Telomere length elongation by epigenetic modifier drugs: A potential mechanism by which cancer develops chemo-resistance

Abstract

Telomeres are specialised structures localized at the ends of eukaryotic linear chromosomes. Telomeres play an important role in protecting chromosome ends by preventing the activation of DNA damage response (DDR). In normal cells, telomeres become shorter with each cell division, which eventually leads to cellular senescence. Cancer cells, avoid telomere shortening by activating mechanisms that maintain telomere lengths. Most normal cells reach replicative senescence after 90 population doublings and then enter telomere crisis. This suggest that many premalignant cells lose their ability to keep dividing before they can accumulate additional mutations. Two mechanisms are known: telomerase reactivation and alternative lengthening of telomere (ALT). 80% -85% of cancer cells activates telomerase. The telomerase enzyme consists of two subunits, telomerase reverse transcriptase (TERT) and telomerase RNA (TR). ALT is active in around 15-20 % of cancer cells and maintains telomere lengths via alternative lengthening of telomere via homologous recombination. A common feature of ALT-positive human cells is the presence of C-circles and promyelocytic leukemia (PML) nuclear bodies (ALT-associated PML (APBs)). POLD3, a subunit of DNA polymerase has also been shown to be essential for ALT activity. The hypomethylating drug 5-aza-2′-deoxycytidine (5-aza) is widely used in the treatment of haematological malignancies. Moreover, cancer cells rapidly become resistant to the drug, leading to relapse. The study presented in this thesis investigated the short term (72 h) and long term (over one month) effects of 5-aza on telomere biology in breast cancer cells, with a focus on ALT activity and POLD3 expression. Telomere maintenance mechanisms were investigated using qPCR, c-circle and TRAP assays. The TIF (Telomere dysfunction induced foci) was used to detect DNA damage at telomeres and immunofluorescence for the detection of ALTassociated PML. POLD3 gene expression was quantified using qPCR and western blotting. Short-term 5-aza treatment modestly altered telomerase activity while significantly promoting ALT activation, which was accompanied by increased expression of POLD3. Silencing of POLD3 using siRNA in 5-aza treated cells resulted in telomere shortening. Long-term exposure to 5-aza led to the development of drug resistance, with cells acquiring the ability to proliferate at higher concentrations of 5-aza, and reduced sensitivity to doxorubicin, alongside increased migratory capacity. However, resistant cells exhibited enhanced sensitivity to radiotherapy. Collectively, our findings demonstrate that low-dose (10 uM) and prolonged exposure to 5- aza promotes ALT- dependent telomere maintenance through POLD3 upregulation, contributing to cancer cell survival and therapeutic resistance. These findings identify a novel mechanism by which cancer cells develop resistance to epigenetic therapy and also highlight telomere maintenance pathways as promising targets to improve treatment outcomes. Resistance to 5-aza significantly compromises the efficacy of combination therapies, including those involving chemotherapy and radiotherapy