Increased risk of cervical cancer associated with cyclin D1 gene A870G polymorphism

Cancer Genetics and Cytogenetics 160 (2005) 49–54

Increased risk of cervical cancer associated with cyclin D1 gene A870G polymorphism Raquel Catarinoa, Ana Matosa,b, Daniela Pintoa, Deolinda Pereirac, Roge´ria Craveirod, Andre´ Vasconcelosa, Carlos Lopesa, Rui Medeirosa,* a

Molecular Oncology Unit, Portuguese Institute of Oncology, Rua Dr. Anto´nio Bernardino de Almedia, Porto 4200-072, Portugal b Gynaecology Department, Ju´lio Dinis Maternity, Largo Maternidade Ju´lio Dinis, Porto 4050-371, Portugal c Medical Oncology Department, Portuguese Institute of Oncology, Rua Dr. Anto´nio Bernardino de Almedia, Porto 4200-072, Portugal d Radiobiology Department, Portuguese Institute of Oncology, Rua Dr. Anto´nio Bernardino de Almedia, Porto 4200-072, Portugal Received 27 July 2004; received in revised form 19 November 2004; accepted 30 November 2004

Abstract

Human papillomavirus (HPV) plays a major role in the etiology of cervical cancer. However, a complex correlation between viral and cellular genes is necessary for cell cycle control deregulation in the progression to invasive cervical cancer (ICC). Cyclin D1 (CCND1) is an important positive regulator of the G1/S phase of the cell cycle. The CCND1 gene is located at 11q13 and is often altered in human cancers. We analyzed the A870G CCND1 polymorphism by polymerase chain reaction/ restriction fragment length polymorphism analysis in 246 women including 50 cases with highgrade squamous intraepithelial lesions of the cervix (HSIL), 93 with ICC, and 103 healthy women. The GG genotype was associated with a 4.32-fold higher risk for the development of HSIL [adjusted odds ratio (aOR) ⫽ 4.32, 95% confidence interval (CI) 1.50–12.46, P ⫽ 0.0067), and a 3.26fold increased risk for the development of ICC (aOR ⫽ 3.26, 95% CI 1.42–7.53, P ⫽ 0.006). The proportion of cervical cancer cases attributable to the GG CCND1 genotype was 17.26%. This study indicates that the A870G CCND1 polymorphism could act as a cofactor of HPV in the initiation of cervical carcinogenesis, particularly in the transformation zone of HPV-infected women, supporting evidence for a genetic factor in ICC risk. 쑖 2005 Elsevier Inc. All rights reserved.

1. Introduction Invasive cervical cancer (ICC) is the most common gynecologic malignancy worldwide and one of the most common causes of death in women. In Portugal, cervical cancer has an age-standardized incidence rate of 17 per 100,000 women and is responsible for 4.2% of malignant deaths [1]. The primary cause in the development of ICC is infection with human papillomavirus (HPV). More than 90% of squamous cervical cancers contain HPV DNA [2]. HPV-16 and -18 have two transcriptional units, E6 and E7, which encode proteins important for cell immortalization [3]. Integration of HPV DNA into the host genome results in the constitutive expression of the viral oncoproteins E6 and E7, which deregulate cell cycle control through interaction with oncogenes

* Corresponding author. Tel.: ⫹351-22-5084000, ext. 5414; fax: ⫹35122-5084001. E-mail address: [email protected] (R. Medeiros). 0165-4608/05/$ – see front matter 쑖 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2004.11.017

and tumor-suppressor gene products, initiating the crucial step for tumorigenesis [4,5]. The E7 oncoprotein binds to retinoblastoma protein (RB) and to the related pocket proteins p107 and p130, leading to functional inactivation of these proliferation regulators, which ultimately allows unchecked cell cycle progression in cells infected with HPV-16 or -18 [4,6,7]. Epidemiological and clinical data suggest that human papillomavirus, especially HPV-16 and -18 (high-risk HPV), play a major role in the etiology of cervical cancer [8]. However, some researchers acknowledge that HPV is a necessary but not a sufficient factor in the etiology of ICC. The progression from HPV infection to cancer involves other environmental and host factors. Single-nucleotide polymorphism markers should be considered in the determination of the combinations of genetic factors involved in precancerous changes to cervical cancer [9]. It is unclear whether the association of these environmental and genetic factors with cervical cancer reflects secondary associations also attributable to infection, if they are independent risk factors, or

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even if they act as cofactors of HPV infection in the induction of cervical carcinogenesis [10]. A frequent target in transitional cell carcinogenesis is the deregulation of G1/S phase progression in the cell cycle, which is regulated by cyclins, cyclin-dependent kinases, and their inhibitors [11]. Cyclin D1 (CCND1) is a key regulator of cell cycle progression, an important positive regulator of the G1/S phase, and a demonstrated oncogene [12–14]. CCND1 is located at chromosome 11q13, and alterations in this gene have been described in several cancers [15–21]. CCND1 regulates cell cycle progression by activating cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), which in turn phosphorylate RB. This reaction inactivates RB and is postulated to lead to progression through a G1–S checkpoint, committing the cell to DNA replication [22,23]. It has been reported recently that CCND1 mRNA is alternatively spliced to produce two transcripts (a and b) that are present simultaneously in a variety of normal tissues and cancer cells [24,25]. Betticher and colleagues [25] identified a single base pair (bp) polymorphism (A870G) in CCND1, and CCND1 genotypes have been significantly associated with carcinogenesis and clinical outcome in a variety of cancers [26–32]. Alterations in this gene, such as gene amplifications and overexpression, have been observed in cervical cancer patients [33–36]. The genetic association between the CCND1 A870G polymorphism and genetic susceptibility to cervical cancer has not yet been investigated. The aim of our study is to determine if CCND1 genotypes are associated with genetic susceptibility to cervical cancer.

2. Materials and methods 2.1. Patients We tested the association between the CCND1 A870G polymorphism and risk of cervical cancer using a case–control study. One-hundred forty-three patients were selected for this study: 50 patients with histologically confirmed highgrade intraepithelial squamous lesions of the cervix (HSIL) and 93 with histologically confirmed invasive cervical carcinoma (ICC). Patients were admitted to the Portuguese Oncology Institute from 1997 to 2003. The median age at diagnosis was 46 years (standard deviation 12.5). The control group consisted of 103 healthy women, with a median age of 55 (standard deviation 16.3), without clinical history of cancer, and from the same geographic area as the case group. All samples were taken after informed consent, according to the declaration of Helsinki.

of the A870G polymorphism of CCND1 was carried out essentially as described previously [25]. The PCR reactions consisted of nearly 0.2 µg of genomic DNA, 30 pmol of each primer, 0.2 mmol/L of each dNTP, 1.5 mmol/L MgCl2, 1 × Taq buffer, and 1 unit of Taq DNA polymerase to a final volume of 50 µL. Primers used in the analysis were CY26 (5′GTG AAG TTC ATT TCC AAT CCG C 3′) and CY27 (5′ GGG ACA TCA CCC TCA CCC TCA CTT AC 3′). Thirtyfive cycles were performed, consisting of an initial heating at 95⬚C for 10 minutes to activate the enzyme, followed by 35 cycles of denaturation at 94⬚C for 1 minute, annealing at 55⬚C for 1 minute, and extension at 72⬚C for 1 minute, with a final extension step at 72⬚C for 2 minutes. PCR products (15 µL) were digested with 1 unit ScrF1 at 37⬚C for 4 hours and then visualized by electrophoresis on 3% agarose containing 0.5 µg/mL ethidium bromide. The 167-bp PCR product generated is not cut by ScrF1 if the A allele is present, whereas the product from the G allele is cut to produce fragments of 145 and 22 bp (Fig. 1). 2.3. Statistical analysis Analysis of data was performed using the computer software SPSS for Windows (version 12.0) and Epi Info (version 6.04) (SPSS Inc, Chicago, IL). Chi-square analysis was used to compare categorical variables, and a 5% level of significance was used in the analysis. The odds ratio (OR) and its 95% confidence interval (CI) were calculated as a measurement of the association between CCND1 genotypes and cervical cancer risk. Hardy-Weinberg equilibrium was tested by a Pearson goodness-of-fit test to compare the observed versus the expected genotype frequencies. Logistic regression analysis was used to calculate the adjusted OR (aOR) and 95% CI for the influence of CCND1 genotypes in the risk of cervical cancer, with adjustment for age. We calculated the attributable proportion (AP) using the following formula: AP ⫽ PRF × (1-1/OR), where AP is the fraction of disease attributable to the risk factor, PRF

2.2. Polymerase chain reaction/restriction fragment length polymorphism analysis (PCR-RFLP) DNA was extracted from peripheral blood leukocytes from each study subject using a salting out protocol [37]. Detection

Fig. 1. Analysis of the CCND1 genotypes – RFLP of the PCR products. M, 100-bp ladder; cases 1, 3, and 7, CCND1 heterozygous (G/A); cases 2, 4, and 5, homozygous GG; cases 6 and 8, homozygous AA.

R. Catarino et al. / Cancer Genetics and Cytogenetics 160 (2005) 49–54

is the percentage of the risk factor in case subjects, and OR is the odds ratio.

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development of ICC (aOR ⫽ 3.26, 95% CI 1.42–7.53, P ⫽ 0.006). For the ICC patients, the proportion of cervical cancer cases attributable to the GG CCND1 genotype was 17.26%.

3. Results The distribution of CCND1 genotypes among cases and controls and the risk of cervical cancer due to the influence of CCND1 polymorphism are shown in Table 1. The frequency of the G allele was higher in cases (46.9%) than in controls (35.4%), and this difference was statistically significant (P ⫽ 0.011). The frequencies of AA, AG, and GG genotypes were 37.9, 53.4, and 8.7%, respectively, in normal controls, and 30.8, 44.7, and 24.5%, respectively in the case group. The genotype distribution of both groups was in the Hardy-Weinberg equilibrium (P ⫽ 0.672 in the case group and 0.461 in the control group). The analysis of the frequencies of CCND1 genotypes indicates that women carrying two G alleles have a threefold increase in the risk for cervical lesions (aOR ⫽ 3.48, 95% CI 1.57–7.70, P ⫽ 0.0021). The stratification of the analysis according to the patients’ median age (women older than 46 years vs. younger than 46 years) lead to the observation that there is a statistically significant threefold increase of cervical cancer risk for the group of women older than 46 years carrying two G alleles (OR ⫽ 3.20, 95% CI 1.25–8.16, P ⫽ 0.017). No significant differences were found in the frequencies of CCND1 genotypes between different stages of ICC (histologic differentiation), as shown in Table 2. However, women carrying the GG genotype have a 4.32-fold higher risk for the development of high-grade squamous intraepithelial lesions of the cervix (HSIL) (aOR ⫽ 4.32, 95% CI 1.50– 12.46, P ⫽ 0.0067), and a 3.26-fold increased risk for the

4. Discussion Cervical cancer remains a major worldwide health problem, especially in developing countries. During the past decades, the expansion of molecular biology has been of great importance in understanding the basis of cancer development and progression. The mechanism of cervical cancer carcinogenesis is not well understood. Our study is the first report suggesting a role for CCND1 polymorphism in cervical cancer. It is now known that specific types of HPV are the principal etiologic agents for both cervical cancer and its precursors. However, the discrepancy between high rates of HPV infection and low rates of cervical cancer development among women suggests that additional genetic events are necessary for progression to a malignant phenotype, namely alterations in oncogenes and tumor suppressor genes [8]. HPVs encode two proteins, E6 and E7, which are important for cell immortalization. HPV-16 proteins E6 and E7 disrupt cell cycle checkpoints, particularly affecting nearly all cyclin-dependent kinase inhibitors linked to the G1 and G2 checkpoints, in each case through a different mechanism [3]. E7 binds to RB, originating its functional inactivation [4,6]. In quiescent cells, RB is complexed to the transcription factor E2F. Approaching the S phase of the cell cycle, RB becomes hyperphosphorylated, causing release of E2F, which then activates expression of growthassociated genes. E7 bypasses this RB-dependent control by

Table 1 Prevalence and OR of CCND1 genotypes and alleles among control group and patients with cervical lesions (HSIL and ICC) Patients (n ⫽ 103) Allelesa A G Genotype AA AG GG Recessive model AA/AG GG Age ⬍46 years AA/AG GG ⭓46 years AA/AG GG a

Controls (n ⫽ 103)

n

%

n

%

OR

95% CI

P

152 134

53.1 46.9

133 73

64.6 35.4

1.00 1.61

reference 1.09–2.36

0.011

44 64 35

30.8 44.7 24.5

39 55 9

37.9 53.4 8.7

1.00 1.03 3.45

reference 0.59–1.81 1.47–7.56

1.000 0.004

108 35

75.5 24.5

94 9

91.3 8.7

1.00 3.48*

reference 1.57–7.70*

0.0021*

52 16

76.5 23.5

28 2

93.3 6.7

1.00 4.31

reference 0.92–20.10

0.052

56 19

74.7 25.3

66 7

90.4 9.6

1.00 3.20

reference 1.25–8.16

0.017

Total allele numbers in the case group is 286 and in the control group is 206. * P, OR, and 95% CI using logistic regression analysis adjusting for age.

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Table 2 Distribution of CCND1 genotypes among case and control groups and histological stage of ICC CCND1 genotype N (frequency)

Cases Controls (n ⫽ 103) HSIL (n ⫽ 50) ICC (n ⫽ 93) FIGO staging ⬍IIb ⭓IIb

AA/AG

GG

OR

95% CI

P

94 (91.3) 37 (74.0) 71 (76.3)

9 (8.7) 13 (26.0) 22 (23.7)

1.00 4.32* 3.26*

Reference 1.50–12.46* 1.42–7.53*

0.0067* 0.006*

3 (16.7) 7 (26.9)

15 (83.3) 19 (73.1)

1.00 0.543

Reference 0.12–2.46

0.49

Abbreviation: FIGO, International Federation of Gynecology and Obstetrics. * P, OR, and 95% CI using logistic regression analysis adjusting for age.

binding to RB and causing the nonphysiological release of active E2F [38,39]. HPV infection may constitute the initial cause of subsequent genetic alterations leading to the development of cervical tumors, whether the mechanism is due to direct integration of HPV DNA, subsequent interaction with other cellular proteins, or both [40]. The accumulation of genetic abnormalities as a result of alterations in cell cycle checkpoint control is likely to be an important mechanism in the process of cellular immortalization by high-risk HPVs [41]. CCND1 encodes cyclin D1 protein, which is expressed in response to mitogenic signals, promoting transition through the restriction point in the G1 phase of the cell cycle [23]. Southern and Herrington [42] reported the absence of CCND1 expression in the majority of naturally occurring low-grade cervical lesions infected with high-risk HPVs, in contrast with the overexpression of CCND1 in most lesions containing low-risk HPVs. These findings are compatible with the in vitro observation that CCND1 overexpression is not required for G1 phase progression in human keratinocyte cell lines expressing the E6 and E7 proteins of high-risk HPVs [43]. The binding of the E7 protein of high-risk HPVs to the RB protein obviates the requirement of the cell for CCND1 and ensures the unrestricted release of E2F transcriptional factors. This, in turn, leads to S-phase entry, thereby inducing the expression of genes required for cellular and viral synthesis [43]. HPV-16 E7 shows homology with the RB-binding sites of CCND1, consequently inducing the release of E2F transcriptional factors [44]. D-type cyclins bind to the pocket domain of hypophosphorylated RB through their LXCXE sequences, which are shared with several DNA tumor viruses [45]. Several genetic polymorphisms contributing to an individual’s susceptibility to cancer have been studied regarding their association with cervical cancer risk [46,47]. In this study, we analyzed a single-nucleotide polymorphism in CCND1 to evaluate its importance in the development of cervical cancer. The A870G polymorphism at codon 242 within the conserved splice donor site of exon 4 of the gene appears to modulate the splicing of CCND1 mRNA,

originating two transcripts (a and b) that are present in a variety of tissues [25,48,49]. The transcript a is identical to the reported CCND1 cDNA [12]. Transcript b, however, fails to splice at the exon 4/intron4 boundary, does not contain exon 5, and terminates downstream of exon 4. The main difference in the cyclin D1 proteins encoded by the two transcripts (a and b) is in the C-terminal PEST-rich region (destruction box) encoded by exon 5, which is responsible for rapid intracellular degradation and turnover of the G1 cyclins [25,48,50]. It has been suggested that the variant A allele is a major source of the variant transcript b in several types of cancer cells [25,29,49]. The AA genotype increases the products of transcript b in tumor tissue cells, resulting in an increase of an altered protein that lacks the PEST region with increased half-life [25,29]. Bae and colleagues [33] reported that CCND1 mRNA and protein expression is decreased in cervical cancer, suggesting that CCND1 expression is regulated at the level of transcription. Moreover, CCND1 expression has been correlated with the CCND1 genotypes, suggesting that the GG genotype is associated with low expression of CCND1 in squamous cell carcinoma [51]. We hypothesize that if the availability of CCND1 in the cell is reduced, there is less CCND1 to interact with the RB in a quantitative manner. Since highrisk HPV E7 protein shows homology with the RB-binding sites of CCND1, the reduced levels of this cyclin in the cell may facilitate the interaction of the HPV E7 protein with the RB. Therefore, CCND1 may not compete with HPV protein E7 for the same binding site in RB, endorsing the binding of E7 to this cell cycle regulator, with consequent release of the transcriptional factors E2F and induction of the expression of genes required for cellular and viral synthesis. Our results suggest that women carrying the CCND1 GG genotype have increased risk for the development of cervical lesions (aOR ⫽ 4.32 for HSIL and aOR ⫽ 3.26 for ICC), and are consistent with previous findings suggesting that the CCND1 GG genotype is associated with cancer development. Matthias and colleagues [27,52] reported that the CCND1 GG genotype was associated with poorly differentiated tumors of the head and neck and reduced the disease-free

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interval in laryngeal and pharyngeal carcinomas independently from tumor differentiation, providing a link between CCND1 A870G alleles with CCND1 expression and clinical outcome in squamous cell carcinoma of the head and neck. A recent report [51] found a significant trend between a reduction in the proportion of CCND1-expressing cells within the tumors of patients with CCND1 GG. Another recently published study reported a correlation between GG genotype and increased susceptibility for laryngeal tumor development [53]. However, controversial results have been reported regarding the role of CCND1 genotypes in cancer development [26,28– 30,32]. The mechanism for this association is unknown. Although the G allele splices less of transcript b than the A allele, individuals with CCND1 GG may have different cellular levels of CCND1 than subjects with CCND1 AA. Furthermore, these results suggest that the effect of genotype on tumor behavior may exhibit some degree of tissue specificity [27]. It is possible that these conflicting results in part reflect the many different mechanisms through which deregulated expression of CCND1 can occur in cancer. The contribution of genetic polymorphisms to the risk for cervical cancer may be dependent on the studied population, as well as on several environmental and other factors that influence that population. Geographical or ethnic differences have been reported regarding the genotype frequency of several polymorphisms. Our results within the Portuguese population are consistent with a recently published study in our population [53]. To the best of our knowledge, this is the first study that demonstrates an association between the A870G CCND1 polymorphism and risk for cervical cancer. Our results suggest that the A870G CCND1 polymorphism could act as a cofactor of HPV in the initiation of cervical carcinogenesis, particularly in the transformation zone of HPV infected women, supporting evidence for a genetic factor on ICC risk. Further studies may include the analysis of other genetic polymorphisms (CYP2E1, GST, ecNOS, ARStuI, VDR) that have been already associated with cancer risk [54–58] to characterize the genetic profile of cervical cancer susceptibility. Moreover, to assess the gene–virus interaction, additional studies should include an ideal control group of HPV-positive women without cervical cancer. The determination of a genetic profile may help to explain the observation that not all high-risk, HPV-infected HSIL lesions will progress to invasive carcinomas. Larger-scale molecular studies are needed to confirm the role of A870G polymorphism of CCND1 in cervical cancer. These results can lead to a better understanding of cyclin D1 influence on cancer, the biologic mechanisms of cervical cancer, and in the definition of a genetic profile for this disease.

Acknowledgments The authors thank the Liga Portuguesa Contra o Cancro (Portuguese League against Cancer), Centro Regional do

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Norte for their support. We gratefully acknowledge funding of this work by the Ministry of Health of Portugal (CFICS – 206/2001). Ana Matos is currently affiliated with Ju´lio Dinis Maternity.

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