Abstract
Aim: The aim of this study was to evaluate the contribution of human mouse double minute 2 (MDM2) gene polymorphisms to the risk of Taiwan lung cancer. Materials and Methods: In this case-control study, the association of MDM2 rs2279744 genotypes with lung cancer risk was investigated among 358 lung cancer patients and 716 age-, gender- and smoking status-matched controls in Taiwan. Results: The percentages of MDM2 rs2279744 GT and GG genotypes were 50.0% and 27.4% in lung cancer group and 50.0% and 26.5% in control group, respectively [odds ratio (OR)=1.03 and 1.07, 95% confidence interval (CI)=0.75-1.43 and 0.75-1.53, respectively]. The analysis about allelic frequency showed that G allele at MDM2 rs2279744 conferred a non-significant increased cancer risk (OR=1.03, 95%CI=0.86-1.24). Conclusion: Polymorphisms of MDM2 rs2279744 may play a role in lung carcinogenesis. However, the studied genotypes were not shown as predictors of lung cancer susceptibility.
According to GLOBOCAN 2018 statistics about cancer occurrence and mortality reported by the International Agency for Research on Cancer, lung cancer is the first cause of cancer death, accounting for about 2.1 million newly-diagnosed cases and 1.8 million deaths worldwide (1). Thus, it is urgently important for translational scientists to figure out effective methodologies predicting and preventing the risk of lung cancer, improving prognosis and extending the survival periods for the lung cancer patients.
The initiation and development of lung cancer is related to complicated factors including environmental, lifestyle, and genomic discrepancies, among which cigarette smoking behavior is the most risk one (2). Nevertheless, 10% to 25% of the lung cancer patients are non-smokers worldwide, and this fact indicates that the personal genomic factors also play as determiners of carcinogenesis for personal lung cancer etiology (3). In Taiwan, the incidence and mortality of lung cancer has occupied the third and first places among the common cancers for decades (4). In Taiwan, although several clinically useful genomic biomarkers for the prediction, diagnosis, and prognosis of lung cancer risk have been proposed during the recent years (5-10), the investigations about clinically practical genomic markers and the intracellular mechanisms are still in urgent need.
Mouse double minute 2 homolog (MDM2; also known as E3 ubiquitin-protein ligase), encoded by the MDM2 gene in human genome, acts as an E3 ubiquitin ligase, recognizing the N-terminal trans-activation domain at p53, and also suppresses the activation of p53 transcription. The most common single nucleotide polymorphism (SNP) of MDM2 gene is known as “rs2279744, “SNP309”, and “SNP309T>G”; it has been examined for its role in cancer etiology and treatment responses. MDM2 rs2279744 has been identified as a T to G substitution at the nucleotide 309 in the first intron, regulating the binding affinity between MDM2 and transcription factor Sp1 (11). In literature, MDM2 rs2279744 has been reported to be closely related to accelerated tumor formation in both hereditary and sporadic cancers and to serve as a rat-limiting factor during tumorigenesis (11). Thus, it is hypothesized that the genomic variant may critically determine the expression level of MDM2 and influence individual's susceptibility to several types of cancer. In literature, several studies have examined the contribution of MDM2 rs2279744 genotypes to various tumors, while the results are not so conclusive among the different ethnicities investigated and various types of cancer (12-14). Some studies have proposed a direct association between MDM2 rs2279744 and cancer risk (15-20); however, other reports have not confirmed this finding (21-24).
Summary of selected demographics of the 358 patients with lung cancer and the 716 matched controls.
In 2008, Liu and his colleagues have reported that there is no significant association between MDM2 rs2279744 genotypes and lung cancer risk among Caucasians (25). The group of Hancox reported that the G allele at MDM2 rs2279744 was associated with not only higher MDM2 expression levels, but also with a reduced p53 response, and hence more DNA damage and the corresponding tissue destruction. Interestingly, the G allele was associated with accelerated smoking-related decline in lung function (26). According to the literature mentioned above, MDM2 genotype and its expression may contribute to the etiology of lung cancer risk. Therefore, the aim of the current study was to reveal the genotypes of MDM2 rs2279744 among Taiwanese and evaluate their association with lung cancer risk, in Taiwan.
Materials and Methods
Selection of lung cancer patients and controls. Three hundred and fifty-eight patients with lung cancer were histologically confirmed and recruited at the medical center in central Taiwan with the approval of the Institutional Review Board of China Medical University Hospital (DMR100-IRB-284), as previously described (5, 6, 10). Briefly, the exclusion criteria of the cases were: history of any other malignancy and pulmonary diseases, such as chronic obstructive pulmonary disease (COPD), pneumothorax, and asthma. During the same period, 760 healthy volunteers were selected from the databank of Health Examination Cohort of China Medical University Hospital with more than 15,000 individuals as controls, matched for their age (differences less than 5 years), gender, and smoking behavior. The exclusion criteria of the control group included previous malignancy, metastasized cancer from other known or unknown origin, and any genetic or familial diseases. The controls and cases were all Taiwanese; their selected recorded characteristics are summarized in Table I.
Genotyping conditions for MDM2 rs2279744. Extraction of genomic DNA from peripheral blood leukocytes was conducted within 24 h with the QIAamp Blood Mini kit (Blossom, Taipei, Taiwan) as usual (27, 28). Then the DNA was quantified, stored long-term at −80°C, diluted and aliquoted for genotyping as a working stock at −20°C as is routinely conducted (29, 30). The MDM2 genotyping working flow including the designing of the forward and reverse primer sequences, the selection of corresponding restriction endonucleases, and the polymerase chain reaction (PCR) cycling condition set-up were previously described (31). Briefly, the cycling conditions of PCR procedure were set as: one cycle at 94°C for 5 min; followed by 35 cycles of 94°C for 30 s, 59°C for 30 s and 72°C for 30 s; last, an extension at 72°C for 10 min. The PCR products were harvested immediately after the final extension, and subjected to DNA electrophoresis. The sequences for the forward and reverse primers were 5’-GTTTTGTTGGACTGGGGCTA-3’ and 5’-CTGCGATCATCCGGACCT-3’, respectively. After the first check of successful PCR amplification procedures, all the PCR products were enzymatically digested by MspA II restriction endonuclease (New England Biolabs, Beverly, MA, USA) and then separated and identified of their MDM2 genotypes using 3% agarose gel electrophoresis. All the genotypic processing was repeated by at least two expert scientists in our team independently and blindly, as expected, the results were 100% concordant to each other.
Distributions of MDM2 rs2279744 genotypic frequencies among lung cancer patients and healthy controls.
Statistical analysis methods. The typical unpaired Student's t-test was applied to compare the distributions of continuous variables such as ages between the lung cancer patients and the control groups. The Pearson's Chi-square was applied in comparison of the distributions of the MDM2 genotypes among the investigated subgroups in comparison. The associations between MDM2 polymorphic genotypes and lung cancer risk were estimated by calculating the odds ratios (ORs) and the responding 95% confidence intervals (CIs) within logistic regression analysis. Statistically, any comparison with p-value less than 0.05 was identified as significantly different to each other.
Results
The distributions of age, sex and smoking status for the 358 lung cancer patients and 716 non-cancer healthy controls, are summarized and compared in Table I. In addition, the histology of the lung cancer patients are also collected at the bottom of Table I. Since we have applied the matching strategies focusing on age, gender and smoking status to recruit those 716 non-cancer healthy subjects as controls, the comparison results showed that there was no difference in respect to the distributions of age, gender and smoking behavior between the case and control groups (p-values all >0.05) (Table I). Among the lung cancer patients, 60.9% (218/358) were adenocarcinoma type, 29.6% (106/358) were squamous cell carcinoma type, and 9.5% (34/358) were other types.
The distributions of the MDM2 rs2279744 genotypes among the 358 lung cancer patients and 716 non-cancer healthy controls are presented in Table II. Overall, the genotypes of MDM2 rs2279744 were non-differentially distributed between the case and control groups (p for trend=0.9348) (Table II). In detail, neither the MDM2 rs2279744 GG nor the GT was associated with altered lung cancer risk, compared to the wild-type TT genotype (crude OR=1.03 and 1.07, 95%CI=0.75-1.43 and 0.75-1.53, p=0.8238 and 0.7135, respectively). After adjusting the confounding factors, the trends were still the same (adjusted OR=1.06 and 1.14, 95%CI=0.78-1.39 and 0.81-1.66, respectively). In the dominant model, there was still a non-significant association between the GT+GG genotype of MDM2 rs2279744 and lung cancer risk, compared to the TT wild-type genotype before and after adjusting those confounding factors.
So as to confirm the findings deduced from Table II, we have further conducted the analysis of allelic frequency distributions for the MDM2 rs2279744 and the results are shown in Table III. In consistence with the idea that MDM2 rs2279744 genotype was not associated with lung cancer risk, the percentage of allele G was 52.4% among the lung cancer patients, non-significantly higher than that of 51.5% among the non-cancer controls (crude and adjusted ORs=1.03 and 1.06, respectively) (Table III).
Discussion
The role of MDM2 in carcinogenesis can be both p53-dependent and p53-indepnedent. First, MDM2 is originally recognized as an oncoprotein which can directly bind to tumor suppressor p53 and down-regulate its tumor suppressive capacity. After binding with p53, MDM2 enhances the expression of p53 and the p53-mediated transactivation of MDM2, which in turn team up together to suppress the growth of soft tissue sarcomas, osteosarcomas and esophageal sarcomas (32, 33). Therefore, in the very beginning, the oncogenic properties of MDM2 were identified as p53-dependent. However, there are also some investigations showing that MDM2 is able to directly interact with other tumor suppressor proteins in p53-deficient cells (34, 35). In the same context, elevated expression of MDM2 by Ras/Raf/MEK/MAP kinase cascade has been observed in p53-deficient cells (36). All these findings indicate that the oncogenic function of MDM2 may be p53-independent.
Regarding the regulation of the MDM2 protein level, the case is more complicated. It has been found that MDM2 expression levels might be regulated at the post-transcriptional level (37, 38). The aforementioned literature all supported the idea that the complex molecular mechanisms underlying the regulation network of MDM2 gene at DNA, RNA and protein levels during tumorigenesis are largely unrevealed. Considering that MDM2 rs2279744 may closely be responsible for the regulation of MDM2 protein expression in addition to its interaction with p53, it is of great value to figure out its contribution to cancer susceptibility determination.
Allelic frequencies for MDM2 rs2279744 polymorphisms among lung cancer patients and healthy controls.
In the study, we have firstly provided solid evidence for that variant GT or TT genotype at MDM2 rs2279744 was not associated with Taiwan lung cancer risk (Table II). Also, allele T or G may not serve as a predictor for lung cancer occurrence (Table III). According to previous lung cancer genomic studies, whether MDM2 rs2279744 has a contribution to lung carcinogenesis is still controversial. In detail, some positive findings have supported that the GT and GG genotypes at MDM2 rs2279744 are associated with higher cancer risk than the wild-type TT genotype (15-20), whereas, other reports have shown no significant association with altered cancer risk (21-24). In Taiwanese population included in the present study, the MDM2 rs2279744 homo-variant GG carriers, but not GT, were found to have an increased CRC risk compared with those carrying TT genotype (31). However, MDM2 rs2279744 genotypes were not associated with lung cancer risk (Tables II and III).
Cigarette smoking is the major environmental risk factor for lung cancer, especially for non-small cell lung cancer (NSCLC) (39). Several compounds in cigarettes may induce DNA damage, thus contributing to carcinogenesis (40, 41). The tumor suppressor protein p53 plays a crucial role in the prevention of cancer initiation through cell cycle arrest, DNA repair, and apoptosis in case of DNA repair failure (42-44). MDM2 gene acts as a negative regulator of p53, and overexpression of MDM2 has been reported in many types of cancer tissues (45). In this study, we investigated the associations between the functional single nucleotide polymorphism of MDM2 rs2279744 and the risk of lung cancer in a large Taiwanese population. An overall association was not found (Tables II and III), and gene-smoking interaction analyses indicated that the variant genotypes were associated with higher risk of lung cancer neither in never smokers nor in ever smokers (data not shown). It has been reported by Liu and his colleagues that although the overall MDM2 rs2279744 genotypes are not associated with lung cancer risk among Caucasians, there is a statistically significant interaction between smoking and MDM2 genotypes. Their stratified analysis according to individual pack-years of smoking status demonstrated that the GG genotype, compared to wild-type T/T genotype, contribute to higher risk for never and mild (<30 pack-years) smokers, while to lower risk for moderate (30-57 pack-years) and heavy smokers (≥58 pack-years) (25). Further investigations are warranted to elucidate the interaction among MDM2 genotype, phenotype and smoking status.
There are several limitations of the current study. Despite the power and the representativeness of the current study, with 358 lung cancer patients and 358 controls are examined as well, a larger number of the overall population would be necessary to further examine the contributions of specific genotypes to lung cancer risk and prognosis. In addition, lung cancer patients can be further classified according to characteristics, such as gender and smoking status, or according to their cancer type, to improve the clinical applicability of the study results.
In conclusion, MDM2 rs2279744 was not associated with lung cancer risk in a representative Taiwanese population. In the near future, we are going to validate the combined effects of p53 and MDM2 on Taiwan lung cancer risk. Validation of our findings in larger population-based studies and in different ethnicities are needed.
Acknowledgements
The Authors appreciate the team of Hsia, Y.-C. Lin, T.-C. Shen and others in collection of all the participants. The perfect technology and efforts from Yu-Ting Chin, Tai-Lin Huang, Yu-Hsin Lin and Yi-Ru Huang are appreciated. This study is supported by the grant from China Medical University Hospital and Asia University (CMU108-ASIA-02).
Footnotes
Authors' Contributions
Research design: Lin YC and Chang WS; patient and questionnaire summaries: Lin YC, Shen TC, Li CH and Hsia TC; experimental work: Wang YC, Li HT and Hsiau YC; statistical analysis: Wang ZH, Wu CN and Gong CL; article writing: Tsai CW and Bau DT; review and revision: Chang WS, Tsai CW and Bau DT.
This article is freely accessible online.
Conflicts of Interest
All the Authors have declared no conflicts of interest regarding this study.
- Received January 13, 2020.
- Revision received January 29, 2020.
- Accepted February 3, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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