## 4 Role of Ap4A: Functions in Normal and Cancer Cells
### Cell Growth and Proliferation  
Cancer cells are known for their ability to undergo significant metabolic reprogramming to sustain their rapid growth and proliferation. This reprogramming is crucial for meeting the high demands for energy and biosynthetic precursors required for tumor development. Thus, Ap4A plays a pivotal role in this metabolic adaptation, influencing both energy homeostasis and the activity of metabolic enzymes. In cancer cells, Ap4A levels are often dysregulated, leading to excessive cell division and tumor growth, suggesting its role in malignant transformation and proliferation. In addition, emerging evidence suggests that Ap4A contributes to tumor development by promoting angiogenesis, facilitating metastasis, and enhancing resistance to apoptosis [24].  
For instance, Ap4A modulates cyclin-dependent kinases (CDKs) and other regulators, affecting cell proliferation. Cell cycle progression is a tightly regulated process orchestrated by a sequential activation of CDKs and their regulatory partners, the cyclins. The cell cycle consists of four main phases: G\({}_{1}\) (cell growth), S (DNA synthesis), G\({}_{2}\) (preparation for mitosis), and M (mitosis). Progression through these phases depends on the activation of specific cyclin-CDK complexes, which are themselves regulated by phosphorylation, proteolysis, and CDK inhibitors [32].  
In normal cells, two major checkpoints govern the fidelity of cell division: the G\({}_{1}\)/S checkpoint, ensuring DNA integrity before replication, and the G\({}_{2}\)/M checkpoint, which prevents mitotic entry in the presence of damaged or unreplicated DNA [33]. A key feature of tumor cells is the decoupling of DNA damage from checkpoint responses. While normal cells undergo senescence or apoptosis when checkpoint fidelity fails, tumor cells with defective checkpoints continue to divide, accumulating mutations that drive oncogenesis [34; 35]. Thus, in breast carcinoma cell lines, it was found that knocking down _NUDT2_ led to reduced cell proliferation and an accumulation of cells in the G\({}_{0}\)/G\({}_{1}\) phase of the cell cycle, further highlighting Ap4A's role in regulating cancer cell growth [36].  
Further research showed that by inhibiting WNT/\(\beta\)-catenin pathway, which regulates cell proliferation, differentiation, and migration, Ap4A can suppress the transcription of genes that promote tumor growth and metastasis [37]. Through its ability to downregulate WNT/\(\beta\)-catenin signaling, diadenosine tetraphosphate exerts an additional level of control over oncogenic pathways, further positioning it as a key regulator of cancer progression.  
Early studies proposed that Ap4A, synthesized by DNA ligase III, delays the initiation of new DNA replicons (the starting points for DNA replication). This delay is believed to prevent replication, giving cells time to repair potential DNA damage, particularly in ovarian cancer cell lines [3]. Such a mechanism could limit uncontrolled proliferation, positioning Ap4A as a potential negative regulator of cell cycle progression in ovarian cancer. Ap4A is synthesized under stress conditions, such as DNA damage, where it inhibits the initiation of DNA replication, contributing to the maintenance of genomic integrity [3; 38]. DNA ligase III synthesizes Ap4A, which acts as a primer for ADP-ribosylation by PARP. The ADP-ribosylated Ap4A binds to the Ap4A-binding site on the protein associated with DNA polymerase-a-primase and inhibits it, possibly by reducing its interaction with DNA. ADP-ribosylated Ap4A also diffuses to other replication forks and inhibits DNA polymerase-a-primase there [39]. It was observed that cells depleted of certain DNA repair proteins such as poly (ADP-ribose) polymerase (PARP1), showed increases in Ap4N (N = adenosine, guanosine, uridine, or cytidine), up to 14-fold Ap4A, preventing replication fork progression.