8-oxoguanine is a major base lesion in DNA or in nucleotides caused by oxidative stress, and is highly mutagenic because it can pair with adenine as well as cytosine. Adenine DNA glycosylase, encoded by the human mutY homolog gene, MUTYH, excises adenine in the nascent strand when inserted opposite 8-oxoguanine in template DNA, and thus suppresses mutagenesis caused by 8-oxoguanine that has accumulated in DNA due to oxidative stress. Several germ-line mutations in MUTYH are predisposed to MUTYH-associated polyposis, an autosomal recessive disorder characterized by multiple colorectal adenomas and carcinomas. Loss of function of MUTYH leads to an accumulation of somatic mutations in APC and KRAS genes, resulting in the development of adenomas/carcinomas. We recently demonstrated that accumulation of 8-oxoguanine in nuclear and mitochondrial DNA triggers two distinct cell death pathways that are independent of each other. Both pathways are initiated by the accumulation of MUTYH-generated single-strand breaks (SSBs) in nuclear or mitochondrial DNA. Our findings indicate that MUTYH-induced cell death due to oxidative stress results in an efficient elimination of mutagenic cells that have accumulated high levels of 8-oxoguanine in their DNAs. It is most likely that loss of function of MUTYH in stem or progenitor cells in the intestinal epithelium of MUTYH-associated polyposis patients results in escape from programmed cell death; however, accumulated 8-oxoguanine causes various mutations in APC or KRAS genes in these proliferative cells, thereby promoting tumorigenesis. We thus propose that MUTYH suppresses tumorigenesis under conditions of oxidative stress by inducing cell death and by suppressing mutagenesis. (Cancer Sci 2011; 102: 677–682)
Reactive oxygen species (ROS), which are generated as byproducts of mitochondrial respiration and as anti-infectious molecules in host defense, or as a consequence of exposure to environmental agents, are known to oxidize nucleic acids. Accumulation of such oxidation damage in DNA often causes mutagenesis, resulting in carcinogenesis.(1) 8-Oxoguanine (8-oxoG) is one of the major oxidation base lesions in nucleotides and in DNA,(2) and it has been considered to cause A:T to C:G and G:C to T:A transversion mutations because it can pair with adenine as well as cytosine (Fig. 1).(3,4) To avoid such mutagenesis, mammals, including humans, have an elaborate error-preventing system with three distinct enzymes (Fig. 1B). Oxidized purine nucleoside triphosphatase, encoded by mutT homolog-1 (MTH1 or NUDT1) hydrolyzes 8-oxo-dGTP to the monophosphate form and pyrophosphate,(5) thus avoiding their utilization by DNA polymerases. 8-OxoG DNA glycosylase, encoded by OGG1, excises 8-oxoG paired with cytosine in DNA,(6) while adenine DNA glycosylase encoded by mutY homolog (MUTYH) removes adenine from DNA inserted opposite template 8-oxoG.(7,8)
Analyses of knockout (KO) mice for each of the three genes demonstrated that these three enzymes play major roles in the suppression of spontaneous mutagenesis and tumorigenesis (Fig. 1B).(9–13) Among the three human homologs of these genes, only mutations in MUTYH have been identified in patients with familial cancer. In particular, a recessive form of familial adenomatous polyposis without a germ-line mutation in APC has been described,(14–16) and is now referred to as MUTYH-associated polyposis (MAP). We have shown that Mutyh-KO mice are highly susceptible to intestinal adenoma/carcinoma,(13) thus providing experimental evidence for the association between biallelic germ-line MUTYH mutations and MAP.
We recently demonstrated that accumulation of 8-oxoG in nuclear DNA and in mitochondrial DNA triggers two distinct cell death pathways that are independent of each other, both of which are dependent on base excision repair (BER) initiated by MUTYH.(17,18) In this review, we propose that MUTYH functions as a molecular switch for programmed cell death under conditions of oxidative stress and, thus, efficiently suppresses tumorigenesis as well as mutagenesis.
The human MUTYH gene encodes multiple transcripts and isoforms of the MUTYH protein
The human MUTYH gene is located on the short arm of chromosome 1, spans 11.2 kb and contains 16 exons (Fig. 2A).(7,16) There is an overlapping gene, target of EGR1, member 1 (TOE1) at the 5′ end of MUTYH and transcription of these two genes proceeds in opposite directions.(19) In human cells, transcription of MUTYH is initiated from three distinct exon 1 sequences producing three types of primary transcripts, α, β and γ with different 5′-untranslated regions. From these three primary transcripts, more than 15 transcripts (α1 to α5; β1 to β5, β ESTs; γ1 to γ4) are generated by alternative splicing in exon 1β and exon 3 (Fig. 2B), and thus more than nine different isoforms of human MUTYH (hMUTYH) protein are expected to be produced (Fig. 2C).(16,20)
We and others demonstrated that type α3 MUTYH mRNA is a major MUTYH transcript that encodes the most abundantly expressed mitochondrial hMUTYH, namely isoform 2 with a mitochondrial targeting signal at the amino terminus (aa 1–14) (Fig. 2C, top).(20,21) On the other hand, hMUTYH isoform 4 encoded by type β3, β5 or γ3 mRNA is the most abundant nuclear isoform. It is translated from an initiation codon corresponding to the second AUG (15th codon) in the type α3 mRNA and lacks the mitochondrial targeting signal.(20,21)
Slupska et al.(8) demonstrated that expression of the major mitochondrial isoform 2 in Escherichia coli complements the mutator phenotype in a mutY mutant, and showed that purified recombinant isoform 2 has a DNA glycosylase activity that can excise adenine opposite 8-oxoG (Fig. 1B). We reported that, as expected, recombinant preparations of nuclear isoform 4 of hMUTYH and mouse MUTYH (mMUTYH) efficiently excise adenine opposite 8-oxoG, and showed that both possess an ability to efficiently excise 2-hydroxyadenine (2-OH-A), an oxidized form of adenine, opposite guanine.(20,22,23)
Mutagenesis and carcinogenesis in Mth1, Ogg1 and Mutyh knockout mice
In comparison with wild-type mice, Mth1-KO mice exhibited a 3.6-fold increased spontaneous mutation rate of A:T to C:G transversions,(24) and also a threefold increase in the incidence of spontaneous liver tumors, with a smaller increase in the incidence of lung and stomach tumors, approximately 1.5 years after birth.(9) In 1.5-year-old Ogg1-KO mice, a fivefold increase in the occurrence of spontaneous lung adenoma/carcinoma was observed, in which the frequency of G:C to T:A transversions as well as accumulation of 8-oxoG in their genomes increased fivefold compared with wild-type mice.(10,12)Mutyh-KO mice had a fourfold increase in the frequency of G:C to T:A transversions and exhibited a 5.3-fold increased occurrence of spontaneous adenoma/adenocarcinoma, especially in small intestine and colon, compared with wild-type mice.(13) In Mutyh-KO mice treated with KBrO3, a strong oxidant, the occurrence of small intestinal tumors increased more than 70-fold in comparison to wild-type mice.(13) Mutation analysis of tumor-associated genes amplified from intestinal tumors that had developed in Mutyh-KO mice after exposure to KBrO3 confirmed G:C to T:A transversions in either Apc or Ctnnb1 but not in Kras (exon 2) or Trp53 (exons 5–8).(25)
Deficiencies in both Mutyh and Ogg1 were reported to predispose 65.7% of the double-KO mice to tumors, predominantly lung and ovarian tumors, and lymphomas.(11) Subsequent analyses identified G:C to T:A transversions in a remarkable 75% of the lung tumors at an activating hot spot, codon 12, of the Kras oncogene, but no mutations were identified in adjacent normal tissues. On the other hand, no tumors were formed in the lungs of mice lacking both Ogg1 and Mth1, despite an increased accumulation of 8-oxoG in these mice.(10) These observations indicate that MUTYH functions as the strongest tumor suppressor among the three enzymes, even in the absence of OGG1 and MTH1 functions. Recently, we observed that Mth1/Ogg1/Mutyh triple-KO mice are highly susceptible to spontaneous tumorigenesis in various tissues (our unpublished observations),(10,26) probably because MUTYH has the strongest tumor suppressor function among the three gene products as discussed above.
Familial alterations in the human MUTYH gene have been reported to be possible causative mutations for certain types of autosomal recessive colorectal adenomatous polyposis.(14–16,27) The colorectal phenotype of MAP resembles that of attenuated familial adenomatous polyposis, usually from 10 to a few hundred adenomas occur; the mean age at diagnosis is 45 years. Present estimates indicate that MAP accounts for approximately 1% of all colorectal cancer; this might increase as more patients are tested for mutations in MUTYH.(16,28) Although patients with MAP have no germ-line mutation in the APC gene, somatic mutations in the APC gene were found exclusively in tumor tissue and most of them were G:C to T:A transversions. A similar spectrum of KRAS mutations was also found in patients with MAP.(29)
To date, more than 80 germ-line mutations have been found in MUTYH alleles in patients with colorectal polyposis and carcinomas.(16,28) Biallelic MUTYH mutations have been found to be associated with a 93-fold increased risk of colorectal cancer, mono-allelic mutations also have been proposed to confer an elevated risk, but this assertion is controversial.(27,30,31)
Missense mutations in MUTYH, Y165C and G382D (residue numbers in isoform 2) are the two most common mutations accounting for approximately 80% of Caucasian patients with MAP.(16,28) The Y165C and G382D mutations have never been found in Korean and Japanese populations,(32–34) and thus an ethnic difference in the pattern of MUTYH mutations has also been suggested. For instance, Y165C and G382D in Caucasians, E466X in Indians and Y90X in Pakistanis have been found (Fig. 2C).(35,36) Biochemical studies using recombinant mitochondrial hMUTYH (isoform 2) protein with the Y165C or G382D mutation revealed that the rate of adenine removal for these variants is 30–40% of the wild-type hMUTYH (isoform 2).(37) In addition, the hMUTYH (G382D) mutant showed reduced ability to suppress the mutator phenotype of the E. coli mutY mutant, while hMUTYH (Y165C) has no ability to suppress the phenotype.(37)
In Japanese APC-negative patients with adenomatous polyposis, seven mutations were identified in MUTYH: P18L, G25D, G272E, A359V, Q324H, 1389G.C, and IVS15-40G.C.(34) Homozygous MUTYH mutations account for approximately 10% of Japanese patients with adenomatous polyposis. G272E may be one of the high-risk mutations for the development of adenomatous polyposis in East Asia, because mMUTYH (G257E) mutant protein which corresponds to the G272E variant protein has no detectable adenine DNA glycosylase activity.(34)
In MAP patients, mutations in the nuclear forms of hMUTYH must be responsible for increased mutation rates in APC or KRAS in the nuclear genome. Goto et al.,(38) therefore, examined adenine DNA glycosylase activity of 14 different variants of recombinant nuclear hMUTYH (isoform 4) protein, all of which had been identified in patients with colorectal polyposis and/or colorectal cancer. The adenine DNA glycosylase activity of the variants with I195V, G368D, M255V and Y151C substitutions (residue numbers in isoform 4) was 66.9, 15.2, 10.7 and 4.5% of the level of wild-type isoform 4, respectively. The activity of the variants with R154H, L360P, P377L, 452delE, R69X and Q310X mutations was also severely impaired, similar to that of the D208N mutant in which an essential aspartate (residue D208) for the DNA glycosylase activity (corresponding to D222 in isoform 3 in Fig. 2C) was substituted with asparagine (N). Variants with V47E, R281C, A345V and S487F substitutions, on the other hand, retained the adenine DNA glycosylase activity at comparable levels to that of wild-type hMUTYH (isoform 4).
MUTYH can excise not only adenine opposite 8-oxoG, but also 2-OH-A incorporated opposite guanine in the template strand.(20,23) A previous study in our laboratory indicated that a mutant mMUTYH (G365D) that corresponds to hMUTYH (G368D) (isoform 4) could not suppress the elevated spontaneous mutation rate in the Hprt gene of Mutyh-KO embryonic stem cells.(39) mMUTYH (G365D) almost completely retained its DNA glycosylase activity, excising adenine opposite 8-oxoG; however, it possessed only 1.5% of the wild-type activity for excising 2-OH-A opposite guanine.(23) Our results imply that the reduced repair capacity for excising 2-OH-A opposite guanine of variant hMUTYH (G368D) (isoform 4) may account for the occurrence of somatic G:C to T:A transversions in the APC or KRAS genes observed in colon cancer patients with the G368D mutation.
MUTYH is a molecular switch for programmed cell death under conditions of oxidative stress
In Mth1-KO mouse embryonic fibroblasts, accumulation of 8-oxoG in nuclear and mitochondrial DNAs was highly increased by exposure to oxidative stress, resulting in mitochondrial dysfunction and finally cell death. Expression of human MTH1 protein, however, efficiently suppressed this cell death (Fig. 3A).(40) These results clearly indicate that the 8-oxoG accumulated in nuclear and mitochondrial DNAs induces cell death; however, it is not clear which form of DNA is involved, nuclear or mitochondrial, or how such programmed processes are executed.
To distinguish the biological effects of 8-oxoG accumulation in nuclear and mitochondrial DNA, we established cells that accumulate 8-oxoG selectively in either nuclear or mitochondrial DNA by the mutually exclusive expression of a nuclear or mitochondrial form of human OGG1 protein.(41) These proteins selectively excise 8-oxoG opposite cytosines in DNA in Ogg1-KO mouse cells.(17,18) The increased accumulation of 8-oxoG in nuclear DNA caused poly(ADP-ribose) polymerase-dependent nuclear translocation of apoptosis-inducing factor (Fig. 3B, top panel), while the increased accumulation of 8-oxoG in mitochondrial DNA caused mitochondrial dysfunction followed by Ca2+ efflux and activation of calpains (Fig. 3B, bottom panel). Both types of cell death were accompanied by increased accumulation of SSBs in the respective DNA. All of these events were efficiently suppressed by knockdown of MUTYH, resulting in escape from both types of cell death.(17) We therefore propose that MUTYH functions as a molecular switch for the two types of programmed cell death when 8-oxoG accumulates in either nuclear or mitochondrial DNA.
It has been shown that MUTYH in mammalian cells functions in a replication-coupled manner (Fig. 4),(42) and that MUTYH is associated with proliferating cell nuclear antigen (PCNA), replication protein A (RPA), MutS homolog 6 (MSH6) and RAD9-RAD1-HUS-1 (9-1-1) complex, as well as with other proteins involved in BER in the nucleus (Fig. 2C).(16,28) Therefore, in nuclear DNA, MUTYH selectively recognizes adenine from the nucleotide pool incorporated in the nascent strand opposite 8-oxoG (Figs 1B,4). On the other hand, the adenine in template DNA opposite 8-oxoG, resulting from incorporation of 8-oxoG from the nucleotide pool by a DNA polymerase, is not excised by MUTYH (Fig. 1B).(42) Recently, it has been shown that DNA polymerase λ efficiently inserts cytosine opposite 8-oxoG after adenine excision by MUTYH, thus ensuring the faithful repair of A:8-oxoG mispairing.(43)
Under oxidative stress, however, accumulation of 8-oxoG in template DNA can be highly increased, and MUTYH might induce futile BER cycles because an adenine can be reinserted opposite an 8-oxoG during BER by other DNA polymerases such as pol β and pol κ (Figs 3A,4).(44–46) The futile BER cycle would cause persistent accumulation of SSBs in the nascent strand, because there are many apurinic/apyrimidinic (AP) endonucleases or AP lyases that efficiently incise AP sites repeatedly generated by MUTYH. Persistent accumulation of SSBs in nuclear DNA continuously activates poly(ADP-ribose) polymerase and causes prolonged accumulation of poly(ADP-ribose) polymer or depletion of nicotinamide adenine dinucleotide and ATP. Under these conditions, processing of mitochondrial apoptosis-inducing factor and its nuclear translocation is promoted, thus initiating cell death (Fig. 3B, top panel).(47,48)
In mitochondria, MUTYH might function independently of replication because mitochondria lack replication coupling factors. Therefore, in mitochondria, MUTYH can excise adenine opposite 8-oxoG regardless of its origin.(18) We thus suggest that the accumulation of 8-oxoG in mitochondrial DNA results in the excessive formation of SSBs in both strands of DNA through MUTYH-initiated BER. This would cause mitochondrial DNA depletion, resulting in mitochondrial dysfunctions, such as ATP depletion and opening the membrane permeability transition pore. These lead to Ca2+ efflux from mitochondria causing activation of the Ca2+-dependent proteases (calpains) in the cytoplasm. Activated calpains induce lysosomal rupture and cell death (Fig. 3B, bottom panel).(5,17)
Tumor suppression and cell death induced by MUTYH
We propose that MUTYH primarily suppresses tumorigenesis by inducing death of pre-mutagenic or pre-cancerous cells that, under oxidative stress, accumulate high levels of 8-oxoG in either the nuclear or mitochondrial DNA, thus eliminating them from stem cell or progenitor populations (Fig. 3B). In the absence of MUTYH, such pre-mutagenic or pre-cancerous cells can survive with an increased mutation rate in proto-oncogenes or tumor suppressor genes due to an increase in 8-oxoG levels, and thus, under oxidative stress, Mutyh-KO mice and MAP patients are highly susceptible to tumorigenesis.
Gushima et al.(49) recently reported that increased accumulation of 8-oxoG in nuclei and altered expression of hMUTYH are observed in the mucosa of patients with ulcerative colitis (UC) or UC-associated neoplasia in comparison with non-UC affected mucosa. The mucosa from patients with UC-associated neoplasia or UC without neoplasia exhibited strong cytoplasmic expression and attenuated nuclear expression of hMUTYH when compared with patients unaffected by UC. Mutations in KRAS but not in MUTYH were detected in two of the UC-associated neoplasia cases, one of which showed a G:C to T:A transversion mutation and attenuated nuclear staining of hMUTYH. It is well known that the inflamed mucosa of UC is highly exposed to oxidative stress. It is therefore reasonable to assume that accumulation of 8-oxoG in the nuclear DNA is responsible for induction of KRAS mutations, thus indicating that MUTYH plays a role in tumor suppression in patients with UC as well as in MAP patients. In particular, in rapidly-proliferating cells, nuclear isoforms of MUTYH may initiate cell death by sensing adenine opposite 8-oxoG during replication of nuclear DNA, thus suppressing tumorigenesis (Fig. 3B, top panel).
We wonder whether accumulated 8-oxoG in mitochondrial DNA and MUTYH-initiated cell death might also contribute to the tumor suppression. Because mitochondrial DNA is always replicated even in non-proliferative cells, mitochondrial isoforms of MUTYH may also initiate death of slowly-proliferating stem cells by sensing adenine opposite 8-oxoG in mitochondrial DNA when those cells encounter increased oxidative stress (Fig. 3B, bottom panel).
This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant number: 20013034 to YN) and from the Japan Society for the Promotion of Science (grant numbers: 22501014 to SO, 22221004 to YN and SO).