In this report, we showed that, after adjustment for known breast cancer risk factors, shorter telomere lengths on chromosomes Xp and 15p were associated with an increased risk of breast cancer in pre-menopausal women. These data support our hypothesis that women who have telomere length deficiency on specific chromosome arms are at an increased risk of breast cancer. Our results are consistent with the concept by previous reports showing that telomere dysfunction plays a role in breast cancer development (19
A key feature necessary for telomere integrity is the maintenance of telomeric DNA at a critical length that allows assembly of the protective end structures. Current knowledge indicates that the telomere length and structure are tightly regulated by several distinct but cooperative mechanisms: (i) replenishment of telomere DNA by telomerase (a ribonucleoprotein reverse transcriptase) (22
); (ii) HR-mediated telomere maintenance or ALT (23
); (iii) length regulation through epigenetic modification of telomeric and subtelomeric DNA (24
); and (iv) negative regulation of the telomere length by a trimming mechanism (25
). Telomeres are thought to play a key role in tumor suppression by limiting the number of times a cell can divide, even in the presence of oncogenic mutations. Deficiency in telomere length maintenance is particularly relevant to carcinogenesis because hyper-proliferative cells could lead to progressive telomere shortening, ultimately generating uncapped telomeres that fuse with each other and leading to genomic instability that promotes malignant transformation. However, it is unclear whether it is the shortest telomeres or the mean telomere length that triggers the telomere dysfunction-associated responses. Several lines of evidence favor the concept that a subset of shortest telomeres triggers the telomere dysfunction-associated responses. First, it has been shown that individual dysfunctional telomeres are detected by the cells as DNA damage, triggering a cellular response (26
). Crossing telomerase knockout mice having short telomeres with those having long telomeres revealed that the loss of telomere function occurs preferentially on the shortest telomere and that the shortest telomeres, rather than the average telomere length, elicit a cellular response (27
). Second, several studies demonstrated that chromosome arms carrying the shortest telomeres were more often found in telomere fusions, leading to chromosomal instability (28
). Third, in humans, chromosome-specific telomere lengths are highly variable between chromosomal arms (31
). The potential implication of this chromosome arm-specific telomere length polymorphism is that chromosome arms bearing the shortest telomeres may predispose to the chromosome alterations and therefore have an impact on the evolution of tumors. Given such previous evidence, it is surprising that so little research has investigated the length of individual telomeres and its relationship to human disease. So far, there are only two recent small studies examined association between telomere lengths on a few chromosome arms and the risk of breast (34
) and esophageal (35
) cancers. The short telomere length on chromosome 9p was found to be significantly associated with the breast cancer risk (34
) and short telomeres on chromosomes 17p and 12q were found to be significantly associated with an increased risk of esophageal cancer (35
). To the best of our knowledge, the present study is the first that examined the association between all 92 individual telomeres in the human genome and risk of breast cancer. Our data suggest that short telomere lengths on chromosomes Xp and 15p are associated with the breast cancer risk in pre-menopausal women. Our data also revealed that telomere lengths between non-homologous telomeres were not correlated and are likely independent telomere-associated parameters that may carry information of clinical importance for cancer patients.
The comprehensive telomere analysis approaches used in the present study allowed us to examine the associations between telomere length variations and breast cancer risk. Since homogenous protection for all chromosome ends is required to prevent telomere dysfunction, we hypothesized that a high degree of telomere length variation represents a deficiency in telomere maintenance that is related to cancer susceptibility. Our data indicated that greater telomere length differences between homologous telomeres on chromosomes 9p, 15p and 15q were associated with the breast cancer risk in pre-menopausal women. To the best of our knowledge, the present report is the first to introduce HTLD as a novel phenotype of telomere deficiency and to have identified greater HTLD on chromosomes 9p, 15p and 15q as potential new risk factors for breast cancer in pre-menopausal women.
The present study also revealed that WCTLV among 46 non-homologous telomeres is associated with the breast cancer risk in pre-menopausal women. These data provided further new evidence that greater telomere length heterogeneity may contribute to an increased breast cancer risk in pre-menopausal women. A substantial number of human malignant tumors utilize ALT, a telomerase-independent telomere length maintenance mechanism that involves HR, to maintain telomere length. These include bone and soft tissue sarcomas, glioblastomas and carcinomas of the lung, kidney, breast and ovary (17
). ALT-mediated telomere length maintenance is characterized by highly heterogeneous telomeric DNA length (36
). Within an ALT+ cell, there are telomeres that ranged from <2 kb to >50 kb in length, and often several chromosome ends lacking any telomere signal (37
). Given the important role of HR in normal telomere biology, it is possible that dysregulation of HR-assisted telomere maintenance may result in increased telomere length variations between individual telomeres in somatic cells, resulting in increased risk of cancer. Additionally, recent data indicated that the telomere length is negatively regulated by a mechanism that trims telomeric repeats from the chromosome ends, preventing excessive long telomeres (25
), indicating that telomere lengths are carefully regulated by multiple mechanisms to maintain the telomeres at an ‘optimal’ length (not too long, not too short), essential for normal telomere homeostasis. Therefore, increased telomere length variation is likely representing deficiencies in certain aspect of telomere regulation machinery, leading to telomere instability and increased cancer risk. Our discoveries that greater telomere length variations between homologous telomeres and across all chromosome ends in a cell are associated with an increased risk of breast cancer in pre-menopausal women provided first evidence to support this hypothesis.
Our data indicated that the associations between telomere deficiencies and breast cancer risk in pre-menopausal women only involve a handful of chromosome arms (Xp, 9p, 15p and 15q). The reason why these chromosomal arms were involved is unknown, possibly related to telomere-mediated dysregulation of genes that reside on those chromosome arms and that are also involved in breast carcinogenesis. For example, telomere lengths are shown to be the critical players in regulating epigenetic modification of regional chromatin and these telomere-related epigenetic changes could result in epigenetic dysregulation of oncogenes and/or tumor suppressor genes (38
). Deficiency in 9p telomeres could potentially affect the stability of chromosome 9p, where the CDKN2A
locus (also known as the INK4a/ARF
locus) locates at 9p21. The CDKN2A
locus encodes two proteins, p16INK4a
, that regulate two critical cell cycle regulatory pathways: the p53 pathway and the retinoblastoma pathway (41
). Inactivation of the CDKN2A
locus removes an important barrier to tumor progression and 9p21 is a frequent target of inactivation by deletion or aberrant DNA methylation in a wide variety of human cancers (43
), including breast cancer (44
). Despite its importance in tumor suppression and considerable research, the cause of CDKN2A
inactivation by deletion or aberrant promoter methylation is currently unknown. To the best of our knowledge, no study has investigated whether chromosome arm-specific telomere dysfunction induces inactivation of specific tumor suppressor genes or amplification of oncogenes in human or model organisms. However, these are important biological questions that could reveal a critical step in carcinogenesis, thus warranting further investigation.
Results from the present study indicated that the association between chromosome arm-specific telomere deficiencies and breast cancer risk is restricted to pre-menopausal women. In post-menopausal women, there is only a suggestive association between the short telomere length on chromosome 15p and a decreased breast cancer risk. However, it should be noted that none of the associations in post-menopausal women was statistically significant after considering correction for multiple comparisons. Thus, future larger studies are needed to define the role of telomeres in breast cancer among post-menopausal women.
Given that this is a case–control study, a theoretical concern is that the telomere length in lymphocytes is affected by case status (reverse causality). Data by previous studies and by us indicated that the mean overall telomere length of blood leukocytes in breast cancer patients was not significantly shorter than in healthy women controls (11
), suggesting there is no significant shortening of blood leukocyte telomere length associated with having breast cancer. Although previous studies (47
) suggested that chemotherapy and/or radiotherapy can induce telomere shortening in leukocytes, all the blood samples in our study were drawn before any chemotherapy and radiotherapy treatments. Thus, reverse causality is not a plausible explanation for our observed associations. Our study is limited by its moderate sample size and does not have sufficient statistical power to detect any small to moderate associations (i.e. OR < 2.0). After Bonferroni's correction for multiple comparisons, observed case–control differences were only borderline significant for the HTLD on chromosomes 9p and 15p (Table ). Thus, future larger studies in independent populations are needed to confirm our findings and to generate more precise estimation on risk association.
In summary, the present study revealed that the short telomere length on chromosomes Xp and 15p, greater length differences between homologous telomeres on chromosomes 9p, 15p and 15q and greater WCTLV were associated with an increased risk of breast cancer in pre-menopausal women. Our data also suggest that telomere length variations between homologous telomeres or within a somatic cell are likely distinct phenotypes of telomere deficiencies. These new discoveries have potentially important clinical implications. If confirmed in future studies, telomere-related parameters are likely to be a useful panel of blood-based biomarkers for breast cancer risk assessment, given their strong associations with the breast cancer risk. These data also provided new clues on the possible role of telomeres in breast carcinogenesis.