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Oncogene (2004) 23, 1–8
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REVIEW
Generating mutations but providing chemosensitivity: the role of
O6-methylguanine DNA methyltransferase in human cancer
Manel Esteller*,1 and James G Herman2
1
Cancer Epigenetics Laboratory, Molecular Pathology Program, Spanish National Cancer Center (CNIO), Melchor Fernandez
Almagro 3, Madrid 28029, Spain; 2Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins School of
Medicine, Bunting-Blaustein Cancer Research Building, 1650 Orleans, Room 543, Baltimore, MD 21231, USA
O6-methylguanine DNA methyltransferase (MGMT) is a
key enzyme in the DNA repair network. MGMT removes
mutagenic and cytotoxic adducts from O6-guanine in
DNA, the preferred point of attack of many carcinogens
(i.e. methylnitrosourea) and alkylating chemotherapeutic
agents (i.e. BCNU, temozolamide, etc.). Hypermethylation of the CpG island located in the promoter region of
MGMT is primarily responsible for the loss of MGMT
function in many tumor types. The methylation-mediated
silencing of MGMT has two consequences for cancer.
First, tumors with MGMT methylation have a new
mutator phenotype characterized by the generation of
transition point mutations in genes involved in cancer
etiology, such as the tumor suppressor p53 and the
oncogene K-ras. Second, MGMT hypermethylation
demonstrates the possibility of pharmacoepigenomics:
methylated tumors are more sensitive to the killing effects
of alkylating drugs used in chemotherapy. These recent
results underscore the importance of MGMT in basic and
translational cancer research.
Oncogene (2004) 23, 1–8. doi:10.1038/sj.onc.1207316
Keywords: O6-methylguanine DNA methyltransferase;
MGMT; CpG island hypermethylation; transition
mutations; alkylating agents
Understanding MGMT function
DNA repair is an important area of research in cancer
biology. Most attention has been focused on the
mismatch, recombination and excision repair pathways.
While these pathways are of clear importance to the
development and progression of human cancer, other
important mechanisms of DNA repair have not been as
extensively studied. The O6-methylguanine DNA
methyltransferase (MGMT) gene is one such less studied
pathway affecting DNA repair capacity and involved in
the biology of cancer formation and behavior.
*Correspondence: M Esteller;
E-mail: [email protected] or [email protected]
MGMT (EC2.1.1.64, also known as O6-alkylguanine
DNA alkyltransferase) is a DNA repair protein that
removes mutagenic and cytotoxic adducts from
O6-guanine in DNA (Pegg, 1990; Pegg et al.,
1995). Alkylation of DNA at the O6 position of
guanine is an important step in the formation
of mutations in cancer, primarily due to the tendency
of the O6-methylguanine to pair with thymine during
replication, resulting in the conversion of guanine–
cytosine to adenine–thymine pairs in DNA (Coulondre
and Miller, 1977). Furthermore, the O6-alkylguanineDNA adduct (especially the O6-chloroethylguanine)
may crosslink with the opposite cytosine residues,
blocking DNA replication (Erickson et al., 1980).
MGMT protects cells against these lesions, transferring
the alkyl group from the O6-guanine in DNA to an
active cysteine within its own sequence in a reaction that
inactivates one MGMT molecule for each lesion
repaired (Pegg, 1990). The alkylated MGMT protein
then becomes detached from DNA and is targeted for
degradation by ubiquitination (Srivenugopal et al.,
1996). Thus, the ability of a cell to withstand such
damage is directly related to the number of MGMT
molecules that it contains and to the rate of de novo
synthesis of MGMT.
Although MGMT can act at different rates on a
wide variety of O6-alkyl groups (and even in a minor
degree over the O4-methylthymine), the majority of
studies have focused on the endogenous substrate
O6-methylguanine. Of the more than 12 different types
of nitrogen and oxygen adducts of purine and pyrimidine bases produced by alkylating agents, O6-methylguanine is, in fact, one of the least abundant, but of
most importance. O6-methylguanine affects cytosine
methylation (Hepburn et al., 1991), the binding of
transcription factors (Bonfanti et al., 1991), the recombinogenic capacity (White et al., 1986) and may inhibit
DNA replication or cleavage if located in replication
origins (Ceccotti et al., 1993) or topoisomerase-I
sites (Pourquier et al., 2001). This review will
focus particularly on how MGMT prevents the formation of transition mutations in DNA in human
tumorigenesis and its role in modulating the sensitivity
of neoplasms to the alkylating agents used in current
chemotherapy.
Role of MGMT in cancer
M Esteller and JG Herman
2
Promoter hypermethylation of MGMT causes its loss in
human cancer
The amounts of MGMT protein differ according to
cellular type (Gerson et al., 1986). The levels and activity
of MGMT in healthy cells are regulated by its protein
phosphorylation status (Srivenugopal et al., 2000), the
binding of the E6 papillomavirus oncoprotein (Srivenugopal and Ali-Osman, 2002) and the action of p53
(Harris et al., 1996), glucocorticoid hormone (Biswas
et al., 1999) and other transcription factors over its 50 CpG island, which includes a classical promoter without
TATA and CAAT boxes and a 59 bp enhancer element
located at the first exon–intron boundary (Harris et al.,
1991) (Figure 1). A single report has described mutations in the MGMT gene (Wang et al., 1997). However,
there are many reports of individual differences in
MGMT activity in tumors. Several germline variants
affecting the MGMT gene have been described (Imai
et al., 1995; Rusin et al., 1999; Inoue et al., 2000;
Egyhazi et al., 2002), but few studies have addressed the
functional relevance of these nucleotides changes (Imai
et al., 1995; Edara et al., 1996). No extensive analysis of
the MGMT genotype and haplotypes distribution in
healthy and cancer population exists.
The MGMT protein is decreased in some tumors with
respect to their normal tissue counterpart (Gerson et al.,
1986, Citron et al., 1992). A subset of tumor cell lines,
termed Mer, completely lack MGMT activity (Day
et al., 1980). As loss of expression is not commonly due
to deletion, mutation or rearrangement of the MGMT
gene (Day et al., 1980, Fornace et al., 1990, Pieper et al.,
1990) or mRNA instability (Kroes and Erickson, 1995),
other causes for loss of activity are expected. Methylation of cytosine in CpG dinucleotides is the main
epigenetic modification of DNA in normal mammalian
cells (Esteller and Herman, 2002a). The human MGMT
gene possesses a CpG island in the 50 portion of the gene
(Harris et al., 1991). Hypermethylation of normally
unmethylated CpG islands in the promoter regions of
many genes, including p16INK4a, p14ARF, VHL, BRCA1,
hMLH1 and E-cadherin, correlates with its loss of
transcription (Esteller et al., 2001a, 2002b). Hypermethylation of the MGMT CpG island as the cause of
MGMT transcriptional silencing in cell lines defective in
O6-Methylguanine DNA Methyltransferase (MGMT)
MGMT protein (aprox. 22 kDa)
I
II
III
IV
V
CpG Island: Hypermethylated in Certain Tumors Types
MGMT Gene (aprox. 170 Kb)
: Described Polymorphisms of Unknown Significance
Figure 1 Genomic structure of the MGMT gene. Exons are
indicated as gray boxes (I–V)
Oncogene
MGMT CpG Island Methylation Analysis
NHL1 NHL2
U M U M
NHL1
NHL2
Retention
Loss
MGMT Protein Expression Analysis
MGMT mRNA (950 nucleotides)
Gene Locus
10q26
O6-methylguanine repair has been demonstrated (Costello et al., 1996; Qian and Brent, 1997; Watts et al.,
1997; Danam et al., 1999; Esteller et al., 1999a). As for
other genes inactivated by CpG island methylation,
hypermethylation of the promoter region is accompanied by histone hypoacetylation and methylation
(Kondo et al., 2003), binding of specific methyl-binding
proteins (Fraga et al., 2003) and loss of nucleosome
positioning (Patel et al., 1997), all of these alterations
rendering a ‘closed’ chromatin state that prevents gene
transcription (Ballestar and Esteller, 2002). As additional proof of causality, in vitro treatment of cancer
cells with demethylating drugs restores MGMT expression (Qian and Brent, 1997; Esteller et al., 2000a).
To study the relevance of the promoter hypermethylation of the MGMT gene in vivo in cancer patients, we
examined a large series of more than 500 primary
human tumors and corresponding normal tissues for
MGMT aberrant methylation using methylation-specific PCR and its relation with MGMT expression
(Esteller et al., 1999a) (Figure 2). MGMT function is
lost frequently in association with hypermethylation of
the promoter region in a wide spectrum of human
tumors (Esteller et al., 1999a). Our initial findings
demonstrated a specific profile of MGMT hypermethylation in human cancer that indicated gliomas,
Figure 2 MGMT promoter hypermethylation correlates with its
gene silencing: an example in NHL. The CpG island methylation
status of the MGMT gene was carried out by methylation-specific
PCR. Briefly, DNA is modified by sodium bisulfite, which modifies
the unmethylated cytosines to uracil, but it does not affect the
methylated cytosines, followed by a PCR reaction using primers
specific for either methylated or the modified unmethylated DNA.
The presence of a visible product in lanes U indicates the presence
of unmethylated genes of MGMT; the presence of a visible product
in lanes M indicates the presence of methylated genes of MGMT.
The analysis demonstrates that NHL1 is unmethylated and
expresses MGMT determined by immunohistochemistry, while
NHL2 is methylated and lacks MGMT expression
Role of MGMT in cancer
M Esteller and JG Herman
3
lymphomas, colon, head and neck and non-small-cell
lung carcinomas as the main tumor targets for the
epigenetic inactivation of MGMT (Esteller et al.,
1999a). This profile, thanks to the popularization of
bisulfite-PCR techniques (Fraga and Esteller, 2002), has
been expanded in additional reports of MGMT inactivation in other tumor types (Toyooka et al., 2001;
Virmani et al., 2001; Choy et al., 2002; Lee et al., 2002;
Maruyama et al., 2002; Smith-Sorensen et al., 2002),
summarized in Figure 3. This aberrant MGMT methylation has been correlated with the loss of MGMT
protein (Esteller et al., 1999a, 2002c; Herfarth et al.,
1999.), lack of mRNA expression (Esteller et al., 2000a;
Yin et al., 2003) and loss of enzymatic activity (Herfarth
et al., 1999). The constellation of tumor types where
MGMT hypermethylation can be invoked has also been
further understood with its analysis in a comprehensive
panel of 70 human cancer cell lines (Paz et al., 2003).
Furthermore, the CpG island hypermethylation-associated silencing of MGMT occurs very early in human
tumorigenesis, such as in small colon adenomas (Esteller
et al., 2000a), strongly supporting its relevant role in
carcinogenesis. MGMT promoter hypermethylation has
also been demonstrated in the serum DNA of lung
cancer patients (Esteller et al., 1999b), and head and
neck cancer patients (Sanchez-Cespedes et al., 2000) as
well as other biological fluids, including sputum
(Palmisano et al., 2000) and saliva (Rosas et al., 2001).
Silencing of MGMT causes a new mutator pathway in
human cancer
The causes of mutations in oncogenes and tumor
suppressor genes are multifactorial (Hussain and Harris,
1998). Exogenous and endogenous compounds are
known to cause DNA damage (Hussain and Harris,
2000), including deletions, insertions and base substitutions, either transversions (change of purine to pyrimidine or vice versa) or transitions, change or purine to
Pattern of Methylation-Mediated Silencing of MGMT in Human Primary Tumors
50
%
40
30
20
10
Testi
cular
(n=6
9
Colo
n (n= 1)
Retin
323)
obla
stom
a (n=
23)
Glio
3
ma (
Head
n=21
and N
3)
eck (
n=11
6)
Cerv
5
ix (n
=19)
Ly m
phom
a (n=
61)
NSC
LC ( 7
n
Esop
hagu =83)
s (n=
14)
Stom
ach ( 9
n=60
)
SCL
C (n
=43)
Panc
reas 11
(
n
=
1 8)
Mela
nom
Chola
a (n=
ngio
18)
carcin
oma 13
Kidn (n=79)
ey( n
=12
Leuk
emia 15)
(n=3
1)
Blad
der (
n=44
Men
)
ingio
ma ( 17
n=26
)
Brea
st (n
=
36)
19
Ovar
y
( n=2
Endo
3)
metr
ium
(n=1
7)
Live 21
r (n=
5 9)
Pros
tate (
n=10
231)
0
Figure 3 Distribution of CpG island promoter hypermethylation
of MGMT across human primary tumors of 23 different cell types.
Y-axis, percentage of neoplasms with MGMT hypermethylation;
X-axis, the tumor types studied with the total number of samples
analysed between parentheses
another purine or pyrimidine to another pyrimidine
(Strauss, 1992). Well-known sources of the spontaneous
generation of point mutations include: deamination of
cytosine and 5-methylcytosine to uracil and thymine,
respectively; depurination; DNA polymerase infidelity;
and oxidative damage from endogenously produced free
radicals (Hussain and Harris, 1998; Strauss, 1992).
Abnormalities in DNA repair and replication have also
long been considered as key elements in the genesis of
mutations. Several mechanisms can be invoked to
contribute to the infidelity of DNA synthesis, including
imbalances in deoxynucleotide triphosphate pools,
mutations in DNA polymerase-a and slippage of DNA
polymerase at nucleotide repeats (Liu et al., 1983; Phear
et al., 1987; Kunkel et al., 1993). However, the
importance of each mechanism and gene(s) involved is
often unknown.
One such mutator pathway in human cancer has been
clearly defined. Germline mutations in the two DNA
mismatch repair (MMR) genes hMLH1 and hMSH2 are
the genetic abnormalities responsible for the vast
majority of hereditary nonpolyposis colorectal carcinoma cases (Bocker et al., 1999), where the presence of
deletions and insertions in microsatellite sequences is a
common hallmark. However, in the nonfamilial cases,
the presence of microsatellite instability is attributable
to methylation-associated silencing of hMLH1 as
indicated by the extremely tight association between
both phenomena (Kane et al., 1997; Esteller et al., 1998,
2000b; Herman et al., 1998; Fleisher et al., 1999), the
restoration of MMR proficiency using demethylating
agents (Herman et al., 1998) and the early appearance of
hMLH1 hypermethylation, occurring in premalignant
stages (Esteller et al., 1999c). The epigenetic silencing of
hMLH1 then leads to mutations in target genes such as
BAX or TGFRbII (Markowitz, 2000).
Methylation-mediated silencing of MGMT causes
another strong and important mutator pathway in
human cancer that it is more prevalent than microsatellite instability. The persistence of O6-methylguanine
adducts, resulting from alkylating agents, may cause
DNA polymerase to misread the base pairing because of
the altered hydrogen-bonding properties of a base that
contains an additional methyl or ethyl group. Thus, O6methylguanine is read as an adenine and mispairs with
thymine (Horsfall et al., 1990). Supporting these data,
the most common mutations caused by alkylating
agents are G : C to A : T transitions (Horsfall et al.,
1990), exemplified in the frequent generation of G to A
transitions in the oncogene K-ras when the carcinogen
N-methylnitrosourea (that forms O6-methylguanine
adducts) is used in experimentally induced tumor
systems (Sukumar et al., 1983; Mitra et al., 1989).
Avoidance of the mutagenic effect is directly related to
the presence of a functional MGMT protein (Pegg et al.,
1995). In vitro assays show that endogenous MGMT
expression protects mammalian cell lines from spontaneous G : C to A : T transitions in the aprt gene
(Aquilina et al., 1992). Animal models also show that
transgenic mice overexpressing MGMT are protected
against O6-methylguanine-DNA adducts caused by
Oncogene
Role of MGMT in cancer
M Esteller and JG Herman
4
methylnitrosourea (Dumenco et al., 1993) and against G
to A mutations in K-ras in aberrant colorectal crypt foci
and lung tumors (Zaidi et al., 1995; Liu et al., 1999).
This link between MGMT inactivation and K-ras
transition mutations in human tumors was first observed
in 2000 (Esteller et al., 2000a). Ras oncogenes acquire
transforming activity following single-point mutations
within their coding sequences. Mutations in naturally
occurring ras oncogenes have been localized in codons
12, 13, 59 and 61. Alteration of codon 12, GGT, is the
most common change. Substitution of the wild-type
Glycine 12 by any other amino acid results in oncogenic
activation of this molecule (Barbacid, 1987). Although
ras mutation is the most common oncogenic alteration in
human cancer (Barbacid, 1987; Bos, 1989), the incidence
of K-ras activation varies widely among carcinomas. Kras mutation is rare in human primary breast carcinomas, but occurs in approximately half of colorectal
carcinomas. This mutation distribution strongly resembles the pattern of MGMT promoter hypermethylation
described (Esteller et al., 1999a): while MGMT aberrant
methylation is not present in breast carcinomas where Kras mutations are extremely rare, it occurs in approximately 40% of cases of colorectal carcinomas associated
with loss of MGMT expression, and is also frequent in
non-small-cell lung carcinoma (Esteller et al., 1999b)
where K-ras mutations are frequent. MGMT promoter
hypermethylation was an early event in human colorectal tumorigenesis linked to the appearance of G to A
mutations in the K-ras oncogene (Esteller et al., 2000a)
(Figure 4). The association between MGMT methylation
and K-ras mutations has now been reported not only in
colon cancer in (Whitehall et al., 2001) but also in gastric
and gall bladder cancers (Park et al., 2001; Kohya et al.,
2003).
How the loss of the DNA Repair Gene MGMT may lead
to G to A mutations in the K-ras and p53 Genes ?
NORMAL CELL
CANCER CELL
Unmethylated, Transcribed
Methylated, Silenced
MGMT Gene
Wild –Type
Codon
Promutagenic
Lesion
Dietary Nitrates,
Alkylating Agents
----GO6mGT-----
----GGT----MGMT
Promutagenic
Lesion
----GO6mGT----06mG
is
read as
Adenine
Mutant Codon
----GAT-----
Figure 4 Methylation-mediated silencing of MGMT causes a
mutator phenotype: a model of how the epigenetic loss of MGMT
leads to the generation of transition mutations in p53 and K-ras.
Left, a normal cell with a functional MGMT protein is able to
repair the presence of O6-methylguanines (induced by endogenous
and exogenous agents) in its DNA; right, tumors with MGMT
inactivation due to promoter hypermethylation accumulate O6methylguanines that are misread as adenines, causing transition
point mutations
Oncogene
The tumor suppressor p53 is the most commonly
mutated gene in human cancer, and transition mutations
constitute the most common p53 mutations (Greenblatt
et al., 1994; Pfeifer, 2000). Approximately 52% of the
mutational events are missense transitional changes,
and, of this subset, B72% are G : C to A : T transitions
(Greenblatt et al., 1994). The profile of the mutational
spectrum varies according to tumor type. Lung and
head and neck tumors of smokers have a higher number
of transversions, whereas colorectal tumors have the
highest rate of transition mutations, reaching 70% of
the total number of p53 mutations (Greenblatt et al.,
1994). These last mutations occur frequently in CpG
dinucleotides, which are normally methylated (Pfeifer,
2000; Rideout et al., 1990; Tornaletti and Pfeifer, 1995),
through increased rates of spontaneous deamination at
methylcytosine, although other mechanisms are also
conceivable. However, 17% of p53 mutations are
transition mutations in non-CpG dinucleotides, where
this causality cannot be invoked (Greenblatt et al.,
1994). Thus, G : C to A : T changes in p53 in non-CpG
and CpG dinucleotides could be attributable, in part, to
a defect in MGMT that allows the persistence of O6methylguanine and its reading as an adenine (Figure 4).
Promoter hypermethylation of MGMT was strongly
linked to the presence of G : C to A : T transition
mutations in p53, particularly in non-CpG dinucleotides
(Esteller et al., 2001b) in colon cancer and has also been
found in glioma (Nakamura et al., 2001; Yin et al.,
2003), liver (Zhang et al., 2003) and non-small-cell lung
carcinomas (Wolf et al., 2001).
Yet to be classified is the particular source of
promutagenic adducts in the O6 position of guanine
not repaired by MGMT in each tumor type. For
colorectal tumors, the alkylating agents causing the
promutagenic lesion may be provided from dietary
nitrates reduced in the proximal colon by bacteria, by
nitrosation of amines and amides derived from protein
catabolism (Ward et al., 1989; Bartsch et al., 1990;
Rowland et al., 1991). For other cancers, these carcinogens are ever more speculative. Not all adducts are
repaired with the same efficacy. O6-ethylguanine is
removed faster than the O6-methylguanine, perhaps
preventing ras mutations in certain tumor types more
exposed to these xenobiotics (Engelberg et al., 1998).
Among the glial tumors, MGMT epigenetic inactivation
(Esteller et al., 1999a) and MGMT loss or reduced
activity (Hongeng et al., 1997; Silber et al., 1998) are
common features, and these epigenetic events associate
with the presence of transition mutation in p53
(Nakamura et al., 2001; Yin et al., 2003). Yet,
carcinogens exposure is not frequently associated with
the development of brain tumors.
Methylation-mediated silencing of MGMT marks tumors
sensitive to alkylating agents
Alkylating agents are among the most widely used
chemotherapeutic drugs in human cancer (Colvin and
Role of MGMT in cancer
M Esteller and JG Herman
5
Hilton, 1981; Teicher, 1997). Several alkylation sites in
DNA have been described as the targets of the action of
these compounds, with the most frequent site the O6
position of guanine. This modification can produce
DNA interstrand crosslinks (Teicher, 1997), and this
base is the preferred point of attack in the DNA of
numerous alkylating chemotherapeutic drugs, such as
BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea), ACNU
(1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea), procarbazine, streptozotocin or
temozolamide. However, the toxicity of alkylating
agents is reduced in the presence of MGMT by rapidly
reversing the formation of adducts at the O6 position
of guanine (Ludlum, 1990; Pegg et al., 1995),
thereby averting the formation of lethal crosslinks.
Thus, MGMT activity is a major mechanism of
resistance to alkylating drugs (Ludlum, 1990; Pegg
et al., 1995).
In gliomas, enhanced sensitivity to the action of
alkylating agents was initially suggested in the subgroup
of patients with reduced MGMT activity (Silber et al.,
1993, 1998; Belanich et al., 1996; Jaeckle et al., 1998;
Gerson, 2002). Following the demonstration that CpG
island hypermethylation of MGMT was the main cause
behind its loss in gliomas (Esteller et al., 1999a), we
thought that glioma tumors hypermethylated at MGMT
would be more sensitive to the action of these alkylating
agents, since their DNA lesions could not be repaired in
the cancer cell and so this would lead to cell death. We
gave proof of principle for this hypothesis and
effectively MGMT promoter hypermethylation predicts
a good response to chemotherapy, greater overall
survival and longer time to progression in glioma
patients treated with BCNU (Esteller et al., 2000c)
(Figure 5). The methylation status of the MGMT
promoter was more predictive of the outcome of
carmustine treatment than the grade of the tumor, the
Karnofsky performance status or the patient’s age
(Esteller et al., 2000c).
The potential of MGMT to predict the chemoresponse of human tumors to alkylating agents is not
limited to BCNU-like alkylating agents, but it may
extend to other drugs such as cyclophosphamide
(Mattern et al., 1998; Cai et al., 1999; Friedman et al.,
1999; Gamcsik et al., 1999), but not cisplatin (Mattern
et al., 1998). This has been demonstrated in vivo in
diffuse large-cell lymphomas treated with chemotherapeutic regimens including the alkylating agent cyclophosphamide (Esteller et al., 2002c), where MGMT
hypermethylation was the strongest predictor of overall
survival and time to progression, and was far superior to
classical clinical factors such as the International
Prognostic Index (IPI) (Esteller et al., 2002c).
It is important to note that MGMT hypermethylation
alone, without treatment with an alkylating agent, is not
a good prognostic factor. In fact, it is a poor prognostic
factor (Hayashi et al., 2002; Kohya et al., 2002;
Brabender et al., 2003), probably due to the finding
that patients with epigenetic silencing of MGMT
accumulate more mutations (p53 and K-ras). This is
an example of the difference between predictive and
Figure 5 Overall survival (panel a) and time to the progression of
disease (panel b) among patients with gliomas treated with
carmustine, according to the methylation status of the MGMT
promoter. Both overall survival and the time to the progression of
disease were significantly greater in the group of patients with
methylation of the MGMT promoter than in the group without
methylation. The association was independent of the type of tumor,
the patient’s age and the Karnofsky score for performance status
prognostic markers. Prognostic markers suggest a
difference in outcome that is independent of the
treatment received, including the possibility of no
treatment. An example of prognostic marker includes
the above-mentioned IPI that summarizes variables
related to tumor stage (stage itself, extranodal sites
and LDH level) as well as host factors (age and
performance status). Predictive markers, on the other
hand, predict a response, and thereby often survival
differences, related to a specific form of therapy. Thus,
while both types of markers can predict differences in
survival, predictive markers potentially provide information leading to treatment decisions. MGMT methylation is definitively a negative prognostic marker, but a
positive predictive marker.
Oncogene
Role of MGMT in cancer
M Esteller and JG Herman
6
ALKYLATING AGENTS
(BCNU, Cyclophosphamide )
OPTION:
CHEMOPROTECTION
BY MGMT VECTORS ?
NORMAL CELLS
MGMT UNMETHYLATED
TOXICITY
(Bone Marrow)
OPTIONS:
OTHER DRUGS !!!!
CANCER CELLS
MGMT UNMETHYLATED
CHEMORESISTANT
TUMOR
MGMT INHIBITORS:
06-BG
RYBOZYMES
RNAi
CONSIDERATIONS:
CANCER CELLS
MGMT METHYLATED
MMR DEFICIENT TUMORS ?
SELECTION OF RESISTANT
CHEMOSENSITIVE
CELLS ?
TUMOR
Figure 6 How the different methylation backgrounds of MGMT
affect the response to alkylating drugs and possible therapeutic
strategies. Tumors unmethylated at MGMT are less sensitive to the
action of alkylating drugs and the use of other types of
chemotherapeutic agents and the inhibition of MGMT is
recommended. Tumors methylated at MGMT respond well to
the chemotherapy with alkylating agents. One requirement would
be the necessity of a functional MMR pathway and one caveat
would be the possible selection of resistant cellular subclones
MGMT methylation, or lack of expression, may
determine the best chemotherapy strategy, a practical
example of pharmacogenomics, or, in this particular
case
‘pharmacoepigenomics’
(Weinstein,
2000)
(Figure 6). One might explore increasing the sensitivity
of the ‘resistant’ tumors, those without MGMT
inactivation, to alkylating agents (Middleton and
Margison, 2003). The development of the MGMT
inhibitor O6-benzylguanine (O6-BG) (Dolan et al.,
1990; Dolan and Pegg, 1997) is being investigated for
this purpose. O6-BG is an MGMT substrate that, by its
binding to the protein in a ‘suicide’ reaction, inactivates
MGMT. While this inhibitor has been found to enhance
primarily the response to alkyl-nitrosoureas both in vitro
and in vivo (Dolan et al., 1990; Dolan and Pegg, 1997),
O6-BG has been shown to increase sensitivity to
cyclophosphamide metabolites as well (Cai et al., 2001).
Future studies should extend the use of determining
MGMT methylation or gene expression as a marker of
response to alkylating drugs in other tumor types:
excellent candidates are non-small-cell lung cancer, head
and neck tumors and colorectal carcinomas, where
MGMT methylation is relatively frequent (Figure 3). In
the case of colorectal carcinoma, MGMT methylation
does not confer sensitivity to the killing action of
alkylating agents if the tumor is also MMR deficient
(Kawate et al., 1998, 2000). This phenomenon occurs in
approximately 10% of colon tumors and in this case the
presence of simultaneous methylation-mediated silencing of hMLH1 and MGMT confers resistance to cell
death and may increase the mutation rate of that
neoplasm.
Future for MGMT
In the last few years, biological and pathological data
have, with the previous knowledge of the biochemistry
of MGMT, raised awareness about the importance of
this molecule and DNA repair of alkylation damage.
The wide, but specific, spectrum of human primary
tumors, having methylation-associated loss of MGMT
has opened two important areas of research. Cancers
with MGMT hypermethylation accumulate more point
mutations than other tumors. This has been exemplified
in the oncogene K-ras and the tumor suppressor gene
p53, but may extend to a long list of genes yet to be
discovered. Second, aberrant methylation of MGMT
identifies tumors with higher sensitivity to alkylating
agents. This assay may help us to design more targeted
therapies and stimulate the research of new ways to
overcome the resistance of the unmethylated tumors.
Acknowledgements
The research in Dr Esteller’s laboratory was sponsored by the
Health Department of the Spanish Government, the I þ D þ I
SAF 2001-0059, CAM08.1/0010.1/2001 Grants and the International Rett Syndrome Association. JGH is an Evelyn
Grollman Glick scholar, whose research was supported by
grants from the National Cancer Institute (CA84986
P50CA58184).
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