Understanding the mechanism that regulates LINE-1 retrotransposons in breast cancer cells
Creators: name: Chen, Long
Publisher: name: The Australian National University
Year: 2013
Subjects: subject: QH452.3.C54 2013
subject: Transposons
subject: Mobile genetic elements
subject: Genetic disorders Molecular aspects
subject: Cancer cells
subject: Breast Cancer
subject: Genomes

description: The human genome contains large numbers of mobile genetic elements such as repeat elements and retrotransposons. When expressed, they can cause genomic instability and induce different genetic disorders. Genomic instability is thought to be a major driving force of cancer initiation and progression. Although, it has not been completely resolved whether genomic instability is a consequence of tumour progression or an active process that drives tumour evolution, an unstable genome can be indicative of poor prognosis for some types of cancer. In humans, the most predominant and the only active autonomous retrotransposon is long interspersed nuclear element 1 (LINE-1 or L1). The expression of LINE-1 elements can impact the human genome in a variety of ways, e.g. by creating insertional mutations, deletions, and double-stranded DNA breaks or by affecting the expression of nearby genes. LINE-1 elements have been reported to be active in neurons, germlines, transformed somatic cell lines, and various types of cancer cells, but strictly limited in most primary cells and tissues. In order to investigate the relationship between the activity of LINE-1 elements and the progress of cancer formation, breast cancer was chosen as the subject of this study because of easy availability of the different stages and grades of breast cancer cells and tissues. As expected, LINE-1 elements were found to be expressed in all types of breast cancer cells studied in this project, but not in non-cancerous epithelial cell lines or normal breast tissues. Interestingly, the expression pattern of LINE-1 elements was significantly different in breast cancer cells and tissues. Non-invasive breast cancer cells showed higher expression levels compared to the highly invasive cancer cells. These findings suggest that LINE-1 expression reaches a peak in the early stages of breast cancer, and decreases as the disease progresses into invasive states. Notwithstanding the discovery of differential LINE-1 expression between normal and cancer cells, LINE-1 expression is thought to be regulated by a variety of genome defense mechanisms. However, the pathways that activate or regulate the expression of LINE-1 elements in humans are not yet clearly understood. It has been shown that the majority of cancer and cancer{u00AD}derived cell lines, which have high levels of LINE-1 expression, are associated with loss of DNA methylation at the LINE-1 5'-UTR promoter regions. It has been proposed that hypomethylation of genome-wide DNA results in epigenetic changes that might derepress LINE-1 expression. In addition, a flurry of recent research in the fly and mouse revealed that small non-coding endogenous siRNAs silence LINE-1 expression by targeting the LINE-1 promoters at the transcriptional and/ or post-transcriptional level, suggesting that endo-siRNAs may play a role in regulation of LINE-1 elements. However, direct evidence for the existence of such endo-siRNAs is still lacking in human cells. Based on the evidence mentioned above, we hypothesized that LINE-1 activation is closely associated with hypomethylation of the promoter region of LINE-1 elements, and failure of expression of endo-siRNAs that might enforce the epigenetic silencing of LINE-1 elements through RNAi pathways. To validate this hypothesis, the DNA methylation profiles of LINE-1 promoters were analysed by two different methods in several breast cancer cells. As expected, the DNA methylation of LINE-1 promoters are high in normal breast cells and significantly low in breast cancer cells. The levels of DNA methylation are also inversely proportional to the expression of LINE-1 elements. Using deep sequencing of small RNA libraries from normal and breast cancer cells, our laboratory has recently identified a set of naturally occurring antisense endo-siRNAs that were differentially expressed in these cells. Notably, the identified endo-siRNAs are perfectly complementary in sequence to the LINE-1 bidirectional promoters. The existence of such endo{u00AD}siRNAs was investigated by Northern blot analysis in a wide range of cancer cells. These endo-siRNAs are significantly depleted in almost all the breast cancer cells tested in this study, correlating with the uncontrolled expression of LINE-1 elements. In addition, the functions of these differentially expressed endo-siRNAs were characterized by DNA methylation analysis in various types of breast cancer cells. In this study we demonstrated that the overexpression of endo-siRNAs in cancer cells resulted in marked silencing of LINE-1 expression through global hypermethylation, including gain of new DNA methylation at the LINE-1 5'-UTR promoter regions. The finding that endo-siRNAs can repress LINE-1 activity through DNA methylation provides a new insight into the function of endo-siRNAs in the silencing of retrotransposons and the maintenance of genome integrity in human cells. In addition to exploring the mechanisms that regulate LINE-1 elements, this study also investigated the functional implications of LINE-1 expression and its subcellular localization in various stages and grades of clinical breast cancer tissues. Clinicopathologic parameters and survival outcomes were investigated in association with the cytoplasmic and nuclear expression of LINE-1 elements using univariate and multivariate statistical analysis. The expression of LINE-1 elements is markedly higher in less-invasive DCIS tissues than that of highly invasive IDC tissues (p&lt;0.001). Interestingly, tumours with high nuclear expression of LINE-1 elements were significantly associated with lymph node metastases (p = 0.001) and worse patient survival (p&lt;0.0001) than those with cytoplasmic expression. Our study suggests that there is a considerable heterogeneity in expression of LINE-1 elements in breast tumour tissues, which relate to the biological and clinical differences in overall patient survival. Overall these studies show that expression of LINE-1 may possibly serve as a potential marker for early breast cancer diagnosis and prognosis. In conclusion, this thesis made three important findings. First, DNA methylation and endo-siRNAs are involved in the regulation of human LINE-1 expression. Second, endo-siRNAs are upregulated in normal breast cells that can silence LINE-1 expression through DNA hypermethylation of the LINE-1 promoters. In contrast, the expression of endo-siRNAs are depleted in breast cancer cells. Third, nuclear but not cytoplasmic expression of LINE-1 is associated with poorer survival in patients with invasive breast cancers. The significance of this thesis study is that it not only provides us with a deeper understanding of the mechanism involved in the regulation of human LINE-1 elements, but also provides new insights for using this element as a possible biomarker in clinical use.