Abstract 3788: Contributions of novel nuclear nucleoside transporters, HNP36 and HNP32, to DNA synthesis during cell cycle

Abstract

Abstract Nucleosides are multi-functional and essential compounds in cells, acting as signalling molecules extracellularly and as the fundamental building blocks of nucleic acids intracellularly. Nucleosides are hydrophilic molecules and require nucleoside transporters (NTs) for translocation across membranes (Young et al. 2013). NTs are also the route of entry of a large class of clinically important nucleoside analog drugs used in anti-cancer treatments. Despite their clinical importance, NTs are poorly understood in terms of regulation, role and contribution to drug efficacy and nucleoside physiology. The SLC29 family of NTs possesses 4 isoforms including equilibrative nucleoside transporter 2 (ENT2; SLC29A2), which, in humans, is observed as a full length protein at the plasma membrane and intracellularly as splice variants (William & Lanahan 1995; Mangravite et al. 2003). The physiological relevance of these splice variants is not known but a correlation between mitogenic signalling and their presence in proximity to the nucleus suggested a novel cellular role for hENT2 at the nuclear envelope. Therefore, we hypothesized that increased expression of the nuclear isoforms of ENT2 under proliferative conditions contributes to translocation of nucleosides required for DNA synthesis into the nucleus during cell cycle. To investigate this hypothesis, we confirmed the presence of novel human ENT2 spliced variants (HNP36/HNP32) at the nuclear envelope and determined that these proteins were functional nucleoside and nucleobase transporters by heterologous expression of variants in oocytes. Human tissues and cell lines possess ubiquitous expression of ENT2 isoforms (based on qPCR) and we observed that there is increase in expression of the nuclear variants in proliferative cancer cells compared to healthy tissues confirming a correlation between proliferation and presence of the isoforms. Furthermore, HNP36/HNP32 appear to recruit full-length ENT2 to the nuclear envelope to form functional heteromers based on co-immunoprecipitation and confocal microscopy. Finally, we demonstrate that knockdown of HNP36/32 leads to a striking decrease in cell proliferation and a dysregulation of cell cycle such that cells are arrested on S phase which confirms that nuclear ENT2 isoforms are critically important during cell proliferation and needed for effective DNA synthesis. In conclusion, we show here that two novel ENT2 splice variants play a role in supporting cellular proliferation through a mechanism that involves the recruitment and formation of oligomers including the full-length ENT2 at the nuclear envelope. We propose a novel model for nuclear trafficking of nucleosides during DNA synthesis in cell proliferation, in which nuclear isoforms of ENT2 play a significant role and, therefore, may be a potential new target in cancer therapeutics. Citation Format: Natalia Grañe-Boladeras, W.J.Brad Hanna, Marçal Pastor-Anglada, Imogen R. Coe. Contributions of novel nuclear nucleoside transporters, HNP36 and HNP32, to DNA synthesis during cell cycle. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3788. doi:10.1158/1538-7445.AM2015-3788