Post-transcriptional regulation of glutaminase enzyme by HuR and its relationship with high glutaminolytic levels in tumors

Abstract

The genetic and metabolic particularities of tumor cells are crucial to their high rates of cell division, normally associated with an increase on glutamine consumption used for energy and cellular biosynthetic blocks (lipids, proteins and nucleic acids) production. The first step on the glutamine metabolism is performed by the glutaminase enzyme and produces glutamate. The glutaminase has three isoforms derived from two different genes: GLS encodes the Kidney-Type Glutaminase (KGA) and Glutaminase C (GAC), products of alternative splicing, and GLS2, which encodes Liver-type Glutaminase (LGA). Preliminary data from our laboratory indicated that KGA and GAC (divergent only by the C-terminus) are more expressed in breast tumor in comparison to adjacent non-transformed tissues. Moreover, GAC isoform was shown to have higher mRNA and protein levels than KGA in breast cancer cell lines compared to normal breast epithelial cells. Our studies pointed to HuR protein as a potential regulator of GAC mRNA stability. HuR is highly expressed in cancer cells and could be related to GAC increased protein levels of in tumor tissues. During the master's degree period, preliminary bioinformatics analysis of microarray databanks indicated a heterogeneous correlation between HuR, GAC and KGA accordingly to the tissue type analyzed. An expanded evaluation with additional microarray and RNA-seq data will be performed to further validate the findings. Subcellular HuR location may be involved with this heterogeneity, as HuR acts in several mRNA regulatory mechanisms such as splicing, stability and translation modulation. HuR silencing in PC-3 prostate cancer cell line using viral system increased GAC and decreased KGA protein levels, while V5-6xHis-HuR construction overexpression led to diminished GAC and increased KGA levels. Reporter assays using luciferase activity directed by the 3'UTR of these isoforms and in silico evaluation of regulatory sequences within the intron 14 (decisive in the alternative splicing) suggested that HuR is potentially involved on the GLS alternative splicing and KGA selection. The HuR's role on the GLS alternative splicing will be assessed by a bichromatic reporter plasmid containing the RG6 minigene-intron 14 construct. The enzymatic activity of glutaminase as well as the extra and intra-cellular levels of nutrients, notably glutamine, will be evaluated by NMR after knocking down or over-expressing HuR. All experiments will be conducted with PC-3, DU-145 cell lines and non-transformed immortalized iPrEC. Preliminary pull-down results showed direct interaction between purified recombinant HuR and the 3'UTR GAC. Confirmation of these results and R-EMSA of the 3' UTR full length and/or subclones constructs will confirm this interaction and define the involved regions. Finally, in vitro experiments and animal models will be used to verify the correlation between obesity/inflammation (through NFkB/HuR) and glutaminase activity. (AU)