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- Materials and Methods
- Disclosure Statement
This study was conducted to determine the impact of a functional tandem repeat minisatellite (MNS16A) polymorphism in the telomerase reverse transcriptase (TERT) gene on the risk of lung cancer, as well as on survival of patients with non-small-cell lung cancer (NSCLC). The effect of the MNS16A variable number of tandem repeat (VNTR) polymorphism on the risk of lung cancer was evaluated in a case–control study that consisted of 937 lung cancer patients and 943 healthy controls. The effect of the polymorphism on survival outcome was evaluated in 703 patients with surgically resected NSCLC. Compared with the VNTR-302 allele, the VNTR-243 allele was associated with a significantly increased risk of lung cancer (adjusted odds ratio, 1.55; 95% confidence interval [CI], 1.07–2.25; P = 0.02). In addition, the genotypes carrying at least one VNTR-243 allele were associated with a significantly increased risk of lung cancer compared with the genotypes with no VNTR-243 allele (adjusted odds ratio, 1.61; 95% CI, 1.09–2.38; P = 0.02). In contrast to the effect of the polymorphism on the risk of lung cancer, the genotypes carrying at least one VNTR-243 allele were associated with a significantly better overall survival in patients with surgically resected NSCLC (adjusted hazard ratio, 0.51; 95% CI, 0.28–0.93; P = 0.03). These findings suggest that the MNS16A VNTR polymorphism in the TERT gene has dual, conflicting roles in lung carcinogenesis. This polymorphism may increase the risk of lung cancer development, and may improve survival in lung cancer patients. (Cancer Sci 2011; 102: 144–149)
Telomeres are specialized nucleoprotein complexes composed of long arrays of double-stranded TTAGGG repeats, a G-rich 3′ single strand overhanging, and associated telomere-binding proteins.(1,2) Telomeres function to cap the ends of chromosomes and protect chromosomes from degradation, end-to-end fusion and atypical recombination; thus, telomeres are essential for maintaining the integrity and stability of chromosomes.(3,4) In addition to the end protective function, the progressive shortening of telomeres serves as a “mitotic clock”, which limits the lifespan of somatic cells.(3,5) Telomeres are maintained by telomerase, a ribonucleoprotein complex that consists of the catalytic component telomerase reverse transcriptase (TERT) and a telomerase RNA template. TERT synthesizes TTAGGG repeats from the RNA template and is the key determinant of telomerase activity.(6–9)
It has been proposed that telomere shortening and telomerase play dual roles in carcinogenesis. During the early phase of cancer development, telomere shortening can induce chromosomal instability, which is perpetuated through fusion–bridge–breakage cycles that increases the risk of cancer development. In this phase, telomerase, the main positive regulator of telomere length, helps to stabilize chromosomal instability and thus reduce cancer development.(10–12) In support of this hypothesis, several studies have reported that individuals with constitutionally short telomeres have a higher risk of developing cancers.(13–15) In addition, it has been reported that humans with dyskeratosis congenita, a hereditary disease with a partial telomerase deficiency, have an increased risk of developing malignant lesions.(16,17) In contrast to the early phase of cancer development, after telomerase is re-activated and the telomere attrition is stabilized, short telomeres inhibit tumor progression. In this later phase of cancer development, activation of telomerase can immortalize transformed cells and thereby promote cancer growth.(10–12) Indeed, several studies have demonstrated that the levels of telomerase expression/activity in cancer tissues correlate with advanced stage and poor prognosis in human cancers.(18,19)
Several studies have documented considerable inter-individual variation in telomere length and telomerase activity among healthy individuals of the same age.(20–22) Like many other phenotypic traits, functional polymorphisms in the TERT gene contribute to variation in the telomerase activity in the general population. Recently, the polymorphic tandem repeats minisatellite, MNS16A, in the downstream region of the TERT gene locus was demonstrated to influence the expression of antisense TERT mRNA.(23) Moreover, this MNS16A variable number of tandem repeat (VNTR) polymorphism has been reported to be associated with the risk of breast cancer.(24) Therefore, to investigate the role of the MNS16A VNTR polymorphism on the risk of lung cancer, we conducted a case–control study in a Korean population. In addition, we examined the effect of the MNS16 VNTR polymorphism on the survival outcome of patients with lung cancer.
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- Materials and Methods
- Disclosure Statement
Telomerase plays an important role in the development and progression of lung cancer. Therefore, it is assumed that functional polymorphisms that affect TERT gene expression or activity might contribute not only to the susceptibility to lung cancer but also to the prognosis of patients with lung cancer. In the present study, we investigated the effect of the MNS16A VNTR on the risk of lung cancer and the prognosis of patients with surgically resected NSCLC. Interestingly, the VNTR-243 allele was associated with an increased risk of lung cancer, whereas it was associated with a better survival outcome in patients with lung cancer. These findings suggest that alteration of the antisense TERT mRNA expression level by the MNS16A VNTR has a dual, conflicting role in lung carcinogenesis. Specifically, the MNS16A VNTR-243 allele may increase the risk of lung cancer development and may improve survival in lung cancer patients.
Wang et al.(23) reported that the MNS16A VNTR is located in the putative promoter region of the antisense TERT mRNA transcript and identified four different sized alleles (VNTR-333, -302, -272 and -243). The core sequence of MNS16A VNTR is a 23 bp tandem repeat or a 26 bp sequence with a CAT insertion. Wang et al.(23) found that the VNTR-302 allele, which contains two 23 bp repeats and three 26 bp repeats, has a significantly lower promoter activity and antisense TERT mRNA expression when compared with the VNTR-243 allele, which contains one 23 bp repeat and two 26 bp repeats. Based on this finding, they suggested that promoter activity depends on the length of the MNS16A VNTR and the 26 bp sequence with a CAT insertion functions as a repressor for the promoter of antisense TERT mRNA. There is growing evidence that naturally occurring antisense RNA transcripts negatively regulate gene expression by modulating sense RNA transcription, pre-mRNA splicing and mRNA stability, transport and translation.(26,27) Therefore, although the biological function of the antisense TERT mRNA remains unknown, it is likely that the antisense TERT mRNA acts as a repressor of TERT expression. Given that the VNTR-243 allele has a significantly higher antisense TERT mRNA expression compared with the VNTR-302 allele,(23) it can be expected that the VNTR-243 allele has reduced TERT expression and thus contributes to carcinogenesis. In accordance with this hypothesis, it has been reported that the MNS16A VNTR is associated with the risk of breast cancer and malignant glioma, as well as the prognosis of patients with brain cancer.(24,28,29)
A major strength of the present study was that the MNS16A VNTR was evaluated in relation to the susceptibility to lung cancer and prognosis of lung cancer patients, which may have a higher value in unraveling the role of the MNS16A VNTR during the multi-step process of carcinogenesis. It has been proposed that telomerase may play both anti- and pro-tumorigenic roles depending on the phase of carcinogenesis. In the earlier phases of cancer development, telomerase can help to control chromosomal instability by maintaining telomere length and suppress cancer initiation. In contrast, in the later phases of cancer development, telomerase is abnormally upregulated and facilitates tumor progression.(10–12,30,31) In agreement with this proposal, we demonstrated dual roles of the MNS16A VNTR in lung carcinogenesis: the higher production allele VNTR-243, for antisense TERT mRNA, was associated with an increased risk of lung cancer, whereas it was associated with a better survival outcome of patients with lung cancer, an opposing effect on the risk of lung cancer.
Few studies have investigated the MNS16A VNTR in relation to human cancers. Wang et al.(24) have reported that the VNTR-243 and VNTR-272 alleles are associated with a significantly increased risk of breast cancer compared with the VNTR-302 allele in a Chinese population. In addition, Wang et al.(29) have reported that the genotypes with at least one VNTR-302 or VNTR-333 allele exhibited a worse survival outcome compared with the homozygous genotypes of the VNTR-243 or VNTR-272 allele in non-Hispanic white patients with glioblastomas multiforme. These two studies are in agreement with our findings that the VNTR-243 allele was associated with an increased risk of lung cancer, whereas it was associated with a better survival outcome in patients with lung cancer. However, although the VNTR-272 allele was combined with the VNTR-243 allele as one group in the previous studies,(24,29) we combined the VNTR-272 and VNTR-302 alleles as one group. Like the VNTR-302 allele, the VNTR-272 allele contains three 26 bp repeats, a repressor for promoter of antisense TERT mRNA. In light of the putative function of the VNTR-272 allele, therefore, it is more reasonable that the VNTR-272 allele is combined with the VNTR-302 allele as one group.
In the present study, we detected a joint effect of MNS16A VNTR genotypes and smoking on the risk of lung cancer. Such a finding is biologically plausible because smoking has been shown to induce telomere shortening and to increase telomerase activity;(32,33) therefore, polymorphisms, including the MNS16A VNTR that can influence telomere shortening, may have a synergistic effect with smoking on lung cancer development.
There is growing evidence that inherited genotypes might affect the disease outcome by influencing the biological characteristics of the disease or an individual’s response to a specific therapy. In view of this point, the present study was aimed at evaluating the role of the MNS16A VNTR polymorphism as a prognostic factor in patients with lung cancer. SCLC is a rapidly progressive malignancy, which is usually a systemic disease at the time of initial presentation. Consequently, in the case of SCLC, surgery is reserved for selected patients and chemotherapy with or without radiotherapy is the standard therapy for most patients. Therefore, the survival analysis of the present study was limited to patients with NSCLC who underwent curative surgical resection. Future studies are needed to evaluate the role of the MNS16A VNTR as a predictive factor for survival outcome in SCLC and NSCLC patients receiving a specific therapy.
Genetic polymorphisms often show a lot of ethnic variation. In the present study, the frequencies of the VNTR-302, -272 and -243 alleles among the healthy controls were 0.944, 0.030 and 0.025, respectively, which were significantly different from those of healthy Chinese (0.947, 0.012 and 0.041, respectively).(24) Therefore, further studies are needed to clarify the association between the TERT MNS16A VNTR and lung cancer in diverse ethnic populations.
In conclusion, we found that the TERT MNS16A VNTR polymorphism exerts a dual, conflicting effect on the risk and prognosis of lung cancer. This finding suggests that a VNTR polymorphism in the TERT gene can be used not only as a marker for the genetic susceptibility to lung cancer, but also as a prognostic marker for patients with lung cancer. Our findings need to be validated by further functional studies as well as well-designed larger studies with diverse ethnic populations.