mutagens, such as HAA that randomly react with DNA, are responsible for CRC. In our discussion, it is assumed that the chemical modification of DNA is random. This is not absolutely accurate since HAA and other alkylating agents do show a modest sequence selectivity in terms of their formation and repair. However, the selectivity for HAA’s and small alkylating agents is not at CpG sequences.50−54 DNA repair can also be impacted by sequence but the effect is relatively small and cannot explain the remarkable preference for CpG and CpApG sequences.55 As mentioned above, the frequency of truncating G → T transversions in APC is below what would be predicted due to random mutations and not elevated as predicted if HAA’s adducts were a cause of mutated APC (Table 2). The age of CRC diagnosis in both the U.S. and China populations also is insensitive to the nature of the nonsense mutation (Figure 2), which is contrary to what would be expected in populations exposed to putative mutagens that induce CRC. The focus of the current work is on the etiology of driver mutations in CRC. There must be other genetic and environmental factors that can increase CRC incidence or reduce the latency as indicated by the increasing frequency of CRC in younger patients55 and the different prevalence of CRC in different countries. It should be noted that there are examples in mice (e.g., C57Bl/6 mice) where chemical-induced mutagenesis in colonic stem cells does not result in tumors. In these mice, exposure to an inflammatory agent (e.g., DSS) after the mutagen is required, although DSS itself does not produce stem cell mutations.33,56 Therefore, generation of a driver mutation in an oncogene or tumor suppressor gene may not be the rate-limiting step in CRC. Identifying these presumably nongenotoxic factors may be the Achilles heel that can be targeted by therapeutics since the processes of chemical deamination and polymerase errors30 that afford the major bulk of the somatic mutations are not targetable.

CRC and (ii) that the pathway that is important for APC mutations must be ineffective in generating missense mutations at the critical β-catenin sequences required for oncogene activation (Figure 1). There are 1031 potential codons in APC coding for Glu, Lys, Ser, Gly, Arg, Leu, Tyr, Trp, Gln, and Cys that can be converted into a stop codon by a single nucleotide alteration (Table 3).15 The C → T transitions at CGA Arg codons, which generate nonsense mutations in APC, account for 43% of the total nonsense mutations observed in CRC. However, only 3% are predicted based on the total number of Arg CGA codons in APC that potentially can be converted into a stop codon.15 C → T transitions at Gln (CAG → TAG) codons are also seen at >2-fold above predicted.15 This pattern of transitional mutations at CpG and CpApG sequences is suggestive of deamination of 5-methylcytosine (5-mC) that is likely to be present at CpG sequences in the APC exons.45 Methylation at CpApG sites is less uniform, and we conservatively estimate that ∼3% of the C’s in CAG codons are methylated.46 Deamination of 5-mC will not yield activating β-catenin mutations simply because there are no CpG or CpApG sites in the region of β-catenin that affect its sensitivity to phosphorylation by GSKβ3 (Figure 1). Hydrolytic deamination would yield 1.8 × 10−5 T•G mismatches/5-mCpG site/year at CGA Arg and CpApG Gln codons:15,47 5.8 × 10−13 deaminations/s at 5‐mC × 3.15 × 107s /y

There are 54 CGA Arg codons and 162 CAG Gln codons in the two APC alleles that are hot spots for C → T nonsense mutations. If all of the CpG sites and 3% of the CpApG are methylated, there will be 78 codons in APC that potentially can be converted to a stop codon by deamination. Therefore, a mean of 4.2 × 105 T•G mismatches at CGA and CAG codons in APC are produced in human colonic stem cells per year: [(54 CGA + 3% × 162 CAG sites)/APC gene] × [1.8 × 10−5 T•G mismatches/CpG site /colonic stem cell/y] × [4 × 108 APC alleles/colon] = 4.2 × 105 T•G mismatches

The repair of T•G mismatches by enzymes (e.g., thymineDNA glycosylase) would reduce this number. If only 10% of the T•G mismatches were converted into mutations,48,49 there would be ∼4.2 × 104 5-mC → T truncating nonsense mutations in APC alleles accumulated and distributed in colonic stem cells per year. In 50 years, hydrolytic deamination of 5-mC would result in ∼2.1 × 106 nonsense mutations distributed in both alleles of APC in colonic stem cells. The statistical probability that one stem cell will have at least one truncating mutation at age 50 y is [(2.1 × 106 inactivated APC alleles/stem cell) /(2 × 108 stem cells/colon)] = 1.1 × 10−2 inactivated APC alleles/colon

The chance of two inactivated APC alleles/colon at age 50 due to deamination is

6. METHODS 6.1. Patients and Mutation Data. In total, 166 APC mutations across 179 CRC samples were downloaded from the TCGA Data Portal. The sequencing and quality control procedures were recently described.44 One-hundred thirty-eight APC mutation across 91 CRC patients were downloaded from the ICGC Data Portal.42 Deidentified

(1.1 × 10−2)2 = 1.1 × 10−4

Thus, 0.011% of the population at age 50 may bear a colonic stem cell with no functional APC due to deamination. 1373

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patient information, including age at diagnosis, tumor stage and grade, and surgical outcome, was also downloaded. Only the nonsense point mutations in APC gene were studied. Written consent was obtained from all live patients. The access to the TCGA database is approved by the dbGaP.

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7. STATISTICAL ANALYSIS Standard statistical tests were used to analyze the clinical and genomics data including the Chi-squared test and student t test. Significance was defined as a p-value of less than 0.05. Analyses were primarily performed using R 2.10.0 (R Foundation for Statistical Computing (http://www.r-project.org/) and SPSS version 18 (SPSS Inc., Chicago, Illinois).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 412-383-9593. ORCID

Barry Gold: 0000-0002-2610-978X Funding

This work was funded by the School of Pharmacy, University of Pittsburgh. Notes

The authors declare no competing financial interest. Biographies Dr. Da Yang is an Assistant Professor in the Department of Pharmaceutical Sciences and the Center for Pharmacogenetics at the University of Pittsburgh. He completed his Ph.D. in pharmacology and genomics at the Harbin Medical University in China and an Odyssey postdoctoral fellowship at the University of Texas MD Anderson Cancer Center. Dr. Min Zhang is an Adjunct Research Assistant Professor in the Department of Pharmaceutical Sciences at the University of Pittsburgh. She completed her Ph.D. in bioinformatics at the Harbin Medical University of China and postdoctoral training at both the National Center for Toxicological Research (NCTR) and the University of Texas MD Anderson Cancer Center. Dr. Barry Gold is a Professor in the Department of Pharmaceutical Sciences at the University of Pittsburgh. He was the Chair of that department from 2005−2017. Prior to Pittsburgh, he was professor and associate director of the Eppley Institute for Research in Cancer at the University of Nebraska Medical Center. He completed his Ph.D. in organic chemistry at the University of Nebraska−Lincoln and did postdoctoral training at the University of Toronto.

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ABBREVIATIONS AOM, azoxymethane; dG-C8-PhIP, 2-amino-9-(2R,5R)-4hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-8-(1-methyl6-phenyl-1H-imidazo[4,5-b]pyridin-2-yl-amino)-1H-purin-6(9H)one; HAA, heterocyclic aromatic amines; 5-mC, 5methylcytosine; 6-mG, O6-methylguanine; PhIP, 2-amino-1methyl-6-phenylimidazo(4,5-b)pyridine

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