Low-Fidelity Compensatory Backup Alternative DNA Repair Pathways May Unify Current Carcinogenesis Theories

ABSTRACT: 

The somatic mutation carcinogenesis theory has dominated for decades. The alternative theory, tissue organization field theory, argues that the development of cancer is determined by the surrounding microenvironment. However, neither theory can explain all features of cancer. As cancers share the features of uncontrolled proliferation and genomic instability, they are likely to have the same pathogenesis. It has been found that various DNA repair pathways within a cell crosstalk with one another, forming a DNA repair network. When one DNA repair pathways is defective, the others may work as compensatory backups. The latter pathways are explored for synthetic lethal anticancer therapy. In this article, we extend the concept of compensatory alternative DNA repair to unify the theories. We propose that the microenvironmental stress can activate low-fidelity compensatory alternative DNA repair, causing mutations. If the mutation occurs to a DNA repair gene, this secondarily mutated gene can lead to even more mutated genes, including those related to other DNA repair pathways, eventually destabilizing the genome. Therefore, the low-fidelity compensatory alternative DNA repair may mediate microenvironment-dependent carcinogenesis. The proposal seems consistent with the view of evolution: the environmental stress causes mutations to adapt to the changing environment.

KEYWORDS :

• alternative DNA repair
• carcinogenesis
• microenvironment
• somatic mutation
• synthetic lethality
• tissue organization field

A number of carcinogenesis theories have been proposed in the past several decades [1]. According to these theories, the development of cancer can essentially be attributed to either the intrinsic defect of the cell itself or the microenvironment that surrounds the cell. These two theories are also sometimes called the somatic mutation and tissue organization field theories, respectively [1,2]. Both theories are supported by a large amount of evidence [2,3]. It has been debated for many years over which theory is more consistent with experimental or clinical observations [4–7]. Recently, attempts have been made to reconcile the theories, arguing that the microenvironmental stress can affect genomic stability [8]. However, this latest theory does not explain what mediates the microenvironment-dependent genomic instability.

Hanahan and Weinberg have summarized several phenotypic hallmarks of cancer [9,10]. Various types of cancers share, more or less, these hallmarks, particularly uncontrolled cell proliferation and genomic instability (Box 1). Moreover, the development of cancer is usually a multistep process, involving a series of genetic alterations occurring in genes, such as oncogenes and tumor suppressor genes. Additionally, a prominent feature of cancer is DNA repair defect that has been attributed to genomic instability [3]. These common phenotypes and genotypes of cancers suggest that many cancers may share the same underlying pathogenesis.

Traditionally, DNA repair pathways are classified as individual entities and each DNA repair pathway is composed of a defined set of DNA repair proteins, such as nonhomologous end joining (NHEJ) complex KU70/KU86/DNA-PKcs. However, numerous studies have shown that DNA repair pathways, together with DNA damage checkpoints and other associated cellular processes, cross-communicate with one another, forming a DNA repair network [13,14]. At the molecular level, individual repair proteins, such as PARP1 and DNA polymerase η, are not limited to one specific DNA repair pathway; rather, they can partner with repair proteins from other DNA repair pathways to resolve DNA damage [15–20].

The cross-communication among DNA repair pathways indicates that DNA damage can be channeled to an alternative DNA repair (AltRep) pathway when the normally designated DNA repair (NormRep) pathway is absent [3,21]. Therefore, the AltRep is adopted by the cell to resolve DNA damage as a compensatory backup in the case that the NormRep is defective or deficient relative to the amount of DNA damage. This phenomenon actually occurs to many types of cells [22–29]. As the AltRep pathway is a substitute for the NormRep, it is not always of high fidelity [15]. Actually, the low-fidelity AltRep pathways can cause genetic alterations, such as nucleotide mutation and chromosome translocation [16,22–29]. The compensatory AltRep pathway has recently been exploited for synthetic lethal anticancer therapy to improve the efficacy of anticancer chemotherapy [21].

We propose that it is the low-fidelity compensatory AltRep pathways that mediate the microenvironmental stress-dependent genomic instability, as well as drive multistep carcinogenesis. We will present evidence that the microenvironmental stress can not only activate or inhibit DNA repair pathways but also cause mutation, an indication of unfaithful processing of DNA, most likely by the low-fidelity DNA repair.

Altered activity of DNA repair pathways by exogenous, microenvironmental or intracellular stress

A tumor is often surrounded by a milieu, called tumor microenvironment, which is consisted of a variety of host cells, soluble factors and structural components (Box 2). Besides exogenous and intracellular stress, many soluble molecules or pathological conditions associated with the microenvironmental stress, such as growth factors, cytokines and chronic inflammation, may alter DNA repair or even cause mutations.

DNA damaging agents

Human cells are frequently exposed to DNA damaging agents that arise from exogenous sources, surrounding microenvironment or intracellular processes that occur under normal or pathological conditions. Free radicals are generated within the cell via metabolism. They can cause at least six types of DNA damage: base loss, base deamination, base alkylation, base dimerization, base oxidation and single-strand breakage (SSB) [35]. On the other hand, a variety of DNA damages are produced by exogenous agents targeting DNA, such as exogenous oxidants, ionizing radiation, radiomimetic drugs, UV light, DNA topoisomerase I and II poisons, and DNA crosslinking drugs. Additionally, a large number of chemicals that healthy subjects are exposed to environmentally or occupationally may induce DNA damage, leading to the upregulation of specific DNA repair pathways. Styrene, a chemical used for the production of polystyrene plastics and resins, or styrene metabolites may cause DNA adducts or SSBs and activate specific DNA repair pathways, such as base excision repair (BER) [36,37]. It should be noted that DNA damages induced by various types of DNA damaging agents are often nucleotide-, sequence- or site-specific as evidenced by numerous studies [38–41]. If the amount of DNA damage inflicted by these agents exceeds the capacity of the high-fidelity NormRep, the low-fidelity AltRep pathway may intervene to rescue the cell [15].

Growth factors, their receptors & associated signal transducers

Epidermal growth factor may induce ERCC1 expression to stimulate nucleotide excision repair (NER) [42]. Upon irradiation, EGFR, accompanied by DNA-PKcs, is translocated to the nucleus, leading to the enhanced DNA-PK activity [43]. Actually, EGFR signaling may affect overall double strand break (DSB) repair by regulating NHEJ [44].

Insulin may upregulate ERCC1 expression as well [45]. Insulin and IGF-1 slow the fast rejoining of DSBs, leading to misrepair and dicentric chromosome aberrations [46]. IGF-1 and IGF-2 regulate p53 response to DNA damage [47,48]. IGF-1 also inhibits cisplatin-induced DBS repair [49]. On the other hand, IGF-1 may enhance homologous recombination (HR) repair of DSBs [50]. Insulin-like growth factor 1 receptor inhibits hyperglycemia-induced DNA strand breaks by promoting HR repair [51]. Additionally, IGF binding protein-6, a growth inhibitory protein, is translocated to the nucleus to be associated with KU80, altering DNA repair [52].

Signal transducers may also regulate DNA repair pathways. MAPK signaling may positively or negatively regulate HR [53]. The effect of EGFR on NHEJ is mediated via the MAPK signaling pathway [44]. Likewise, the upregulation of ERCC1 expression by insulin is transduced by the Ras/ERK-dependent signaling pathway [45].

Hormones

Besides insulin and the related molecules discussed above, other hormones may also alter DNA repair. Estrogen is well known to damage DNA, activate BRCA1 and stimulate cell proliferation. The β receptor of estrogen mediates nuclear interaction between insulin receptor substrate 1 and RAD51, leading to the inhibition of HR-directed DNA repair [54].

Oncogenes

c-MYC directly regulates the transcription of NBS1 [55]. Deregulated c-MYC can partially impair p53-mediated DNA damage responses [56]. The overexpression of proto-oncogene myc disrupts DSB repair, causing chromosomal translocations [57].

The oncogene-encoded BCR–ABL can stimulate rad51 expression, directly interacts with RAD51, phosphorylates it, and promotes it to interact with BLM helicase [58,59]. Consequently, BCR–ABL increases DNA repair, causes drug resistance and is believed to account for BCR–ABL-stimulated mutagenic NHEJ/HR repair [58–60]. BCR–ABL may also enhance the expression and nuclear localization of DNA helicase WRN, which may promotes mutagenic HR repair and single strand annealing (SSA) [61]. Like BCR–ABL, a number of oncogenic kinases may promote mutagenic SSA [62].

Apoptosis-related proteins

Antiapoptotic BCL-2 potently inhibits the repair of abasic DNA lesions induced by nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) through upregulation of c-MYC and APE1 [63]. Simultaneously, BCL-2 enhances NNK-induced mutation frequencies by downregulating mismatch repair (MMR) efficiency [64]. BCL-2 also suppresses MMR [65] or NHEJ, which is associated with elevated genetic instability [66]. Additionally, overexpression of BCL-2 inhibits γ-ray-induced HR repair pathways, causing elevated mutation frequencies [67]. Overexpression of antiapoptotic BCL-2 or BCL-X(L) inhibits HR induced by UV-C, γ-ray or mutant p53 [67]. Apoptotic trigger anti-CD95 antibody may initiate translocations within the MLL gene via NHEJ [68]. The proapoptotic BAX and BID may also regulate DNA repair by repressing HR [69]. Many other apoptosis-related proteins have been found to inhibit HR as well [69,70].

Cytokines & inflammatory molecules

Many cytokines and inflammatory molecules can damage DNA or alter the DNA repair network, including DNA damage checkpoints. JAK2, a tyrosine kinase that is activated downstream of several cytokine receptors following ligand binding, stimulates HR and genetic instability [71]. Activated neutrophils may induce the hMSH2-dependent G2/M checkpoint arrest and replication errors [72]. TGF-β1/Smad3 counteracts BRCA1-dependent DNA repair [73]. IL-6 can inhibit p53 expression through the methylation of the promoter region of TP53 gene [74]. The inflammatory cytokine macrophage migration inhibitory factor can negatively regulate p53 activity [72]. Helicobacter pylori and proinflammatory cytokines, such as TNF-α, can induce aberrant expression of activation-induced cytidine deaminase (AID), leading to the mutation in TP53 [75]. AID may also be induced by TGF-β and the Th2-driven cytokines IL-4 and IL-13 that are activated in human inflammatory bowel diseases [72].

Viral proteins

The T-antigen of SV40 virus is well known to inactivate tumor suppressor p53. The T-antigen of the human polymavirus JCV promotes DNA misrepair via inhibiting RAD51-dependent HR [76,77].

Epigenetic factors

The expression of MMR gene hMLH1 is inhibited following hypermethylation, predisposing the host cell to mutations [78]. Karpinets and Foy have summarized that the epigenetic alteration may lead to the abnormality of tumor suppression genes and genes related to DNA repair [79]. Dysregulation of DNA methylation may change the expression levels of DNA repair-related proteins. The quantitative changes of the proteins may lead to the misrepair of DNA damage as discussed below.

Besides dysregulations of DNA methylation, non-coding RNAs have also been found to regulate DNA repair [80]. Knockdown of miRNA processor DICER or Ago reduces checkpoint response following UV damage. Treatment of noncanonical small RNAs with RNase interferes repair factor foci. Moreover, genomic instability may be promoted by miRNA-34c overexpression.

Chronic inflammation & other microenvironmental stressors

Colotta et al. have explicitly reviewed that chronic inflammation may induce low-fidelity AltReps via direct DNA damage or inflammatory molecules such as cytokines [72]. Additionally, activated neutrophils may inhibit NER [81]. Chronic inflammation may also cause the overexpression of BER enzyme AAG or APE1. The latter can process damaged base, as well as indirectly affect several DNA repair pathways, such as MMR, by modifying a number of transcription factors [82,83]. Both AAG and APE1 are positively associated with microsatellite instability (MSI) [72].

Hypoxic stress, as well as low pH, impairs DNA repair, causing elevated mutation rates [84]. HIF-1α, a hypoxia-responsive transcription factor, induces genetic instability by inhibiting MSH2 and MSH6 expression [72]. Additionally, hypoxic cells upregulate two miRNAs, miR-210 and miR-373. miR-210 suppresses the levels of RAD52 whereas miR-373 reduces the expression of RAD23B and RAD52. The suppression of RAD52 and RAD23B is associated with genomic instability [72]. Hypoxia also downregulates the expression of RAD51 at the transcription level, resulting in reduced HR [85]. Likewise, chronic hypoxia downregulates the expression of HR proteins [86].

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade the components of the extracellular matrix [87]. MMPs may stimulate genomic alterations and compromise genomic integrity [87]. Recently, MMP-9 has been found to interact with NHEJ protein KU at the cell surface [88].

Contact inhibition may also disrupt the DNA repair network. Exposure to anchorage-independence impairs RAD51-involved HR repair [89].

Senescence

Senescence can be triggered by DNA damage or strong mitogenic signals [90]. The senescent cells secrete many cytokines, growth factors and proteases that have autocrine or paracrine activities, a phenomenon known as senescence-associated secretory phenotype [91]. These factors, as discussed above, may activate the DNA repair pathways. It should be emphasized that the senescence-associated cytokine secretion usually does not occur after transient DNA damage responses but only after the establishment of sustained DNA damage [92].

Metabolism & obesity

Hyperglycemia may induce SSBs and DSBs mediated by reactive oxygen species [51]. Likewise, the oxidation of carbohydrates and lipids may produce reactive carbonyl species that may break DNA strands, crosslink protein–DNA and cause mutations [93,94]. Actually, the metabolism of carbohydrates may alter DNA repair. A systems biology study on yeast shows that there is a correlation between the glucose or fructose metabolic-associated enzymes and the upregulation of proteins of several DNA repair pathways [94].

An increased ratio of NAD⁺ to NADH may lead to the upregulation of BRCA1 expression. Low NAD⁺:NADH ratio, which may result from a high caloric diet or obesity, can downregulate BRCA1 [95]. Moreover, NAD⁺ may regulate the activity of SIRT1 [96], a deacetylase that can promote RAD51-independet HR or regulate DNA helicase WRN [97,98,97].

Possible pathogenesis for mutation caused by microenvironmental stress via AltRep pathways

As discussed above, microenvironmental stress can activate or inhibit the DNA repair pathway. If the activation or inhibition of the DNA repair pathways occurs under an undesignated condition, such as an undesignated cell phase, it may cause mutation. RAD51-mediated HR is considered as a high-fidelity repair pathway as it uses the template of the homologous sequences of the sister chromatid to repair at the G2 phase of the cell cycle. However, overexpression of RAD51 or activation of RAD51-mediated HR at G1 phase of the cell cycle causes mutations [99].

Additionally, the sustained presence of the microenvironmental stress is likely to lead to the persistent upregulation or overexpression of DNA repair pathways. A number of studies show that the repair by the overexpressed DNA repair pathways often cause mutations [16,100–101]. This is probably because the overexpressed DNA repair pathways, likely via competitive inhibition, hijack the damaged DNA that would have been repaired by the high-fidelity NormRep.

It should be noted that not all the microenvironmental stresses necessarily cause any cells to misrepair DNA under any conditions. It seems that whether the DNA misrepair occurs is often cell-specific, depending on a number of factors, such as the specific status of the cell [99].

Theory of AltRep-driven carcinogenesis

Evidence of AltRep as the driver of multistep carcinogenesis

Carcinogenesis is generally considered a multistep process involving genetic alterations to many proto-oncogenes and tumor suppressor genes. Although not absolute, genetic alterations to these genes usually follow a defined order: the genetic alteration to a given gene usually occurs only after another given gene is altered first.

In B-cell lymphomas, translocations that link c-myc or other proto-oncogenes to the igh locus usually do not occur unless AID, p53, ATM, NBS1, ARF, KU70, ligase 4, XRCC4 or PARP1 loses its function first. Moreover, it has been illustrated that an AltRep, which is activated to substitute the above dysfunctioned DNA repair pathways, induces the translocation [102–105]. Similarly, in colorectal cancer associated with MutYH-associated polyposis, guanine-to-thymine transversion of APC or k-ras is induced by a low-fidelity AltRep that replaces the mutated DNA glycosylase. Under normal conditions, the wild-type glycosylase can prevent the transversion by faithfully restoring normal base pairing [106,107].

These studies show that the AltReps may account for the ordered genetic alterations of oncogenes and tumor suppressor genes during multistep carcinogenesis.

The low-fidelity AltRep-driven carcinogenesis theory

Under the condition of excessive and/or sustained microenvironmental stress, such as excessive DNA damage and chronic inflammation, the low-fidelity AltRep is altered to induce mutations to adapt to the changing environment. Mutations to proto-oncogenes or cell proliferation-related genes can not only stimulate cell proliferation but also modulate the DNA repair network. On the other hand, mutations to DNA repair or tumor suppression genes may generate defective DNA repair proteins, which cause another round of altered DNA repair. Cells harboring mutations that have survival advantage over microenvironmental stress are selected. They proliferate to replace other unmutated, disadvantaged dying cells, to repopulate and remodel the tissue. The sustained or repeated microenvironmental stress causes a vicious cycle consisting of the stress-induced cell stimulation, altered DNA repair by low-fidelity AltReps, mutations, selection and expansion. The vicious cycle leads to the activation of many low-fidelity AltReps, eventually destabilizing the genome, as well as causing cancer.

The theory suggests that the mutator phenotype or genomic instability should not always be overt at the early stage of carcinogenesis because only a limited number of low-fidelity AltReps are activated as a result of limited rounds of the vicious cycle. The theory also suggests that the mutator phenotype or genomic instability is not required for the development of cancer at the early stage of carcinogenesis. Rather, cells rely more on the environmental stress to support the activation of the AltReps. Indeed, it is reported that the induction of the mutator phenotype is not always a prerequisite for cancer development [108]. For some highly proliferative tissues, sufficient cell division may allow for the accumulation of mutations that provide a selective advantage and clonal expansion [108].

The required supportive microenvironment for the development of cancer may be the reason that a tumor usually cannot survive if it is transplanted to another recipient host or tissue if the microenvironment, including the immune response, of the recipient host or tissue does not provide the tumor cell-specific supportive soluble molecules, such as growth factors and cytokines, on which the tumor cell depends.

The low-fidelity AltRep-driven carcinogenesis theory is also consistent with the view of evolution: the environmental stress causes mutations of the cell, rendering the cell the ability to adapt to the changing environment.

Substantiation & reconciliation of somatic mutation & tissue organization field theories

The environmental stress-activated low-fidelity AltRep theory may substantiate and reconcile the current two major carcinogenesis theories, the somatic mutation theory and the tissue organization field theory. The low-fidelity AltReps introduce mutations into the genome, conferring a mutator phenotype. On the other hand, the low-fidelity AltRep can be activated by a large number of factors present in the microenvironment. Therefore, the low-fidelity AltRep mediates the microenvironment-dependent genomic instability, as well as the multistep development of cancer via the vicious cycle.

Implications of AltRep-driven carcinogenesis

Triads contributing to cancer development

The current carcinogenesis theory centers on the activation of the low-fidelity AltRep, which can be induced by either excessive DNA damage or sustained microenvironmental stress. Therefore, the AltRep, excessive DNA damage and sustained microenvironmental stress-induced cell stimulation constitute the triads that contribute to the development of cancer.

Requirement of specific amount of DNA damage or cell stimulation for cancer development

It seems that the amount of DNA damage may determine whether the low-fidelity AltRep pathway is activated. Transient, limited damage may be restored by the high-fidelity NormRe pathway. On the other hand, extraordinarily excessive DNA damage may be lethal to the cell [109,110]. Therefore, only the appropriate amount of DNA damage may activate the AltRep without killing the cell. This may explain why human cells are constantly exposed to DNA damage, but the incidence of cancer is relatively limited. Indeed, the appropriate amount of DNA damage has been found to be critical for carcinogenesis [111]. Likewise, transient cell stimulation, such as acute inflammation, does not seem to cause cancer. As discussed above, chronic inflammation, on the other hand, is likely to constantly upregulate the DNA repair pathway, which can competitively hijack and misrepair DNA damage.

Conditional low-fidelity AltRep

Whether an AltRep is of low fidelity depends on many factors, such as the status of the cell and the differentiation type of the cell. The high-fidelity NormRep pathway for a specific type of DNA damage can cause mutations when it is adopted to repair other undesignated types of DNA damages or under undesignated conditions. RAD51-mediated HR is considered as high-fidelity repair pathway as it uses the template of the homologous sequences of the sister chromatid to repair at G2 phase of the cell cycle. However, overexpression of RAD51 or activation of RAD51-mediated HR at G1 phase of the cell cycle causes mutations [99].

Factors determining selection of specific AltRep pathways

The choice of a specific DNA repair pathway to address the damaged DNA depends on many factors, including the development stage [112–114], types of differentiations [115,116], cell cycle phases [113,117–118], the proliferating status of the cell [119], types of damages [120–125], the location of the damage on the chromosome [126], relative amount between the damaged DNA and repair proteins [127], relative concentrations of individual repair proteins of the repair complex [15–16,128], relative levels of the four deoxyribonucleotides that compose DNA [129], the post-translational modification of the repair protein [130], the amount of DNA damage [131], the interference of a viral protein [132] or the types of the damages adjacent to the original damage 133]. The choice of a specific DNA repair pathway may also result from a direct qualitative modification of a repair protein itself or quantitative regulations via repair protein expression. Post-translational modifications of APE1 may alter its endonuclease activities or binding ability to other repair proteins, such as XRCC1. On the other hand, APE1 may redox-modify several transcription factions, such as NF-κB and p53, which are associated with many DNA repair genes [82–83,134].

The multiple options of the repair pathways available to the cell indicate that different cancer cells, even within the same tumor, may resort to different AltReps. This may be the foundation for the heterogeneity of the cancer cells within the same tumor, as well as the different responses to the same anticancer drug for different cells within the same tumor or different tumors of the same patient. This suggests that patient- or even tumor cell-specific chemotherapeutic regimens are required.

Nonthreshold characteristics of cancer development

According to the AltRep theory, each error introduced into the oncogene, tumor suppression gene or other related genes by the AltRep, such as a nucleotide mutation and a misligation of two broken ends, drives the cell one step further on its way to cancer as in the case of mutations in k-ras, apc or TP53 for some colon cancers and igh–c-myc translocation for B-cell lymphomas [135]. Therefore, the development of cancer is a gradual process. It does not seem to have a threshold for the numbers of mutations, beyond which the cell suddenly becomes malignant. Neither does it seem that the mutation ceases once the cell has become malignant. Rather, the development of cancer is a nonstop evolving multistep process for the cell to adapt to the changing environment. It should be noted that the number of mutations required for a cell to develop into a biologically or clinically overt tumor are not the same for various types of tumors [136].

Progressive changes in AltRep pathways or cell stimulating signals along the course of cancer development

The development of cancer is a gradual process. Initially, only a limited number of genes related to AltReps and cell stimulation are involved. Therefore, at the early stage, mutations may not be evident. This may explain why the early phases of cancer development seem to be far from being exclusively cell autonomous, heavily dependent on environmental influences, and can actually be interpreted as adaptive reactions to the changed environment [137,138]. However, the vicious cycle leads to the activation of secondary, tertiary or even further AltReps. At the late stages of carcinogenesis, the early AltReps are replaced by the more efficient AltRep pathway with even lower fidelity, which gradually cause overt genomic destabilization or mutator phenotype. Apparently, correction of the initial or early AltRep may not always reverse or halt cancer development as demonstrated by the study that restoring MMR does not inhibit the formation of reciprocal translocation in the colon cancer cell [139]. Likewise, the cell-stimulating molecules that the cancer cell lives on may also change along the process of carcinogenesis. Cancer cells can eventually develop a phenotype that can grow independent of the growth factors that are required for the cells at the early stage of cancer development [140–143].

The progressive changes in AltReps or cell-stimulating molecules along the course of cancer development indicate that the chemotherapeutic regimens should change accordingly along the course of cancer treatment.

Two classes of cancer according to AltRep-driven carcinogenesis theory

According to the AltRep theory, a cell with the defective NormRep, as well as a cell under sustained stress, can activate the low-fidelity AltRep. Therefore, cancer and the development of cancer, especially at the early stage, can be classified into two types. One is primarily due to defective DNA repair, such as those developed from DNA repair-defective syndromes, whereas the other is caused by the sustained microenvironmental stress-induced cell stimulation, such as those resulting from chronic inflammation. As a result, both genotoxic and nongenotoxic stresses may activate the AltRep, causing cancer. Apparently, the simultaneous presence of the defective DNA repair and stress expedites the development of cancer, evidenced by the observation that DNA repair-defective carcinogenesis is more effective if a cell stimulating stress is also present [144]. Likewise, the stress resulting from cell injury, by tumor promoters, or by natural hormones can enhance the mutagenic effects of genotoxic agents [145,146]. This is also supported by the earlier study that the mutagenicity of N-ethyl-N-nitrosourea to the liver is dramatically enhanced by partial hepatectomy, which stimulates cell proliferation [147].

Noncontinuous, cumulative characteristics of carcinogenic mutations

Sustained cell stimulation or excessive DNA damage may activate the low-fidelity AltRep to cause mutations. However, if its presence is transient, the number of mutations as well as the number of mutated genes may be limited, not sufficient to transform the cell. The mutated genes may turn silent. These ‘pre-existing’ mutated genes related to DNA repair or cell stimulation can be re-activated later if the microenvironment changes [148–151]. Therefore, the process of multistep carcinogenesis may not necessarily be continuous; rather, it is cumulative. The noncontinuous, cumulative characteristics of carcinogenetic mutations is probably important for cancer prevention because it implies that avoiding exposure to carcinogens or blocking cell-stimulation signaling may halt the development of cancer.

It will be interesting to investigate whether the relationship between the development of breast cancer and the early exposure to estrogen may follow a similar path, which may guide the strategies to prevent breast cancer.

Specific, nonrandom genetic mutations

Several studies have indicated that the target genes selected by the AltRep are not random but quite specific. The analysis of combinatorial mutational patterns of various types of cancers show that the cell or the type of tissue determines which oncogenes, tumor-suppressor genes or their combinations are mutated [152]. Moreover, the number of the candidate genes that are mutated is limited [152]. Likewise, the MMR-deficient cells show that the mutation frequency of the Real Common Target genes, the genes that promote tumor cell growth, are much higher than that of the bystander or survivor genes, the genes that are not under growth selection or the genes that are essential to cell survival [153]. As a matter of fact, MSI of most MMR-defective colorectal cancers occurs in genes concerning DNA damage response signaling, DSB repair and MMR, but not those related to NER or BER [154]. The nonrandomness is consistent with the purpose of mutations, the survival advantage. Random mutations are more likely to damage, rather than improve, the function of the genome [155]. Only the mutation that confers a phenotype with survival advantage is required by the cell. Mutations to other genes, such as the housekeeping genes, may be lethal to the cell and should be avoided.

The nonrandom, specific selection of the genes mutated by the AltRep pathway indicates that once an AltRep is known, the corresponding mutated signal transduction-related genes are also known, or vice versa [152]. This may be important for the combination chemotherapy. It indicates that if the AltRep of a tumor is identified, the signal transducers are also narrowed down, or vice versa. Simultaneous inhibition of the AltRep and the signal transducer may enhance the efficacy of anticancer drug therapy. Moreover, as the number of the AltRep and the corresponding signal transducers is limited [154], the number of the drug targets is limited as well.

Cancer development in the context of the surrounding tissue

In the context of a tissue, environmental stress kills sensitive cells, leaving resistant cells behind. The dying cells, as well as the inflammatory cells following cell death, release various types of growth factors and cytokines which, if sustainedly present, can stimulate the proliferation or activate the AltRep pathways of the resistant cells [138,156]. Activating the AltRep pathway assists the resistant cell to mutate for survival whereas the cell stimulation promotes the proliferation of the resistant cell to replace the neighboring cells dying from the environmental stress. This indicates that the induction of cell death of the normal cells may be required for the mutated cell to proliferate to facilitate repopulation or remodeling of the tissue [157]. Interestingly, many tumor promoters, such as phorbol esters, phenobarbital and orotic acid, have been found to be cytotoxic or mitoinhibitory to their target tissues [138].

The requirement of the death of the sensitive cells for the development of cancer indicates that it is probably important to selectively kill cancer cells without impairing the surrounding normal cells during the treatment of cancer.

Interpretations of cancer stem cells, cancer-contributing germ-line mutations & cancer-associated polymorphisms by low fidelity AltRep

Cancer stem cells (CSCs) are proposed to be a distinct population of cells within a tumor, possessing the ability to self-renew and generate into multiple cell types. Several studies have found that CSCs manifest altered DNA repair [158]. MMR-deficient mouse embryonic stem cells are more resistant to several types of DNA damages, which may be due to the elevated function of other DNA repair pathways that cause mutations [159]. These MMR-deficient cells are more prone to cancer, suggesting that the substitutive AltReps may contribute to the development of cancer among CSCs. The low-fidelity DNA repair pathways can be promoted by intracellular oncogenes, such as BCR/ABL [158]. However, except hypoxia that can regulate p53 expression, not much work has been done concerning whether the tumor microenvironment may induce AltReps. On the other hand, the tumor microenvironment is required to support CSCs [160].

Patients with DNA repair-defective syndromes or defective DNA repair genes, such as Fanconi syndrome and mutated brca1 gene, are prone to cancer. Germline mutations compromise NormReps. As a result, the cell with germline mutations in gene related to DNA repair is more likely to resort to AltReps to address damaged DNA, leading to the vicious cycle that causes cancer.

Polymorphisms of several DNA repair-related genes have been reported to be associated with increased risk of developing cancer [161]. Like mutations, polymorphisms may also, more or less, affect DNA repair proteins [162,163]. Therefore, cells containing cancer-associated polymorphic DNA repair genes are more likely to resort to cancer-contributing AltRep pathways.

Conclusion & future perspective

We have proposed that low-fidelity AltReps drive the carcinogenesis process. Our proposal seems able to unify the somatic mutation and tumor microenvironment theories that are currently predominant in the field of cancer research.

The low-fidelity AltRep-driven carcinogenesis theory is centered on the low-fidelity AltRep pathway that can be activated by excessive or sustained DNA damage as well as by microenvironmental stress. Therefore, the low-fidelity AltRep pathway, excessive or sustained DNA damage and microenvironmental stress constitute the cancer-contributing triad.

Prospectively, the conclusions suggest future strategies to prevent or treat cancer, as well as the rationales for therapeutic strategies. For example, inhibiting the sustained actions of the microenvironmental stress, such as chronic inflammation, may retard cancer development. This may be the reason that the nonsteroidal anti-inflammatory drugs have chemopreventive effects. Similarly, hepatic or ovarian cancer associated with chronic inflammation might also benefit from the same principle.

The AltRep proposal also indicates that only those signal transducers that can activate the cell-specific low-fidelity AltRep pathway may be selected as the molecular targets. Moreover, for a molecularly targeted drug to have improved efficacy, it should be combined with a DNA damaging agent that can induce the type of DNA damage, the repair of which requires the specific low-fidelity AltRep activated by the signal transducer that can be inhibited by the molecularly targeted drug. Likewise, the efficacy of synthetic lethality may only be obtained by inhibiting the low-fidelity compensatory AltRep either directly or via blocking the environmental stress-induced signal transducers.

Box 1. Possible mechanisms of gene amplifications and gene deletions.

• Gene amplifications and gene deletions are two common types of genomic instabilities frequently observed for cancer cells. They indicate that DNA is unfaithfully processed

• DNA repair pathways, such as those repairing DNA double strands, are required for generating gene amplications and gene deletions. The bridge–breakage–fusion model is generally accepted to interpret gene amplifications. Additionally, several models have been proposed to interpret circular extrachromosomal double minutes and episomes [11]

• A number of models have been suggested to account for gene deletions, including losses from translocation, chromosomal crossovers within a chromosomal inversion, unequal crossing over and breaking without rejoining [12]

Box 2. Tumor microenvironment.

• The tumor microenvironment is consisted of a variety of host cells, soluble factors and structural components, such as extracellular matrix

• The host cells that may interact with the tumor include cancer-associated fibroblasts, endothelial cells, pericytes, adipocytes, tumor-associated macrophages, dendritic cells, basophils, mast cells, polymorphonuclear neutrophils, eosinophils and natural killer cells

• These host cells may secret a large numbers of growth factors, cytokines and other soluble factors, such as vascular endothelial growth factors and interleukins, which may stimulate angiogenesis, prolong survival, remodel metabolism, induce proliferative activities or enhance migration potential of tumor cells [30–32]

• Morphologically, blood vessels within a tumor often show aberrant structure. They stimulate tumor growth, as well as metastasis [33]

• Tumors can stimulate their own innervation, a phenomenon called neoneurogenesis [34]. Moreover, a number of neuropeptides and neurotransmitters present in the tumor microenvironment can influence tumor progression, suppression of immune responses, vascularization and migration

• The interactions between the tumor and the tumor microenvironment are not unilateral but mutual [34]. Tumor cells can also influence the microenvironment by secreting a large number of soluble factors that can recruit inflammatory cells to the surrounding microenvironment, stimulate angiogenesis and induce neoneurogenesis

EXECUTIVE SUMMARY

Altered activity of DNA repair pathways caused by microenvironmental stress

• The alternative DNA repair serves as a backup for the normally designated high-fidelity DNA repair in case the latter is defective or deficient relative to the amount of DNA damage.

• The alternative DNA repair is of low fidelity and may cause mutations.

• Many factors associated with microenvironmental stress may activate the alternative DNA repair, even causing mutations.

Possible pathogenesis for mutations caused by microenvironmental stress via low-fidelity alternative DNA repair pathways

• The low-fidelity alternative DNA repair may cause mutations if it is activated at undesignated conditions or if it is overexpressed to competitively inhibit the normally designated high-fidelity DNA repair.

Theory of alternative DNA repair-driven carcinogenesis

• Sustained or repeated microenvironmental stress causes a vicious cycle consisting of the stress-induced low-fidelity alternative DNA repair, mutation, selection and expansion of the mutated cells.

• Rounds of the vicious cycle may activate more types of low-fidelity alternative DNA repair pathways, eventually destabilizing genome and causing cancer.

Substantiation & reconciliation of somatic mutation & tissue organization field theories

• Sustained or repeated excessive microenvironment stress may cause somatic mutations to oncogenes, tumor suppressor genes or DNA repair genes, etc.

• The low-fidelity alternative DNA repair pathway mediates the environmental stress-dependent genomic instability.

Implications of alternative DNA repair-driven carcinogenesis

• A specific amount of DNA damage or stress that exceed the normal DNA repair capacity but are below the cytotoxic threshold result in the generation of mutations by the alternative DNA repair pathway.

• The normally designated high-fidelity DNA repair pathway for a specific type of DNA damage may be conditionally mutagenic if it is activated to repair an undesignated type of DNA damage.

• Selection of a specific DNA repair is determined by a large number of factors, such as amount of DNA damage, development stages, etc.

• The development of cancer is a nonstop evolving process.

Conclusion & future perspectives

• The low-fidelity alternative DNA repair pathway drives multistep carcinogenesis.

• The low-fidelity alternative DNA repair pathway-driven carcinogenesis theory reconciles and substantiates the somatic mutation and tumor microenvironment theories.

• The low-fidelity alternative DNA repair pathway, excessive DNA damage and sustained stress constitute a carcinogenic triad.

• The proposed theory suggests future strategies for cancer prevention or patient management.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.