Equilibrative nucleoside transporter 2 (ENT2) is a bidirectional transporter embedded in the biological membrane and is ubiquitously found in most tissue and cell types. ENT2 mediates the uptake of purine and pyrimidine nucleosides and nucleobase besides transporting a variety of nucleoside‐derived drugs, mostly in anticancer therapy. Since high expression of ENT2 has been correlated with advanced stages of different types of cancers, consequently, this has gained significant interest in the role of ENT2 as a potential therapeutic target. Furthermore, ENT2 plays critical roles in signaling pathway and cell cycle progression. Therefore, elucidating the physiological roles of ENT2 and its properties may contribute to a better understanding of ENT2 roles beyond their transportation mechanism. This review is aimed at highlighting the main roles of ENT2 and at providing a brief update on the recent research.

Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into these mutations’ molecular properties and implications in tumorigenesis. The generation of such models harboring the alterations is feasible with the CRISPR-based gene editing technology. Moreover, the development of new CRISPR applications, particularly DNA base and prime editing, has considerably improved the precision and versatility of gene editing. Here, we employed the prime editing tool to revert a TP53 missense C > T mutation (L194F) in a T47D luminal A breast cancer cell line. In parallel, this prime editing tool was also utilized to introduce the L194F mutation in HEK293T cells. To assess the prime editing efficiency in both cell lines, we first performed Sanger sequencing in the prime-edited cells pool and single cell-derived clones. However, the Sanger sequencing approach did not detect any base substitution in these cell lines. Next, by employing the more sensitive amplicon target sequencing, we managed to identify the expected substitution in these T47D and HEK293T cells, albeit the editing efficiency was low. In light of these findings, we discussed the technical aspects and provided suggestions for improve the prime editing workflow and efficiency for future experiments.

Although purine metabolism is one of the most impacted pathways in colorectal cancer (CRC), little is known about the role of equilibrative nucleoside transporter 2 (ENT2) in CRC development and its association with the altered purine metabolism pathway. This study aimed to determine the role of ENT2 in altered purine metabolism in the early and late stages of CRC using CRISPR/Cas9 gene editing tools and a variety of functional experiments. The expression of ENT2 was significantly higher (P < 0.001) in all CRC cell lines as compared to the normal colon cells. The two CRC cell lines with the highest ENT2 expression, the early stage HT29 cells and the late stage DLD1 cells, were knocked out (KO) using the CRISPR/Cas9 tool. The hypoxanthine (HPX) level and the xanthine oxidase (XO) activity were significantly higher in both HT29/KO and DLD1/KO single cell-derived clones (P < 0.01). The increase in HPX level and XO activity were associated with an elevation in the reactive oxygen species (ROS) level. These data suggest that the ENT2 KO elevated the ROS levels induced apoptosis and impaired the cell proliferation of the early stage of CRC cell line, i.e., HT29/KO clonal cells. In this context, targeting ENT2 gene might be a potential strategy in CRC treatment by increasing the production of ROS and hence, inducing the apoptosis pathway.