Expression of shelterin and shelterin-associated genes in breast cancer cell lines

Abstract

Mammalian telomeric DNA consists of tandem repeats of the sequence TTAGGG associated with a specialized set of proteins, known collectively as Shelterin. These telosomal proteins protect the ends of chromosomes against end-to-end fusion and degradation. The objective of this project was to investigate whether expression of Shelterin and Shelterin-associated proteins are altered, and influence the protection and maintenance of telomeres, in breast cancer cells. Initial findings showed that most of the Shelterin and Shelterin-associated genes were significantly down-regulated (at the mRNA expression level) in a panel of ten breast cancer cell lines. Epigenetic alterations to DNA (methylation at CpG Islands) and histones can result in altered expression of genes. Further investigations showed that the promoter region of POT1 was partially methylated in the breast cancer cell line, 21NT. To support these observations, a DNA methylation inhibitor, 5-aza-CdR, and a histone deacetylation inhibitor, TSA, were used in an attempt to reactivate the expression of silenced genes. This work generated novel findings. Treatment with 5-aza-CdR and TSA resulted in the highest recovery of TIN2 and POT1 mRNA levels at both short-term (48 and 72 hours) and long-term (3 weeks) treatment of the breast cancer cell line, 21NT cells. In addition, POT1 promoter methylation was analysed before and after treatment of 21NT cells. Bisulphite sequencing data were consistent with the mRNA expression results, showing up-regulation of POT1, as all methylation sites were demethylated after the treatment of 21NT cells with 5-aza-CdR. These studies also showed for the first time that both the short-term (72 hours) and 3 weeks treatment of 21NT cells with 5-aza-CdR was able to increase telomere lengths (using four measurement methods, i.e. TRF, q-PCR, flow-FISH and iQFISH). Breast cancer cell lines expressed low levels of several telosomal mRNAs and that this down-regulation was found to be due in part to promoter methylation. Methylation was shown to be relieved through treatment of the cells with 5-aza-CdR and TSA; specifically, POT1 was shown to be up-regulated to a higher extent compared with other Shelterin genes. Given that previous studies involved over-expression of POT1 in telomerase-positive cells to demonstrate telomere length elongation, we addressed the possibility that over-expression of POT1 may affect telomere length in 21NT breast cancer cells. The results showed that the average telomere length of the POT1 over-expressing clones was increased by 2 to 3 kb compared with 21NT non-transfected and empty vector controls. The study also demonstrated that increased telomere length (by ectopic over-expression of POT1) is not due to a direct effect of telomerase enzyme activity. One explanation for this could be that POT1 may induce a negative regulator of telomerase activity to maintain telomere length. Taken together, the results generated in this project suggest that POT1 may control a localised activation of telomerase enzyme at the telomere end, and regulate stability of the Shelterin complex.