Examination of the queuine-insertase RNA modificaton in breast cancer

Abstract

Queuine is a bacterial-derived metabolite that is harvested by eukaryotes and incorporated into tRNA at GUN anticodon sequences (N=A/C/G/U) by displacing the nuclear encoded guanine base by the queuine-insertase enzyme. Despite efforts over 40 years a specific function for the queuine modification remains elusive. The queuine modification is absent in many human cancers. Ominously, depletion of the modification correlates with poor prognosis and decreased patient survival. There are hints in the literature that the depletion of queuine leads to an altered metabolism and increased proliferative rates, but these results are complicated by undefined cell culture conditions. Here, a completely chemically defined serum-free, queuine-free culture medium was developed for the growth of the triple negative breast cancer cell line, MDA-MB-231. The incorporation of queuine into the RNA of the MDA-MB-231 cells was examined with saturating concentrations observed at 50 nM by 24 hours. Queuine modification of the RNA of MDA-MB-231 cells did not affect their proliferation or metabolism. However a stimulation of proliferation in response to superphysiological concentrations of queuine (25-150 μM) was observed, while higher concentrations again (250-750 μM) were inhibitory to proliferation. The proliferative effects observed at super-physiological concentrations were considered to be due to a separate mechanism to the RNA modification. In an effort to expose the function of the RNA modification, a number of queuine analogues (synthesised by our collaborators) were examined. In vitro screening assays were developed to assess analogues as substrates of the queuine-insertase enzyme and as substrates of the HPRT purine salvage enzyme. These analogues were anticipated to be specific antimetabolites to queuine-insertase enzyme and to act as anticancer agents. One such analogue, MC4a, was found to be a specific substrate of the queuine-insertase enzyme and was incorporated into the RNA of MDA-MB-231 cells.