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Protective effect of Glutathione S-Transferase T1 gene on melanoma risk in presence of

CDKN2A mutations, MC1R variants and host-related phenotypes.

V. CHAUDRU (1), MT. LO (1), F. LESUEUR (2,3), K. LAUD (4), H. MOHAMDI (1), C.

MARIAN (5), M. BARROIS (2), A. CHOMPRET (2), MF. AVRIL (6), F. DEMENAIS (1), B.

BRESSAC-DE PAILLERETS (2)

(1) INSERM U794, Université d’Evry, Evry, (2) Service de Génétique and FRE2939 CNRS,

Institut Gustave Roussy, Villejuif, (3) Genetic Susceptibility Group, IARC, Lyon, (4) Unité

830 INSERM, Institut Curie, (5) Biochemistry Department, University of Medecine and

Pharmacy, Romania, (6) AP-HP Hôpital Cochin, Université René Descartes Paris 5, Paris

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ABSTRACT

UV exposure is the major environmental risk factor of cutaneous melanoma mainly by

generating reactive oxygen species (ROS) causing molecular damages. Glutathione S-

transferases (GSTs) are enzymes playing an important role in detoxifying products of

oxidative stress, but only few case-control studies have investigated the effect of allelic

variants of GST genes on melanoma risk and have led to controversial results. The effect of

these GST genes in melanoma-prone families segregating CDKN2A mutations has not yet

been studied. We examined the effect of GSTP1, GSTM1 and GSTT1 genotypes on melanoma

risk in 25 melanoma-prone families with CDKN2A mutations, in presence of other risk factors

including MC1R gene variants, sun exposure, pigmentation characteristics and nevus

phenotypes. Logistic regression analyses were applied to 195 subjects genotyped for all three

genes investigated, using a generalized estimating equations (GEE) approach to take into

account correlations among family members. Different models were considered, which

allowed testing for the effect of each GST variant while including successively MC1R, sun

exposure and host factors in the regression and adjusting for age, sex and CDKN2A mutation

status. No significant effect of null GSTM1 allele and GSTP1 variants (p.I105V or p.A114V)

on melanoma risk was found. However, risk of melanoma was significantly decreased in

subjects carrying at least one null GSTT1 allele as compared to subjects carrying two active

alleles (ORadjusted for age, sex, CDKN2A = 0.41, 95% CI 0.18-0.94) and even more when the number of

MC1R variants, that is associated with increased melanoma risk, was entered into the model

(ORadjusted for age, sex, CDKN2A, MC1R = 0.24, 95% CI 0.15-0.58). Moreover, an additional independent

significant effect of dysplastic nevi which increased melanoma risk, was found. One possible

explanation for the observed protective role of GSTT1 deficiency on the development of

melanoma might be the promotion of cell death induced by the accumulation of deleterious

genetic events, secondary to accumulation of ROS or glutathione (GSH) depletion, in

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melanocytes. Ou The observed protective role of GSTT1 deficiency on the development of

melanoma would need to be interpreted in the context of the other genes, CDKN2A, MC1R

and potentially those underlying dysplastic nevi, that influence melanoma risk and are

involved in multiple interacting pathways.

Key words: Glutathione S-Transferase, CDKN2A mutations, MC1R variants, Melanoma

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INTRODUCTION

Cutaneous malignant melanoma (CMM) is a complex disease resulting from genetic and

other risk factors. Epidemiological studies have shown that exposure to sunlight is the major

environmental risk factor associated with melanoma, while high numbers of melanocytic nevi

(clinically banal and/or atypical (dysplastic)), hair color, eye color, skin color, extent of

freckling and skin reactions to sun exposure are the major host factors [Tucker and Goldstein,

2003 for a review].

Approximately 10% of malignant melanomas are observed in a familial setting. Up-to now,

CDKN2A gene (9p21; MIM# 600160) is the major high-risk melanoma susceptibility gene.

Germline CDKN2A mutations have been observed in approximately 20 - 40 percent of

melanoma-prone families from around the world [Goldstein et al., 2007a]. Although

CDKN2A mutations confer a substantial risk for melanoma, there is a proportion of mutation

carriers who do not develop melanoma. Penetrance of CDKN2A mutations has been found to

vary across continents, reaching 58% in Europe, 76% in the United States and 91% in

Australia by 80 years of age [Bishop et al., 2002]. These variations could be due to

differences in genetic background, host-related characteristics and/or sun-exposure behaviour.

On the one hand, poor tanning ability, sunburns, dysplastic nevi and/or high number of nevi

were characterized as host factors increasing risk of melanoma in families with CDKN2A

mutations [Chaudru et al., 2004; Goldstein et al., 2000]. On the other hand, genetic variation

at the melanocortin- receptor (MC1R) gene (16q24, MIM# 155555) was consistently found to

increase CDKN2A penetrance [Box, 2001; Chaudru et al., 2005; Goldstein et al., 2005; Van

der Velden et al., 2001]. MC1R plays a key role in the pigmentation process. Some MC1R

variants were reported to be associated with red hair and fair skin [Sturm et al., 2001 for a

review] and to influence melanoma risk independently of skin type, UV exposure and clinical

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risk factors by case-control studies [Kennedy et al., 2001; Matichard et al., 2004; Palmer et

al., 2000]. Joint analysis of host-related factors, sun exposure and MC1R in French families

with CDKN2A mutations have shown that CDKN2A penetrance is influenced significantly by

independent effects of MC1R variants and dysplastic nevi [Chaudru et al., 2005].

Besides MC1R, other low-risk genes have been reported to be associated with melanoma

by a few case-control studies, but the results are most often controversial [Fargnoli et al.,

2006; Hayward, 2003]. These genes include the glutathione S-transferase genes (GSTP1

MIM# 134660, GSTM1 MIM# 138350, GSTT1 MIM# 600436) whose products detoxify

electrophilic xenobiotics and inactive endogenous metabolites generated during oxidative

stress, such as the reactive oxygen species (ROS) occurring after excessive ultraviolet (UV)

exposure [Hayes et al., 2005]. Human GST genes have been shown to be polymorphic. These

polymorphisms are either single nucleotide polymorphisms (SNPs) in the coding regions

(GSTP1), or gene deletions (GSTT1 and GSTM1). SNPs in exon 5 (p.I105V) and exon 6

(p.A114V) of the GSTP1 gene have been reported as resulting in reduced enzymatic activity

[Ali-Osman et al., 1997], whereas homozygous deletion of GSTM1 and GSTT1 genes result in

total absence of the isoenzymes.

The aim of the present study was to examine for the first time the effects of GSTM1,

GSTT1 and GSTP1 polymorphisms on melanoma risk in 25 melanoma-prone French families

with CDKN2A mutations, while taking into account the effects of MC1R variants,

environmental and host-related factors.

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MATERIALS AND METHODS

Ascertainment of families and data collection

The 25 melanoma-prone families available for the present study have been recruited from

the Department of Dermatology at the Institut Gustave Roussy (IGR) and other French

hospitals forming the French Familial Melanoma Study Group. This sample includes five

additional families to our previous sample used to assess the effects of host factors and MC1R

on CDKN2A penetrance [Chaudru et al, 2005] and will be briefly described.

Eligible melanoma probands, from whom families were ascertained, were white

subjects living in France for more than ten years who had a newly diagnosed and

histologically confirmed melanoma. Family data were collected by interviewing the probands

on their first-degree, second-degree and third-degree relatives and included demographic

characteristics (gender, date of birth, and if deceased, age at death and cause of death) and the

occurrence of melanoma and any other cancer, along with the age at diagnosis. Confirmation

of melanoma diagnosis was sought through medical records, review of pathological material

and/or pathology reports. Only histologically confirmed melanoma patients were considered

as affected in the analyses. Physical examination for nevus counting was conducted by a

dermatologist in all probands and 70% of probands’ relatives. A questionnaire, recording data

on various risk factors, was completed by the probands and relatives seen in the hospital and

was distributed by probands to all other relatives. The data recorded on the questionnaire

included skin color (pale or dark), eye color (blue, green or dark), hair color (red, blond, light

brown, dark brown or dark), number of nevi (<10, 10-50, >50 nevi), presence of

atypical/dysplastic nevi (moles ≥5 mm in diameter with irregular margins and variegated

color, only for those examined by a dermatologist), degree of exposure to sunlight (low,

medium or high) during holidays, leisure time and work time, artificial UV exposure (yes or

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no), long stay (>1 year) in a sunny country, skin reactions to sunlight evaluated by the ability

to tan (low, medium, or high) and propensity to sunburn (low, medium or high). Information

on risk factors was obtained in all probands and at least 70% of all living relatives.

Written informed consent was obtained prior to participation under an Institutional

Review Board-approved protocol.

Genotyping

Genomic DNA from participants was extracted from peripheral blood lymphocytes, using the

QIAamp DNA Blood mini kit (QIAGEN, Hilden, Germany). A total of 18 different CDKN2A

mutations and 12 non-synonymous MC1R variants were detected in our 25 families (see

[Chaudru et al, 2004; 2005] for a list of CDKN2A mutations and MC1R variants). The gene

copy number variation for GSTT1 and GSTM1 were determined using an adapted protocol

from the Quantitative Multiplex PCR of Short fluorescent Fragments (QMPSF) method

[Casilli et al., 2002]. In brief, short exonic fragments of GSTT1 (exon 5) and GSTM1 (exons 4

and 5) were PCR-amplified in a multiplex PCR. An additional fragment, corresponding to

exon 3 of GAPDH was co-amplified, as a control. Sequences of primers and probes used for

QMPSF are available upon request. PCR fragments were then separated by electrophoresis on

the ABI Prism© 3730 Genetic Analyzer (Applied Biosystems, Foster City, California).

Electropherograms were superposed on those generated from control DNA by adjusting to the

same level the peaks obtained for the control amplicon, and the surface of the corresponding

peaks were then compared among the different samples. Using this approach, the

homozygous active and the heterozygous active genotypes for GSTT1 and GSTM1 could be

distinguished. For GSTP1 variants p.I105V (rs1695) and p.A114V (rs1138272), genotyping

was carried out using Taqman® according to manufacturer’s instructions. Primers and probes

were supplied directly by Applied Biosystems as Assays-by-Design™ and are available upon

request.

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Statistical analyses

Comparisons of GSTP1, GSTM1, and GSTT1 genotypes frequencies in affected and

unaffected members of the melanoma-prone families were first carried out by Fisher exact

test. Crude odds-ratios were computed to estimate the effects of these genotypes on melanoma

risk.

We then used logistic regression to assess the effects of each GST gene on melanoma risk

by examining the following models: (1) GST in presence of CDKN2A mutation status; (2)

GST in presence of CDKN2A and number of MC1R variants (or presence of RHC (Red Hair

Color) variants or NRHC (non-RHC) variants); (3) GST in presence of CDKN2A and by

entering MC1R, sun exposure, pigmentation and nevus phenotypes in the model using a

stepwise procedure. Age and sex were included in all regression models. We used a

generalized estimating equation (GEE) approach to take into account correlations among

family members. The measure of association between melanoma risk and risk factors was the

odds-ratio [OR] with 95% confidence interval [CI]. Prior to the multivariate logistic

regression analysis conducted under model (3), we assessed the association of sun exposure,

pigmentation and nevus phenotypes with GSTT genotypes in unaffected family members

using χ2 and Fisher exact tests. No association of any GSTT genotype with any of these

factors was found whereas, as reported in our previous study [Chaudru et al., 2005], MC1R

was significantly associated with hair color, skin color and tanning ability.

For each GST gene, the three genotypes were first investigated, the baseline category being

the homozygous for the active allele (GSTM1 or GSTT1) or the most frequent allele (GSTP1).

Heterozygous and homozygous genotypes for null allele (GSTM1 or GSTT1) or less frequent

allele (GSTP1) were then pooled, since they showed similar effects and because of sample

size. Regarding the CDKN2A mutation status, the baseline category included non-carriers

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while the at-risk category included heterozygous mutation carriers (there was no homozygous

carriers in our sample). Since the risk of melanoma was found to increase with the number of

MC1R variants across populations [Goldstein et al., 2007b; Demenais, personal

communication], a MC1R variable was created by assigning subjects to one of the three

following categories: MC1R consensus homozygous subjects versus subjects with 1 MC1R

variant versus subjects with at least two MC1R variants. A RHC variable (pooling p.R151C,

p.R160W, p.D84E and p.D294H variants which are associated with red-hair color) and a

NRHC variable (pooling all non-RHC variants) were also considered: the baseline category

included MC1R consensus homozygous subjects while the at-risk category included subjects

with either at least one RHC variant (and no NRHC variant) or at least one NRHC variant

(and no RHC variant). Environmental and host-related covariates were dichotomized with the

baseline and at-risk categories being defined as follows: sun exposure (low or medium versus

high), hair color (dark or dark brown versus light brown, blond or red), skin color (dark

versus pale), eye color (dark versus blue or green), propensity to sunburn (low or medium

versus high), ability to tan (high or medium versus low), total nevi (≤50 versus >50 nevi) and

dysplastic nevi (absent versus present). Note: où retrouve-t-on ces variables dans les résultats?

Peut-on expliquer l’analyse stepwise qui conduit au Table 3? – ces variables sont mentionnées

quand on parle de l’analyse stepwise (model 3 ci-dessus) et quand on présente les résultats de

cette analyse (dernier paragraphe des résultats)

All analyses were performed using the SAS package (V9.1).

RESULTS

The 25 melanoma-prone families (comprising a total of 306 subjects) included 195

CDKN2A genotyped subjects, 96 of them being CDKN2A mutation carriers (comprising 53

melanoma patients and 43 unaffected individuals). Among the 99 non-mutation carriers, three

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subjects had melanoma while all other ones were unaffected. The mean age at diagnosis of

melanoma patients (38.56 ± 11.70 years) and mean age at examination of unaffected subjects

(40.93 ± 20.58 years) did not differ significantly (p=0.65). The risk of melanoma was

significantly higher in females than in males (p= 0.03; OR adjusted for CDKN2A = 2.17; 95%

CI, 1.09-4.31). Sex was thus included in all analyses.

The GSTP1, GSTM1, and GSTT1 genotypes as well as MC1R genotypes were determined

in all 195 individuals genotyped for CDKN2A. All GST and MC1R variants were in Hardy-

Weinberg equilibrium (p> 0.05). Regarding MC1R, 85 subjects (43.6 %) carried one MC1R

variant and 69 had at last two variants (35.4 %), these proportions being 28.6% and 49.6% in

affected subjects and 58.9% and 25.9% in unaffecteds (p<0.001, for the difference between

affecteds and unaffecteds).

The distribution of GST genotypes in all subjects and CDKN2A mutation carriers according

to melanoma status is shown in table 1. The frequency of GST genotypes did not differ

significantly between affected and unaffected subjects (p ≥ 0.14). When examining the odds-

ratios, heterozygous and homozygous for null GSTM1 or GTT1 allele showed a similar

decreased melanoma risk, although not significant. Pooling these two genotypic categories led

to a significant protective effect of having at least one GSTT1 null allele on melanoma risk in

the whole sample (p = 0.05; OR=0.52; 95% CI, 0.27-1.00). Heterozygous and homozygous

for either GSTP1 variant were also pooled given the small number of homozygous, but no

significant effect was detected.

The effect of GST genes on melanoma risk was then assessed by logistic regression that

first incorporated in the model age, sex and CDKN2A mutation status and then MC1R (table

2). Prior to that analysis, we confirmed the significant effects of CDKN2A mutations (p<10-5)

and MC1R variables (p=0.006 for number of MC1R variants, p=0.029 for RHC, and p=0.036

for NRHC) on melanoma risk. The melanoma risk was not significantly modified by GSTM1

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null allele nor by GSTP1 p.I105V or p.A114V variant (table 2). However, carrying at least

one GSTT1 null allele decreased melanoma risk significantly. This protective effect was

higher when MC1R was added to a model including CDKN2A as compared to a model with

CDKN2A alone: OR = 0.24 (95% CI, 0.15-0.58; p=0.001) with CDKN2A and MC1R vs OR =

0.41 (95% CI, 0.18-0.94; p=0.035) with CDKN2A alone. This decrease in melanoma risk

associated with GSTT1 null allele was similar in presence of RHC or NRHC variants,

although being more significant with NRHC variants (p = 0.008 with NRHC variants vs

p=0.046 with RHC variants). To investigate further the higher decrease in melanoma risk

when MC1R was incorporated in the regression model, we tested for interaction between

MC1R and GSTT1 variables but no significant interaction was evidenced. However, when

estimating odds-ratios by stratifying on number of MC1R variants, we observed a lower

melanoma risk associated with GSTT1 null allele in carriers of less than two MC1R variants as

compared to subjects having two or more variants (test of homogeneity between ORs being

borderline significant, p = 0.06). When all these analyses were restricted to CDKN2A

mutation carriers, similar results were obtained (results not shown). Note BBr et Fle: nous

suggérons d’enlever les sous catégories RHC vs NRHC (mat et methods, résultats, tableau 2)

car les chiffres deviennent trop faibles pour être analysés dans la partie CDKN2A carriers :

nous pensons que c’est intéressant de montrer RHC et NRHC/nous avons supprimé la partie

du tableau avec les mutation carriers et indiqué dans le texte que les mêmes resultats étaient

obtenus. Dans la Table 2 pourquoi n’analyse-t-on pas MC1R “1 variant” vs “2 variants”,

comme dans la table 3?; c’est l’analyse qui a été faite dans la table 2 , une footnote a été

ajoutée à la table 2 pour préciser.

Finally, effects of MC1R, GSTT1, host factors (hair colour, skin colour, eye colour,

inability to tan, sunburns, nevus phenotypes) and sun exposure on melanoma risk were

investigated by a stepwise procedure while adjusting for age, sex and CDKN2A mutation

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status (table 3). This multivariate analysis showed that the best fitting model included the

effects of MC1R variants (OR=10.17 for at least two MC1R variants, P<0.001), GSTT1 null

allele (OR=0.24; P=0.005), and dysplastic nevi (OR=7.21; P=0.004). Although non

significant, the next factor to enter into the model was the high number of nevi (OR=3.02;

P=0.08).

DISCUSSION

This is, to our knowledge, the first study investigating the modifier effect of GSTs

polymorphisms in melanoma-prone families segregating CDKN2A mutations. Our study

shows a significant protective effect of null GSTT1 allele on melanoma risk in these families.

This protective effect was enhanced when the effect of MC1R variants was added to the

model that included age, sex and CDKN2A mutation status. Moreover, besides these genes, an

additional independent effect of dysplastic nevi that increased significantly melanoma risk

was found. Regarding the other GST genes, GSTM1 null allele decreased melanoma risk

similarly to GSTT1, although not significantly, while either p.I105V or p.A114V GTSP1

variant did not modify melanoma risk.

Few case-control studies have investigated the effect of the glutathione S-transferase genes

on melanoma risk and have led to controversial results. No significant effect of GSTT1 was

found by two previous Slovenian and German case-control studies [Dolzan et al., 2006;

Mössner et al., 2007]. However, in the Slovenian study, the GSTT1 null genotype led to a

decrease in melanoma risk, although not significantly (OR=0.74, 95% CI 0.40-1.34, [Dolzan

et al., 2006]). Note that the genotyping method in that study did not allow to detect

heterozygous carriers of GSTT1 gene deletion, which were therefore part of the baseline

category while, in our study, the decrease in melanoma risk reached significance by pooling

homozygous and heterozygous carriers of GSTT1 null allele. Ne peut-on pas raccourcir,

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puisqu’on ne peut rien comparer (différente méthode de génotypage et différente population

étudiée?) – Ceci a été raccourci.

The decrease in odds-ratio associated with the effect of GSTT1 was enhanced when MC1R

was added to the regression model including CDKN2A as compared to the model with

CDKN2A alone. No significant interaction between MC1R and GSTT1 on melanoma risk

could be detected. However, estimates of ORs associated with GSTT1, which were lower in

subjects with less than two MC1R variants than in those with two variants or more, suggest

such interaction which may become significant in larger samples. The protective effect of

GSTT1 was of the same order of magnitude when considering separately RHC and NRHC

variants. The higher significance level observed in presence of NRHC as compared to RHC

variants may be accounted for by differences in sample size (149 and 115 subjects examined

in presence of NRHC and RHC variants respectively). Moreover, the stepwise analysis

showed that dysplastic nevi increased significantly melanoma risk independently from the

three genes (CDKN2A, MC1R and GSTT1) that already influenced risk. This shows that genes

underlying nevus formation and transformation may also play a role in addition to the genetic

pathways involved in cell cycle, pigmentation, DNA repair or oxidative stress.

Regarding the GSTM1 gene, five case-control studies failed to show an effect of the

GSTM1 null genotype on melanoma risk [Dolzan et al., 2006; Heagerty et al., 1994; Kanetsky

et al., 2001; Mössner et al., 2007; Shanley et al., 1995], while this latter genotype led to a

significant 2-fold increase in risk in a Spanish case-control study [Lafuente et al., 1995]. The

GSTM1 null genotype was also reported to confer a two-fold increase in melanoma risk in a

subset of American subjects with red/blond hair, that increase being higher (10-fold increase

in risk) when these subjects carried both GSTM1 null and GSTT1 null genotypes [Kanetsky et

al., 2001]. However, this effect was not confirmed in a subset of German red/blond hair

individuals [Mössner et al., 2007]. These results differ from those observed in our sample

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where a decrease in risk, although not significant, was found in subjects carrying at least one

GSTM1 null allele. As observed for GSTT1, the decrease in OR was higher when MC1R was

included in the model and was similar whether considering number of MC1R variants, RHC

or NRHC variants. Pooling the effects of both GSTM1 and GSTT1 gene (having at least one

GSTT1 or GSTM1 null allele) led also to a reduced melanoma risk which remained not

significant (results not shown).

To our knowledge, only one Slovenian case-control study has investigated the role of

GSTP1 gene on melanoma risk [Dolzan et al., 2006] and found that none of GSTP1 genotypes

influenced significantly melanoma risk as in our study.

The finding of an association between the GSTT1 null allele and a decreased risk of

melanoma was somehow unexpected but has to be interpreted in the context of CDKN2A

mutations and MC1R variants. These three genes are involved in multiple pathways with

complex interactions between these pathways and it has been reported that key genetic factors

underlying melanoma development and progression have pleiotropic effects which may be

totally opposite according to the factors with which they interplay at a given time of the

cellular processes. Increased UV exposure is regarded as the major environmental factor

contributing to melanoma. Pleiotropic effects of UV irradiation on skin cells include direct

DNA damage and formation of ROS. ROS can directly attack DNA or can cause DNA

damage following reaction with membrane lipids [Hayes et al., 2005]. GSTs are a family of

isoenzymes that catalyze the conjugation of tripeptide glutathione (GSH) to exogenous and

endogenous chemicals (including products of oxidative stress), thus rendering these products

more water soluble and minimizing the injurious events caused by these products [Afaq et al.,

2001; Franco et al., 2007]. Genetic variations of these enzymes have been postulated to

influence the risk of melanoma and other skin cancers related to UV exposure [Afaq et al.,

2001]. However, ROS are also involved in the regulation of many signaling pathways which

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can lead to proliferation, differentiation and stress adaptation but also apoptosis [Wittgen and

Kempen, 2007]. The α-melanocyte-stimulating hormone (α-MSH), acting through MC1R,

plays a major role in the pigmentation process which involves melanin synthesis that guards

against the photodamaging effects of UV and acts as a scavenger of ROS [Miyamura et al,

2007]. However, melanin can also turn into a prooxidant under oxidative stress as a result of

inflammation or higher metabolic processes [Wittgen and Kempen, 2007]. Besides its effects

on pigmentation, α-MSH is involved in antiapoptotic and DNA repair pathways [Kadekaro et

al, 2005; Miyamura et al., 2007] and has also been shown to potentiate the expression of

CDKN2A gene product (p16) after UV exposure [Pavey and Gabrielli, 2002]. MC1R

expression is also regulated by oxidative products [Garcia-Borron et al., 2005]. The positive

and negative feedbacks among these various factors underlie the complexity of the

mechanisms involved.

One possible explanation that could be put forward is that the active GSTT1, acting as a

catalyzer of GSH conjugation, would increase risk of developing melanoma. Indeed,

mammalian cells have evolved protective mechanisms, such as GSH conjugation, to minimize

injurious events that result from toxic chemicals and normal oxidative products of cellular

metabolism. It has been shown that GSH depletion to 20-30% of total GSH levels can impair

the conjugation defence against the toxic actions of such compounds and become detrimental

to cellular processes [Reed, 1990]. The combined conjugation activities of GSTs may lead to

GSH depletion and thus, instead of protecting, the GSTs may expose cells to oxidative DNA

damage and associated mutagenic lesions. This latter hypothesis is supported by a recent

study showing that GSH depletion is involved in apoptosis, and that this process is

independent of ROS formation [Franco et al., 2007]. Je ne comprends pas cette dernière

phrase. L’explication indiquée dans les phrases précédentes, tirée de l’article cancer du sein /

GSTM1, indique que la déplétion de GSH favoriserait les mutations et donc un cancer mais

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pas de apoptose ou peut-être clarifier cette phrase??? D’autre part, comme tout l’explication

de la depeletion de GSH vient de l’article du cancer du sein, ne vaudrait-il pas mieux la mettre

après avoir exposé les résultats du ca du sein, dire que les auteurs ont suggéré cette hypothèse

et qu’elle pourrait aussi s’appliquer au melanome ??

Because unrepaired DNA damages appear to play a central role in carcinogenesis, the

effect of GST genes had been widely studied in different types of cancer (such as breast,

colon, lung, and brain cancers). Although homozygous deletion for GSTM1 slightly increased

lung cancer risk and homozygous deletion for GSTM1 and GSTT1 slightly increased head and

neck cancer risk, controversial results were found for other cancers by epidemiological studies

[Hayes et al., 2005; Parl, 2005 for reviews]. Interestingly, one study [Roodi, 2004] has

indicated a protective effect of the GSTM1 deletion on breast cancer. Caucasian women

carrying two active GSTM1 alleles had a significant 2.8-fold increased risk of breast cancer,

compared to the ones carrying the GSTM1 null genotype, the same trend being observed in

African-American women without reaching significance. This in agreement with our finding

of decreased melanoma risk associated with carrying at least one GSTT1 null allele which has

the same type of effect as GSTM1, the mode of action of these genes depending potentially on

their level of expression in different tissues and their interactions with tissue-specific factors.

The high frequency of GSTM1 and GSTT1 deficiency in various populations, together with

the small risk conferred by the GST genes, when a significant association with any cancer was

found, suggests a complex role of these genes in carcinogenesis. Other studies are needed to

confirm the present findings and to disentangle the role of GST genes in cancer susceptibility.

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_________________________________________________________________________

Previous version / biology

Biological hypotheses can be put forward in an attempt to explain the significant protective

effect of GSTT1 null allele on melanoma risk as evidenced in the present genetic context of

CDKN2A mutations and MC1R variants. Increased UV exposure is regarded as the major

environmental factor contributing to melanoma. Pleiotropic effects of UV irradiation on skin

cells include direct DNA damage and formation of ROS. ROS can directly attack DNA or can

cause DNA damage following reaction with membrane lipids [Hayes et al., 2005].

Accordingly, genetic variations of enzymes involved in the detoxification of oxidative stress

products have been postulated to influence the risk of melanoma and other skin cancers

related to UV exposure [Afaq et al., 2001]. GSTs are a family of isoenzymes that catalyze the

detoxification of substrates of ROS and the conjugation of the tripeptide glutathione (GSH) to

minimize injurious events that results from toxic chemicals and normal oxidative products of

cellular metabolism [Afaq et al., 2001; Franco et al., 2007]. The GST genes are expressed in

melanocytes, and inhibition of GSTs in melanoma cell lines has been shown to diminish their

capacity to scavenge the reactive metabolites produced during melanin synthesis [Fruehauf et

al., 1998]. Moreover, it has been shown that the α-melanocyte-stimulating hormone (α-MSH)

is able to block UVB radiation-induced apoptosis of normal human melanocytes in vitro while

it does not block UV radiation-mediated apoptosis in nucleotide excision repair-deficient

fibroblasts [Böhm et al., 2005]. This points out the crucial role of GSTs detoxifying function

when ROS are accumulating in the melanocyte during the tumorigenesis. However, the

findings of an association between the GSTT1 null allele and a reduced risk of melanoma was

somehow unexpected. This is observed in the particular genetic context of CDKN2A

mutations and MC1R variants, all these genes being involved in multiple pathways with

complex interactions between these pathways. Experiments in whole-skin organ cultures have

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shown that α-MSH, acting through MC1R, potentiates the expression of CDKN2A gene

product (p16) after UV exposure (Pavey and Gabrielli, 2002). The α-MSH protein, besides its

effect on pigmentation, plays a role in apoptotic/antiapoptotic and DNA repair pathways

(Kadekaro et al, 2005; Miyamura et al., 2006). Reactive oxygen species are involved in the

regulation of many signaling pathways which can lead to proliferation, differentiation and

stress adaptation but also apoptosis (Wittgen and Kempen, 2007).

A first hypothesis would be that the absence of GSTT1 protein induces an increase of cell

death when the number of deleterious genetic events (such as UV-induced DNA damages) in

melanocytes is accumulating, thus preventing the transformation to cancerous state. UV-

induced DNA damage could be BRAF oncogenic somatic mutations as it has been shown

recently that BRAF mutations are a characteristic feature of non-CSD melanoma in

individuals carriers of two variant MC1R alleles, such mutation could be the result of A>T

transversion secondary to an increased of ROS. [Landi et al., 2006]. Therefore, the protective

effect of the GSTT1 null genotype on the risk of melanoma may be particularly relevant for

the carriers of MC1R at risk variants. Indeed, in our series, the risk of melanoma as

significantly decreased in subjects carrying at least one GSTT1 null allele, especially when the

number of MC1R variants was entered in the model.

One alternative hypothesis would be that the active GSTT1, acting as a catalyzer of GSH

conjugation, is associated with an increased risk of developing melanoma. Indeed,

mammalian cells have evolved protective mechanisms, such as GSH conjugation, to minimize

injurious events that result from toxic chemicals and normal oxidative products of cellular

metabolism. It has been shown that GSH depletion to 20-30% of total GSH levels can impair

the conjugation defence against toxic actions of such compounds and become detrimental to

cellular processes [Reed, 1990]. This later hypothesis is support by a recent study showing

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that GSH depletion is involved in apoptosis, and that this process is independent of ROS

formation [Franco et al., 2007].

Because unrepaired DNA damages appear to play a central role in carcinogenesis, the

effect of GST genes had been widely studied in different types of cancer (such as breast,

colon, lung, and brain cancers). Although homozygous deletion for GSTM1 slightly increased

lung cancer risk and homozygous deletion for GSTM1 and GSTT1 slightly increased head and

neck cancer risk, controversial results were found for other cancers by epidemiological studies

[Hayes et al., 2005; Parl, 2005 for reviews]. Interestingly, one study [Roodi, 2004] has

suggested a protective effect of the GSTM1 deletion on breast cancer. Subjects carrying two

active GSTM1 alleles had increased risk of breast cancer, compared to subjects carrying the

GSTM1 null genotype: the relative risk adjusted for age was 2.83 (95% CI 1.45-5.59) in

Caucasian women while the increased risk was not significant in African-American women

(the relative risk adjusted for age was 1.66, 95% CI 0.66-4.20).

The high frequency of GSTM1 and GSTT1 deficiency in different populations, together

with the small risk conferred by the GST genes, when a significant association with any

cancer was found, suggests a complex role of these genes in carcinogenesis, and other studies

are needed to confirm the present findings and to disentangle the role of GST genes in cancer

susceptibility.

___________________________________________________________________

ACKNOWLEDGMENT

We thank the members of the families who have so willingly participated in this study and the

clinicians who identified these families. This work was funded by Ligue Contre le Cancer

(Comité du Val de Marne 2003-2004 and Ligue Nationale PRE2005.LNCC/FD1), ARC (grant

N°3222, 2003), and INCA - Cancéropole Ile de France (melanoma network RS#13). FL was

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recipient of a postdoctoral fellowship from the Fondation pour la Recherche Médicale

(UFP20051105597).

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Table 1

Distribution of GST genotyped by melanoma affection status and crude odds-ratio associated to the GST genes in the total sample and in

the subset of CDKN2A mutation carriers

Carriers and non carriers of CDKN2A mutations carriers of CDKN2A mutations

(N=195) (N=96)
No. of No. of
GST genotypes genotyped Cases/Controls Odds-Ratio 95% CI genotyped Cases/Controls Odds-Ratio 95% CI
subject (%) subject (%)
GSTM1b
+/+ 24 (12.9) 9/15 1.00 - 15 (15.6) 8/7 1.00 -
+/- 93 (47.3) 28/65 0.72 0.28- 47 (48.9) 27/20 1.18 0.37-
-/- 78 (39.8) 19/59 0.54 1.83 34 (35.4) 18/16 0.98 3.80
0.20- 0.29-
1.42 3.33
pa =0.43 pa =0.96
GSTT1b
+/+ 54 (27.7) 21/33 1.00 - 29 (30.2) 20/9 1.00 -
+/- 105 (53.8) 27/78 0.54 0.27- 49 (51.0) 25/24 0.47 0.18-
-/- 36 (18.5) 8/28 0.45 1.10 18 (18.7) 8/10 0.36 1.23
0.17- 0.11-1.22
1.17
pa=0.14 pa =0.18
GSTP1
p.I105V (A>G): A/A 91 (46.7) 24/67 1.00 - 44 (45.8) 23/21 1.00 -
A/G 92 (47.2) 27/65 1.16 0.61- 43 (44.8) 25/18 1.27 0.54-
G/G 12 (6.1) 5/7 1.99 2.22 9 (9.4) 5/4 1.14 2.96
0.58- 0.27-
6.88 4.83
pa =0.49 pa =0.91

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p.A114V (C>T): C/C 170 (87.2) 50/120 1.00 - 86 (89.6) 47/39 1.00 -
C/T 25 (12.8) 6/19 0.76 0.29- 10 (10.4) 6/4 1.25 0.33-
T/T 0 (0) - - 2.01 0 (0) - - 4.73
- -
pa =0.64 pa =1.00
a
: p = p-value associated to the Fisher exact test in order to compare GST allele frequencies in affected and non affected members of melanoma-
prone families
b
: + = active allele, - = null allele

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Table 2

Effect of GST genes on melanoma risk by taking into account successively age, sex, CDKN2A, and MC1R in the total sample of carriers

and non carriers of CDKN2A mutations.

Adjustment model
Age, sex, CDKN2A Age, sex, CDKN2A, Age, sex, CDKN2A, Age, sex, CDKN2A,
MC1Ra RHC NRHC
OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI)
Gene tested
p-value p-value p-value p-value
N=195 N=195 N=115b N=149b
GSTT1 c 0.41 [0.18-0.94] 0.24 [0.15-0.58] 0.29 [0.09-0.98] 0.33 [0.14-0.75]
+/- or -/- 0.035 0.001 0.046 0.008
GSTM1 c 0.84 [0.35-2.01] 0.42 [0.15-1.11] 0.46 [0.09-2.25] 0.45 [0.12-1.67]
+/- or -/- 0.699 0.081 0.338 0.230
GSTP1p. I105V 1.22 [0.63-2.35] 1.28 [0.57-2.89] 1.36 [0.42-4.46] 1.55 [0.72-3.32]
C/T or T/T 0.550 0.545 0.607 0.264
GSTP1 p.A114V 0.99 [0.42-2.33] 1.29 [0.41-4.09] 4.02 [0.49-33.20] 0.82 [0.21-3.20]
A/G or G/G 0.987 0.661 0.196 0.771
a
: the MC1R variable includes two dummy variables : a variable were the baseline category included MC1R consensus compared to subject with

1 MC1R variant and a second variable were the baseline category included MC1R consensus compared to subject with at least 2 MC1R variants.
b
: When the RHC and NRHC variables were considered, some subjects were excluded from the analyse since the baseline category included

MC1R consensus homozygous subjects while the at-risk category included subjects with either at least one RHC variant (and no NRHC variant)

or at least one NRHC variant (and no RHC variant) .

c
: + = active allele, - = null allele

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Table 3

Outcomes of the unconditional logistic regression analyses selecting the effects of MC1R gene, GSTT1 gene, and host factors (nevi

phenotypes) by a stepwise procedure in the total sample of carriers and non carriers of CDKN2A mutations.

Effect of the following variable on melanoma risk

1 MC1R variant ≥2 MC1R variants GSTT1 b Presence of High number

+/- or -/- Dysplastic nevi of nevi

(DN) (HNN)
a a a a a
Model OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI)
p-value p-value p-value p-value p-value
1. MC1R 1.42 [0.46-4.42] 6.24 [1.68-23.21] - - -
0.546 0.006
2. GSTT1 added to model 1 1.33 [0.42-4.21] 7.68 [2.16-27.33] 0.24 [0.10-0.58] - -
0.627 0.002 0.001
3. DN added to model 2 1.34 [0.42-4.23] 10.17 [2.88-35.91] 0.24 [0.09-0.66] 7.21 [1.91-27.28] -
0.623 <0.001 0.005 0.004
4. HNN added to model 3 2.00 [0.57-7.10] 12.45[3.79-40.89] 0.25 [0.08-0.83] 5.38 [1.38-20.92] 3.02 [0.88-10.29]
0.281 <0.001 0.024 0.015 0.078
a
: Adjusted odds-ratio for age as continuous variable, sex and CDKN2A status
b
: + = active allele, - = null allele

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