Telomere length and genetic analyses in population-based studies of endometrial cancer risk

Introduction

Telomeres are structures at the ends of linear chromosomes composed of proteins complexed to long hexameric (TTAGGG)_n DNA repeats charged with the critical role of maintaining structural integrity ¹. Due to limitations in lagging strand synthesis, human telomeres shorten by 50-100 bp per mitotic division ². Therefore, telomeres are analogous to a “molecular clock” reflecting the number of divisions a cell has undergone ³. When telomeres shorten to a critical length the cell enters replicative senescence. However, according to the telomere hypothesis of carcinogenesis, if the Rb and p53 signaling pathways have been inactivated, cell division continues further eroding telomeres with a concurrent increase in genomic instability. Upon reaching crisis, a second proliferation block characterized by gross chromosomal aberrations, the vast majority of these cells undergo apoptosis. A rare cell may escape by re-expressing telomerase reverse transcriptase (TERT), which may facilitate tumorigenesis. Reactivation of telomerase is detected in >90% of human tumors, making it one of the most common abnormalities in cancer cells ¹.

Endometrial cancer, the most common gynecologic malignancy among women in the US, arises from the epithelial lining of the uterine corpus. Younger age at menarche, older age at menopause, and nulliparity have been associated with endometrial cancer risk ⁴ suggesting a higher number of total lifetime menstrual cycles may lead to accelerated telomere erosion in endometrial cells. To date, data to support this hypothesis are limited. A few small studies found shortened telomere length in endometrial tumor tissue compared to adjacent normal ^5-7, whereas another did not find significant differences in length between adjacent normal, endometrial hyperplasia and endometrial cancer ⁸. It is difficult for such studies to distinguish whether short telomeres trigger early carcinogenic events or are a byproduct of the cancer cell's high proliferative rate.

Telomere length in peripheral blood leukocytes (PBL) has emerged as a potential biomarker for chronic disease risk. Short telomere length in PBL has been associated with increased risk of some cancers (e.g. head and neck, esophageal, lung, renal cell, and bladder) ^9-13, but not all (melanoma and breast) ^14-18. Our study is the first to examine the relationship between relative telomere length (RTL) and endometrial cancer risk using 2 nested case-control studies, the Nurses’ Health Study and the Women's Health Study. We also investigated whether common variation at 5 telomere maintenance gene loci (TERT, TNKS2, POT1, TERF1, TERF2) were associated with endometrial cancer risk.

Materials and Methods

Results

Descriptive characteristics of the study populations have been published ²³. Briefly, mean age at biospecimen collection was similar among cases and controls as expected from the matched design. In both cohorts, cases had greater mean BMI at diagnosis and were more likely to have never smoked than controls. Most participants were postmenopausal at diagnosis. Among postmenopausal women, cases were more likely to have used postmenopausal hormones.

Based on 1181 controls with blood specimens that were successfully assayed, we examined the relationship of RTL with several endometrial cancer risk factors (Table I). RTL showed significant inverse relationships with age (P_trend = 0.03) and BMI at blood draw (P_trend = 0.003). Weight gain from age 18 until blood draw among NHS participants showed a significant inverse association with RTL (P_trend = 0.001). Among all controls, we found a highly significant positive association with age at menarche (P_trend < 0.001), where women who had a later age at menarche tended to have longer RTL. RTL distributions did not differ by smoking status (p=0.41). RTL was also not associated with cigarettes per day among ever smokers, oral contraceptive use, or age at menopause (P_trend ≥ 0.34).

To minimize the potential for reverse causation, we restricted RTL analyses to 279 women with incident endometrial cancer diagnosed after blood collection and their 791 matched controls. We did not observe a significant relationship between RTL and endometrial cancer risk (P_trend = 0.37; Table II). Women in the shortest quartile of RTL (1^st quartile) had a multivariate adjusted OR = 1.20 (95% CI = 0.73 – 1.96) compared to women in the longest quartile of RTL (4^th quartile). This risk was attenuated when restricted to women postmenopausal at diagnosis (OR = 1.04, 95% CI = 0.61 – 1.79).

Using blood and buccal cell DNA samples from all women (674 cases, 1685 controls), we assessed whether endometrial cancer risk was associated with single nucleotide polymorphisms (SNPs) at telomere maintenance gene loci. Based on a Bonferroni corrected p-value of 0.0012, departure from Hardy-Weinberg equilibrium in the control populations was evident for 6 of the 41 SNPs genotyped [RS2242652 (TERT), rs2736098 (TERT), rs11972248 (POT1), rs929365 (POT1), rs1530941 (TNKS2), rs166134 (TERF2)] and thus were excluded from analyses. We observed positive multivariate-adjusted per allele increases in endometrial cancer risk with rs2736122 (TERT; OR = 1.18, 95% CI = 1.01 – 1.38) and RS12412538 (TNKS2; OR = 1.16, 95% CI = 1.00 – 1.34) (Table III). Too few minor allele carriers of RS6882077 (TERT), RS6989159 (TERF1), and RS6989493 (TERF1) were available for analysis. In multivariate analyses, p-values for tests of heterogeneity comparing NHS and WHS results were >0.05 for all except one SNP, RS153045 (TERF2). No association with endometrial cancer risk was observed for RS153045 (OR = 0.94, 95% CI = 0.80 – 1.11) in the NHS, whereas a positive association with risk was observed (OR = 1.45, 95% CI = 1.02 – 2.08) in the WHS.

Oxidative stress preferentially damages telomeres, contributing to telomere shortening ²⁴. Obesity and smoking are associated with an increase in systemic oxidative stress ^{25, 26} and, as discussed below, age at menarche may serve as a proxy for adolescent adiposity. Thus, we conducted exploratory analyses of SNP-environment interactions with smoking, BMI, and age at menarche. One additive interaction was observed at p < 0.05 between RS401681 (TERT) and BMI. However, when stratified by BMI, RS401681 was not associated with endometrial cancer risk.

Our rationale for investigating common variation at these loci was based on the notion that genetic variants tagged by these markers influence protein expression levels and therefore would have an effect on telomere length. To test our assumption, we examined the relationship between the SNPs in our study and RTL. We did not find significant associations between the SNPs analyzed and RTL, adjusted for age and study.

Discussion

Since the endometrial surface is a highly proliferative tissue, according to the telomere hypothesis of carcinogenesis, one might expect that women with shorter RTL would be at greater risk of endometrial cancer. We did not find evidence to support an association with RTL as measured in peripheral blood leukocytes among 279 incident invasive endometrial cancer cases and 791 matched controls nested within the Nurses’ Health Study and Women's Health Study. Women in the shortest RTL quartile were not at significantly greater risk compared to women in the longest RTL quartile (OR = 1.20, 95% CI = 0.73 – 1.96; P_trend = 0.37). Although, our power was limited by the small sample size having only 35% power to detect a significant trend.

DNA from 674 endometrial cancer cases and 1685 matched controls were genotyped for tag SNPs across gene loci containing the core proteins that localize to telomeres in order to shape and tightly regulate the length of telomeres^{27, 28}. An elevation in risk was observed among women carrying variant alleles of RS2736122 at the TERT locus (P=0.03) and RS12412538 at the TNKS2 locus (P=0.05). However, after adjusting the significance level using either a Bonferroni correction or the less conservative False Discovery Rate procedure ²⁹, neither of these SNPs reached significance. RS153045 at the TERF2 locus displayed significant heterogeneity between the studies. This SNP was not associated with endometrial cancer risk in NHS, but showed a 45% increased risk per variant allele in WHS. We believe the heterogeneity was due to chance. After adjustment for multiple comparisons, Rafnar et al observed that RS401681 at the TERT-CLPTM1L locus was significantly associated with basal cell carcinoma (OR=1.25), lung (OR=1.15), bladder (OR=1.12), prostate (OR=1.07), and cervical cancer (1.31), but not with breast (OR=0.98) or endometrial cancer (OR=1.21) ²⁰. We had 80% power to detect an OR=1.21 between RS401681 and endometrial cancer risk. Our study supports a null association between RS401681 and endometrial cancer risk. RTL was not associated with the SNPs analyzed in this study, which is not entirely surprising given the constraint on nucleotide diversity observed in these genes ³⁰.

This is the first population-based investigation of the relationship between RTL, telomere-related gene SNPs and endometrial cancer risk. Whereas some studies found evidence for telomere attrition within endometrial tumors compared to matched normal adjacent tissue^5-7, a recent report on telomere dynamics during uterine carcinogenesis did not find consistent telomere shortening among endometrial cancer samples compared to surrounding normal tissue. An increase in telomere length was observed in roughly a third of patients ⁸. Additionally, Type I endometrial cancers, which make up the majority of endometrial tumors, generally lack defects in the p53 gene and associated chromosomal instability ³¹, two key components of the telomere hypothesis of carcinogenesis ¹. Over 95% of cases in our study were classified as Type I endometrial tumors. Our results and observations from these prior studies suggest telomere shortening may not be a major mechanism of Type I endometrial carcinogenesis.

We observed statistically significant inverse correlations with age, BMI, and weight gain from age 18. Telomere length decreases with age as a result of the end replication problem. Obesity, a state of chronic inflammation and oxidative stress ²⁵, is believed to contribute to telomere attrition ³², which has been observed in some prior studies of women ^33-36. We also observed a significant association between longer RTL and later age at menarche (P_trend < 0.001). We do not have a plausible biological reason to believe the onset of menarche would have a direct effect on telomere length or vice versa. The positive correlation between RTL and age at menarche was attenuated slightly, but still significant after adjusting for BMI at diagnosis (data not shown). Since adolescent body fatness has been inversely associated with age at menarche ^{37, 38}, the observed RTL association with age at menarche may actually reflect an association with adolescent body size.

We cannot be certain that RTL measured in blood reflects telomere length in endometrial tissue. However, telomere lengths are highly synchronized in fetal tissues ³⁹ and at birth among white blood cells, umbilical artery cells, and skin cells^{40, 41}. Though variation in telomere length increases with age, studies that have compared telomere length of blood DNA with that of matched skin ^{42, 43}, synovial tissue ⁴³, or fibroblasts ⁴⁴ in older participants have found significant correlations between the pairs of tissues. Interindividual variation in telomere length far exceeds the variation between different tissues from the same individual^{40, 41}. This suggests blood serves as an adequate proxy for non-malignant endometrial tissue.

Our analyses benefit from the nested case-control design as cases and controls were drawn from well-characterized relatively homogeneous populations limiting selection bias. To prevent invalid risk estimates due to the limitations of retrospective case-control studies, such as cancer treatment and/or the disease itself influencing telomere length, we restricted telomere length analyses to endometrial cancer cases diagnosed after blood collection. Cases were followed for a median of 5.9 years prior to diagnosis (range: 1 month to 14.3 years). Estimates were similar after excluding cases diagnosed within 12 months of blood draw (n=30).

Due to recent evidence suggesting racial differences in telomere length dynamics ⁴⁰, we restricted our analyses to white women as most women in the NHS and WHS are Caucasian, limiting the generalizability of our results. Recent studies have also demonstrated differences in telomere attrition rates between individuals, which were mainly dictated by baseline telomere length^{40, 45-47}. We were not able to assess telomere attrition rate as a risk factor since we were only able to measure telomere length at one point in time. Nevertheless, Nordfjäll et al. ⁴⁷ found comparable telomere attrition rates among individuals who later developed cancer and those who did not. Since RTL distributions were similar among cases and controls within our study, we have reason to believe attrition rates between these two groups would be alike as well.

In summary, we did not observe a significant elevation in endometrial cancer risk associated with shorter telomere lengths. We observed a nominal elevation in endometrial cancer risk associated with genetic variants at the TERT and TNKS2 loci. Overall, our data provide little support for leukocyte RTL as a biomarker of endometrial cancer risk among white women. Additional prospective epidemiologic studies are needed to confirm our findings as well as explore the relationship of telomere length dynamics in other racial and ethnic groups.