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Impact of MDM2 SNP309T>G Polymorphism: Increased Risk of Developing Non Small Cell Lung Cancer and Poor Prognosis in Indian Patients

Jamsheed Javid1, AB Rashid Mir1, Imtiyaz Ahamad1, Shazia Farooq1, Prasant Yadav1, Mariyam Zuberi1, Masroor M1, Ray PC1, Julka PK2, Anant Mohan3, Maqbool Lone4, Banday MA5 and Alpana Saxena1*

1Molecular Oncology Lab, Department of Biochemistry, Maulana Azad Medical College and Associated hospitals, New Delhi, India

2Department of Radiotherapy, All India Institute of Medical Sciences, New Delhi, India

3Department of Medicine, All India Institute of Medical Sciences, New Delhi, India

4Department of Radiation Oncology, SKIMS, Srinagar, India

5Associate Professor, Department of Medical Oncology, SKIMS, Srinagar, India

*Corresponding Author:
Alpana Saxena
Director, Professor and Head
Department of Biochemistry
Maulana Azad Medical College and Associated Hospitals
New Delhi 110002, India
Tel: +91-9868937401
E-mail: [email protected],
[email protected]

Received date: August 16, 2012; Accepted date: September 22, 2012; Published date: September 24, 2012

Citation: Javid J, Rashid Mir AB, Ahamad I, Farooq S, Yadav P, et al. (2012) Impact of MDM2 SNP309T>G Polymorphism: Increased Risk of Developing Non Small Cell Lung Cancer and Poor Prognosis in Indian Patients. J Cancer Sci Ther 4: 341-346. doi: 10.4172/1948-5956.1000165

Copyright: © 2012 Javid J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Background: MDM2 is an important negative regulator of the TP53 pathway, over expressed in many cancers as oncoprotein. Polymorphisms in the promoter region of the MDM2 gene have been shown to alter protein expression and may, thus play an important role in carcinogenesis.
 
Aim and methods:
To test our hypothesis that the MDM2 promoter polymorphisms are associated with risk of non small cell lung cancer, we conducted a hospital-based, case–control study of 136 Indian patients diagnosed with NSCLC and 136 cancer-free controls and investigated the association between genetic variation in the promoter region of MDM2 (c.–51309G4T, rs2279744:g.G4T) and the risk of developing NSCLC by tetra-primer ARMS-PCR and ASO-PCR.
 
Results:
Compared with the MDM2-2580TT genotype, we found that the MDM2-309G variant genotypes were associated with an increased risk of NSCLC in Indian patients [OR 3.88 (1.82-8.27) RR 1.94 (1.27-2.96) RD 32.6 (15.7-49.6) p 0.0004 for GG and OR 2.60 (1.49-4.57) RR 1.52 (1.20-1.93) RD 23.16 (10.3-36.0) p 0.0009 for GT genotype]. GG genotype was found to be associated with poor survival outcome of NSCLC patients and in addition significant association was observed with stage (p 0.01) and metastasis status (p 0.002) of NSCLC patients.
 
Conclusion: Genetic polymorphism in cell cycle regulatory genes MDM2 contribute to the risk of developing NSCLC in Indian Patients. In addition G allele was associated with an increased risk and poor survival outcome than T allele.

Keywords

MDM2; SNP309T>G polymorphism; Non small cell lung cancer; Tetra-primer ARMS-PCR and ASO-PCR; Amplification refractory mutation system; Allele specific oligonucleotide

Abbreviations

MDM2: Murine Double Minute 2; ARMS: Amplification Refractory Mutation System; ASO: Allele Specific Oligonucleotide; NSCLC: Non Small Cell Lung Cancer

Introduction

Lung cancer, the most common form of malignancy leading to the major cause of cancer related deaths around the world [1] including India. Lung cancer constituted 14.4% of all cancers in a review of 9210 consecutive autopsies by Banker [2]. Cigarette smoking constitutes 80% of the attributable risk to lung cancer, but only a small proportion of smokers will develop lung cancer, suggesting that there is an interindividual variation in genetic susceptibility to lung cancer in the general population [1]. The human homologue of MDM2 located on chromosome 12q13-14 with genomic size of 34 kb is a negative regulator of the tumor suppressor gene p53 [3]. The encoded protein is a nuclear phosphoprotein that binds to p53 and inhibits p53-dependent transcription [4]. Over expression of this gene can result in excessive inactivation of p53, which enables damaged cells to escape the cellcycle checkpoint control and become carcinogenic [5,6]. MDM2 has also been shown to promote tumor growth in a p53-independent manner through interaction with transcriptional factors of the E2F family [7], inhibition of the Rb growth regulatory function [8] and inhibition of G0/G1-S-phase transition in normal cells [9]. In tumors, the overexpression of MDM2 mRNA and proteins can substitute for p53 inactivation in the absence of p53 mutations [10] and is thus, often associated with the clinical behavior of the tumors, such as cancer progression and treatment response [11].

MDM2 polymorphisms in a regulatory region, such as the promoter, may alter its transcriptional activities, there by affecting p53 tumor suppression and carcinogenesis in humans. Therefore, MDM2 functional promoter polymorphisms probably contribute to inter individual variation in susceptibility to lung cancers as evidenced by an early age of onset [12]. Thus present study aimed to investigate the effect of the MDM2 promoter polymorphism (309T/G) on the risk and susceptibility of NSCLC in Indian patients.

Materials and Methods

Study population

Clinically confirmed NSCLC patients were selected from an on going molecular study of NSCLC Conducted in Department of Biochemistry, Maulana Azad Medical College & associated hospitals, New Delhi. The study includes 136 NSCLC patients and 136 healthy controls frequency matched with age (±5years) and gender. Patients with a history of previous cancer or metastasized cancer from other organs except lung were excluded. All controls, like the cases, were the residents of North India. The study was approved by the institutional ethics committee, MAMC, New Delhi. Written informed consent was obtained from all subjects. Patient follow up was obtained through of hospital records as well as by direct patient contact.

Genotype analysis

Genomic DNA was extracted from blood samples using DNA sure blood mini kit (Nucleo-pore Genetix brand) according to the manufacturer’s protocol. MDM2 (NM_ 002392.2) c.-5+309T/G, rs 2279744:g.T/G genotypes were analyzed using ASO [13] and tetra primer ARMS-PCR [14]. Genotyping was performed without the knowledge of the case/control status of the study subjects. The amplification was accomplished with a 25 ul reaction mixture containing 5 ul of 20 ng template DNA, 0.25 ul of 25 pmol each Primers, 2.5 ul 10 mM dNTPs, 1.5 ul of 20 mM MgCl2, 0.3 ul of 5 U/ul Taq polymerase with 2.5 ul of 10X Taq Buffer (Fermantas). The amplification conditions were 10 min of initial denaturation at 95°C; 40 cycles at 95°C for 45s, 66.7°C for 45s and 72°C for 45s with a final 10 min extension step at 72°C. ASO- PCR amplification reaction and conditions were same as above except 60°C of annealing temperature with ASO specific. PCR products were visualized on ethidium bromide stained 2% agarose gel (Figure 1A and 1B).

cancer-science-therapy-ASO-PCR

Figure 1a: ASO PCR MDM2 T309G: L–100bp ladder, L2, L–Homozygous TT, L4, L5-Homozygous GG, L6, L7–Heterozygous GT, and L8–Negative control.

cancer-science-therapy-ARMS-PCR

Figure 1b: ARMS PCR MDM2 T309G: L1-100bp ladder, L2, L3-Homozygous GG, L4, L5–Homozygous TT, L6, L7-Heterozygous GT and L8–Negative control.

Statistical analysis

Differences in select demographic variables and MDM2 genotype frequencies between the cases and controls were evaluated by using the Chi–square test. The associations between MDM2 variant genotypes and risk of lung cancer were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) from both univariate and multivariate logistic regression analyses. Fisher Exact Test was performed for the values below 5. Statistical difference was considered significant for P values<0.05.

Results

General characteristics of study population

The demographic variables among cases and healthy controls are summarized in (Table 1). Briefly among NSCLC cases 112 were male and only 24 were females. Smokers constitute more than 75 percent with 39.6% as current and 60.4 as ex-smokers, who had left smoking from more than 6 months. Only few patients (13.2%) were with some significant family history of lung cancer or any other cancer.Histological studies revealed 79 cases with Squamous cell carcinoma, 57 cases with adenocarcinoma. Two age groups were made, patients with age <45 included 24 cases and >45 included 112 cases. More than 60% of the patients were in advanced stages with 25% cases positive for distant metastasis.

 Variable NSCLC patients (%) Healthy Controls (%)
Total No. 136 136
Sex
Males 112(82.3) 112(82.3)
Females 24(17.7) 24(17.7)
Age (Years)
≤45 22(16.2) 22(16.2)
>45 114(83.8) 114(83.8)
Mean ± SD age (Y) 57.39 ±10.9 56 ±10.3
Smoking Status
Non Smoker 35(24.3) 136(100)
Smokers 101(75.7)  
Current smokers 40(39.6)  
Ex-smokers 61(60.4)  
Smoking Level (Pack Year)  
Mild (≤10) 10(9.9)  
Moderate (≤40) 45(44.6)  
Heavy (>40) 46(45.5)  
Histological Type  
SCC 79(58.1)  
ADC 57(41.9)  
TNM Stage  
Early (I & II) 51(37.5)  
Advanced (III & IV ) 85(62.5)  
Distant Metastasis  
Positive 34(25.0)  
Negative 102(75.0)  
Family History of any Cancer  
Significant 18(13.2)  
Non Significant 118(86.8)  
Smoking Type  
Cigarette 29(28.7)  
Bidi 13(12.9)  
Huka 26(25.7)  
Cigarette+Bidi 11(10.9)  
Cigarette+Huka 22(21.8)  
Cytological Type  
Squamous cell carcinoma  
Well differentiated 43(54.4)  
Moderately Differentiated 22(27.8)  
Poorly Differentiated 14(17.7)  
Adenocarcinoma  
Well differentiated 11(19.3)  
Moderately Differentiated 16(28.1)  
Poorly Differentiated 30(52.6)  

Table 1: Distribution of Selected Characteristics among NSCLC patients.

Allele and genotype distribution

The genotyping results are shown in Table 2A. The frequencies of MDM2-TT, -TG, and -GG genotypes among patients were significantly different compared to healthy controls (Chi square-16.6, df 2 & p-0.0003), with the GG homozygotes being significantly overrepresented among patients compared to controls (22.0% vs.11.8%) The frequency of the genotypes TT, TG and GG among healthy controls were 42.6%, 45.6% and 11.8% and NSCLC patients were 20.6%, 57.4% and 22.0% respectively. The frequencies of MDM2-TT, -TG, and -GG genotypes with respect to TNM stage (p 0.01) and metastasis status (p 0.002) among patients were statistically significant. whereas their was not any significant difference with respect to other parameter’s like gender, age and Smoking status (Table 2B).

Genotype TT TG GG T allele frequency G allele frequency
Patients (n=136) 28(20.6) 78(57.4) 30(22.0) 0.49 0.51
Controls (n=136) 58(42.6) 62(45.6) 16(11.8) 0.65 0.35
Chi-Square=16.55 df=2 P<0.0003      

Table 2A: Genotype frequencies of MDM2 309 T/G among NSCLC cases and controls.

Variables TT Genotype n(%) TG Genotype n(%) GG Genotype n(%) T allele frequency G allele frequency Chi-Square df P –value
Gender
Males 26(23.2) 65(58.0) 21(18.8) 0.52 0.48 5.3 2 0.06
Females 2(8.3) 13(54.2) 9(37.5) 0.35 0.65      
Age at diagnosis (Years)
≤45 3(13.6) 15(68.2) 4(18.2) 0.48 0.52     0.56
>45 25(21.9) 63(55.3) 26(22.8) 0.5 0.5      
Histological Type
SCC 19(24.1) 41(51.8) 19(24.1) 0.5 0.5 2.41 2 0.29
ADC 9(15.8) 37(64.9) 11(19.2) 0.48 0.52      
TNM Stage
Early Stage (I&II) 10(19.6) 36(70.6) 5(9.8) 0.55 0.45 8.09 2 0.01
Advanced Stage (III& IV) 18(21.1) 42(49.5) 25(29.4) 0.46 0.54      
Metastasis
Positive 10(29.4) 11(32.4) 13(38.2) 0.46 0.54 12.03 2 0.002
Negative 18(17.6) 67(65.7) 17(16.7) 0.5 0.5      
Family History of any Cancer
Significant 9(50.0) 8(44.4) 1(5.6) 0.72 0.28     0.004
Non Significant 19(16.1) 70(59.3) 29(24.6) 0.46 0.54      
Smoking Status    
Non Smoker 4(11.4) 24(68.6) 7(20.0) 0.46 0.54 3.05 2 0.22
Smokers 24(23.7) 54(53.5) 23(22.8) 0.5 0.5      
Smoking Level ( Pack Year)
Mild (≤10) 0(0) 9(90.0) 1(10.0) 045 0.55 10.3 4 0.036
Moderate (≤40) 10(22.2) 27(60.0) 8(17.8) 0.52 0.48      
Heavy (>40) 14(30.4) 18( 39.0) 14(30.4) 0.5 0.5      
Smoking Type
Cigarette 8(27.6) 16(55.2 ) 5(17.2) 0.55 0.45     0.99
Huka 7(27.0) 14(53.8) 5(19.2) 0.54 0.46      
Bidi 3(23.0) 8(61.6) 2(15.4) 0.54 0.46      
Cigarette+Bidi 1(9.0) 4(36.4) 6(54.6) 0.27 0.73     0.08
Cigarette+Huka 9(40.9) 12(54.5) 5(22.7) 0.58 0.42      
Cytological Type
Squamous Cell Carcinoma
Well Differentiated 11( 25.5) 22(51.2) 10(23.3) 0.51 0.49     0.56
Moderately Differentiated 7(31.8 ) 10(45.5) 5(22.7) 0.55 0.45      
Poorly Differentiated 1(7.1 ) 9(64.3 ) 4(28.6) 0.39 0.61      
Adenocarcinoma
Well Differentiated 2(18.2) 9(81.8 ) 0(0 ) 0.59 0.41     0.07
Moderately Differentiated 0(0) 12(75.0) 4(25.0) 0.38 0.63      
Poorly Differentiated 7(23.3) 16(53.3) 7(23.3) 0.5 0.5      

Table 2B: Associations and stratification analysis of MDM2 309T/G polymorphism and NSCLC.

MDM2 309 T/G polymorphism and NSCLC risk

An unconditional logistic regression was used to estimate associations between the genotypes and risk of lung cancer (Table 3). It was found that an increased risk of lung cancer was associated with the MDM2 G allele in an allele dosage-dependent manner. Compared to the TT genotype, the ORs 2.60 (1.12-3.88) and 3.88 (0.96-5.97) RR 1.52 (1.49-4.57) and 1.94 (1.27-2.96), RD 23.16 (10.3-36.0) and 32.6 (15.7-49.6) for the TG and GG genotypes, suggesting a possible dominant effect of this polymorphism.

MDM2 G2580T Genotype Cases (n=136) Control (n=136) OR*(95% CI) RR** RD*** P value
TT(ref) 28 58 1.00 1.00    
TG 78 62 2.60(1.49-4.57) 1.52(1.20-1.93) 23.16(10.3-36.0) 0.0009
GG 30 16 3.88(1.82-8.27) 1.94(1.27-2.96) 32.6(15.7-49.6) 0.0004
TG+GG 108 78 2.87(1.68-4.91) 1.61(1.29-2.01) 25.5(13.3-37.7) 0.0001

Table 3: Risk of lung cancer associated with the MDM2 (-309T/G) genotypes.

MDM2 genotypes and survival analysis

Follow-up of patients regarding survival was performed with median duration of 11.25 months (range, 0.5-127.5 months). A total of 84 patients suffered cancer related deaths with mean follow up time of 10.73 months and for the patients who survived the follow up period (censored patients), the follow-up time was 17.61 months. It was observed that the MDM2 309GG (Figure 2A) and GT+GG (Figure 2B) genotype was significantly associated with poor survival p<0.0001. Patient’s survival was calculated using log-rank (Mantel–Cox) test. The estimated median survival time for patients with 309 TT, TG, GG+TG and GG genotype were 35.5, 13.5, 11.0 and 6.0 months respectively.

cancer-science-therapy-NSCLC-patients

Figure 2: Kaplan-Meier survival curves for NSCLC patients with respect to MDM2 (309T/G) polymorphism.

Discussion

In the present study, we examined whether genetic polymorphisms in MDM2 is associated with the risk of developing NSCLC. Our results obtained by analyzing 136 NSCLC patients and 136 controls demonstrate that the functional polymorphisms in the MDM2 promoter have a significant impact on the risk of developing NSCLC. The GG genotype was associated with the approximately 3.9 fold increased risk of NSCLC and was more predominant in subjects who were in advanced stage (p<0.01) with positive metastasis status at diagnosis (p<0.002). The frequency of GG allele was more among patients with squamous-cell carcinoma than adenocarcinoma of NSCLC. Patients of old age group (>45y) and females also showed higher frequency of risk allele (309GG). Higher frequency of GG genotype was found in patients who smoke cigarette as well as bidi as their smoking habit. Individuals with at least one 309T allele were at decreased risk of overall NSCLC as compared to those with 309GG genotype. Histologically poorly differentiated SCC and moderately differentiated ADC patients showed higher percentage of 309GG genotype.

The observations in this study are biologically plausible and consistent in several ways with previous studies. MDM2 negatively regulates the TP53 pathway that targets TP53 for degradation, and over expression or amplification of MDM2 has been frequently observed in many human cancer types, including lung cancer [13,15-18]. The high expression level of MDM2 is associated with the susceptibility to carcinogenesis. Over expression of mdm2 in the murine mammary epithelium resulted in mammary tumors [19], MDM2-transgenic mice developed tumors, produced an average of four fold more mdm2 in various tissues relative to non transgenic mice [20].

MDM2 has been shown to interact with several key tumor suppressors, including Rb [11], p53-family protein, p73 [21], the growth suppressor p14/p19 [22] and p53 [23]. Over expression of MDM2 and the subsequent expression of p21 are induced by p53 in response to DNA damage caused by exposure to environmental carcinogens, leading to an arrest of cell cycle, which in turn allows sufficient time for DNA repair [24]. Over expression of MDM2 was correlated with a decreased level of p21 expression in epithelial breast cancer but with an increased level of p21 in oral squamous cell carcinoma.

MDM2G309T polymorphism occurs near the element believed to direct the intronic p53-response promoter activity of the gene, thus resulting in increased levels of MDM2 RNA and protein in an in vitro functional assay [12]. The G allele is correlated with a higher promoter activity which increases the binding affinity of Sp1 to the promoter of MDM2 [12], resulting in increased MDM2 expression which causes high rate of P53 protein degradation and may result in decreased apoptosis and ultimately the cancer. °Thus individuals carrying G allele may be at higher risk for developing NSCLC.

Various reports suggest an increased risk of lung cancer for the G allele of the MDM2 SNP309 in populations of various countries [14,25,26] (Figure 3). Our results were similar to the report of Zhang et al. [15].

cancer-science-therapy-World-scenario

Figure 3: World scenario of MDM2 309GG genotype in lung cancer.

Present study found that the risk was higher for squamous cell lung cancer may probably induced by tobacco smoke [27]. Significant death was observed in cells with the MDM2 TT genotype (20-35% of the total cell population) but not in cells with the MDM2 GG genotype (only 2-3% of the total cell population) after etoposide treatment to induce DNA damage, which activates the TP53 pathway and leads to DNA repair, cell cycle arrest, and apoptosis [12]. In addition MDM2 polymorphism was significantly associated with the poor survival outcome of patients with 308GG genotype and the median survival times were found to be of just 6 months. To the best of our knowledge, this is the first case-control study to investigate the polymorphism in cell cycle regulatory gene MDM2 (309T/G) and its association with NSCLC risk among Indian Patients [28-32].

Conclusion

In conclusion, present study shows that genetic polymorphism in cell cycle regulatory genes MDM2 contribute to the risk of developing NSCLC in Indian Patients. In addition 309GG genotype was associated with an increased risk than 309TT genotype. Study further suggest that 309T/G polymorphism in MDM2 gene might be a useful genetic marker in peripheral blood to determine susceptibility to advanced stage with distant metastasis status of NSCLC. However, because this is the first report concerning the MDM2 polymorphism and the risk of NSCLC in the Indian population, independent large population-based prospective studies for more rigorous analyses of subgroups are needed to validate our findings.

Acknowledgement

We thank Regional Cancer Center SKIMS, Srinagar and All India Institute of Medical Sciences, New Delhi, for assistance in recruiting the subjects.

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