Thiyab, Asraa
(2025)
Polyadenylation as a target for the treatment of breast cancer.
PhD thesis, University of Nottingham.
Abstract
Polyadenylation is a fundamental step in gene regulation and mRNA maturation. It is the addition of multiple adenosine residues to the 3`end of an RNA to form a poly(A) tail. Poly(A) tails play a significant role in mRNA stability, nuclear export, and translation control. Cordycepin (3′-deoxyadenosine) is a natural product reported to have anti-cancer, anti-inflammatory, and other therapeutical effects in pre-clinical studies. It’s an adenosine analogue that lacks the 3`-hydroxyl group and is phosphorylated in the cell to form cordycepin triphosphate (cordTP). Its mechanism of action includes cordTP ability to directly inhibit polyadenylation in both cells and cell free systems, and incorporation into a growing polyadenylation reaction, thus acting as a chain terminator. However, cordycepin was also found to mediate the repress growth related signal transduction including the inactivation of mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase (PI3K) and Akt kinase. Many PI3K inhibitors show significant anti-tumour activity, while previous work in the de Moor lab showed that cordycepin can inhibit Akt and mTOR1 probably through affecting PI3K inhibition. Limited data indicate that polyadenylation factor knockdown does affect PI3K signalling, which suggests that polyadenylation is a new potential target to interfere with PI3K signalling. Developing other polyadenylation inhibitors and comparing their effects could validate polyadenylation as a target for cancer therapy.
To test the effects of compounds and cordycepin on polyadenylation, an assay suitable for screening potential polyadenylation inhibitors is required. This study describes the development of a high-throughput poly(A) assay (HbpA Assay) capable of directly detecting changes in polyadenylation on a micro-titer plate. This assay included the use of purified poly(A) polymerases (PAP), and mammalian PAP from MCF-7 nuclear extracts (both cell-free assay (CFA) and pre-treated assay (PTA)). The selected compounds (adenosine analogues, kinase inhibitors, and anti-cancer drugs) were tested for their effect on gene expression by RT-qPCR, and on polyadenylation in gel electrophoresis, the HbpA assays and in situ hybridisation.
By means of qPCR and effects on gene expression, cordycepin treatment inhibited relative mRNA expression of growth factor-independent mRNA markers and transcription factors, while it’s analogue 3`-aminocordycepin showed similar effects to that of cordycepin and 8-aminoadenosine. This altogether suggests these compounds repress gene expression of MCF-7 cell lines in cancer-related genes in a similar manner to cordycepin.
In HbpA assays, the triphosphates of these nucleoside significantly inhibited polyadenylation by E.coli and yeast PAP. The Pre-Treated Assay (PTA) and Cell-Free Assay (CFA) illustrated the significant inhibitory effects of cordycepin on the poly(A) tail in different PAP and that this inhibition was similar to 3`-aminocordycepin, 8-aminoadenosine, fludarabine and to the PI3K inhibitor LY294002.
Through Fluorescent In Situ Hybridization (FISH) with oligo d(T), cordycepin treatment was found to exhibit similar reduction in nuclear poly(A) RNA as fludarabine, 8-aminoadenosine, and compared to the nuclear reduction with LY294002 and PARP inhibitor Olaparib on poly(A) RNA. Similarly, these compounds reduced cytoplasmic poly(A) RNA, likely through direct inhibition of RNA processing pathways and its subsequent downstream effects leading to DNA condensation and cell apoptosis.
The above findings indicate that cordycepin inhibitory effects on gene expression of growth and proliferation genes associated to the PI3K pathways. This inhibition was also found in other adenosine analogues and is possibly due to the different modification such as the absence of 3`-OH group in 3`-aminocordycepin, 3`-deoxyinosine and cordycepin and the C8 modification attributing to the chain termination of polyadenylation in 8-aminoadenosine.The effects of cordycepin on polyadenylation was similar to that of adenosine analogues using the HbpA and FISH assay, confirming that these effects are most likely through direct/indirect blocks of polyadenylation and/or polyadenylation factors which gives out its therapeutic effects in various disease models; and not a second mode of action that causes the signal transduction effects. Additionally, cordycepin inhibited polyadenylation in the PTA more significantly than the CFA and suggests inhibition is by chain termination and sequestration of 3`polyadenylation factors. Furthermore, FISH data concluded cordycepin mainly reduced nuclear poly(A) RNA while cytoplasm poly(A) RNA was less affected, which suggests its repressive effects on the production of a mature RNA and reduction in poly(A) RNA for nuclear export. The effects of cordycepin and LY294002 on the poly(A) tail suggest that polyadenylation and the PI3K signalling pathway are functionally intertwined. These data altogether suggests that cordycepin, 3`-deoxyinosine, 3`-aminocordycepin and 8-aminoadenosine all are polyadenylation inhibitors and affect growth factor dependent gene expression in similar manner, suggesting that polyadenylation is indeed a target for anti-cancer drugs. Further screening using the HbpA assay with purified human PAP will identify more such compounds and a systematic study of their effects on gene expression and signal transduction will prove the link between polyadenylation and growth factor signalling.
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