Nature Reviews Cancer 2011 11(3), 211-218

Despite the intensive efforts and substantial advances that have occurred through focus-ing on improving treatments, cancer is still a leading cause of death worldwide. Since the idea of cancer chemoprevention was introduced by Sporn1 and Wattenberg et al.2, the hope for overcoming cancer has started to change from one of treatment to one of prevention. The World Health Organization (WHO) indicates that one-third of all cancer deaths are preventable and that diet is closely linked to cancer prevention (see REF. 3 for a review). On the basis of this idea, and numerous epidemiological findings, atten-tion has centred on dietary phytochemicals as an effective intervention in cancer devel-opment. However, the failure of large-scale clinical trials in the 1990s, including the Alpha-Tocopherol, Beta-Carotene trial4 and the Carotene and Retinol Efficacy Trial5, has raised doubts that diet-based cancer prevention can be clinically successful. Because these trials were not designed on mechanism-based preclinical studies, iden-tifying cancer preventive dietary agents that have specific molecular or cellular targets is thought to be an essential way forwards.

Similar to the development of cancer therapeutic drugs, the development of recent cancer preventive agents is based on the discovery of precise molecular targets (see REF. 6 for a review). Because of their success in clinical trials, drugs such as tamoxifen7–9 and finasteride10–13, which have specific molecular targets, have been approved by the US Food and Drug Administration (FDA) as cancer preventive agents. In this Opinion article, we discuss phytochemical-derived drug discovery and mechanisms by which these compounds can modulate distinct tar-get proteins that are involved in oncogenic

signalling. We also address the progress, limitations and future directions in target-driven phytochemical cancer prevention research.

Phytochemical-derived anticancer drugsThe effort to develop anticancer drugs began in the 1950s. In 1955, the US National Cancer Institute (NCI) established the Cancer Chemotherapy National Service Center to screen natural and synthetic com-pounds that effectively induced cancer cell death14. One of the most successful FDA-approved drugs to come from this initiative is paclitaxel15 (Taxol; Bristol-Myers Squibb), which was isolated from the bark of the Pacific Yew tree. Examples of FDA-approved anticancer therapeutic agents derived from phytochemicals and their identified mecha-nisms of action are listed in Supplementary information S1 (table). These data show that phytochemicals with precisely defined mechanisms of action based on molecular target identification can be linked to successful drug discovery.

Phytochemicals, such as resveratrol16–18, (–)-epigallocatechin gallate (EGCG)19–21, [6]-gingerol 22,23 and myricetin24–26, have been reported to directly modulate various molecular signal transduction pathways, and research efforts have centred on the effects of phytochemicals on signalling cascades that are known to induce cancer cell death or to inhibit cancer cell proliferation. However, the specific molecular and cellular targets need to be identified.

Target discovery methodsThe overall strategy for discovering molecu-lar targets of phytochemicals can involve several approaches depending on whether

the starting point is a known oncoprotein or a phytochemical with proven anticancer activity (FIG. 1). The first is a target-based approach beginning with target protein selection on the basis of data ideally obtained from an RNA interference (RNAi) screen (reviewed in REFs 27–29), as well as in vitro, ex vivo and in vivo models. Potential phyto-chemical candidates can then be selected by in silico virtual screening, based on natural chemical libraries30. An in silico screen requires diverse ligand databases, such as the ZINC database (see Further information)31, which contains ‘ready-to-dock’ searchable libraries of 4.6 million three-dimensional compounds ranging from hit-to-lead can-didates to FDA-approved compounds with vendor links to order31. The Asinex database (see Further information) is another exten-sive library of searchable natural compounds that are also available for purchase.

The X-ray crystallographic structures of a protein of interest either alone or bound to an inhibitor or ligand are crucial in the process of identifying protein–small molecule interactions and designing more specific inhibitors. Protein crystal structures that have been resolved are deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) protein data bank (PDB; see Further information)32. However, if a protein crystal structure with a resolu-tion average of about 2.0 Å does not exist, or the structure has not been resolved, homology modelling methods are used to create a suitable structure with which to work. Homology modelling between pro-teins requires that they possess a minimum protein sequence identity of 30%33, and the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) web service (see Further information) provides many of the tools necessary for protein sequence searching and align-ments34. In addition, the National Center for Biotechnology Information (see Further information) provides databases on protein structures and sequences.

A second approach is a compound-based method that begins with the selection of putative cancer preventive phytochemical candidates based on previous research stud-ies. From the selected compounds, the most effective candidates are taken forwards as lead compounds using screening methods such as cell transformation, cell proliferation and kinase or reporter gene assays. After selecting lead compounds, candidate target proteins that directly interact with the lead compounds are identified by ‘reverse dock-ing’ on known protein crystal structures

O P i n i O n

Molecular targets of phytochemicals for cancer preventionKi Won Lee, Ann M. Bode and Zigang Dong

Abstract | Although successful for a limited number of tumour types, the efficacy of cancer therapies, especially for late-stage disease, remains poor overall. Many have argued that this could be avoided by focusing on cancer prevention, which has now entered the arena of targeted therapies. During the process of identifying preventive agents, dietary phytochemicals, which are thought to be safe for human use, have emerged as modulators of key cellular signalling pathways. The task now is to understand how these chemicals perturb these pathways by modelling their interactions with their target proteins.

P e r s P e c t i v e s

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Nature Reviews | Cancer

• RNAi screening • In vitro screening• Ex vivo screening• In vivo screening

• Kinase assays • Pull-down binding

assays• In vivo animal studies

• Cell transformation assays

• Reporter gene assays• ELISA

• X-ray crystallography• NMR• Point mutation

Identify target protein

In silico virtual screeningbased on variouschemical libraries

Identify candidate compounds

Reverse dockingproteomic tools

Identify lead compound

Identify candidate target proteins

Known protein andknown inhibitor library

Shape similaritysearch

Identify similar compound

Docking and scoring

Docking and scoring

Docking and scoring

Identify best fit candidate compounds

Validation

Binding site identification

Putative chemopreventive phytochemicals Clinical trials

Chemical libraries

or proteomic tools35,36. Reverse dock-ing involves flexible molecular receptors around rigid transition state models of the catalyst-free asymmetric reaction (referred to as TS models), whereas normal docking explores the configuration of a small mol-ecule in the confines of a large receptor37. To identify potential target proteins of the lead candidate compounds, protein databases, such as the Potential Drug Target Database (PDTD) (see Further information), which contains structural information such as the position and conformation of the active site for more than 830 known or potential protein drug targets, are commonly used37. Proteomic tools involve lead compounds that are chemically immobilized onto an affinity resin or are attached to a biotin molecule.

Protein extracts are applied to these com-pounds, and liquid chromatography analysis is used to separate the proteins bound to the compounds. The natural product–protein adducts can then be identified using two-dimensional gel electrophoresis and mass spectroscopy (MS)/MS analysis.

Finally, shape-based virtual screening of chemical libraries can be used to search for chemicals in a library that are structur-ally similar to known inhibitors of a given protein. For shape similarity screening, PHASE38, a software application tool that is part of the Schrödinger Suite molecular modelling software package, is used to search for three-dimensional shape complementa-rity and to examine the common elements of ligands with a reference molecule. Similar

chemicals are identified and then ‘docked’ into the target protein by computer, ena-bling detailed protein–ligand interactions to be obtained.

All three approaches require the compu-tational processes of docking and scoring using known and hypothetical drug targets on a protein, coupled with databases of virtual chemical compounds. In docking, various algorithms are used to position a chemical from a virtual library into a speci-fied target site or sites on the protein of inter-est. The objective of molecular docking is to determine the binding interactions between two molecules — either protein to protein or protein to ligand. Once a compound is docked, it is then scored using mathemati-cal models. Scoring estimates the chemical interactions, such as binding strength and energy state, between the ligand and pro-tein to assist in ranking the efficacy of the compound being scored39–41. From these approaches, candidate phytochemicals that directly interact with target proteins can be identified. The cancer preventive effects and interactions of selected phytochemicals must then be confirmed in validation steps using laboratory experiments, such as cell trans-formation assays, pull-down assays, reporter gene assays and animal studies. To further verify the specific interaction and binding site of a selected phytochemical and its target protein, X-ray crystallography, nuclear mag-netic resonance (NMR) and protein point mutation methods are used. Finally, modula-tion of candidate pathway and target pro-teins needs to be validated in patients, with tissue analysis before and after exposure to the agent, ideally in preneoplastic lesions. Promising candidate chemopreventive phyto chemicals can then be fully evaluated in clinical trials to determine their suitability for use as cancer preventive agents.

Protein targets of dietary phytochemicalsSeveral proteins have been identified as specific targets of some phytochemicals (TABLE 1). Representative signalling pathways targeted by various phytochemicals include the MAPK pathways, the oncogenic AKT pathway and proteins involved in cell cycle progression (FIG. 2).

Interfering with the MAPK signalling path-ways. MEK1 is an important downstream component of oncogenic RAS signalling and thus is potentially a good target for disrupt-ing MAPK signalling. The development of pharmacological inhibitors of MEK1, such as PD184352, has shown that MEK1 pos-sesses a unique binding pocket adjacent

Figure 1 | Strategy for identifying preventive agents and molecular targets. Three approaches can be used to elucidate the specific binding modes between a phytochemical and its target proteins. Molecular docking studies can identify the most promising structures that bind to a protein target. A second approach entails the use of reverse docking studies such as chemical immobilization. In this case, a natural or synthetic compound that is an effective inhibitor of cancer growth in vitro or ex vivo is docked into every known protein X-ray crystal structure, and the best protein candidates are chosen. Shape-based virtual screening of chemical libraries can be used to search for chemicals in a library that are structurally similar to known inhibitors of a given protein. Using these processes, candidate modulators of target proteins and potential target proteins of lead compounds can be identified. After discovering possible combinations of phytochemicals and target proteins, the binding is confirmed by complete validation processes, including cell transformation assays, reporter gene assays, kinase assays and immunoreactivity assays. The verified binding mode is then analysed by X-ray crystallo-graphy, nuclear magnetic resonance (NMR) and point mutation to identify the specific binding sites. ELISA, enzyme-linked immunosorbent assay; RNAi, RNA interference.


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Table 1 | Examples of molecular target discoveries of chemopreventive phytochemicals

Binding phytochemical Chemical structure Molecular targets Chemopreventive effect refs

3′,4′,7-trihydroxyisoflavone

O

OOH

OH

HO

PI3K EGF-induced cell proliferation and transformation

51

Cyclin-dependent kinase 2 (CDK2) and CDK4

5-deoxykaempferol

O

O

OH

HO

OH SRC UVB-induced two-stage skin carcinogenesis

53

Ribosomal S6 kinase 2 (RSK2)

UVB-induced COX2 and VEGF expression

PI3K

6-gingerol

HO

OCH3

O OH Leukotriene A4 hydrolase

(LTA4H)

Xenograft tumour volume of human HCT116 colon cancer cells

23

Caffeic acid

HO

OH

OH

O FYN UVB-induced COX2 expression 56

Cyanidin

O+

OH

HO

OH

OH

OH

RAF UVB-induced COX2 expression 57

Mitogen-activated protein kinase kinase 4 (MKK4)

MEK1

Cryptotanshinone

O

O

O Signal transducer and activator of transcription 3

Human prostate cancer cell proliferation

86

Deguelin

O

OO

O CH3

OCH3

OH

H Heat shock protein 90 Xenograft tumour volume of human lung, head, neck, stomach and prostate cancer cells

87

Delphinidin

O+

OH

HO

OH

OH

OH

OH FYN TNFα-induced COX2 expression 55

RAF TPA-induced cell transformation 88

MEK1

ERKs

MKK4 UVB-induced COX2 expression 59

PI3K

(–)-Epigallocatechin gallate

O

O

HO

OH

OH

OH

OH

O

OH

OH

OH

FYN EGF-induced cell transformation 20

Insulin-like growth factor 1 receptor

Cell proliferation and transformation 71

Glucose-regulated protein 78

Etoposide-induced breast cancer cell death and drug resistance

21

Heat shock protein 90 TCDD-mediated gene induction in hepatoma cells

72

ζ-chain-associated protein kinase 70

Leukaemia proliferation 19

Ras-GTPase-activating protein SH3 domain-binding protein 1

Anchorage-independent growth of human and mouse lung cancer cell lines

73




Table 1 (cont.) | Examples of molecular target discoveries of chemopreventive phytochemicals

Binding phytochemical Chemical structure Molecular targets Chemopreventive effect refs

Equol

O

OH

HO

MEK1 TPA-induced cell transformation 45

Fisetin

O

O

OH

HO

OH

OH

CDK6 Kinase activity 89

Kaempferol

O

O

OH

HO

OH

OH

SRC UVB-induced two-stage skin carcinogenesis

54

RSK2 RSK2-mediated cancer cell proliferation

60

PI3K EGF-induced cell transformation 90

Luteolin

O

O

OH

HO

OH

OH

OH SRC UVB-induced two-stage skin carcinogenesis

52

Protein kinase Cε

Myricetin

O

O

OH

HO

OH

OH

OH

OH

FYN UVB-induced two-stage skin carcinogenesis

24

RAF UVB-induced MMP9 activity and expression

26

MKK4 TNFα-induced VEGF expression 58

MEK1 TPA- or EGF-induced cell tranformation 44

PI3K UVB-induced angiogenesis 25

Janus kinase 1 Cell transformation 91

Procyanidin B2

O

OH

HO

OH

OH

OH

O

OH

HO

OH

OH

OH

MEK1 TPA-induced cell transformation 92

Quercetin

O

O

OH

HO

OH

OH

OH RAF TPA-induced cell transformation 43

MEK1

PI3K TNFα-induced MMP9 activation 93

Arsenite-induced COX2 expression 94

Resveratrol

OH

OH

HO COX2 Human colon cancer cell proliferation 16

LTA4H Xenograft tumour volume of human

pancreatic cancer cells41, 68

COX2, cyclooxygenase 2; EGF, epidermal growth factor; MMP9, matrix metalloproteinase 9; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TNFα, tumour necrosis factor-α; UV, ultraviolet; VEGF, vascular endothelial growth factor.




Nature Reviews | Cancer

Nucleus

Stimuli

Cytoplasm

SRC

FYN

RAS

RAF

MEK

ERK

RSK

p38

MKK3 orMKK6

MKK4 orMKK7

JNK

MSK

PI3K

AKT

PHAS40Raptor

mTORGene expression

Cell proliferation

p21

p53p27

CDK2 CDK2Cyclin E Cyclin A

to its ATP-binding site42, and computer modelling has indicated that several phyto-chemicals, including quercetin43, myricetin44 and equol45, could dock with this allosteric pocket. In fact, quercetin showed a much stronger inhibitory effect against MEK1 kinase activity than the well-known MEK1 inhibitor, PD098059 (REF. 43).

These inhibitors of MEK1 have also helped in understanding the binding proper-ties of diverse and related phytochemicals that are known to inhibit the same target. Experimentally, an analogue of resvera-trol (RSvL2) was shown to strongly bind MEK1, whereas resveratrol bound only very weakly17. When PD098059 binds MEK1, it is oriented in the allosteric site so that it forms a hydrogen bond with the backbone amide of Ser212 with its carbonyl oxygen, an electrostatic interaction with the backbone of val127 and edge-to-face aromatic stack-ing with Phe209 (REF. 42). The mechanism of allosteric inhibition of MEK is attributed to the inhibitor being able to stabilize the inactive conformation of the activation loop and deform the catalytic site46. The flavones, quercetin and myricetin, which

contain numerous polar hydroxyl groups, were not expected to preferentially bind in the extremely hydrophobic pocket in which other MEK inhibitors bind. These com-pounds were found to bind in the opening of the allosteric site and interact with the metal ion. Overall, the docked energies reproduced the trend that was found experimentally and, therefore, compared with resveratrol and quercetin, the addition of the one or two hydroxyl groups in RSvL2 and in myrcetin, respectively, seems to increase their bind-ing affinity with MEK (Z.D., unpublished observations). These results support the idea that a subtle difference in phytochemical structure, such as the addition of hydroxyl groups, will affect the binding affinity of a compound with a target protein.

Suppressing AKT signalling. Tumour-specific changes in metabolism were dis-covered by Warburg in the 1920s. These metabolic changes consist of increased glucose consumption, decreased oxida-tive phosphorylation and lactate pro-duction47 — characteristics of aerobic glycolysis. AKT and mTOR are key players

in reprogramming metabolic pathways in cancer cells. AKT is also thought to be involved in pathways that control the avail-ability of nutrients acting through AMP-activated protein kinase (AMPK), which controls glucose and lipid metabolism by sensing changes in nutrient and extracel-lular energy levels. This suggests that the AKT-mediated oncogenic pathway could be regulated by nutrients. PI3K is an upstream regulator of Akt–mTOR signalling and also interacts with several phytochemicals. Based on X-ray crystallography, quercetin and myricetin48 have been shown to directly bind and suppress PI3K activity, but so have other protein targets, as discussed above.

Intervening with cell cycle progression. Regulating cancer cell proliferation is crucial for chemoprevention. Cyclin-dependent kinases (CDKs), which are essential proteins for cell cycle progression, bind with cyclins to form CDK–cyclin complexes49. Many CDK inhibitors (CDKIs), such as the p21 and p27 proteins, attenuate formation of these complexes and block cell cycle pro-gression50. Several phytochemicals can also function as CDKIs. For example, 3′,4′,7- trihydroxyisoflavone, which is a metabolite of the soybean isoflavone daidzein, is a direct inhibitor of CDK2 and CDK4 (REF. 51).

Regulation of other oncogenic pathways by phytochemicals. In addition to the pathways mentioned above, several other pathways involved in cancer development are potential targets for phytochemicals. Pull-down assays using a specific phytochemical conjugated to cyanogen bromide (CNBr)-activated beads have led to the identification of numerous novel protein targets, including SRC52–54, FYN20,24,55,56, RAF17,26,43,57, mitogen-activated protein kinase kinase 4 (MKK4)57–59 and ribosomal S6 kinase 2 (RSK2)53,60,61 (TABLE 1). Leukotriene A4 hydrolase (LTA4H) is an example of an inflammation-related pro-tein that is implicated in tumorigenesis62,63. LTA4H catalyses the rate-limiting step in the biosynthesis of LTB4, a potent inducer of inflammation64–67. LTA4H was recently dis-covered through the reverse docking method as a direct target protein of [6]-gingerol and directly bound with Glu271 of LTA4H to inhibit its activity in vitro23. Furthermore, the inhibition of LTA4H protein expression was associated with decreased colon can-cer development in vivo23, indicating that the effects of gingerol are more complex than simple inhibition of enzyme activ-ity. Resveratrol has also been reported to directly bind and inhibit LTA4H

68.

Figure 2 | representative oncogenic signalling pathways. When stimulated, RAS activates inflammation-related proteins, such as ERKs, p38 and JUN N-terminal kinase (JNKs), to trigger down-stream signalling effectors. Along with the signalling pathway, metabolism-related pathways are also turned on. Activated SRC, FYN or RAS lead to activation of PI3K and AKT, and consequently turn on downstream signals. In addition, activation of AKT leads to the translocation of p27, a cell cycle inhibi-tor, from the nucleus to the cytosol and degradation of the protein. This event ensures formation of CDK–cyclin complexes to activate cell cycle progression. CDK, cyclin-dependent kinase; MKK, mitogen-activated protein kinase kinase; MSK, mitogen- and stress-activated protein kinase; PRAS40, proline-rich AKT substrate of 40 kDa; RSK, ribosomal S6 kinase; S6K, S6 kinase.




Another oncoprotein that is directly tar-geted by phytochemicals is the insulin-like growth factor 1 receptor (IGF1R). IGF1R can form heterodimers with the insulin receptor, thereby mediating insulin signal-ling, and has been implicated in the develop-ment of lung, breast and prostate cancers69,70. EGCG was reported to regulate cell transfor-mation of several cancer cell lines, including HeLa and MCF-7 cells through the direct binding and inhibition of IGFIR71.

Present limits and future prospectsThe importance of multi-target inhibitors. Using a combination of agents or a multi-targeted approach that provides synergistic or additive preventive effects when the agents are combined could theoretically permit the administration of the lowest active dose of each agent and therefore lower the potential for adverse side effects. Phytochemicals are known to inhibit a number of diverse tar-gets. For example, EGCG has been reported to directly bind to IGF1R, glucose-regulated protein 78 (REF. 21), FYN20, HSP90 (REF. 72), ZAP70 kinase19, Ras-GTPase-activating pro-tein SH3 domain-binding protein 1 (REF. 73) and the intermediate filament protein vimentin74. This apparent promiscuity poses a challenge in determining which protein is the physiological target that is bound first and which protein is the most important target. In addition, compounds with only a small difference in molecular structure can lead to substantial differences in the target protein, as discussed above for the inhibi-tors of MEK1. However, this promiscuity might prove to be a great advantage in the prevention of cancer. Cancer cell growth is driven by multiple signalling pathways and arises through a complex, multistep process by which cancer cells acquire characteristics of unlimited proliferation potential, lack of response to growth signals and resistance to cell death75,76. Identifying the most important players or biomarkers in carcinogenesis has been difficult because of the heterogeneity of cancer and the constantly changing can-cer genome. For example, tumours that are drug sensitive can at the same time develop drug-resistance mutations77. Downstream pathways can be aberrantly activated to cause resistance to therapies78,79. Therefore, blocking only one specific signalling path-way might not be sufficient to suppress the growth of cancer cells. Resveratrol is known to induce apoptosis through the induction of p53 phosphorylation and also suppresses AP1 and cyclooxygenase 2 activities, leading to cancer cell death16,18,80. These varied activi-ties suggest that foods containing resveratrol

and other compounds, such as myricetin, quercetin and kaempferol, could be benefi-cial in helping to prevent cancer. As multiple pathways are targeted by phytochemicals, potentially affecting both epithelial and stromal cells, another approach would be to use high-throughput methods in parallel, preferentially studying proteomic profiling, to define candidate targets. Even more prag-matically, protein signatures might serve as surrogate biomarkers for the specific targets identified. Ideally, this approach could be conducted using human preneoplastic tissue from patients who have a high risk of cancer development and who have entered cancer prevention or screening trials: comparisons could be made between baseline and post-treatment biopsy samples.

It was hoped that targeted therapies would prove to have fewer side effects; how-ever, this seems not to be the case. For exam-ple, ATP-competitive kinase inhibitors of BRAF(v600E) can have opposite effects by functioning as either inhibitors or activators of oncogenic signalling pathways, depend-ing on the cellular context81. Phytochemicals have a relatively low specificity towards single target proteins when compared with synthetic inhibitors, and this might be an advantage for developing phytochemicals as multiple inhibitors. Considering that phytochemicals seem to have relatively low toxicity and that chemopreventive agents will need to be used for a prolonged period, muti-targeting phytochemicals might be a solution for effective chemoprevention.

Personalized phytochemical cancer preven-tion. Every individual has a different and unique risk of cancer incidence, prognosis and response to treatment. Indeed, the neces-sity of personalized therapy for the treat-ment of lung or breast cancer, for example, is well established82,83. Such reports indicate a trend towards personalized cancer treat-ment as a requirement for effective cancer therapy. Conversely, a personalized diet for cancer prevention has not been as well docu-mented, although prescribing a personalized diet for cancer prevention might make sense. Unfortunately, this is not likely to happen soon because, beyond unhealthy eating and lack of exercise, a valid link between diet and cancer has been elusive.

However, owing to individual differences, a thorough analysis of distinct personal charac-teristics for the application of proper chemo-preventive agents could be capable of providing health benefits, especially in high-risk popula-tions. Cancer prevention has been categorized into three main types: primary, secondary

and tertiary84,85. Briefly, primary prevention is the avoidance of carcinogens, such as tobacco or ultraviolet radiation exposure. Secondary prevention includes screening for and detect-ing premalignant lesions, which is probably most important for high-risk populations, such as germline BRCA mutation carriers85. This might be a niche for phytochemical-based chemoprevention. In addition, the tertiary prevention aspect of preventing recurrence or a second primary tumour85 might also be a cancer prevention type for which personalized phytochemical prevention could be useful. Each type of cancer has its own particular char-acteristic genes and proteins that regulate its growth. Therefore, knowing the specific genes or protein targets of a phyto chemical chemo-preventive agent would increase the prob ability of the agent exerting efficacy in these high-risk individuals.

Although many reports have suggested benefits and targets of phytochemicals, these reports mainly rely on cell and animal models. In order to apply phytochemicals as personalized cancer preventive agents, the effects of phytochemicals in humans will need to be assessed. In the future, personalized prevention methods using phytochemicals could have a crucial role in cancer prevention, especially in high-risk populations. Rigorous research in identifying molecular targets and conducting human studies with phytochemi-cals would provide an enhanced approach to personalized cancer prevention.

Ki Won Lee is at the Department of Bioscience and Biotechnology, Konkuk University, Seoul 143‑701,

Republic of Korea, and the Department of Agricultural Biotechnology, Seoul National University, Seoul

151‑921, Republic of Korea.

Ann M. Bode and Zigang Dong are at The Hormel Institute, University of Minnesota,

Austin MN 55,912, USA.

K.W.L. and A.M.B. contributed equally to this work.Correspondence to Z.D.

e‑mail: [email protected]

doi:10.1038/nrc3017Published online 10 February 2011

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AcknowledgementsThis article was supported by grants from the US National Institutes of Health CA077646, CA111536, CA111536, CA120388, ES016548 and R37CA081064, The Hormel Foundation, and the Leap Research Program (No. 2010-0029,233) and the World Class Institute Program founded by the Korea Research Foundation, Ministry of Education, Science and Technology, Republic of Korea.

Competing interests statementThe authors declare no competing financial interests.

FURTHER inFORMATiOnZigang Dong’s homepage: http://www.hi.umn.edu/zgdong.htmlAsinex database: http://www.asinex.com/eMBL-eBi: http://www.ebi.ac.uk/embl/National center for Biotechnology information: http://www.ncbi.nlm.nih.gov/PDB: http://www.pdb.org/pdb/home/home.doPotential Drug target Databast (PDtD): http://www.dddc.ac.cn/pdtd/index.phpZiNc database: http://zinc.docking.org

SUPPLEMEnTARY inFORMATiOnsee online article: S1 (table)

all linkS are aCtive in the online pdf

organisms to cell death or cancer in mam-malian species1. DNA damage-induced mutation is an extensively documented route to mutagenesis for replicating cells. This is caused by DNA polymerases encounter-ing damaged bases and inserting a non-complementary nucleotide opposite the lesion that gives rise to a permanent and heritable change in the DNA sequence2,3. This replication-centric model for the initia-tion of mutagenesis has provided a plethora

of information for understanding major routes of mutagenesis for organisms existing under conditions of cell growth and divi-sion and has contributed substantially to our understanding of a host of biological events, including the origin of genetic variability, evolution and the development of cancer4,5.

However, the majority of cells living outside the artificial, growth factor-rich environment of a laboratory do not undergo continuous cycles of replication and growth but are instead more likely to exist in a non-proliferative or slow-growing state6. For example, several organs of multicellular organisms, such as the heart or brain, are comprised primarily of non-dividing cells, the lifespan of which is limited by functional degeneration of their normal physiology. Therefore, it follows that a large proportion of an organism’s tissues that are exposed to exogenous and endogenous DNA damaging agents are likely to be quiescent or slowly replicating cells, and these exposed cells are probably the origin of some tumours. Therefore, the physiological maintenance of cells and organisms is likely to be largely dependent on the fidelity of both transcription and translation.

There are numerous possible pathways for generating erroneous proteins that do not involve DNA replication (FIG. 1). At the level of translation, errors can occur through incor-rect amino acid incorporation, slippage of the translational machinery or absence of tRNA modifications resulting in misreading of the mRNA7. Together, these errors occur once for every 1,000 to 10,000 codons translated8, which renders synthesis of a functional pro-tein from an mRNA noticeably error prone. Nonetheless, lapses in translational fidelity or post-translational processing can functionally alter proteins and possibly change the physi-ology of the cell9,10, which could be crucial for cancer initiation or progression11.

At the level of transcription, some dam-aged ribonucleotides with altered pairing specificities can be incorporated into the nascent mRNA by RNA polymerases (RNAPs), thus leading to a mutant transcript that is translated into erroneous proteins12. Another possibility is direct damage to the transcript itself. In this case, damaged ribo-nucleotides can lead to altered specificities of codon–anticodon recognition such that incorrect amino acids can be incorporated. Direct damage to the transcript has been proposed to explain the aetiology of some human diseases, including neurodegenera-tive syndromes and the development of sev-eral types of cancer13–15. In addition, RNAPs may also misincorporate ribonucleotides

g E n O M i c i n S TA b i L i T Y i n c A n c E R — O P i n i O n

Transcriptional mutagenesis: causes and involvement in tumour developmentDamien Brégeon and Paul W. Doetsch

Abstract | The majority of human cells do not multiply continuously but are quiescent or slow-replicating and devote a large part of their energy to transcription. When DNA damage in the transcribed strand of an active gene is bypassed by a RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process is known as transcriptional mutagenesis and, as discussed in this Perspective, could lead to the production of mutant proteins and might therefore be important in tumour development.

Nearly every aspect of cellular behaviour and properties can be altered by the produc-tion of erroneous proteins. This situation holds true for cells of every living organism from the simplest prokaryote to the most complex metazoan species. Amino acid sub-stitutions or deletions are causes of changes to protein structure and function that are responsible for a large variety of biological outcomes, which range from conferring an advantageous ability to grow for unicellular




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Nature Reviews Cancer 2011 11(3), 211-218